Journal: Science Advances

Article Title: Sustainable synthesis of amino-cellulose nanofibers for biomaterial platforms

doi: 10.1126/sciadv.adx4556

Figure Lengend Snippet: Morphological characterizations: ( A ) digital photograph of 0.2% (w/v) A-CNF suspension (carboxylated cellulose:PEI molar ratio, 1: 1); ( B ) atomic force microscopy (AFM) micrograph (scale bar, 400 nm); ( C and D ) transmission electron microscopy (TEM) micrographs at different magnifications (scale bars, 0.5 μm and 200 nm); ( E ) diameter distribution histogram; ( F ) digital photograph of 0.2% (w/v) A-CNF suspension (carboxylated cellulose:PEI molar ratio, 1: 2); ( G ) AFM micrograph (scale bar, 400 nm); ( H and I ) TEM micrographs at different magnifications (scale bars, 0.5 μm and 200 nm); ( J ) diameter distribution histogram; ( K ) TEM visualization of high aspect ratio (scale bar, 0.5 μm). Physicochemical properties: comparison of ( L ) aspect ratio and ( N ) amine content of A-CNF prepared in optimal conditions with literature values. Data are detailed in table S1 ( – ). Quantitative analysis: comparison of ( M ) amine content of A-CNF, based on three independent measurements. Error bars denote SD. Stability and scalability assessments: ( O ) photographs of A-CNF suspension pre– and post–8 months of storage; ( P ) pilot production tests of 1 wt % A-CNF demonstrating scalability of the production process. LCA and TEA analyses of A-CNF: ( Q ) At an electricity price of US$0.1 per kWh, the per-ton synthesis cost of A-CNF via this method and the full process cost breakdown were calculated. These were compared with expenditures for quaternization reaction, amidation reactions, and the market price of CTS per ton. ( R ) For A-CNF and CTS production, environmental impacts of the quaternization reaction, amidation reaction, and the proposed synthesis method were analyzed across 10 categories (including global warming, human health, and terrestrial ecosystems). Data were standardized to enable consistent, objective evaluation of environmental outcomes.

Article Snippet: Before imaging, samples were coated with platinum under a 30-mA current for 40 s. Transmission electron microscopy images were captured using a JEM-2100 microscope (JEOL, Tokyo, Japan).

Techniques: Suspension, Microscopy, Transmission Assay, Electron Microscopy, Comparison

Journal: Science Advances

Article Title: Sustainable synthesis of amino-cellulose nanofibers for biomaterial platforms

doi: 10.1126/sciadv.adx4556

Figure Lengend Snippet: ( A ) Schematic and scanning electron microscopy (SEM) images show the cross-sectional views of A-CNF scaffold with varying solid contents. Insets provide digital photographs of each scaffold (scale bars, 500 μm). ( B ) Micro-CT reconstructed three-dimensional images depict the microstructures of voids within the A-CNF scaffold. ( C ) Variations in SSA and pore dimensions as a function of solid content in the scaffold. ( D ) Analysis of void structure connectivity within A-CNF and CTS scaffolds. ( E ) Comparative analysis of porosity and density metrics for A-CNF scaffold versus CTS scaffold at different solid content levels. ( F ) Graphical representation of water absorption and retention capacities for A-CNF and CTS scaffolds. ( G ) Comparison of the biostability of A-CNF and CTS scaffolds across varying solid contents, supplemented with digital photographs documenting changes over specified time periods. h, hours. ( H ) Antimicrobial properties of the scaffolds are demonstrated through bacterial inhibition of E. coli and S. aureus in the presence of the A-CNF scaffold, compared to a control group. ( I ) The influence of solid content on the compressive strength and Young’s modulus of the A-CNF scaffold. ( J ) Ashby plots comparing the compressive strength and specific strength of the A-CNF scaffold to four other documented scaffolds. ( K ) A comparative overview of Young’s modulus in the A-CNF scaffold with natural in vivo tissues, illustrating a broad range of tissue stiffness from soft tissues with sub-megapascal modulus to hard tissues like bone. The modulus values of common tissue engineering materials such as 8% acrylamide (AM) gel, tissue culture polystyrene, and titanium (used in dental and bone implants) are included for comparison. ( L ) Details on the gel casting preparation method used to mold the A-CNF scaffold into various biomimetic soft tissue forms (scale bars, 1 cm). All data were analyzed from three independent measurements. Error bars represent SD.

Article Snippet: Before imaging, samples were coated with platinum under a 30-mA current for 40 s. Transmission electron microscopy images were captured using a JEM-2100 microscope (JEOL, Tokyo, Japan).

