Deep tissue profiling of Populus stem at single nucleus level reveals uncharacterized cell types and cell-specific gene regulatory networks
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The root-tip region of plants, including the root cap, forms the most basal terminal of the root and exhibits a high degree of cellular complexity in terms of morphology, cytological function, and interaction with environmental cues in the soil. Cells in this region follow a developmental trajectory, transitioning from stem cells to meristematic cells, and ultimately to fully differentiated cell types. However, our understanding of root-tip cell-type specific proteomic responses to abiotic stresses, such as drought, particularly under field conditions, remains limited. This study aimed to identify spatially resolved, cell type-specific proteomes in switchgrass (Panicum virgatum) root tips under drought stress. Root tips were collected from seven-year-old, field-grown switchgrass ‘Alamo’ plants excavated under both well-watered and long-term drought conditions. Cell type-specific proteins were identified using laser capture microdissection (LCM) coupled with nanoPOTS (Nanodroplet Processing in One Pot for Trace Samples) and nano-LC-MS proteomics analysis. Five distinct cell types were targeted: (1) cells in the quiescent center and stem cell niche (QuC), (2) protodermal epidermal cells (PEC) in the meristematic zone, (3) epidermal cells in the transition and elongation zones above the root cap (Epi), (4) peripheral root cap cells (PRC), forming 2–3 layers below the PEC and 1–2 layers above the root border cells, and (5) columella root cap cells (Col) comprising of the columella initials and a single underlying layer of cells undergoing active growth. Principal component analysis (PCA) revealed clear separation among the five targeted cell types, confirming distinct proteomic profiles. Proteins predominantly enriched in each cell type were linked to distinct cellular functions, with QuC cells showing involvement in chromosomal behavior, DNA replication, and mitosis—key processes for stem cell niche regulation. Drought stress resulted in alterations of proteostasis, as evidenced by significant decreases in ribosomal proteins and increases in protein synthesis inhibitors. Moreover, drought stress induced unique cell-type–specific proteins involved in phytohormone biosynthesis and signaling pathways, including auxin, cytokinin, and jasmonic acid. In particular, QuC cells were more highly enriched in proteins associated with DNA repair and mitotic processes. Metabolic pathways related to amino acids, carbohydrates, and lipids were differentially affected in a cell-type–dependent manner, whereas general stress-responsive proteins exhibited consistent changes across all five cell types. Overall, this study provides unique spatially resolved, cell-type-specific proteomic profiles in root tips, representing a significant advancement in our understanding of the cellular mechanisms underlying plant responses to drought stress in natural field conditions.
Catfish production is the primary aquaculture sector in the United States, and the key cultured species is channel catfish (Ictalurus punctatus). The major causes of production losses are pathogenic diseases, and the spleen, an important site of adaptive immunity, is implicated in these diseases. To examine the channel catfish immune system, single-nuclei transcriptomes of sorted and captured IgM + cells were produced from adult channel catfish. Three channel catfish (~1 kg) were euthanized, the spleen dissected, and the tissue dissociated. The lymphocytes were isolated using a Ficoll gradient and IgM + cells were then sorted with flow cytometry. The IgM + cells were lysed and single-nuclei libraries generated using a Chromium Next GEM Single Cell 3’ GEM Kit and the Chromium X Instrument (10x Genomics) and sequenced with the Illumina NovaSeq X Plus sequencer. The reads were aligned to theI. punctatusreference assembly (Coco_2.0) using Cell Ranger, and normalization, cluster analysis, and differential gene expression analysis were carried out with Seurat. Across the three samples, approximately 753.5 million reads were generated for 18,686 cells. After filtering, 10,637 cells remained for the cluster analysis. The cluster analysis identified 16 clusters which were classified as B cells (10,276), natural killer-like (NK-like) cells (178), T cells or natural killer cells (45), hematopoietic stem and progenitor cells (HSPC)/megakaryocytes (MK) (66), myeloid/epithelial cells (40), and plasma cells (32). The B cell clusters were further defined as different populations of mature B cells, cycling B cells, and plasma cells. The plasma cells highly expressedighmand we demonstrated that the secreted form of the transcript was largely being expressed by these cells. This atlas provides insight into the gene expression of IgM + immune cells in channel catfish. The atlas is publicly available and could be used garner more important information regarding the gene expression of splenic immune cells.
