Journal: Vaccines

Article Title: Three-Component Subunit Vaccine Induces Protective Immunity Against Mycoplasma pneumoniae in Mice

doi: 10.3390/vaccines14040330

Figure Lengend Snippet: Recombinant protein design, construction, and purity validation. ( A ) Schematic diagram of recombinant protein constructs. Mutation sites are indicated by short black lines. ( B – H ) SDS-PAGE, Western blot, and Superdex™ 200 Increase 10/300 GL purification results for recombinant proteins. ( B ) P1 (~165 kDa); ( C ) P40/90 complex (~117 kDa); ( D ) CARDS-WT (~69 kDa); ( E ) CARDS-E132A (~69 kDa); ( F ) CARDS-E132Q (~69 kDa); ( G ) CARDS-H36A (~69 kDa); ( H ) CARDS-R10A (~69 kDa). Left lane: protein marker; right lane: purified recombinant protein.

Article Snippet: The coding sequences for P1 (GenBank I D: X06871 ), P40 (GenBank ID: AF125204 ), P90 (GenBank ID: AF125205 ), and CARDS-WT (GenBank ID: AY189944 ) were retrieved from the NCBI database based on the Mycoplasma pneumoniae standard strain ATCC M129.

Techniques: Recombinant, Biomarker Discovery, Construct, Mutagenesis, SDS Page, Western Blot, Purification, Marker

Journal: Vaccines

Article Title: Three-Component Subunit Vaccine Induces Protective Immunity Against Mycoplasma pneumoniae in Mice

doi: 10.3390/vaccines14040330

Figure Lengend Snippet: Toxicity screening of CARDS mutants and purity analysis of recombinant proteins. ( A ) Dose–response curves of TNF-α secretion from RAW264.7 cells treated with CARDS-WT and various mutants, used for screening attenuated mutants. ( B – H ) HPLC chromatograms of recombinant proteins on TSKgel G2000SWXL column. Purity values are indicated above the main peaks: ( B ) CARDS-E132A (78.88%), ( C ) CARDS-E132Q (73.47%), ( D ) CARDS-H36A (86.99%), ( E ) CARDS-R10A (82.72%), ( F ) P1 (87.65%), ( G ) P40/90 (96.76%), ( H ) CARDS-WT (91.77%).

Article Snippet: The coding sequences for P1 (GenBank I D: X06871 ), P40 (GenBank ID: AF125204 ), P90 (GenBank ID: AF125205 ), and CARDS-WT (GenBank ID: AY189944 ) were retrieved from the NCBI database based on the Mycoplasma pneumoniae standard strain ATCC M129.

Techniques: Recombinant

Journal: Vaccines

Article Title: Three-Component Subunit Vaccine Induces Protective Immunity Against Mycoplasma pneumoniae in Mice

doi: 10.3390/vaccines14040330

Figure Lengend Snippet: Formulation, immunization schedule, and antigen-specific IgG antibody responses of the three-component M. pneumoniae vaccine. ( A ) Formulation scheme showing the 6 experimental groups, their respective vaccine and adjuvant combinations. ( B ) Immunization schedule showing time points for immunizations (days 0, 14, 28) and serum collection (days 13, 27, 41). ( C – F ) Serum titers (log10) of specific IgG antibodies against P1, P40/90, CARDS-WT, and CARDS-Mut following single antigen immunizations (days 0, 14, 28) and serum collection (days 13, 27, 41). The saline group served as the negative control. ( G – J ) Serum antibody titers (days 41) against P1, P40/90, CARDS-WT, and CARDS-Mut in mice immunized according to the groups shown in ( A ). ( K ) Comparison of antibody titers (days 41) between the MPtriVa-D group (containing CARDS-WT with dual adjuvant) and the MPtriVb-D group (containing CARDS-Mut with dual adjuvant). ( L ) Comparison of antibody titers (days 41) between the MPtriVb-S (single adjuvant) and MPtriVb-D (dual adjuvant) groups. Data are presented as mean ± SD. p > 0.05 (not significant); *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The coding sequences for P1 (GenBank I D: X06871 ), P40 (GenBank ID: AF125204 ), P90 (GenBank ID: AF125205 ), and CARDS-WT (GenBank ID: AY189944 ) were retrieved from the NCBI database based on the Mycoplasma pneumoniae standard strain ATCC M129.

Techniques: Formulation, Adjuvant, Saline, Negative Control, Comparison

Journal: Nature

Article Title: Cell-type-targeted mitochondrial transplantation rescues cell degeneration

doi: 10.1038/s41586-026-10391-0

Figure Lengend Snippet: Donor mito-mTagBFP2 can be free from endocytic vesicles. HUVECs expressing cell surface GFP and endosome-targeted Rab5a-TagRFP (magenta) transplanted with mito-mTagBFP2 displaying anti-GFP nanobody (cyan). The videos were recorded 6 h and 1 day after mitochondrial transplantation, respectively. Two different cells are shown. Scale bar, 5 mm.