Techniques: Electron Microscopy, Micro-CT, Comparison, Inhibition, Control, In Vivo

Journal: Science Advances

Article Title: Sustainable synthesis of amino-cellulose nanofibers for biomaterial platforms

doi: 10.1126/sciadv.adx4556

Figure Lengend Snippet: ( A ) Schematic of the A-CNF scaffold tailored for organoid culture with adjustable pore sizes to accommodate varying cell sizes. H9c2 and SK-BR-3 (single adult cells) and PHC (adult primary cells) were used to develop heart, mammary, and liver organoids. ( B ) Optical images display hemolysis test results for CTS and A-CNF scaffolds, using deionized (DI) water (positive control, high hemolysis) and PBS (negative control, no hemolysis). ( C ) Quantification of hemolysis ratios for each scaffold group (Error bars show means ± SD; n = 6 repeats). ( D ) Results of CCK-8 assays showing cell inhibition rates (box plot elements: lower/upper boundaries, Q1/Q3; whiskers, max/min; dashed line, mean; n = 6 repeats), and ( E ) cell viability analyses of H9c2, SK-BR-3, and PHC cells cultured on A-CNF scaffolds for 24 hours versus controls (error bars show means ± SD; n = 6 repeats). ( F ) Confocal and three-dimensional (3D) fluorescence microscopy images of H9c2, SK-BR-3, and PHC cells cultured on CTS and A-CNF scaffolds for 48 hours. Live cells stained green (calcein-AM), and dead cells stained red (propidium iodide) (scale bars, 200 μm). ( G ) Schematic illustrating the impact of scaffold pore size on cell behavior. Larger pores may lead to cell crowding and reduced adhesion efficiency, while smaller pores can restrict cell extension and adhesion. Appropriately sized pores enhance cell attachment and promote growth. ( H and I ) Graphs of cell adhesion and proliferation rates for H9c2, SK-BR-3, and PHC cells cultured on A-CNF scaffolds with different pore sizes, showing how scaffold configurations affect cellular responses (error bars show means ± SD; n = 6 repeats). ( J ) Digital photographs of heart (H9c2), mammary (SK-BR-3), and liver (PHC) organoid cultures (scale bars, 5 mm), demonstrating the supportive role of A-CNF scaffolds in maintaining morphology and function of diverse organoids. h, hours.

Article Snippet: Before imaging, samples were coated with platinum under a 30-mA current for 40 s. Transmission electron microscopy images were captured using a JEM-2100 microscope (JEOL, Tokyo, Japan).

Techniques: Positive Control, Negative Control, CCK-8 Assay, Inhibition, Cell Culture, Fluorescence, Microscopy, Staining, Pore Size, Cell Attachment Assay

Journal: Science (New York, N.Y.)

Article Title: The nucleus acts as a ruler tailoring cell responses to spatial constraints

doi: 10.1126/science.aba2894

Figure Lengend Snippet: A: Cortical myosin levels (left) and force response (ΔF, right) to 5 μm confinement of HeLa-Kyoto cells treated with drugs affecting PM tension and extracellular [Ca2+]out (blue) or ER/NE tension and intracellular [Ca2+]in (red). See Table S1 for drug target description and Materials and Methods for drug concentrations. Data are from ≥ 2 experiments (mean ± SD; n = 10 cells per perturbation; see Table S2 for statistics). B: 3D XZ views of the DAPI-stained nucleus at 20, 10, and 5 μm. C: Left, XY views of the nucleus at 10 and 5 μm. Middle and right, measurements of nuclear area and volume at 10 and subsequently 5 μm (n = 10 cells; p value, paired t test). Scale bar, 10 μm. D: Left top, images of the LAP2-GFP-labeled NE confined to 20-to-10-to-5 μm. Scale bar, 5 μm. Left bottom, zoom on a gradually opening nuclear fold. Sscale bar, 2.5 μm. Right, EOPNE at 10 and subsequently 5 μm (upper graph, n = 10 different cells; p value, paired t test) and statistics of EOPNE in cell populations at 20, 10, and 5 μm (lower graph, data are from ≥ 2 experiments; mean ± SD; n = 30 cells per height; p value, unpaired t test). E: Images of the LAP2-GFP-labeled NE and EOPNE quantifications in live cells confined to 5 μm and un-confined to 20 μm (n = 10 cells; p value, paired t test). Scale bar, 5 μm. F: NE fluctuation curves at various confinement heights (h) and quantifications of NE fluctuations at 10 and subsequently 5 μm (n = 10 cells; p value, paired t test) or in cell populations at 20, 10, and 5 μm (mean ± SD; n = 30 cells per height; p value, unpaired t test). G: Images of NUP107-GFP-labeled nuclear pores (NPs) and quantification of inter-NP (NP-NP) distance at 10 and subsequently 5 μm (n = 10 cells; p value, paired t test). Scale bar, 0.5 μm. H: Images of nuclear cPla2-mKate2 signal and quantification of its NE-to-nucleoplasm (NE/NPM) ratio at 10 and subsequently 5 μm (n = 10 cells; p value, paired t test). Scale bar, 1.5 μm.

