Eukaryotic genome replication is surveyed by the S-phase checkpoint, which coordinates sequential origin activation to prevent the exhaustion of poorly defined, rate-limiting replisome components1-3. Here we show that excessive origin firing saturates chromatin-bound proliferating cell nuclear antigen (PCNA)-a sliding clamp for DNA polymerase processivity and Okazaki fragment processing4-thereby restricting further PCNA loading and lagging-strand synthesis when checkpoint control is lost. PCNA-associated factor 15 (PAF15) emerges as a dosage-sensitive regulator of this process5-9. During unperturbed S phase, the entire soluble PAF15 pool binds to chromatin, leaving no reserve to stabilize PCNA under conditions of excessive origin activation. PAF15 binds to PCNA specifically on the lagging strand through a high-affinity PIP motif and occupies the DNA-encircling channel, protecting the clamp and associated enzymes from premature unloading by the ATAD5-RFC complex. Conversely, overexpression of PAF15 or forced redistribution to the leading strand disrupts replisome progression and induces cell death. These detrimental effects are mitigated by Timeless-Claspin, which blocks PAF15-PCNA binding on the leading strand. E2F4-mediated repression fine-tunes PAF15 expression to ensure optimal dosage and strand specificity. These findings reveal a previously unrecognized replisome constraint: when PAF15-PCNA assemblies are exhausted, the S-phase checkpoint globally restricts origin activation, linking a strand-specific rate-limiting mechanism to global replication dynamics.

Delivering hydrophilic macromolecules and particulate systems into skin is limited by the strong transport resistance of the stratum corneum and viable epidermis. Microneedling offers a potential solution, but its usefulness depends on whether a practical treatment protocol can reproducibly generate barrier-bypassing microchannels. Here, we investigated whether an electrically powered microneedling pen, Dermapen, can be used to create such access pathways in excised human skin and whether this improves delivery of liposomal, macromolecular, and particulate model systems. A 12-needle Dermapen protocol generated quantifiable microchannels with a mean measured depth of 450 ± 156 µm, extending well beyond the principal epidermal barrier layers. In Franz diffusion cells, Dermapen pretreatment markedly increased 30 h cumulative recovered fluorescence from Texas Red-dextran-containing liposome dispersions and increased fluorescence signal within the viable epidermis and dermis by cryosection imaging and depth profiling. In intact skin, formulation-dependent differences were more evident, whereas these differences were greatly reduced once microchannels were created, consistent with physical barrier bypass as the dominant mechanism. Microneedling also enhanced intraskin delivery across distinct payload classes, including free 70 kDa dextran, liposome dispersions, and 1 µm fluorescent particles, and brief post-application topical massage further amplified delivery. These findings show that controlled Dermapen-generated microchannels are the primary enabler of deep intraskin drug delivery, while post-application massage can further increase payload deposition. By linking quantified microchannel formation to downstream transport of multiple payload classes, the study provides a practical framework for reproducible device-assisted cutaneous and transdermal delivery research.

The stratum corneum (SC) is the principal barrier to topical and transdermal delivery, yet how lipid-based formulations interact with the SC lipid matrix remains incompletely resolved. Here, we compared conventional lecithin/cholesterol liposomes, hexosomes, ultradeformable liposomes, and a surfactant-only micelle control in ex vivo human skin after 4 h Franz-cell exposure. Depth-resolved Laurdan generalized polarization (GP) imaging of cryosections was used to quantify formulation-induced changes in SC lipid organization, while DiD fluorescence and deuterated-lipid stimulated Raman scattering (SRS) provided orthogonal readouts of formulation-derived material within the tissue. Across formulations, GP decreased relative to matched controls, with the largest effects in the outer SC. Hexosomes produced larger outer-SC GP reductions than conventional liposomes (ΔGP ≈ 0.25-0.33 vs 0.14-0.18). In a separate donor-matched series, ultradeformable vesicles induced stronger SC remodeling than conventional liposomes, whereas Tween 20 micelles produced smaller effects. DiD and SRS signals were predominantly localized to the SC and decreased with depth, with SRS indicating a more surface-weighted distribution for rigid liposomes than for ultradeformable vesicles. Together, these findings identify formulation-dependent SC lipid remodeling, involving lipid redistribution and reduced local packing order, as a plausible mechanism by which lipid-based formulations may contribute to penetration enhancement reported in previous studies.

