Journal: Applied Microbiology and Biotechnology

Article Title: Streptomycetes as platform for biotechnological production processes of drugs

doi: 10.1007/s00253-020-11064-2

Figure Lengend Snippet: SEM pictures of Streptomyces roseochromogenes ATCC 13400 in liquid cultures during hydrocortisone bioconversion at 0, 4, and 48 h, respectively ( a – c ): the accumulation of material on the cell surface is due to the process of steroid bioconversion and it is indicated by the arrows ( c ); SEM pictures of Streptomyces roseochromogenes ATCC 13400 whole cells as immobilized in calcium alginate beads ( d – f ): a tight mycelium was visible in the picture of bead internal view ( f ); SEM pictures of Streptomyces cyanogriseus ATCC 27426 in liquid cultures during secondary metabolite production at 96, 168, and 264 h, respectively ( g – i ): morphology changes with increase of cell tangling, branching, and rugosity are visible. (Mag from × 82 to × 20,000, scale bar from 2 to 200 μm). (Preparation of the samples for SEM analyses: small volumes of culture (1 mL) were pelleted end suspended in 4% formalin in PBS for 18 h, dehydrated in increasing ethanol concentrations (from 30 to 100% for 5–15 min), dried in a critical point dryer, and sputtered with platinum-palladium (sputter coater Denton Vacuum Desk V). Fe-SEM Supra 40 Zeiss (5 kV, detector InLens) and Smart SEM Zeiss software were used for observation

Article Snippet: Fig. 2 SEM pictures of Streptomyces roseochromogenes ATCC 13400 in liquid cultures during hydrocortisone bioconversion at 0, 4, and 48 h, respectively ( a – c ): the accumulation of material on the cell surface is due to the process of steroid bioconversion and it is indicated by the arrows ( c ); SEM pictures of Streptomyces roseochromogenes ATCC 13400 whole cells as immobilized in calcium alginate beads ( d – f ): a tight mycelium was visible in the picture of bead internal view ( f ); SEM pictures of Streptomyces cyanogriseus ATCC 27426 in liquid cultures during secondary metabolite production at 96, 168, and 264 h, respectively ( g – i ): morphology changes with increase of cell tangling, branching, and rugosity are visible. (Mag from × 82 to × 20,000, scale bar from 2 to 200 μm). (Preparation of the samples for SEM analyses: small volumes of culture (1 mL) were pelleted end suspended in 4% formalin in PBS for 18 h, dehydrated in increasing ethanol concentrations (from 30 to 100% for 5–15 min), dried in a critical point dryer, and sputtered with platinum-palladium (sputter coater Denton Vacuum Desk V).

Techniques: Software

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

Article Title: Prokaryotic innate immunity via pattern recognition of conserved viral proteins

doi: 10.1126/science.abm4096

Figure Lengend Snippet: SeAvs3 and EcAvs4 are phage-activated DNA endonucleases. (A) Domain architecture of SeAvs3 and EcAvs4. (B) Alignment of Avs D-QxK nuclease motifs with characterized Cap4 and Mrr representatives. (C, D) Agarose gel analysis of SeAvs3 nuclease function in vitro with a linear dsDNA substrate and (E) cofactor requirements. (F, G) Agarose gel analysis of EcAvs4 nuclease function in vitro with a linear dsDNA substrate and (H) cofactor requirements.

Article Snippet: The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with the following codes: EMD-27421 (SeAvs3-gp19, C2 refinement), EMD-27424 (SeAvs3-gp19, C4-expanded C1 refinement), EMD-27425 (EcAvs4-gp8, C2 refinement), EMD-27422 (EcAvs4-gp8, C2 refinement focused on Mrr), EMD-27426 (EcAvs4-gp8, C4-expanded C1 refinement).

Techniques: Agarose Gel Electrophoresis, In Vitro

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

Article Title: Prokaryotic innate immunity via pattern recognition of conserved viral proteins

doi: 10.1126/science.abm4096

Figure Lengend Snippet: Cryo-EM structures of SeAvs3 and EcAvs4 in complex with their cognate triggers. (A, B) Structure of the SeAvs3-terminase complex. (C, D) Structure of the EcAvs4-portal complex. (E, F) ATP molecule in the STAND ATPase active site of EcAvs4 and SeAvs3. Cryo-EM density is shown as a transparent surface. (G) SeAvs3 Cap4-like nuclease effector domain. (H, I) Active sites for the inward and outward facing protomers of the SeAvs3 Cap4-like nuclease. (J) Equivalent view of the active site of HindIII bound to target DNA with two divalent metal ions (PDB 3A4K). (K) Electrostatic surface potential for the SeAvs3 Cap4-like nuclease and the EcAvs4 Mrr-like nuclease. Active sites are indicated by purple circles. Ideal B-form DNA is modeled on both surfaces based on the crystal structure of HindIII bound to its target (PDB 3A4K). (L) EcAvs4 Mrr-like nuclease effector domain. (M, N) Active sites for the inward and outward facing protomers of the EcAvs4 Mrr-like nuclease. TPR, tetratricopeptide repeat.

