The Amyloid Precursor Protein Modulates the Position and Length of the Axon Initial Segment.

The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the ß-amyloid (Aß) peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aß are intensively studied, yet the normal functions of APP itself have generated less interest. We report here that glutamate stimulation of neuronal activity leads to a rapid increase in App gene expression. In mouse and human neurons, elevated APP protein changes the structure of the axon initial segment (AIS) where action potentials are initiated. The AIS is shortened in length and shifts away from the cell body. The GCaMP8f Ca2+ reporter confirms the predicted decrease in neuronal activity. NMDA antagonists or knockdown of App block the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aß, either fibrillar or oligomeric, has no effect. In culture, APPSwe (a familial AD mutation) induces larger AIS changes than wild type APP. Ankyrin G and ßIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, more so in AD brains. Finally, in humans with sporadic AD or in the R1.40 AD mouse model, both females and males, neurons have elevated levels of APP protein that invade the AIS. In vivo as in vitro, this increased APP is associated with a significant shortening of the AIS. The findings outline a new role for the APP and encourage a reconsideration of its relationship to AD.SIGNIFICANCE STATEMENT While the amyloid precursor protein (APP) has long been associated with Alzheimer's disease (AD), the normal functions of the full-length Type I membrane protein have been largely unexplored. We report here that the levels of APP protein increase with neuronal activity. In vivo and in vitro, modest amounts of excess APP alter the properties of the axon initial segment. The ß-amyloid peptide derived from APP is without effect. Consistent with the observed changes in the axon initial segment which would be expected to decrease action potential firing, we show that APP expression depresses neuronal activity. In mouse AD models and human sporadic AD, APP physically associates with the scaffolding proteins of the axon initial segment, suggesting a relationship with AD dementia.

Analysis of the gut microbiome associated to PVC biodegradation in yellow mealworms.

The potential of invertebrates in the biodegradation of plastic polymers such as polyvinyl chloride (PVC) is receiving increasing attention. The present study is aimed to identify the gut microbiome involved in this degradation in yellow mealworms, i.e., the larvae of Tenebrio molitor Linnaeus. The egested PVC polymer experienced a dramatic reduction in both number average molecular weight (Mn) and weight average molecular weight (Mw) of 99.3% and 99.6%, respectively, whereas FTIR analysis revealed chemical alterations. Mass spectrometry analysis identified two potential degradation products: phthalic acid, di(2-propylpentyl) ester and 2-Propenoic acid, tridecyl ester. Further, we used metagenomic sequencing to elucidate the response of the gut microbiome when transitioning from bran to PVC as a food source, identifying four microorganisms actively involved in PVC degradation. Additionally, metagenomic functional analysis of the gut microbiome identified 111 key gene modules that were significantly enriched. In summary, our findings suggest that yellow mealworms adapt to PVC degradation by modifying their gut microbiome both structurally and functionally.

Flattened chains dominate the adsorption dynamics of loosely adsorbed chains on modified planar substrates.

Herein, the adsorption of polystyrene (PS) on phenyl-modified SiO2-Si substrates was investigated. Different from those for PS adsorption on a neat SiO2-Si substrate, the growth rate (vads) in the linear regime and hads/Rg (hads, thickness of flattened and loosely adsorbed layers on the substrate; Rg, radius of gyration) declined with increasing molecular weight (Mw) of PS and the phenyl content on the modified substrates, while the thickness of the flattened layer (hflat) and its coverage increased with increasing phenyl content. The results indicated that the adsorption of loose chains was controlled by the adsorption of flattened chains, as it only occurred in the empty contact sites remaining after the adsorption of flattened chains. Before approaching quasi-equilibrium (t < tcross), the number of flattened chain contact sites increased due to an enthalpically favorable process and, correspondingly, their spatial positions dynamically changed, which perturbed the adsorption of loose chains. When the adsorption of flattened chains reached quasi-equilibrium (t > tcross), the adsorption of loose chains was determined by the empty contact sites. The coverage of flattened chains and time to reach quasi-equilibrium were increased with more phenyl groups on the substrate, enhancing π-π interfacial interactions and resulting in a decreased adsorption rate and fewer loosely adsorbed chains. Mw-dependent vads and hads/Rg differed on phenyl-modified substrates compared to the neat SiO2-Si substrate owing to fewer empty contact sites for loose chains. The study findings improve our understanding of the mechanism responsible for the formation and structure of the adsorbed layer on solid surfaces.

