Journal: Nature Communications

Article Title: IFN-γ-driven UBE2D3 upregulation impairs antigen presentation pathways and anti-tumor immunity in pancreatic cancer

doi: 10.1038/s41467-025-65762-4

Figure Lengend Snippet: a Hypothesis diagram depicting the crosstalk between immune inflammatory cells and PDAC cells. b Heatmap of transcriptome DEGs in the cellular response to IFN-γ process between KPC-shScramble (Scr) cells and KPC-shUbe2d3-1 (KD1) cells ( n = 3 samples per group). c Tumor growth curves following subcutaneous inoculation of KPC/Panc02 Scr/KD1 cells in syngeneic immunodeficient Rag1 −/− mice and subsequent implantation into immunocompetent C57BL/6 mice. Once tumor reached 1000 mm 3 in Rag1 −/− mice ( n = 4 mice per group), they were excised, and 1–2 mm 3 fragments were further inoculated subcutaneously into immunocompetent C57BL/6 mice ( n = 7 mice per group). d Experimental setup for in vitro CTL-mediated killing of KPC-WT/KO cells. KPC-WT cells were subjected to lentiviral transduction with the indicated sgCtrl (Ctrl)-GFP, sgCtrl (Ctrl)-mCherry, sgUbe2d3-1 (KO1)-mCherry, or sgUbe2d3-2 (KO2)-mCherry Cas9 vectors. These cells were further transiently transfected with the OVA OE pcDNA3 plasmid to construct target cells. OT-I CD8 + T cells were cocultured with either a mixture of KPC OVA -Ctrl-GFP cells and KPC OVA -Ctrl-mCherry cells or a mixture of KPC OVA -Ctrl-GFP cells and KPC OVA -KO-mCherry cells for 24 h ( n = 5 independent experiments per group). e Quantification of tumor-infiltrating CD4 + T cells and CD8 + T cells in subcutaneous KPC Scr/KD1 model ( n = 5 mice per group) and Panc02 Scr/KD1 model ( n = 9 mice per group). f Tumor growth curves from CD4 + or CD8 + T-cell deletion assay ( n = 5 mice per group). g Tumor growth evaluation in humanized NCG mice. Seven days post-PBMC injection, these immunoreconstituted mice were subcutaneously engrafted with Capan-1-Scr or Capan-1-KD1 cells. The level of immune humanization was confirmed by flow cytometric analysis of hCD45 + cells in peripheral blood on days 7 and 21. All mice were sacrificed 24 days after tumor inoculation for tumor weight measurement ( n = 7 mice per group). Statistical significance was determined via the unpaired t test (two-tailed) in ( c – g ). The data are presented as the means ± SDs. Source data are provided in the Source Data file.

Article Snippet: The tumor burdens in the pancreas and liver were examined 21 d after tumor inoculation via IVIS detection of GFP signals (PerkinElmer, Lumina Series III).

Techniques: In Vitro, Transduction, Transfection, Plasmid Preparation, Construct, DNA Deletion Assay, Injection, Two Tailed Test

Journal: Science Advances

Article Title: Structural dynamics-guided engineering of a riboswitch RNA for evolving c-di-AMP synthases

doi: 10.1126/sciadv.adt8165

Figure Lengend Snippet: ( A ) Schematic of PK formation via strand displacement in the presence of a probe. The addition of c-di-AMP dissociates the DNA probe (blue line) from riboCdA, triggering PK formation. ( B ) smFRET histograms for PK-riboCdA in the presence of the probe at 2 mM Mg 2+ with and without 200 μM c-di-AMP. ( C ) Schematic of c-di-AMP addition to dissociate the Cy5-labeled probe from riboCdA at 2 mM Mg 2+ and 37°C. ( D ) Denaturing PAGE images showing dissociation of the Cy5-probe from riboCdA (WT, top) and 2bP1-riboCdA (bottom) upon the addition of c-di-AMP. The gels were excited using a 630 nm fluorescent wavelength. ( E ) Fitting of relative fluorescence unit (RFU) for riboCdA (green), 2bP1-riboCdA (red), and ΔP1-riboCdA (brown) plotted with c-di-AMP concentrations. ( F ) Schematic of inverting riboCdA from OFF to ON biosensor via strand displacement. c-di-AMP, synthesized from DisA Bs , disrupts the stem-loop structure, releasing the SD sequence for ribosome binding. ( G ) In vivo translation activities of stem loops D1 to D8, with D4 exhibiting the highest up-regulation folds. ( H ) Predicted secondary structure of D4, with PK in purple, SD in blue, and the translation initiation codon in red. ( I ) Fluorescent images of agar plates from in vivo translation experiments, showing GFP production regulated by riboCdA (left) and ON-riboCdA (right). ( J ) Comparison of activity among CdaA Lm (cyan), CdaA Bs (brown), DisA Bs (light purple), CdaA Pt (green), CdaS Bs (red), and CdaS Pt (purple) using ON-riboCdA, ON-2bP1-riboCdA, ON-ΔP1-riboCdA, riboCdA, and 2bP1-riboCdA as biosensors.

Article Snippet: The single colonies exhibiting high GFP fluorescence under the ChemiScope 6200 gel imager (Clinx Company, China) were inoculated into 1 ml of LB medium containing kanamycin (50 mg/liter) and grown overnight at 37°C.