Understanding molecular organization across complex biological interfaces is essential for evaluating biomimetic tissues and identifying pathological remodeling. Here, hyperspectral stimulated Raman scattering (SRS) microscopy combined with spectral deconvolution was employed for label-free characterization of native human skin, reconstructed three-dimensional (3D) skin models, and a melanoma skin model. Layer-specific analysis across the stratum corneum, epidermis, and dermis enabled investigation of biochemical organization and compositional heterogeneity. In the stratum corneum, variations in the C–H stretching region revealed differences in lipid chain ordering and lipid-to-protein balance, with native human skin exhibiting higher lipid ordering and greater compositional homogeneity than reconstructed models. Amide I analysis demonstrated altered keratin-associated secondary structure organization in reconstructed skin systems, including increased β-sheet and disordered spectral contributions relative to native tissue. Melanoma-associated skin models exhibited enhanced nucleic-acid-related spectral contributions together with reduced collagen-associated signals in the dermal compartment, consistent with extracellular matrix remodeling. These findings demonstrate that hyperspectral SRS microscopy combined with spectral deconvolution provides a sensitive and label-free platform for molecular-level characterization of complex skin biointerfaces and comparative evaluation of native, engineered, and pathological skin systems.

Synapses are essential for neuronal function and are central to numerous neurological disorders including developmental and neurodegenerative diseases. Synapses structurally constitute a very small proportion of a neuron, and their protein content is difficult to study using whole tissue preparations. Especially studying synapses on the functional level is challenging. To overcome this limitation, synapses can be captured as synaptosomes generated through enrichment of isolated nerve terminals. Such synaptosomes have a re-sealed plasma membrane and can regenerate their membrane potential and perform physiological function, for example neurotransmitter release. Synaptosomes are traditionally enriched from rodent or postmortem human brain tissue, but rodent models lack human-specific synaptic features, and the functionality of synaptosomes from postmortem tissues is limited by the postmortem interval and often only show disease endpoints. Furthermore, due to ethical issues and availability, only a few studies have been conducted on human samples. However, neural organoids (NOs) have emerged as a possible new source for isolation of intact and live human nerve terminals to study human-specific aspects of synaptic transmission. Further, the enrichment of synaptosomes is usually performed using density gradient centrifugation, which requires a lot of starting material. In the present study we developed a method for the enrichment of synaptic structures from human NOs applying a differential centrifugation protocol. We then used mass spectrometry-based quantitative proteomics to document the enrichment of synapse and growth cone specific proteins, and quantitative phosphoproteomics upon KCl stimulation to demonstrate viability and physiological function of the derived synaptic structures. ### Competing Interest Statement The authors have declared no competing interest.

The liver is essential for normal fatty acid utilization during fasting. Circulating fatty acids are taken up by hepatocytes and esterified as triacylglycerols for either oxidative metabolization and ketogenesis or export. Whereas the regulation of fatty acid oxidation in hepatocytes is well understood, the uptake and retention of non-esterified fatty acids by hepatocytes is not. Here, we show that murine hepatic stellate cells (HSCs) and their abundantly expressed plasmalemma vesicle-associated protein (PLVAP) control hepatic substrate preference for fasting energy metabolism. HSC-specific ablation of PLVAP in mice elevated hepatic insulin signaling and improved glucose tolerance. Fasted HSC PLVAP knockout mice showed suppressed hepatic fatty acid esterification into di- and triacylglycerols, shifting fasting metabolism from fatty acid oxidation to reliance on carbohydrates. By super-resolution microscopy, we localized HSC PLVAP to caveolae residing along the sinusoidal lumen, supporting a role for HSCs and PLVAP-diaphragmed caveolae in normal fasting metabolism of the liver.