Article Snippet: The following primers were used: Mm01284324_m1 for PLA2g4 and Mm99999915 for GAPDH as a control. cPLA2a expression was assessed in si-control and si-cPLA2a iDCs.

Techniques: Staining, Labeling

Journal: Science (New York, N.Y.)

Article Title: The nucleus acts as a ruler tailoring cell responses to spatial constraints

doi: 10.1126/science.aba2894

Figure Lengend Snippet: A: Left and middle, cartoons illustrating primary culture of iDCs and their confinement between two parallel surfaces inducing a highly migratory DC phenotype. Right top, iDC velocity measured at 10 (n = 20 cells), 4 (n = 35 cells), and 3 (n = 35 cells) μm confinement height (h). Right bottom, cell velocity (vcell) measured at 3 μm confinement in control (DMSO and wild-type (WT)) vs. cPLA2-inhibited (AACOCF3/AA treatment) or Lmna knockout (KO) cells (n = 20 cells per condition). Data are from ≥ 2 experiments; mean ± SD; p value, unpaired t test. B: Top, temporal color-coded cell tracks from a representative time-lapse movie of control (Ctrl si) and cPLA2a-depleted (Pla2g4a si) LifeAct-GFP-expressing iDCs under 3 μm-confinement. Bottom, statistical analysis of cell velocity (vcell) for control and depleted cells at 4 vs. 3 μm. Data are from ≥ 2 experiments; mean ± SD; n = 20 cells per condition; p value, unpaired t test. Scale bar, 50 μm. C: Representative images of DAPI-stained nuclei (XY view, single confocal slices) and EOPNE quantifications in iDCs at 4 vs. 3 μm. Data are from ≥ 2 experiments; mean ± SD; n = 35 cells per condition; p value, unpaired t test. Scale bar, 5 μm. D: Representative images and quantifications of myosin cortical accumulation in control (Ctrl si) and cPLA2a-depleted (Pla2g4a si) MYH9-GFP-expressing iDCs at 4 and 3 μm. Data are from ≥ 2 experiments; mean ± SD; n = 20 cells per condition; p value, unpaired t test. Scale bar, 15 μm.

Article Snippet: The following primers were used: Mm01284324_m1 for PLA2g4 and Mm99999915 for GAPDH as a control. cPLA2a expression was assessed in si-control and si-cPLA2a iDCs.

Techniques: Control, Knock-Out, Expressing, Staining

Journal: Science (New York, N.Y.)

Article Title: The nucleus acts as a ruler tailoring cell responses to spatial constraints

doi: 10.1126/science.aba2894

Figure Lengend Snippet: A: Left, 3D light-sheet microscopy images of atelopeptide fibrillar bovine dermal collagen (1.7 mg ml−1) lattices. Right, quantifications of the percentage of human melanoma cells A375P able to chemotactically transmigrate through the lattice (1 mm thick) in the presence of the broad-spectrum matrix metalloproteinase inhibitor GM6001 in conditions affecting cell contractility (Y27, BBS, and ML7), NE properties (LMNA si and LBR OE), cPLA2 expression and activity (PLA2G4A si, AA, and PA), and stretch-sensitive calcium release (Gd3+, GsMTx4, 2APB, and Xesto). Data are from ≥ 2 experiments; mean ± SD; n ≥ 300 cells per condition. See Table S3 for drug target description and pairwise statistical comparisons, and Materials and Methods for drug concentrations. B: Scanning electron microscopy images of polycarbonate membranes with 12 (blue) and 8 (red) μm pores (scale bar, 10 μm), and quantifications of the percentage of A375P cells able to chemotactically transmigrate through the pores in the conditions specified in A. Data are from ≥ 2 experiments; mean ± SD; n ≥ 300 cells per condition. See Table S3 for drug target description and pairwise statistical comparisons, and Materials and Methods for drug concentrations. Dotted line in A and B, cell transmigration rate upon global perturbation of actomyosin contractility.

Article Snippet: The following primers were used: Mm01284324_m1 for PLA2g4 and Mm99999915 for GAPDH as a control. cPLA2a expression was assessed in si-control and si-cPLA2a iDCs.

Techniques: Microscopy, Expressing, Activity Assay, Electron Microscopy, Transmigration Assay