Article Snippet: The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with the following codes: EMD-27421 (SeAvs3-gp19, C2 refinement), EMD-27424 (SeAvs3-gp19, C4-expanded C1 refinement), EMD-27425 (EcAvs4-gp8, C2 refinement), EMD-27422 (EcAvs4-gp8, C2 refinement focused on Mrr), EMD-27426 (EcAvs4-gp8, C4-expanded C1 refinement).

Techniques: Cryo-EM Sample Prep

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

Article Title: Prokaryotic innate immunity via pattern recognition of conserved viral proteins

doi: 10.1126/science.abm4096

Figure Lengend Snippet: Structural basis for viral fold recognition by SeAvs3 and EcAvs4. (A) The interface between SeAvs3 and the PhiV-1 terminase. An SeAvs3 surface view is shown in transparency. SeAvs3 is colored from N to C terminus according to the key. (B) AlphaFold or crystal structures of different terminases modeled into SeAvs3. The ATPase and nuclease domains were individually aligned to the PhiV-1 terminase domains. (C, D) Recognition of the PhiV-1 terminase ATPase and nuclease active sites by the SeAvs3 TPR domain. (E) Sequence logos for terminase ATPase Walker A motifs and terminase nuclease active sites. A total of 11,000 terminase sequences were clustered at 30% sequence identity, and motifs were extracted from clusters containing terminases targeted or not targeted by SeAvs3 according to Fig. 2B (see also fig. S21). (F) Plasmid depletion assay for SeAvs3 co-expressed in E. coli with a terminase ATPase or nuclease domain harboring active site mutations. (G) The interface between EcAvs4 and the PhiV-1 portal. An EcAvs4 surface view is shown in transparency. EcAvs4 is colored from N to C terminus according to the key. (H) β-sheet augmentation between EcAvs4 and the portal clip domain. (I) Comparison of the EcAvs4-bound state of the PhiV-1 portal, the cryo-EM structure of the highly homologous T7 portal in its native virion, and AlphaFold models of diverse portals. A top view of the assembled dodecamer of the T7 portal is also shown. TPR, tetratricopeptide repeat; CTD, C-terminal domain.

Article Snippet: The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with the following codes: EMD-27421 (SeAvs3-gp19, C2 refinement), EMD-27424 (SeAvs3-gp19, C4-expanded C1 refinement), EMD-27425 (EcAvs4-gp8, C2 refinement), EMD-27422 (EcAvs4-gp8, C2 refinement focused on Mrr), EMD-27426 (EcAvs4-gp8, C4-expanded C1 refinement).

Techniques: Sequencing, Plasmid Preparation, Depletion Assay, Cryo-EM Sample Prep

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

Article Title: Prokaryotic innate immunity via pattern recognition of conserved viral proteins

doi: 10.1126/science.abm4096

Figure Lengend Snippet: Taxonomic distribution and domain architectures of Avs families. (A) Distribution of avs genes across phyla. The values above the bars indicate the number and percentage of genomes containing each gene. (B) Number of bacterial and archaeal phyla (minimum 100 sequenced isolates) with at least one detected instance of an avs gene. (C) Kernel density plots of the length distribution of Avs proteins, excluding the N-terminal domain. The red lines indicate medians. ****p < 0.0001 (Mann-Whitney). Maximum likelihood tree of representatives of the ATPase + C-terminal domain of (D) Avs2 terminase sensors (n = 1,255) and (E) Avs4 portal sensors (n = 1,089) clustered at 95% sequence identity. See fig. S24 for the trees for Avs1 and Avs3. Stars on the outer ring indicate homologs investigated experimentally in this study. MBL, metallo-β-lactamase; REase, restriction endonuclease; TIR, Toll/interleukin-1 receptor homology domain; SIR2, sirtuin; CMP, cytidine monophosphate; HTH, helix-turn-helix. (F) Anti-phage defense activity of a chimeric Avs4 with transmembrane N-terminal helices from Sulfurospirillum sp. replacing the nuclease domain of EcAvs4.

Article Snippet: The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with the following codes: EMD-27421 (SeAvs3-gp19, C2 refinement), EMD-27424 (SeAvs3-gp19, C4-expanded C1 refinement), EMD-27425 (EcAvs4-gp8, C2 refinement), EMD-27422 (EcAvs4-gp8, C2 refinement focused on Mrr), EMD-27426 (EcAvs4-gp8, C4-expanded C1 refinement).