The role of the molecular weight of the adsorbed layer on a substrate in the suppressed dynamics of supported thin polystyrene films.

The adsorbed layer on a solid surface plays a crucial role in the dynamics of nanoconfinement polymer materials. However, the influence of the adsorbed layer is complex, and clarifying this influence on the dynamics of confined polymers remains a major challenge. In this paper, SiO2-Si substrates with various thicknesses and adsorbed layers of PS with various molecular weights were used to reveal the effect of the adsorbed layer on the corresponding segmental dynamics of the supported thin PS films. Strongly suppressed segmental dynamics of thin PS films were observed for the films supported on thicker adsorbed layers or prepared using higher molecular weight. Neutron reflectivity revealed that the overlap region thickness between the adsorbed layer and the top overlayer increased with increasing thickness and molecular weight of the adsorbed layer, both of which correlate well with the distance over which the polystyrene dynamics were depressed by the adsorbed layer. The results show that the influencing distance of the adsorbed layer is related to the overlap zone formed between the adsorption layer and the upper thin film. The effect of the adsorbed layer molecular weight can be ascribed to the fact that large loops and long tails in the adsorbed layer result in stronger interpenetrations and entanglements between polymer chains in the adsorbed layer and in the overlayer, causing a stronger substrate effect and suppression of the segment dynamics of the supported thin PS films.

Concentric organization of A- and B-type lamins predicts their distinct roles in the spatial organization and stability of the nuclear lamina.

The nuclear lamina is an intermediate filament meshwork adjacent to the inner nuclear membrane (INM) that plays a critical role in maintaining nuclear shape and regulating gene expression through chromatin interactions. Studies have demonstrated that A- and B-type lamins, the filamentous proteins that make up the nuclear lamina, form independent but interacting networks. However, whether these lamin subtypes exhibit a distinct spatial organization or whether their organization has any functional consequences is unknown. Using stochastic optical reconstruction microscopy (STORM) our studies reveal that lamin B1 and lamin A/C form concentric but overlapping networks, with lamin B1 forming the outer concentric ring located adjacent to the INM. The more peripheral localization of lamin B1 is mediated by its carboxyl-terminal farnesyl group. Lamin B1 localization is also curvature- and strain-dependent, while the localization of lamin A/C is not. We also show that lamin B1's outer-facing localization stabilizes nuclear shape by restraining outward protrusions of the lamin A/C network. These two findings, that lamin B1 forms an outer concentric ring and that its localization is energy-dependent, are significant as they suggest a distinct model for the nuclear lamina-one that is able to predict its behavior and clarifies the distinct roles of individual nuclear lamin proteins and the consequences of their perturbation.

Glass transition temperature of single-chain polystyrene particles end-grafted to oxide-coated silicon.

A method based on the PeakForce QNM atomic force microscopic (AFM) adhesion measurement is employed to investigate the glassy dynamics of polystyrene (PS) single-chain particles end-grafted to SiO2-Si substrates with different diameters, D0, of 3.4 nm-8.8 nm and molar masses, Mn, of 8-123 kg/mol. As temperature was increased, the adhesion force, Fad, experienced by the AFM tip on pulling off the single chains after loading demonstrated a stepwise increase at an elevated temperature, which we identified to be Tg based on previous works. Our result shows that Tg of our grafted single chains increases with Mn in a manner consistent with the Fox-Flory equation, but the coefficient quantifying the Mn dependence of Tg is only (36 ± 6)% the value of bulk PS. In addition, the value of Tg in the Mn → ∞ limit is about 25 °C below the bulk Tg but more than 15 °C above that of (untethered) PS nanoparticles with D0 ≈ 100 nm suspended in a solution. Our findings are consistent with Tg of our single chains being dominated by simultaneous effects of the interfaces, which depress Tg, and end-grafting, which enhances Tg. The latter is believed to exert its influence on the glass transition dynamics by a mechanism reliant on chain connectivity and does not vary with chain length.