Article Snippet: For TagBFP2 targeting into the matrix, the mTagBFP2 coding DNA sequence was fused to COX8 matrix-targeting signal peptide, synthesized by Twist Biosciences, and inserted into a pCMV backbone.

Techniques:

Journal: Nature

Article Title: Cell-type-targeted mitochondrial transplantation rescues cell degeneration

doi: 10.1038/s41586-026-10391-0

Figure Lengend Snippet: a. Top, schematic diagram of the construct used for directing a nanobody to the outer membrane of mitochondria. Bottom, a super-resolution image of an HEK293T cell with mitochondria-targeted nanobody detected by anti-alpacaV H H antibodies (magenta). Cell nuclei are labelled with Hoechst (blue). Mitochondria matrix is labelled with dsRed2 (cyan). 3D SIM, three-dimensional structural illumination microscopy. The construct was validated in at least three independent experiments. b. Top, schematic diagram of the construct used for directing GFP to the cell surface. Bottom, a super-resolution image of an HEK293T cell with cell surface-targeted GFP detected by anti-GFP antibodies (green). Cell nuclei are labelled with Hoechst (blue). The construct was validated in at least three independent experiments. c. HEK239T cells transplanted with donor mitochondria displaying the anti-GFP nanobody, two hours after transplantation. HEK293T cells are transfected with cell-surface mCherry (cyan) or GFP (green). Nanobodies are detected by anti-alpacaV H H antibodies (magenta). d. Quantification of the efficacy of the delivery of nanobody-displaying mitochondria two hours after transplantation. n = 6, P < 0.0001 (top) and P = 0.0012 (bottom), two-sided Welch’s t test. e. HEK239T cells transplanted with donor mitochondria displaying the anti-mCherry nanobody, two hours after transplantation. HEK293T cells were transfected with cell-surface GFP (green) or mCherry (cyan). Nanobodies detected by anti-alpacaV H H antibodies (magenta). f. Quantification of the efficacy of the delivery of nanobody-displaying mitochondria, two hours after transplantation. n = 8, P = 0.0003 (top) and P = 0.0012 (bottom), two-sided Welch’s test. g. Live-imaged endothelial cells expressing cell surface GFP and endosome-targeted RAB5A-TagRFP with (top) and without (bottom) transplanted with mito-mTagBFP2 (cyan) displaying anti-GFP nanobody, six hours after mitochondrial transplantation. White arrows, endosome-free donor mitochondria (confirmed in at least three independent experiments). For an example of mito-mTagBP2 in endothelial cells transplanted without the anti-GFP nanobody, see Supplementary Fig. . h. Donor mito-mTagBFP2 (cyan) inside endocytic vesicles (red arrows) labelled with RAB5A-TagRFP (magenta) (from g), six hours after mitochondrial transplantation. i. Donor mito-mTagBFP2 (cyan) free from endocytic vesicles (white arrows) labelled with RAB5A-TagRFP (magenta) (from g), six hours after mitochondrial transplantation. j. Donor mito-mTagBFP2 (cyan) free from endocytic vesicles (white arrows) labelled with RAB5A-TagRFP (magenta), 24 h after mitochondrial transplantation. Outlined region, mito-mTagBFP2 free from endocytic vesicles and lysosomes (yellow). Lysosomes are stained with LysoTracker Deep Red dye. The experiment was repeated at least three times with similar results. k. Quantification of proportion of endosome-free mitochondria by pixel-based co-localization analysis. Mito-mTagBFP2: n = 30; mito-mTagBFP2 + anti-GFP nanobody: n = 52, P = 0.0756, two-sided Mann-Whitney U test. l. Quantification of abundance of endosome-free mitochondria by pixel-based co-localization analysis. The values were normalized to cell size (µm 2 of donor mitochondria area per 1 µm 2 cell area). Mito-mTagBFP2: n = 30; mito-mTagBFP2 + anti-GFP nanobody: n = 52, P < 0.0001, two-sided Mann-Whitney U test. m. Endothelial cells stained with pH-dependent lysosome staining dye pHLys Red (yellow). Cells expressed cell surface GFP and were transplanted with mito-mTagBFP2 displaying anti-GFP nanobody or no binder. In addition, Bafilomycin A1 was used as a positive control for pH acidification change in lysosomes. For mitochondria transplanted conditions, images of cells positive for mito-mTagBFP2 are shown (Supplementary Fig. ). n. Quantification of pH changes in lysosomes relative to untreated condition. Untreated: n = 6; Bafilomycin A1: n = 4; mito-mTagBFP2: n = 4; mito-mTagBFP2 + anti-GFP nanobody: n = 6, Untreated vs. Bafilomycin A1: P = 0.0036, Untreated vs. mito-mTagBFP2: P = 0.8235, Untreated vs. mito-mTagBFP2 + anti-GFP nanobody: P = 0.9648, Welch’s ANOVA test corrected with two-sided Dunnett’s test for multiple comparisons. o. Live-imaged endothelial cell expressing cell surface GFP (green), and transplanted with mito-dsRed2 (cyan) displaying anti-GFP nanobody, four days after mitochondrial transplantation. The cell is outlined with a grey dashed line. The zoomed-in region is outlined with a white dashed square. Two timeframes are shown on the right. The tracked mitochondrion is indicated with a red arrow. The experiment was repeated at least three times with similar results. p. Live-imaged endothelial cell expressing cell surface GFP and transplanted with mito-dsRed2 (cyan) displaying outer membrane anti-GFP nanobody, four days after mitochondrial transplantation. Mitochondria are labelled with 50 nM MitoTracker Deep Red (magenta). The zoomed-in region is outlined with a white dashed square and the tracked mitochondrion is indicated with a white arrow. The experiment was repeated at least three times with similar results. q. Labelling of donor and native mitochondria with MitoTracker Deep Red dye in live-recorded endothelial cells. At the used concentration, the dye stained both native (black) and donor mitochondria (cyan) with stronger enrichment in the native mitochondria. Donor mitochondria positive for matrix-labelled dsRed2 and MitoTracker Deep Red are indicated with red arrows. NS not significant, ** P < 0.01, *** P < 0.001. Data, mean ± s.e.m and median for k, l. Scale bars, 2.5 µm (a, b), 25 µm (c, e), 5 µm (g, h, i, o, p, q), 10 µm (j), 20 µm (m).

