Search for: cells

While spider silk threads mainly consist of a core of partially crystalline silk proteins, it has been found that they also exhibit a very thin skin layer of distinct structure and a coating rich in lipids and glycoproteins. These outer layers are poorly researched, but can be assumed to be a major player governing the interaction of cells with spider silk threads, as observed in cell culture. Here we propose SAXS/WAXS mapping with ultra-high spatial resolution to examine the surface layer of thin cryo-cut sections of different spider silks that have shown different cell guiding behavior in cell culture. This approach allows studying surface layers from two orientations (along and normal to fiber axis) and the cryo-approach minimizes morphological changes. In a recent nano-SAXS/WAXS beamtime at ID13, we obtained very promising data, however with whole threads and with lower resolution. This follow-up work aims to characterize the surface layer systematically. 

With the growing number of single-cell datasets collected under more complex experimental conditions, there is an opportunity to leverage single-cell variability to reveal deeper insights into how cells respond to perturbations. Many existing approaches rely on discretizing the data into clusters for differential gene expression (DGE), effectively ironing out any information unveiled by the single-cell variability across cell-types. In addition, DGE often assumes a statistical distribution that, if erroneous, can lead to false positive differentially expressed genes. Here, we present Cellograph: a semi-supervised framework that uses graph neural networks to quantify the effects of perturbations at single-cell granularity. Cellograph not only measures how prototypical cells are of each condition but also learns a latent space that is amenable to interpretable data visualization and clustering. The learned gene weight matrix from training reveals pertinent genes driving the differences between conditions. We demonstrate the utility of our approach on publicly-available datasets including cancer drug therapy, stem cell reprogramming, and organoid differentiation. Cellograph outperforms existing methods for quantifying the effects of experimental perturbations and offers a novel framework to analyze single-cell data using deep learning.

 

Elephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia.

Seal and human endothelial cells exposed to 1% O₂for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure.

We found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

 

The use of oxygen by cells is an essential aspect of cell metabolism and a reliable indicator of viable and functional cells. Here, we report partial pressure oxygen (pO₂) mapping of live cells as a reliable indicator of viable and metabolically active cells. For pO₂imaging, we utilized trityl OX071-based pulse electron paramagnetic resonance oxygen imaging (EPROI), in combination with a 25 mT EPROI instrument, JIVA-25™, that provides 3D oxygen maps with high spatial, temporal, and pO₂resolution. To perform oxygen imaging in an environment-controlled apparatus, we developed a novel multi-well-plate incubator-resonator (MWIR) system that could accommodate 3 strips from a 96-well strip-well plate and image the middle 12 wells noninvasively and simultaneously. The MWIR system was able to keep a controlled environment (temperature at 37 °C, relative humidity between 70%–100%, and a controlled gas flow) during oxygen imaging and could keep cells alive for up to 24 h of measurement, providing a rare previously unseen longitudinal perspective of 3D cell metabolic activities. The robustness of MWIR was tested using an adherent cell line (HEK-293 cells), a nonadherent cell line (Jurkat cells), a cell-biomaterial construct (Jurkat cells seeded in a hydrogel), and a negative control (dead HEK-293 cells). For the first time, we demonstrated that oxygen concentration in a multi-well plate seeded with live cells reduces exponentially with the increase in cell seeding density, even if the cells are exposed to incubator-like gas conditions. For the first time, we demonstrate that 3D, longitudinal oxygen imaging can be used to assess cells seeded in a hydrogel. These results demonstrate that MWIR-based EPROI is a versatile and robust method that can be utilized to observe the cell metabolic activity nondestructively, longitudinally, and in 3D. This approach may be useful for characterizing cell therapies, tissue-engineered medical products, and other advanced therapeutics.

 

Single-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures. Here, we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D single-molecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin – a protein located in the focal adhesion complex – and actin in human osteosarcoma cells.

 

Inorganic perovskite solar cells (IPSCs) have gained significant attention due to their excellent thermal stability and suitable band gap (~1.7 eV) for tandem solar cell applications. However, the defect‐induced non‐radiative recombination losses, low charge extraction efficiency, energy level mismatches, and so on render the fabrication of high‐efficiency inverted IPSCs remains challenging. Here, the use of 3‐amino‐5‐bromopyridine‐2‐formamide (ABF) in methanol was dynamically spin‐coated on the surface of CsPbI_2.85Br_0.15film, which facilitates the limited etching of defect‐rich subsurface layer, resulting in the formation of vertical PbI₂nanosheet structures. This enabled localized contacts between the perovskite film and the electron transport layer, suppress the recombination of electron‐hole and beneficial to electron extraction. Additionally, the C=O and C=N groups in ABF effectively passivated the undercoordinated Pb²⁺at grain boundaries and on the surface of CsPbI_2.85Br_0.15film. Eventually, we achieved a champion efficiency of 20.80 % (certified efficiency of 20.02 %) for inverted IPSCs with enhanced stability, which is the highest value ever reported to date. Furthermore, we successfully prepared p‐i‐n type monolithic inorganic perovskite/silicon tandem solar cells (IPSTSCs) with an efficiency of 26.26 %. This strategy provided both fast extraction and efficient passivation at the electron‐selective interface.