A Simple Marker-Assisted 3D Nanometer Drift Correction Method for Superresolution Microscopy.

High-precision fluorescence microscopy such as superresolution imaging or single-particle tracking often requires an online drift correction method to maintain the stability of the three-dimensional (3D) position of the sample at a nanometer precision throughout the entire data acquisition process. Current online drift correction methods require modification of the existing two-dimensional (2D) fluorescence microscope with additional optics and detectors, which can be cumbersome and limit its use in many biological laboratories. Here we report a simple marker-assisted online drift correction method in which all 3D positions can be derived from fiducial markers on the coverslip of the sample on a standard 2D fluorescence microscope without additional optical components. We validate this method by tracking the long-term 3D stability of single-molecule localization microscopy at a precision of <2 and 5 nm in the lateral and axial dimension, respectively. We then provide three examples to evaluate the performance of the marker-assisted drift correction method. Finally, we give an example of a biological application of superresolution imaging of spatiotemporal alteration for a DNA replication structure with both low-abundance newly synthesized DNAs at the early onset of DNA synthesis and gradually condensed DNA structures during DNA replication. Using an isogenic breast cancer progression cell line model that recapitulates normal-like, precancerous, and tumorigenic stages, we characterize a distinction in the DNA replication process in normal, precancerous, and tumorigenic cells.

Targeted DNA damage at individual telomeres disrupts their integrity and triggers cell death.

Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising <1% of the genome, and telomere dysfunction has not been established. By fusing the KillerRed chromophore with the telomere repeat binding factor 1, TRF1, we developed a novel approach to generate localized damage to telomere DNA and to monitor the real time damage response at the single telomere level. We found that DNA damage at long telomeres in U2OS cells is not repaired efficiently compared to DNA damage in non-telomeric regions of the same length in heterochromatin. Telomeric DNA damage shortens the average length of telomeres and leads to cell senescence in HeLa cells and cell death in HeLa, U2OS and IMR90 cells, when DNA damage at non-telomeric regions is undetectable. Telomere-specific damage induces chromosomal aberrations, including chromatid telomere loss and telomere associations, distinct from the damage induced by ionizing irradiation. Taken together, our results demonstrate that oxidative damage induces telomere dysfunction and underline the importance of maintaining telomere integrity upon oxidative damage.

Stepwise crystallization and the layered distribution in crystallization kinetics of ultra-thin poly(ethylene terephthalate) film.

Crystallization is an important property of polymeric materials. In conventional viewpoint, the transformation of disordered chains into crystals is usually a spatially homogeneous process (i.e., it occurs simultaneously throughout the sample), that is, the crystallization rate at each local position within the sample is almost the same. Here, we show that crystallization of ultra-thin poly(ethylene terephthalate) (PET) films can occur in the heterogeneous way, exhibiting a stepwise crystallization process. We found that the layered distribution of glass transition dynamics of thin film modifies the corresponding crystallization behavior, giving rise to the layered distribution of the crystallization kinetics of PET films, with an 11-nm-thick surface layer having faster crystallization rate and the underlying layer showing bulk-like behavior. The layered distribution in crystallization kinetics results in a particular stepwise crystallization behavior during heating the sample, with the two cold-crystallization temperatures separated by up to 20 K. Meanwhile, interfacial interaction is crucial for the occurrence of the heterogeneous crystallization, as the thin film crystallizes simultaneously if the interfacial interaction is relatively strong. We anticipate that this mechanism of stepwise crystallization of thin polymeric films will allow new insight into the chain organization in confined environments and permit independent manipulation of localized properties of nanomaterials.

Adaptive optics stochastic optical reconstruction microscopy (AO-STORM) using a genetic algorithm.