Article Snippet: For TagBFP2 targeting into the matrix, the mTagBFP2 coding DNA sequence was fused to COX8 matrix-targeting signal peptide, synthesized by Twist Biosciences, and inserted into a pCMV backbone.

Techniques: Construct, Membrane, Microscopy, Transplantation Assay, Transfection, Expressing, Staining, MANN-WHITNEY, Positive Control, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Exploration of the proxiOME of large subunit ribosomal proteins reveals Acl1 and Bcl1 as cooperating dedicated chaperones of Rpl1

doi: 10.1093/nar/gkag264

Figure Lengend Snippet: The conserved Bcl1 interacts via its WD40 β-propeller with Rpl1. ( A ) Proximity labelling assay with C-terminally miniTurbo-tagged RPL10A (HsRPL10A-miniTurbo) in HeLa cells. The RPL10A bait r-protein and selected enriched proteins are written in bold, the red dot highlights the highly enriched WDR89. ( B ) AlphaFold3 model of the Bcl1–Rpl1 complex. The seven-bladed β-propeller domain of Bcl1 is coloured in green and the C-terminal extension in light green; the position of residue Asn322 (N322) is indicated to better visualize from where the C-terminal extension emanates. ( C ) AlphaFold3 model of the WDR89–RPL10A complex (left) and its structural superposition with the AlphaFold3 model of the Bcl1–Rpl1 complex (right). ( D ) Predicted electrostatic surface potential of Bcl1 (left) and close-up view of two of the three Rpl1 sites, indicating residues predicted to form H-bonds with Bcl1, that are in contact with the negatively charged top surface of the β-propeller (right). ( E–G ) Y2H interaction assays between the full-length Rpl1 and Bcl1 proteins ( E ), between full-length Rpl1 and the C-terminally truncated Bcl1.N366 and Bcl1.N325 variants or the C-terminal extension of Bcl1 (323C) ( F ), and between the indicated Rpl1 mutant variants and either Bcl1 or Acl1 ( G ). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; E, Glu; K, Lys; R, Arg. ( H ) In vitro binding assay between Bcl1.N366 and Rpl1. Bcl1.N366-(His) 6 or Bcl1.N366 and Rpl1b were co-expressed in E. coli and purified by Ni–NTA affinity purification. Proteins were revealed by SDS–PAGE and Coomassie staining (top) or by western blotting using anti-His and anti-Rpl1 antibodies (bottom). Bands corresponding to Bcl1.N366-(His) 6 and Bcl1.N366 or to Rpl1b are indicated by blue or black arrowheads.

Article Snippet: The DNA sequence coding for the Homo sapiens RPL10A protein was PCR-amplified from plasmid pADH111-HsRPL10A (pDK10427), generated by cloning the PCR-amplified RPL10A coding sequence [template pNTI194 (Addgene plasmid #84266)] into the Nde I/ Bam HI-restricted plasmid pADH111-LTV1 (pDK3331), and cloned by Gibson assembly between the Nhe I and Pst I restriction sites of the lentiviral donor vector pSKP-32, a pCW57.1-derived plasmid bearing the MND-Blasticidin resistance cassette instead of the hPGK-puromycin resistance cassette [ ], to generate plasmid pDS79 containing the RPL10A gene under the transcriptional control of a doxycycline-inducible promoter and fused at its 3′ end to sequences encoding the V5 tag, the miniTurbo (MT) biotin ligase, and the HA tag.

Techniques: Residue, Mutagenesis, In Vitro, Binding Assay, Purification, Affinity Purification, SDS Page, Staining, Western Blot