 

Melanocyte stem cells (McSCs) of the hair follicle are a rare cell population within the skin and are notably underrepresented in whole‐skin, single‐cell RNA sequencing (scRNA‐seq) datasets. Using a cell enrichment strategy to isolate KIT+/CD45− cells from the telogen skin of adult female C57BL/6J mice, we evaluated the transcriptional landscape of quiescent McSCs (qMcSCs) at high resolution. Through this evaluation, we confirmed existing molecular signatures for qMcCS subpopulations (e.g.,Kit+,Cd34+/−,Plp1+,Cd274+/−,Thy1+,Cdh3+/−) and identified novel qMcSC subpopulations, including two that differentially regulate their immune privilege status. Within qMcSC subpopulations, we also predicted melanocyte differentiation potential, neural crest potential, and quiescence depth. Taken together, the results demonstrate that the qMcSC population is heterogeneous and future studies focused on investigating changes in qMcSCs should consider changes in subpopulation composition.

 

The building sector accounts for 36% of energy consumption and 39% of energy-related greenhouse-gas emissions. Integrating bifacial photovoltaic solar cells in buildings could significantly reduce energy consumption and related greenhouse gas emissions. Bifacial solar cells should be flexible, bifacially balanced for electricity production, and perform reasonably well under weak-light conditions. Using rigorous optoelectronic simulation software and the differential evolution algorithm, we optimized symmetric/asymmetric bifacial CIGS solar cells with either (i) homogeneous or (ii) graded-bandgap photon-absorbing layers and a flexible central contact layer of aluminum-doped zinc oxide to harvest light outdoors as well as indoors. Indoor light was modeled as a fraction of the standard sunlight. Also, we computed the weak-light responses of the CIGS solar cells using LED illumination of different light intensities. The optimal bifacial CIGS solar cell with graded-bandgap photon-absorbing layers is predicted to perform with 18%–29% efficiency under 0.01–1.0-Sun illumination; furthermore, efficiencies of 26.08% and 28.30% under weak LED light illumination of 0.0964 mW cm⁻²and 0.22 mW cm⁻²intensities, respectively, are predicted.

 

Oligonucleotide therapeutics are becoming increasingly important as more are approved by the FDA, both for treatment and vaccination. Similarly, dynamic DNA nanotechnology is a promising technique that can be used to sense exogenous input molecules or endogenous biomarkers and integrate the results of multiple sensing reactionsin situvia a programmed cascade of reactions. The combination of these two technologies could be highly impactful in biomedicine by enabling smart oligonucleotide therapeutics that can autonomously sense and respond to a disease state. A particular challenge, however, is the limited lifetime of standard nucleic acid components in living cells and organisms due to degradation by endogenous nucleases. In this work, we address this challenge by incorporating mirror‐image, ʟ‐DNA nucleotides to produce heterochiral “gapmers”. We use dynamic DNA nanotechnology to show that these modifications keep the oligonucleotide intact in living human cells for longer than an unmodified strand. To this end, we used a sequential transfection protocol for delivering multiple nucleic acids into living human cells while providing enhanced confidence that subsequent interactions are actually occurring within the cells. Taken together, this work advances the state of the art of ʟ‐nucleic acid protection of oligonucleotides and DNA circuitry for applicationsin vivo.

 

The CDK4/6 inhibitor palbociclib blocks cell cycle progression in Estrogen receptor–positive, human epidermal growth factor 2 receptor–negative (ER+/HER2−) breast tumor cells. Despite the drug’s success in improving patient outcomes, a small percentage of tumor cells continues to divide in the presence of palbociclib—a phenomenon we refer to as fractional resistance. It is critical to understand the cellular mechanisms underlying fractional resistance because the precise percentage of resistant cells in patient tissue is a strong predictor of clinical outcomes. Here, we hypothesize that fractional resistance arises from cell-to-cell differences in core cell cycle regulators that allow a subset of cells to escape CDK4/6 inhibitor therapy. We used multiplex, single-cell imaging to identify fractionally resistant cells in both cultured and primary breast tumor samples resected from patients. Resistant cells showed premature accumulation of multiple G1 regulators including E2F1, retinoblastoma protein, and CDK2, as well as enhanced sensitivity to pharmacological inhibition of CDK2 activity. Using trajectory inference approaches, we show how plasticity among cell cycle regulators gives rise to alternate cell cycle “paths” that allow individual tumor cells to escape palbociclib treatment. Understanding drivers of cell cycle plasticity, and how to eliminate resistant cell cycle paths, could lead to improved cancer therapies targeting fractionally resistant cells to improve patient outcomes.