Single-cell mass spectrometry (MS) is advancing our understanding of metabolic pathways in heterogeneous cell populations; however, many techniques are slow or require disruptive sample preparations. This study evaluated coupling a modified HP D100 single-cell inkjet dispenser with liquid vortex capture-mass spectrometry (D100/LVC-MS). The D100 is a single-cell inkjet dispenser capable of titrating solutions and isolating single cells via disposable cassettes equipped with microfluidic channels and an impedance sensor. The LVC-MS enables high-throughput capture, lysis, and ionization of analytes for mass spectrometric analysis. The D100/LVC-MS system was characterized through titration and single-cell experiments. Propranolol titration demonstrated linearity across a broad concentration range using the D100/LVC-MS system. Additionally, Hep G2 hepatocarcinoma cells and Chlamydomonas reinhardtii algae were used to showcase the D100’s high-throughput or low-buffer-volume single-cell dispensing strategies. The D100/LVC-MS system’s performance was validated by evaluating tamoxifen-induced steatosis in Hep G2 cells. Tamoxifen, associated with nonalcoholic fatty liver disease in breast cancer patients following long-term use, was tested in Hep G2 cells at 20 µM for 72 h against DMSO-treated controls. High-throughput analysis of 500 cells per condition, completed in 25 min per run, demonstrated the system’s efficiency. The D100/LVC-MS system simultaneously quantified tamoxifen and measured triglycerides and phosphatidylcholines. Triglycerides were upregulated in the tamoxifen-treated cells and the results indicated two distinct cell populations, differing in tamoxifen and phosphatidylcholines levels, suggesting heterogeneity within the treated population. In conclusion, these findings highlight the D100/LVC-MS system as a cost-effective, high-throughput platform for single-cell metabolomics and lipidomics, with significant potential for evaluating metabolic alterations.
How early sensory experience during “critical periods” of postnatal life affects the organization of the mammalian neocortex at the resolution of neuronal cell types is poorly understood. We previously reported that the functional and molecular profiles of layer 2/3 (L2/3) cell types in the primary visual cortex (V1) are vision-dependent [S. Chenget al.,Cell185, 311–327.e24 (2022)]. Here, we characterize the spatial organization of L2/3 cell types with and without visual experience. Spatial transcriptomic profiling based on 500 genes recapitulates the zonation of L2/3 cell types along the pial–ventricular axis in V1. By applying multitasking theory, we suggest that the spatial zonation of L2/3 cell types is linked to the continuous nature of their gene expression profiles, which can be represented as a 2D manifold bounded by three archetypal cell types. By comparing normally reared and dark reared L2/3 cells, we show that visual deprivation-induced transcriptomic changes comprise two independent gene programs. The first, induced specifically in the visual cortex, includes immediate-early genes and genes associated with metabolic processes. It manifests as a change in cell state that is orthogonal to cell-type-specific gene expression programs. By contrast, the second program impacts L2/3 cell-type identity, regulating a subset of cell-type-specific genes and shifting the distribution of cells within the L2/3 cell-type manifold. Through an integrated analysis of spatial transcriptomics with single-nucleus RNA-seq data, we describe how vision patterns cortical L2/3 cell types during the critical period.
A method for analyzing single cells by mass spectrometry includes the steps of providing a plurality of cells in a liquid medium and placing the cells and liquid medium in a single cell isolation and ejection system. Liquid medium containing a single cell is released from the single cell isolation and ejection system. The liquid medium and single cell are captured in a capture probe containing a flowing capture probe solvent. The cell is lysed by a lysis inducer in the capture probe to disperse single cell components into the medium. The lysed single cell components are transported to a mass spectrometer, where the lysed single cell components entering the mass spectrometer are spatially and temporally separated from any dispersed components of another single cell from the sample entering the mass spectrometer. Mass spectrometry is conducted on the lysed single-cell components. A system for analyzing single cells by mass spectrometry is also disclosed.
Human Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined. We set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches. We demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 – ULBP1 complex indicates some similarities to the m152-RAE1γ complex.