The resolution of Single Molecule Localization Microscopy (SML) is dependent on the width of the Point Spread Function (PSF) and the number of photons collected. However, biological samples tend to degrade the shape of the PSF due to the heterogeneity of the index of refraction. In addition, there are aberrations caused by imperfections in the optical components and alignment, and the refractive index mismatch between the coverslip and the sample, all of which directly reduce the accuracy of SML. Adaptive Optics (AO) can play a critical role in compensating for aberrations in order to increase the resolution. However the stochastic nature of single molecule emission presents a challenge for wavefront optimization because the large fluctuations in photon emission do not permit many traditional optimization techniques to be used. Here we present an approach that optimizes the wavefront during SML acquisition by combining an intensity independent merit function with a Genetic algorithm (GA) to optimize the PSF despite the fluctuating intensity. We demonstrate the use of AO with GA in tissue culture cells and through ~50µm of tissue in the Drosophila Central Nervous System (CNS) to achieve a 4-fold increase in the localization precision.

Surface structures of poly(methyl methacrylate) films influenced by chain entanglement in the corresponding film-formation solution.

The effects of the properties of casting solution on the surface structure of poly(methyl methacrylate) (PMMA) films were systematically investigated. It was observed that the hydrophobicity of PMMA films increased with increasing viscosity of the corresponding polymer solution regardless of the film-formation techniques that were utilized. The ratio of the C-H symmetric stretching vibrations of methylene groups (hydrophobic components, with a peak at 2910 cm(-1)) to those of the ester methyl groups (relative hydrophilic components, with a peak at 2955 cm(-1)) from sum frequency generation (SFG) vibrational spectra, A2910/A2955, was used as a parameter to evaluate the structure on the film surface, which was related to the surface wettability of the films. The results showed that A2910/A2955 of cast PMMA films increased linearly with ηsp(0.3) (ηsp, the specific viscosity of the casting solution), whereas that of the corresponding spin-coated films showed a linear relationship defined as ηsp(0.3)E(0.26), where E is the average number of entanglement points per molecule (E = Mw/Me). These results indicate that a relative equilibrium conformation on the PMMA film surface, adopted from the perspective of thermodynamics, was easily achieved during film formation, when the conformation of the polymer chains in the corresponding casting solution was close to that in the bulk. For the spin-coated films, the chain entanglement structure in the casting solution was a more important factor for the resulting film to reach a relative equilibrium state, since this structure was in favor of maintaining the pristine conformation in casting solution under centrifugal force during spin-coating. This work may help to enhance the fundamental understanding of the formation of the film surface structure from polymer solution to the resulting solid film, which will affect not only the corresponding surface properties, but also the dynamics of the resulting thin films.

Micelle-templated composite quantum dots for super-resolution imaging.

Quantum dots (QDs) have tremendous potential for biomedical imaging, including super-resolution techniques that permit imaging below the diffraction limit. However, most QDs are produced via organic methods, and hence require surface treatment to render them water-soluble for biological applications. Previously, we reported a micelle-templating method that yields nanocomposites containing multiple core/shell ZnS-CdSe QDs within the same nanocarrier, increasing overall particle brightness and virtually eliminating QD blinking. Here, this technique is extended to the encapsulation of Mn-doped ZnSe QDs (Mn-ZnSe QDs), which have potential applications in super-resolution imaging as a result of the introduction of Mn(2+) dopant energy levels. The size, shape and fluorescence characteristics of these doped QD-micelles were compared to those of micelles created using core/shell ZnS-CdSe QDs (ZnS-CdSe QD-micelles). Additionally, the stability of both types of particles to photo-oxidation was investigated. Compared to commercial QDs, micelle-templated QDs demonstrated superior fluorescence intensity, higher signal-to-noise ratios, and greater stability against photo-oxidization,while reducing blinking. Additionally, the fluorescence of doped QD-micelles could be modulated from a bright 'on' state to a dark 'off' state, with a modulation depth of up to 76%, suggesting the potential of doped QD-micelles for applications in super-resolution imaging.

Super-resolution imaging of T lymphocyte activation reveals chromatin decondensation and disrupted nuclear envelope.