Techniques: Labeling, Fluorescence, Synthesized, Sequencing, Binding Assay, In Vivo, Comparison, Activity Assay

Journal: Science Advances

Article Title: Structural dynamics-guided engineering of a riboswitch RNA for evolving c-di-AMP synthases

doi: 10.1126/sciadv.adt8165

Figure Lengend Snippet: ( A ) Schematic procedures for rapid screening using our designed ON-riboCdA or ON-2bP1-riboCdA biosensors. Site-saturated mutants of DisA Bs are screened on the basis of real-time measurements of GFP fluorescence in the presence of c-di-AMP in E. coli BL21 (DE3). The optimal-performance mutants screened using ON-riboCdA or ON-2bP1-riboCdA biosensors are then sequenced and characterized. ( B and C ) Three and six mutants exhibiting higher enzymatic activities compared to the WT (DisA Bs ) were identified using ON-riboCdA or ON-2bP1-riboCdA biosensors, respectively. ( D ) In vitro activity comparison among DisA Bs , selected superior mutants, and combined mutants. ( E ) HPLC spectra showing the conversion of c-di-AMP from ATP at 30 min using DisA B s (black), E53P (cyan), P+K+L (purple), P+K (green), and E130K (brown).

Article Snippet: The single colonies exhibiting high GFP fluorescence under the ChemiScope 6200 gel imager (Clinx Company, China) were inoculated into 1 ml of LB medium containing kanamycin (50 mg/liter) and grown overnight at 37°C.

Techniques: Fluorescence, In Vitro, Activity Assay, Comparison

Journal: Scientific Reports

Article Title: Development of the membrane ceiling method for in vitro spermatogenesis

doi: 10.1038/s41598-024-84965-1

Figure Lengend Snippet: Optimization of porous membranes used for the MC chip. ( A ) Representative images using an inverted microscope (BZ-X700) of the MC chip made of various porous membranes as indicated. Scale bar = 0.5 mm. ( B ) The ratio of tissue volume expansion and ( C ) the GFP-positive rate over 4 weeks with MC chips consisting of indicated membranes. ** P < 0.01 and P > 0.05 if no indication. Twelve (Cont), 8 (PC0.4), 8 (PC10), 8 (PET0.45), and 8 (PET3) tissues were used for the experiments. Images were taken by IX73 for quantifications.

Article Snippet: In brief, bright field and GFP images were taken by inverted microscopes with a tiling function (BioStation CT; Nikon, Tokyo, Japan, BZ-X700; Keyence Corporation, Osaka, Japan).

Techniques: Inverted Microscopy

Journal: Scientific Reports

Article Title: Development of the membrane ceiling method for in vitro spermatogenesis

doi: 10.1038/s41598-024-84965-1

Figure Lengend Snippet: Optimization of oxygen-permeable materials for device base. ( A ) Representative images showing the visibility of seminiferous tubules collected from GARN adult mice in the device using PMP or FEP. Images were taken by an inverted microscope (BZ-X700). Scale bar = 100 μm. ( B ) The ratio of tissue area expansion and ( C ) the GFP-positive rate for 5 weeks in the device with PDMS, PMP, or FEP base plate or dish. * P < 0.05, **** P < 0.0001, and ns; no significant. ( D ) Representative images of He-PAS staining of testis sections after culturing in the device with PDMS, PMP, or FEP base plate or dish. rST round spermatids, eST elongating spermatids. Scale bar = 200 μm (upper panel) and 50 μm (lower panel). ( E ) Live imaging of in vitro spermatogenesis of the same tubule during cultivation on the PMP bottom plate. The images were taken by BioStation CT. St step of spermatids. d culture days. Scale bar = 50 μm and 10 μm (zoomed in). ( F ) The number of mCherry-positive cells divided by tissue area. Each dot shows the average cell number per tissue area of frames. Images were taken at 5 weeks in the device with PDMS, PMP, and FEP using the tiling function of a microscope (BioStation CT). ** P < 0.01, **** P < 0.0001, and ns; no significant. Seventeen (PDMS), 17 (PMP), 8 (FEP) tissues were used for quantification.

Article Snippet: In brief, bright field and GFP images were taken by inverted microscopes with a tiling function (BioStation CT; Nikon, Tokyo, Japan, BZ-X700; Keyence Corporation, Osaka, Japan).

Techniques: Inverted Microscopy, Staining, Imaging, In Vitro, Microscopy

Journal: Scientific Reports

Article Title: Development of the membrane ceiling method for in vitro spermatogenesis

doi: 10.1038/s41598-024-84965-1

Figure Lengend Snippet: The effect of advanced DMEM/F12 (AD) as the in vitro basal medium in spermatogenesis. ( A ) The ratio of tissue area expansion and ( B ) the GFP-positive rate for 5 weeks in MEMα or AD-based culture medium. There were no significant differences ( P > 0.05). ( C ) Representative images of mCherry-positive cells observed using an inverted microscope (BZ-X700) after 5 weeks cultivated in MEMα or AD-based culture medium. Scale bar = 50 μm. ( D ) The number of mCherry-positive cells divided by tissue area (mm 2 ). Each dot shows the average cell number per tissue area of frames. Images were taken at 5 weeks using the tiling function of a microscope (BioStation CT and BZ-X700). * P < 0.05. Fifteen (MEMα) and 18 (AD) tissues were used for quantification. ( E ) In vitro generated sperm after 5 weeks in AD-based culture medium. The images were taken using an upright microscope (BX53). Flagellated cells with normal (upper panel) or abnormal (lower panel) heads were used for ICSI. Scale bar = 20 μm. ( F ) A picture of obtained offspring by ICSI using in vitro generated sperm.

Article Snippet: In brief, bright field and GFP images were taken by inverted microscopes with a tiling function (BioStation CT; Nikon, Tokyo, Japan, BZ-X700; Keyence Corporation, Osaka, Japan).

Techniques: In Vitro, Inverted Microscopy, Microscopy, Generated