Radiation therapy plays a prominent role in breast cancer treatment, but the high doses of radiation damage both healthy and cancerous cells. Therefore, additional research is needed into combination therapies that could preferentially radiosensitize cancer cells compared to surrounding healthy tissue without causing deleterious side effects. Histone deacetylase inhibitor drugs (HDACis) have been tested as radiosensitizers in both basic research and clinical trials, but the long exposure time typically used in these treatments and the lack of matched healthy cell controls often leave aspects of their mechanism of action unclear. Here, we show that transient (2 h) trichostatin A (TSA) treatment of cancerous and non-tumorigenic breast epithelial cell lines increases immediate DNA damage and decreases long term cell viability in both cell types at high radiation doses. Transient TSA treatment also causes an increase in DNA damage signals after 5 Gy X-rays in other cancer and healthy cell types: A375 melanoma cells and BJ5-ta fibroblasts. This suggests that chromatin decompaction acts to increase cellular vulnerability to initial DNA damage from high doses of radiation in a cell type independent manner that does not rely on changes to DNA repair pathways caused by longer TSA treatment. However, responses to lower doses of radiation and long term survival are more cell type specific: only MCF7 cells experience an effect of TSA on DNA damage after 1 Gy X-ray radiation while MCF10a cells experience somewhat more evident cell viability effects of combined TSA and radiation treatment long term.
Segmented cells are a class of advanced diagnostic devices that enable the measurement of current distribution within the active area of an electrochemical cell, allowing for the evaluation of localized impacts from operating conditions, flow fields and component inhomogeneities. Segmented cell devices have been applied to support R&D efforts for the advancement of polymer electrolyte membrane fuel cell technologies and more recently proton exchange membrane water electrolysis (PEMWE). Beyond current density distributions, segmented cells have been successfully deployed to collect the distribution of other relevant parameters such as high frequency resistance and electrode potentials. Very recently, a promising impedance method has been reported for water electrolysis where a one-dimensional segmented cell is coupled to a multichannel potentiostat, to enable the measurement of local cell impedances, further expanding the diagnostic capabilities of the segmented cell. In this study, we will present recent efforts to accurately measure local impedance characteristics for a PEMWE cell using a two-dimensional segmented cell device coupled with a multichannel potentiostat. The distribution of local resistances, i.e. high frequency, charge transfer, mass transport and catalyst layer, and iR-free overpotentials were successfully measured after eliminating sources of random and systematic error. We will discuss the application of this diagnostic on a cell containing a 1 cm feature simulating an ionomer skin. The results from these tests give insights if such an irregularity needs to be classified as a defect, and highlight the usefulness of coupling impedance spectroscopy with segmented cells for holistic spatial diagnostics of electrochemical devices.
Abstract Immunoisolation strategies that rely on porous membranes play an important role in cell transplantation therapies to protect cells from the host's immune system. These membranes must possess immunoprotective properties while facilitating the transport of nutrients and cell products to maintain the functional integrity of encapsulated cells. An easy and scalable process is described to fabricate a dual function porous polymeric membrane that shields cells against immune cell attack and promotes vascularization to address the nutritional and oxygen requirements of transplanted cells. The fabrication process results in a membrane cross‐section with a gradient of nanopores to micropores that support cell immunoisolation and interfacial vascularization requirements, respectively. The membranes demonstrate excellent cell compatibility and effectively prevent T cell transmigration without compromising glucose diffusion and oxygen permeability. In a murine subcutaneous implantation model, membranes are stable for 60 days and exhibit significantly reduced fibrous capsules, with enhanced vascularization near the membrane. These porous polymeric membranes can potentially be used as pro‐angiogenic immunoprotective membranes for cell transplantation applications where maximizing cell viability and function is of critical importance.
Inhalation of uranium aerosols produced via human activities such as mining can pose a threat to human respiratory systems. Uranium oxide particulates emit short-range alpha particles that elicit DNA and direct damage, beyond associated physiochemical heavy-metal toxicity, to internal epithelial tissues. The availability of reliable in vitro models to study radiation exposure can greatly enhance our ability to understand and combat the biological impacts of exposure. However, the toxicological effects of alpha emissions and/or the oxidation states of uranium particulates vary across different human lung epithelial cell models and have not been systematically compared. We have endeavored to address this limitation by comparing impacts in three different human lung cell models: primary human bronchial and tracheal epithelial cells, primary human small airway epithelial cells, and human adenocarcinoma alveolar basal epithelial cells. Other studies have mainly investigated the toxicity of depleted uranium. Here, we compared the exposure of uranium oxide particulates (U 3 O 8 and UO 3 ) of different enrichment states on the chosen cell systems. Each cell model was exposed to 0.1, 1, 10, 50, 100, and 500 µg/mL of depleted U 3 O 8 , highly-enriched U 3 O 8 , and natural UO 3 particulates for 24 hours in submerged monolayer cultures. We compared viability and superoxide dismutase activity results across cell lines and uranium enrichment/ oxidative states. The results showed that 1) the oxide state of the particulates affected cell viability, implying that uranium’s different oxidation states contribute to different toxicological responses, and 2) each cell model reacts differently when exposed to uranium oxides, which may provide insights into the mechanistic processes associated with the exposure of radiological particulates on different biological systems. For instance, increased uranium enrichment corresponds to increased toxicity for the primary cells, but not for the immortalized cells. Our study shows that a holistic approach that incorporates similarities between model systems and types of radionuclides is required to truly develop empirical solutions for radiation exposure.