BACKGROUND:Despite promising preclinical studies, the application of DNA methyltransferase inhibitors in treating patients with solid cancers has thus far produced only modest outcomes. The presence of intratumoral heterogeneity in response to DNA methyltransferase inhibitors could significantly influence clinical efficacy, yet our understanding of the single-cell response to these drugs in solid tumors remains very limited. METHODS:In this study, we used cancer/testis antigen genes as a model for methylation-dependent gene expression to examine the activity of DNA methyltransferase inhibitors and their potential synergistic effect with histone deacetylase inhibitors at the single-cancer cell level. The analysis was performed on breast cancer patient-derived xenograft tumors and cell lines, employing a comprehensive set of techniques, including targeted single-cell mRNA sequencing. Mechanistic insights were further gained through DNA methylation profiling and chromatin structure analysis. RESULTS:We show that breast cancer tumors and cell cultures exhibit a highly heterogenous response to DNA methyltransferase inhibitors, persisting even under high drug concentrations and efficient DNA methyltransferase depletion. The observed variability in response to DNA methyltransferase inhibitors was independent of cancer-associated aberrations and clonal genetic diversity. Instead, these variations were attributed to stochastic demethylation of regulatory CpG sites and the DNA methylation-independent suppressive function of histone deacetylases. CONCLUSIONS:Our findings point to intratumoral heterogeneity as a limiting factor in the use of DNA methyltransferase inhibitors as single agents in treatment of solid cancers and highlight histone deacetylase inhibitors as essential partners to DNA methyltransferase inhibitors in the clinic.

The aim of the present study was to gain insight into the hydrodynamic characteristics of the relatively simple aquiferous system in specimens of a calcareous syconoid sponge, Urna sp. Data on the morphology and ultrastructure of the sponge combined with measured pumping rates were provided and used for subsequent estimates of the pressure drops of water flow through the aquiferous system. The pumping rates were estimated from microscope video-recordings as the product of osculum-cross sectional area and exhalant jet speed. Estimates are given of the sensitivity of pressure drops to dimensional changes associated with observed dynamic, contractile structures (e.g., osculum, apopyles), as well as possible artefacts introduced in the preparations for ultrastructural studies. The estimated pressure losses showed that the choanocyte pumps provide a pressure of 5.5 ± 3.9 Pa at a pumping rate of 1533 ± 1089 µm3 s−1 per choanocyte. Such high pumping rates, comparable to those of some choanoflagellates, have not been reported for syconoid and leuconoid sponges before. However, the corresponding sponge volume-specific pumping rates (about 10–30 min−1) are comparable to values reported in the literature for small sponges and explants that also have relatively higher pumping rates of choanocytes than larger sponges.

The recent shift towards design of sustainable foods demands for developing advanced micro or more accurately, nanoscopic characterization techniques to study the complex structures formed in these foods. A well-studied and characterized model gel system, incorporating ingredients both from dairy and plant sources was prepared. Super-resolution STED microscopy and fluorescence lifetime imaging microscopy (FLIM) are combined to reveal the microstructure and molecular dynamics of acidified skim milk gels (ASMG) formulated with high-methoxy (HMP) and low-methoxy (LMP) pectins. A significant spatial variation of the fluorescence lifetime (tau) of the viscosity sensitive probe, Viscous Aqua (VA), is observed for all the samples in the sequence: tau(proteins) < tau(interface) < tau(voids). This suggests a more pronounced restriction of molecular dynamics in the void regions relative to the protein-dense regions. The lifetime distribution responds to the incorporation of pectin mainly in the protein regions of the gels. This indicates that pectin mainly localizes to the vicinity of the casein micelles. Spatial autocorrelation analysis of STED images provides novel insights into how pectin alters the ASMGs microstructure reveling distinct patterns of structural organization, with the correlation length xi indicating the thickness of the protein network and the repeat distance lambda representing the size of void regions within the gels. The incorporation of pectin leads to noticeable increases in both these parameters. The increase in xi suggests a thicker, more cohesive protein network, likely due to pectin's interaction with casein micelles, expanding the protein-rich areas. Similarly, the rise in lambda indicates larger void spaces between the protein regions, reflecting a more porous gel structure. The results highlight changes in the organization of water present inside the microstructure as induced by pectin incorporation into these gels and demonstrate the potential of integrating microstructure imaging and molecular dynamics to optimize similar plant and/or dairy based sustainable food systems.