T lymphocyte activation plays a pivotal role in adaptive immune response and alters the spatial organization of nuclear architecture that subsequently impacts transcription activities. Here, using stochastic optical reconstruction microscopy (STORM), we observe dramatic de-condensation of chromatin and the disruption of nuclear envelope at a nanoscale resolution upon T lymphocyte activation. Super-resolution imaging reveals that such alterations in nuclear architecture are accompanied by the release of nuclear DNA into the cytoplasm, correlating with the degree of chromatin decompaction within the nucleus. The authors show that under the influence of metabolism, T lymphocyte activation de-condenses chromatin, disrupts the nuclear envelope, and releases DNA into the cytoplasm. Taken together, this result provides a direct, molecular-scale insight into the alteration in nuclear architecture. It suggests the release of nuclear DNA into the cytoplasm as a general consequence of chromatin decompaction after lymphocyte activation.

An Omni-Mesoscope for multiscale high-throughput quantitative phase imaging of cellular dynamics and high-content molecular characterization.

The mesoscope has emerged as a powerful imaging tool in biomedical research, yet its high cost and low resolution have limited its broader application. Here, we introduce the Omni-Mesoscope, a cost-effective high-spatial-temporal, multimodal, and multiplex mesoscopic imaging platform built from cost-efficient off-the-shelf components. This system uniquely merges the capabilities of quantitative phase microscopy to capture live-cell dynamics over a large cell population with highly multiplexed fluorescence imaging for comprehensive molecular characterization. This integration facilitates simultaneous tracking of live-cell morphodynamics across thousands of cells, alongside high-content molecular analysis at the single-cell level. Furthermore, the Omni-Mesoscope offers a mesoscale field of view of approximately 5 mm 2 with a high spatial resolution down to 700 nm, enabling the capture of information-rich images with detailed sub-cellular features. We demonstrate such capability in delineating molecular characteristics underlying rare dynamic cellular phenomena, such as cancer cell responses to chemotherapy and the emergence of polyploidy in drug-resistant cells. Moreover, the cost-effectiveness and the simplicity of our Omni-Mesoscope democratizes mesoscopic imaging, making it accessible across diverse biomedical research fields. To further demonstrate its versatility, we integrate expansion microscopy to enhance 3D volumetric super-resolution imaging of thicker tissues, opening new avenues for biological exploration at unprecedented scales and resolutions.

Exploring the Molecular Origin for the Long-Range Propagation of the Substrate Effect in Unentangled Poly(methyl methacrylate) Films.

The polymer/substrate interface plays a significant role in the dynamics of nanoconfined polymers because of its suppression on polymer mobility and its long-range propagation feature, while the molecular origin of the long-range substrate effect in unentangled polymer material is still ambiguous. Herein, we investigated the propagation distances of the substrate effect (h*) by a fluorinated tracer-labeled method of two unentangled polymer films supported on silicon substrates: linear and ring poly(methyl methacrylate) films with relatively low molecular weights. The results indicate that the value of h* has a molecular weight dependence of h*∝N (N is the degree of polymerization) in the unentangled polymer films, while h*∝N1/2 was presented as previously reported in the entangled films. A theoretical model, depending on the polymer/polymer intermolecular interaction, was proposed to describe the above long-range propagation behavior of the substrate effect and agrees with our experiment results very well. From the model, it revealed that the intermolecular friction determines the long-range propagation of the substrate effect in the unentangled system, but the intermolecular entanglement is the dominant role in entangled system. These results give us a deeper understanding of the long-range substrate effect.

Loosely Adsorbed Chains Expedite the Desorption of Flattened Polystyrene Chains on Flat Silicon Surface.

Herein, the desorption of the adsorbed chains (including two regions of flattened chains and loosely adsorbed chains) was examined by monitoring the chain exchange kinetics between the adsorbed chains and the top-free chains in a bilayer system by using fluorine-labeled polystyrene (PS). The results indicated that the exchange behavior of PS-flattened chains with the top-free chains is much slower than that of PS-loose chains and has a strong molecular weight (MW) dependence. Interestingly, in the presence of loosely adsorbed chains, the desorption of flattened chains was accelerated greatly and had weaker MW dependency. We attribute the MW-dependent desorption phenomena to the average number of contact sites between polymer adsorbed chains and the substrate, which rapidly increased with increasing MW. Likewise, the desorption of loosely adsorbed chains may provide extra conformational energy to accelerate the desorption of flattened chains.

Enhanced Glass Transition Temperature of Thin Polystyrene Films Having an Underneath Cross-Linked Layer.