Pacific Northwest National Laboratory (PNNL) tested electrolyte coatings from Fuel Cell Enabling Technologies, Inc. (FCET) for use in solid oxide fuel cells (SOFCs). The key technology held by FCET is a process to deposit thin layers of oxide materials, less than 1 μm in thickness. The range of possible materials that can be deposited with their method is broad, but this project focused on the gadolinium-doped ceria (GDC) and yttria-stabilized zirconia (YSZ) electrolytes for SOFCs. Thin, gas tight electrolyte membranes have been a long-sought target in SOFC research. The thinner the electrolyte, the lower the cell resistance, and the higher performance of the cell (or the lower the operating temperature). A YSZ thickness of 1 μm would be a step change from the state-of-the-art, tape-cast electrolytes (~10 μm). An in-house prototype SOFC stack from FCET was first tested. The sealing geometry of the prototype stack was determined to be problematic, and testing shifted to button cells. Anode-supported solid oxide electrolysis cell (SOEC) button cells without an electrolyte layer were produced at PNNL and sent to FCET for coating with electrolyte. Three cells were tested with a gadolinium-doped ceria (GDC) electrolyte applied via spin coating. GDC was chosen for its conductivity at lower temperatures than YSZ. All three cells failed during initial reduction under hydrogen at 600°C. Testing then shifted to YSZ, which is the standard SOFC electrolyte. Several button cells were coated with YSZ and examined with scanning electron microscopy (SEM). A promising coating of ~1 mm thickness was observed under SEM. A similarly coated button cell was tested and failed similarly to previous tests during reduction at 600°C. The YSZ coating appeared dense and uniform in SEM analysis. The roughness of the underlying Ni/YSZ anode is on the order of 1 μm, and that may have compromised the gas-tightness of the coating. Further development is warranted to understand and refine the coating process. Thin YSZ applied via this spin-coating technique could be used as a low-cost, drop-in replacement in large-scale SOFC manufacturing processes, improving cell performance and lowering the cost per watt of SOFCs.
The convective lifecycle, from initiation to maturity and dissipation, is driven by a combination of kinematic, thermodynamic, microphysical, and radiative processes that are strongly coupled and variable in time and space. Weather radars have been traditionally used to provide various convective-cloud characteristics. Here, we analyzed climatological convective-cell radar characteristics to obtain and assess the diurnal cycles of three convective-cell types – shallow, modest deep, and vigorous deep convective cells – that formed in the greater Houston area, using the National Weather Service radar from Houston, Texas, and a multi-cell identification and tracking algorithm. The examined dataset spans 4 years (2018–2021) and covers the warm-season months (June to September) in those years. The analysis showed clear diurnal cycles in cell initiation (CI) consistent with the sea breeze circulation and showed diurnal and normalized lifetime relationships in cell evolution parameters (e.g., maximum reflectivity, echo-top height, Geostationary Operational Environmental Satellite-16 (GOES-16) channel 13 brightness temperature, and the height of maximum reflectivity). The cell evolution is well represented by relationships between (1) the height and value of the maximum radar reflectivity, (2) the minimum GOES-16 channel 13 brightness temperature and the maximum vertically integrated liquid, (3) the maximum reflectivity and columnar-average reflectivity, and (4) the echo-top ascent rate and cell lifetime. The relationships presented herein help to identify the cell lifecycle stages such as early shallow convection, vigorous vertical development, anvil development, and convective core dissipation. GOES-16 Aerosol Optical Depth values are also used as a proxy for cell initiation aerosol concentrations to investigate any potential relationships between initiation location and aerosol concentration. Overall, no significant relationships between initiation location and aerosol concentration were found for the three cell types investigated, but there are some minor differences in the pre-CI aerosol optical depth for vigorous deep convective cells.