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. Impact statement This microfluidic chip designed in this study allows easy application of dynamical stimuli. We show how to fabricate simple and cost-effective microfluidic chips and show the successful generation of reconstructed skin models. The presented data show skin models with a structure similar to native skin. Overall, the chip is an easy tool to study the effect of mechanical forces on skin equivalents.

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.

Cerebral organoids (CBOs) are generated from pluripotent stem cells that undergo neuroectoderm specification and neuronal differentiation in three dimensions. The developing neurons in CBOs migrate and self-organize into cerebral cortex-like layers, mimicking human brain development. CBOs develop according to intrinsic signaling mechanisms and offer unique insights into mechanisms of early human brain development. This process requires coordinated spatiotemporal regulation of protein expression and function, where the latter can be achieved by post-translational modifications (PTMs) on proteins. Despite the importance of proteins in brain development and function, profiling of protein abundance and the involvement of PTMs in CBO development remain underexplored. To gain insight into protein and PTM abundance in CBOs, we performed a high-resolution temporal analysis of CBOs up to day 200 using proteomics, PTMomics and metabolomics. We quantified more than 9,300 proteins and various neurodevelopmentally relevant PTMs (including phosphorylation, lysine acetylation, sialylated N-glycosylation, and cysteine modifications). We demonstrate that protein abundance and dynamic PTMs show significant temporal changes during CBO development related to neuronal differentiation and energy metabolism, whereas calcium signaling is mainly regulated by dynamic PTMs. We further show that synaptic protein content correlated with neurotransmitter levels, and we detected astroglia beyond day 100. Lastly, comparative analysis showed proteomic similarities between CBOs and human fetal brain tissue, supporting the physiological relevance of CBOs. Overall, our study presents a temporal atlas of protein and PTM abundance in CBOs and provides a valuable resource for studying neurodevelopment in neural organoids. ### Competing Interest Statement The authors have declared no competing interest.

Advancing 3D in vitro human tissue models is crucial for biomedical research and drug development to address the ethical and biological limitations of animal testing. Recently, 3D skin models have proven to be effective for studying serious skin conditions, such as melanoma. For these advanced models to be applicable in preclinical studies, thorough characterization is essential to understand their applicability and limitations.In this study, we used bioimaging and RNA sequencing to assess the architecture and transcriptomic profiles of skin models, including models with melanoma. Our results indicated that these models closely mimicked skin morphology and gene expression patterns. The full-thickness (FT) model shows a superior resemblance to the human skin, particularly in basement membrane formation and cellular interactions.The integrity of the skin-like properties and gene expression signatures of both skin and melanoma cells were preserved upon the integration of melanoma cells, establishing these models as robust platforms for cancer research. The responsiveness of the FT melanoma models to vemurafenib treatment was successfully monitored, demonstrating their validity as a reliable, reproducible, and humane tool for pharmacological testing and drug development. Furthermore, the transcriptomic data showed that skin models with cancer spheroids had upregulated genes linked to aggressive and resilient cancer behavior compared to spheroids alone. This emphasizes the importance of the microenvironment in cancer progression and suggests that 3D skin models can serve to uncover mechanisms and therapeutic targets that are not detectable in simpler systems. Statement of Significance This study introduces advanced, ethically sound skin and melanoma models as alternatives to animal testing in drug discovery. By thoroughly characterizing these models using bioimaging and RNA sequencing, we demonstrate their close resemblance to human skin, particularly in full-thickness models. These models not only replicate the complex cellular interactions and gene expression patterns of human tissue but also maintain robustness after melanoma integration. Our findings highlight the potential of these models in revealing cancer mechanisms and therapeutic targets, offering a significant impact on melanoma research and preclinical testing.

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron donating or withdrawing functional groups. Here, we compare the potential of derivatives of Nile Red with such functional substitutions for super-resolution fluorescence microscopy of lipid packing in model membranes and living cells. All studied Nile Red derivatives exhibit cholesterol-dependent fluorescence changes in model membranes, as shown by spectrally resolved stimulated emission depletion (STED) microscopy. STED imaging of Nile Red probes in cells reveals lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C1 (NPC1) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes. We also find small but consistent changes in the fluorescence lifetime of the Nile Red derivatives in NPC1 cells, suggesting altered hydrogen-bonding capacity in their membranes. All Nile Red derivatives are essentially non-fluorescent in water but increase their brightness in membranes, allowing for their use in MINFLUX single molecule tracking experiments. Our study uncovers the potential of Nile Red probes with functional substitutions for nanoscopic membrane imaging.