Techniques: MANN-WHITNEY, Sequencing, Activity Assay

Journal: Journal of Cardiovascular Pharmacology

Article Title: Transient Receptor Potential Vanilloid Type 1 Protects Against Pressure Overload–Induced Cardiac Hypertrophy by Promoting Mitochondria-Associated Endoplasmic Reticulum Membranes

doi: 10.1097/FJC.0000000000001301

Figure Lengend Snippet: TRPV1 activation protected against PE-induced cardiomyocyte hypertrophy in vitro. A, Representative images and analysis of the cell surface area of neonatal rat cardiomyocytes stained with cTnT (green) and DAPI (blue) (n = 5, scale bar = 10 μm). B, The ANP, BNP, β-MHC protein expression in primary cardiomyocytes treated with capsaicin and PE (n = 5). C, Quantitative polymerase chain reaction analyses of the mRNA levels of ANP, BNP, β-MHC in the indicated groups. n = 5 per group. * P < 0.05 compared with control, # P < 0.05 compared with PE group.

Article Snippet: After the nonspecific binding sites were blocked in Tris-buffered saline containing 5% nonfat dry milk for 1 hour, the membranes were incubated at 4°C overnight with primary antibodies, including mouse anti-TRPV1 (1:1000; cat. No. sc-398417; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti–voltage-dependent anion channels (1:1000; cat. No. 10866-1-AP; Proteintech, Wuhan, China), rabbit anti-inositol 1,4,5-trisphosphate receptor (IP3R) (1:1000; cat. No. 19962-1-AP; Proteintech), rabbit anti-P-AMPK (1:1000; cat. No. 8208; Cell Signaling Technology, Danvers, MA), rabbit anti-AMPK (1:1000; cat. No. 5832; Cell Signaling Technology), rabbit anti-MFN2 (1:1000; cat. No. 12186-1-AP; Proteintech), rabbit anti-atrial natriuretic peptide (ANP) (1:500; cat. No. 27426-1-AP; Proteintech), rabbit anti-B-type natriuretic peptide (BNP) (1:500; cat. No. 13299-1-AP; Proteintech), and rabbit anti-beta-myosin heavy chain (β-MHC) (1:500; cat. No. 10799-1-AP; Proteintech).

Techniques: Activation Assay, In Vitro, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Cardiovascular Pharmacology

Article Title: Transient Receptor Potential Vanilloid Type 1 Protects Against Pressure Overload–Induced Cardiac Hypertrophy by Promoting Mitochondria-Associated Endoplasmic Reticulum Membranes

doi: 10.1097/FJC.0000000000001301

Figure Lengend Snippet: TRPV1 activation protected against PE-induced cardiomyocyte hypertrophy in vitro. A, Representative images and analysis of the cell surface area of neonatal rat cardiomyocytes stained with cTnT (green) and DAPI (blue) (n = 5, scale bar = 10 μm). B, The ANP, BNP, β-MHC protein expression in primary cardiomyocytes treated with capsaicin and PE (n = 5). C, Quantitative polymerase chain reaction analyses of the mRNA levels of ANP, BNP, β-MHC in the indicated groups. n = 5 per group. * P < 0.05 compared with control, # P < 0.05 compared with PE group.

Article Snippet: After the nonspecific binding sites were blocked in Tris-buffered saline containing 5% nonfat dry milk for 1 hour, the membranes were incubated at 4°C overnight with primary antibodies, including mouse anti-TRPV1 (1:1000; cat. No. sc-398417; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti–voltage-dependent anion channels (1:1000; cat. No. 10866-1-AP; Proteintech, Wuhan, China), rabbit anti-inositol 1,4,5-trisphosphate receptor (IP3R) (1:1000; cat. No. 19962-1-AP; Proteintech), rabbit anti-P-AMPK (1:1000; cat. No. 8208; Cell Signaling Technology, Danvers, MA), rabbit anti-AMPK (1:1000; cat. No. 5832; Cell Signaling Technology), rabbit anti-MFN2 (1:1000; cat. No. 12186-1-AP; Proteintech), rabbit anti-atrial natriuretic peptide (ANP) (1:500; cat. No. 27426-1-AP; Proteintech), rabbit anti-B-type natriuretic peptide (BNP) (1:500; cat. No. 13299-1-AP; Proteintech), and rabbit anti-beta-myosin heavy chain (β-MHC) (1:500; cat. No. 10799-1-AP; Proteintech).

Techniques: Activation Assay, In Vitro, Staining, Expressing, Real-time Polymerase Chain Reaction