Abstract Background Lipids produced using oleaginous yeast cells are an emerging feedstock to manufacture commercially valuable oleochemicals ranging from pharmaceuticals to lipid-derived biofuels. Production of biofuels using oleaginous yeast is a multistep procedure that requires yeast cultivation and harvesting, lipid recovery, and conversion of the lipids to biofuels. The quantitative recovery of the total intracellular lipid from the yeast cells is a critical step during the development of a bioprocess. Their rigid cell walls often make them resistant to lysis. The existing methods include mechanical, chemical, biological and thermochemical lysis of yeast cell walls followed by solvent extraction. In this study, an aqueous thermal pretreatment was explored as a method for lysing the cell wall of the oleaginous yeast Rhodotorula toruloides for lipid recovery. Results Hydrothermal pretreatment for 60 min at 121 °C with a dry cell weight of 7% (w/v) in the yeast slurry led to a recovery of 84.6 ± 3.2% (w/w) of the total lipids when extracted with organic solvents. The conventional sonication and acid-assisted thermal cell lysis led to a lipid recovery yield of 99.8 ± 0.03% (w/w) and 109.5 ± 1.9% (w/w), respectively. The fatty acid profiles of the hydrothermally pretreated cells and freeze-dried control were similar, suggesting that the thermal lysis of the cells did not degrade the lipids. Conclusion This work demonstrates that hydrothermal pretreatment of yeast cell slurry at 121 °C for 60 min is a robust and sustainable method for cell conditioning to extract intracellular microbial lipids for biofuel production and provides a baseline for further scale-up and process integration. Graphical abstract
Tissues rely on supracellular signals to coordinate their cells over a long range. Two such tissuescale cues are calcium waves and patterns of cell-cell alignment or nematic order. During wound healing, for example, calcium waves propagate across a tissue to guide directed cell migration and reepithelialization. Defects in long-range cell-cell alignment, or nematic orientation, can act to localize morphogenetic events in a tissue. Although these two cues have been considered in isolation, we demonstrate a relationship in epithelial tissue between long-range calcium signaling and the cell’s nematic order: The speed of a wound-induced calcium wave depends monotonically on the angle between the wave vector and cell axis, with maximal wave speed occurring perpendicular to the tissue’s orientation. Including anisotropic di↵usive coupling between cells in a canonical reactiondi↵usion model recapitulates our measured calcium wave dynamics. Our model demonstrates how orientation defects can desynchronize information propagation across a tissue. A calcium wave front is bent around nematic defects, therefore cells the same distance from a wound can receive the calcium signal at di↵erent times. Our work elucidates how spatial patterns in global cell alignment can control collective communication via calcium signaling during development, wound healing, and disease.
Among the various fuel cells, solid oxide cells (SOCs) are the unique type that can principally operate reversibly as either fuel cells to produce electricity or as an electrolyser to split water and produce green hydrogen (H 2 ). Nevertheless, the SOCs' reversibility presents enormous challenges that are manifested by the fast degradation through the cycling between fuel cell and electrolysis mode. While the La 0.8 Sr 0.2 MnO 3 /yttria-stabilized zirconia (LSM/YSZ) air electrode possesses significant advantages in terms of high electrical conductivity and high thermal stability under fuel cell mode, the SOCs with the LSM/YSZ air electrode experience rapid performance degradation with catastrophic electrode delamination shortly after switching from fuel cell to electrolysis mode. To prevent such catastrophic delamination and enable the electrolysis H 2 production, a chemical solution with the designed chemistry of SrFe 2 O 4-d was infiltrated into the LSM/YSZ air electrode. The infiltration immediately mitigates the catastrophic delamination, and the infiltrated cells exhibit significantly improved reversibility upon the electrochemical operation. Nanostructure examination reveals nanoscale cracks and second-phase nanograins formed in the air electrode from the baseline cell. By contrast, no delamination was observed at either the micron or the nanoscale for the infiltrated cell, which is attributed to the increased ion conductivity of the Fe-doped LSM mixed conductor induced by the significant interdiffusion between the LSM backbone and infiltrates. Here, this study presents a viable method for preventing electrode delamination while enhancing the durability of H 2 production and power generation for reversible fuel cell/electrolysis cell operation. It further opens new research directions of modifying the electrochemical activity of the electrode of inherently functional cells through the infiltration of the solutions with different chemistry.