Jellyfish as a potential sustainable food material has recently gained increasing interest. However, with their soft gel-like texture and easy spoilage, it remains challenging to achieve desirable edible structures from jellyfish. The culinary preparation of jellyfish is a complex process and extends beyond conventional cooking methods. In this study, we investigate the transformation of jellyfish into crispy-like structures by manipulating their microstructural and mechanical properties through a solvent-based preparation. The study focuses on the use of "poor solvents", namely ethanol and acetone, and employs rheology measurements and quantitative microscopy techniques to analyze the effects of these solvents on the mechanical properties and microstructure of jellyfish. Our findings reveal that both ethanol and acetone lead to a significant increase in jellyfish hardness and deswelling. Notably, a micro-scale network is formed within the jellyfish matrix, and this network is then mechanically reinforced before a crispy-like texture can be obtained. Our study points to solvent polarity as also being a crucial factor for creating these effects and determines an upper polarity limit in the range of 12.2-12.9 MPa1/2 for added solvents, corresponding to approximately 60% of added ethanol or 70% of added acetone. Our study highlights that solvent-based preparation serves as a "reverse cooking" technique, where mechanical modification rather than traditional softening mechanisms are employed to stabilize and strengthen the microstructures and fibers of jellyfish. By elucidating the underlying mechanisms of solvent-induced stabilization, our findings may facilitate the development of innovative and sustainable culinary practices, paving the way for broader applications of jellyfish and other soft edible materials in the gastronomic landscape.

Intrinsic and acquired resistance to mitogen-activated protein kinase inhibitors (MAPKi) in melanoma remains a major therapeutic challenge. Here, we show that the clinical development of resistance to MAPKi is associated with reduced tumor expression of the melanoma suppressor Autophagy and Beclin 1 Regulator 1 (AMBRA1) and that lower expression levels of AMBRA1 predict a poor response to MAPKi treatment. Functional analyses show that loss of AMBRA1 induces phenotype switching and orchestrates an extracellular signal-regulated kinase (ERK)-independent resistance mechanism by activating focal adhesion kinase 1 (FAK1). In both in vitro and in vivo settings, melanomas with low AMBRA1 expression exhibit intrinsic resistance to MAPKi therapy but higher sensitivity to FAK1 inhibition. Finally, we show that the rapid development of resistance in initially MAPKi-sensitive melanomas can be attributed to preexisting subclones characterized by low AMBRA1 expression and that cotreatment with MAPKi and FAK1 inhibitors (FAKi) effectively prevents the development of resistance in these tumors. In summary, our findings underscore the value of AMBRA1 expression for predicting melanoma response to MAPKi and supporting the therapeutic efficacy of FAKi to overcome MAPKi-induced resistance.

Spider silk fibres have unique mechanical properties due to their hierarchical structure and the nanoscale organization of their proteins. Novel imaging techniques reveal new insights into the macro- and nanoscopic structure of Major (MAS) and Minor (MiS) Ampullate silk fibres from pristine samples of the orb-web spider Nephila Madagascariensis. Untreated threads were imaged using Coherent Anti-Stokes Raman Scattering and Confocal Microscopy, which revealed an outer lipid layer surrounding an autofluorescent protein core, that is divided into two layers in both fibre types. Helium ion imaging shows the inner fibrils without chemical or mechanical modifications. The fibrils are arranged parallel to the long axis of the fibres with typical spacing between fibrils of 230 nm ± 22 nm in the MAS fibres and 99 nm ± 24 nm in the MiS fibres. Confocal Reflection Fluorescence Depletion (CRFD) microscopy imaged these nano-fibrils through the whole fibre and showed diameters of 145 nm ± 18 nm and 116 nm ± 12 nm for MAS and MiS, respectively. The combined data from HIM and CRFD suggests that the silk fibres consist of multiple nanoscale parallel protein fibrils with crystalline cores oriented along the fibre axes, surrounded by areas with less scattering and more amorphous protein structures.