Due to the importance of the interface in the segmental dynamics of supported macromolecule ultrathin films, the glass transition temperature (Tg) of polystyrene (PS) ultrathin films upon solid substrates modified with a cross-linked PS (CLPS) layer has been investigated. The results showed that the Tg of the thin PS films on a silica surface with a ∼5 nm cross-linked layer increased with reducing film thickness. Meanwhile, the increase in Tg of the thin PS films became more pronounced with increasing the cross-linking density of the layer. For example, a 20 nm thick PS film supported on CLPS with 1.8 kDa of cross-linking degree exhibited a ∼35 and ∼50 K increase in Tg compared to its bulk and that on neat SiO2 substrate, respectively. Such a large Tg elevation for the ultrathin PS films was attributed to the interfacial aggregation states in which chains diffused through nanolevel voids formed in the cross-linked layer to the SiO2-Si surface. In such a situation, the chains were topologically constrained in the cross-linked layer with less mobility. These results offer us the opportunity to tailor interfacial effects by changing the degree of cross-linking, which has great potential application in many polymer nanocomposites.

Ultrastructural visualization of chromatin in cancer pathogenesis using a simple small-molecule fluorescent probe.

Imaging chromatin organization at the molecular-scale resolution remains an important endeavor in basic and translational research. Stochastic optical reconstruction microscopy (STORM) is a powerful superresolution imaging technique to visualize nanoscale molecular organization down to the resolution of ~20 to 30 nm. Despite the substantial progress in imaging chromatin organization in cells and model systems, its routine application on assessing pathological tissue remains limited. It is, in part, hampered by the lack of simple labels that consistently generates high-quality STORM images on the highly processed clinical tissue. We developed a fast, simple, and robust small-molecule fluorescent probe-cyanine 5-conjugated Hoechst-for routine superresolution imaging of nanoscale nuclear architecture on clinical tissue. We demonstrated the biological and clinical significance of imaging superresolved chromatin structure in cancer development and its potential clinical utility for cancer risk stratification.

Changes in nuclear pore numbers control nuclear import and stress response of mouse hearts.

Nuclear pores are essential for nuclear-cytoplasmic transport. Whether and how cells change nuclear pores to alter nuclear transport and cellular function is unknown. Here, we show that rat heart muscle cells (cardiomyocytes) undergo a 63% decrease in nuclear pore numbers during maturation, and this changes their responses to extracellular signals. The maturation-associated decline in nuclear pore numbers is associated with lower nuclear import of signaling proteins such as mitogen-activated protein kinase (MAPK). Experimental reduction of nuclear pore numbers decreased nuclear import of signaling proteins, resulting in decreased expression of immediate-early genes. In a mouse model of high blood pressure, reduction of nuclear pore numbers improved adverse heart remodeling and reduced progression to lethal heart failure. The decrease in nuclear pore numbers in cardiomyocyte maturation and resulting functional changes demonstrate how terminally differentiated cells permanently alter their handling of information flux across the nuclear envelope and, with that, their behavior.

Probing Chromatin Compaction and Its Epigenetic States in situ With Single-Molecule Localization-Based Super-Resolution Microscopy.

Chromatin organization play a vital role in gene regulation and genome maintenance in normal biological processes and in response to environmental insults. Disruption of chromatin organization imposes a significant effect on many cellular processes and is often associated with a range of pathological processes such as aging and cancer. Extensive attention has been attracted to understand the structural and functional studies of chromatin architecture. Biochemical assays coupled with the state-of-the-art genomic technologies have been traditionally used to probe chromatin architecture. Recent advances in single molecule localization microscopy (SMLM) open up new opportunities to directly visualize higher-order chromatin architecture, its compaction status and its functional states at nanometer resolution in the intact cells or tissue. In this review, we will first discuss the recent technical advantages and challenges of using SMLM to image chromatin architecture. Next, we will focus on the recent applications of SMLM for structural and functional studies to probe chromatin architecture in key cellular processes. Finally, we will provide our perspectives on the recent development and potential applications of super-resolution imaging of chromatin architecture in improving our understanding in diseases.