Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: In vitro susceptibilities of planktonic S. mutans UA159

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: In Vitro

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: In Vitro

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: Comparative killing kinetics of CLP-4. S. mutans UA159 cultures at a cell density of 6 × 105 CFU/ml were challenged with 5, 10, and 25 μg/ml CLP-4 under conditions of active growth in CDM supplemented with 0.5% (wt/vol) glucose (A) and against growth-arrested cells in CDM lacking any carbon source (B). Samples at time zero were enumerated prior to peptide treatment. Data shown are the means and standard deviations of three biological replicates from three independent experiments.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: CLP-4 prevents S. mutans biofilm formation. (A) Biofilms inoculated with 2 × 107 CFU/ml were grown for 24 h in the presence of CLP-4, chlorhexidine, or erythromycin at concentrations ranging between 0.6× and 2× their respective MICs. Biofilm formation was quantified using crystal violet staining and expressed in percentage relative to untreated control. Shown are the means and standard deviations of three biological replicates from three independent experiments. *, P < 0.05; ***, P < 0.001 compared to untreated control. (B) Corresponding growth curve kinetics showing the MIC of CLP-4 on S. mutans UA159.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: Staining, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: Effects of CLP-4 on preformed biofilms. S. mutans UA159 biofilms were established for 24 h and then treated with increasing concentrations (1× to 10× the MIC) of CLP-4, chlorhexidine, or erythromycin. (A) Antibiofilm activities were assessed by quantifying the cell viability of treated biofilms by colony enumeration on agar plates. The means and standard deviations of three biological replicates from three independent experiments are shown. **, P < 0.01; ***, P < 0.001 compared to untreated control. (B) Biofilms treated with 10× the MICs for each antimicrobial were fluorescently labeled using the LIVE/DEAD BacLight viability stain and visualized by confocal laser scanning microscopy. Shown are the top-down three-dimensional (3D) volume rendering of biofilms at a total magnification of ×400. Bottom images represent optical planes in the xz, and vertical thin images represent yz dimensions. Membrane-compromised bacteria are stained red with propidium iodide, while intact bacteria are stained green with SYTO 9. Areas highlighted by dashed lines indicate regions of interest (ROIs) viewed at a higher magnification. Dimensions shown are 387.5 μm by 387.5 μm by 16 μm. (C) ROIs are presented at ×2,300 magnification. Dimensions shown are 68.1 μm by 68.1 μm by 16 μm.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: Control, Labeling, Staining, Confocal Laser Scanning Microscopy, Membrane, Bacteria

Journal: Molecular pharmacology

Article Title: Site-Directed Mutagenesis of the CC Chemokine Binding Protein 35K-Fc Reveals Residues Essential for Activity and Mutations That Increase the Potency of CC Chemokine Blockade

doi: 10.1124/mol.111.071985

Figure Lengend Snippet: Screening of 35K-Fc mutants by CCR5 DiscoveRx assay. A, dose-response curve showing recruitment of β-arrestin in response to CCR5 activation by RANTES as measured by PathHunter eXpress assay (DiscoveRx). B, RANTES (5 nM) was preincubated for 2 h with the indicated concentration of WT 35K-Fc (●) or MR-Fc (○) and then added to the assay. A and B show mean ± S.E.M. of two technical replicates and are representative of three independent experiments. C, RANTES (5 nM) was preincubated with the indicated 35K-Fc mutant (fixed dose, 15 nM) and then added to PathHunter eXpress CCR5 transfected cells and β-arrestin recruitment measured according to manufacturer’s instructions. Data are shown as the percentage of the response to RANTES alone and are the mean of two independent experiments ± S.E.M., each with two technical replicates for two batches of protein (i.e., four wells per mutant per experiment). Statistical analysis performed by one-way ANOVA and Dunnett’s multiple comparison post test. ***, p < 0.001 relative to WT 35K-Fc.

Article Snippet: Data for all mutants are summarized in . table ft1 table-wrap mode="anchored" t5 caption a7 Mutant RANTES Response (CCR5 DiscoveRx) IC 50 Overall Rank CHO-CCR2 xCelligence CCR5 DiscoveRx CCR2 DiscoveRx CCR7 DiscoveRx MΦ xCelligence % nM WT 35.1 90.0 6.6 ± 0.9 (3) 13.9 ± 1 (3) 12.2 ± 0.4 (2) 3 ± 0.8 (3) 3 Y80A 95.6 >1000 10 R89A 15.1 20.6 3.7 ± 1.7 (4) * 3.1 ± 0.4 (2) *** 4.1 ± 0.1 (2) ** 1 ± 0.4 (3) * 1 D141A 74.3 139.0 4 D141L 80.6 >750 8 D141R 79.4 >700 6 E143A 71.7 5 E143K 93.9 >5000 N.D. (3) N.D. (2) N.D. (3) 11 E143R 92.5 11 V185A 26.5 50.0 2 Y217A 66.5 >5000 7 Y217N 67.5 >1400 6 MR-Fc 91.2 >1400 N.D. (3) N.D. (2) N.D. (3) 9 Open in a separate window N.D., IC 50 could not be determined (i.e., a curve could not be fitted).

Techniques: Activation Assay, Concentration Assay, Mutagenesis, Transfection

Journal: Molecular pharmacology

Article Title: Site-Directed Mutagenesis of the CC Chemokine Binding Protein 35K-Fc Reveals Residues Essential for Activity and Mutations That Increase the Potency of CC Chemokine Blockade

doi: 10.1124/mol.111.071985

Figure Lengend Snippet: Summary of data for 35K-Fc mutants Values are presented as mean ± S.D. Column 2 summarizes data shown in Fig. 3C and indicates the response to 5 nM RANTES (set as 100%) in a CCR5 DiscoveRx assay after preincubation of 5 nM RANTES with 15 nM 35K-Fc. Column 3 gives the IC 50 (mean of two technical replicates) for the indicated 35K-Fc mutant in a single xCELLigence ECIS screening assay with CCR2-transfected CHO cells responding to 10 nM MCP-1. Columns 4 to 7 show the IC 50 from the number of independent experiments as indicated in parentheses. Statistical analysis was performed by unpaired t test. Columns 4 and 5 summarize the data shown in Fig. 4 . Column 6 gives the IC 50 for the indicated 35K-Fc mutant in a DiscoveRx assay with CCR7-transfected CHO cells responding to 10 nM MIP-3 β (CCL19). Column 7 summarizes the data shown in . The final column indicates the overall rank of the mutants (1, most potent chemokine blockade; 11, least potent).

Article Snippet: Data for all mutants are summarized in . table ft1 table-wrap mode="anchored" t5 caption a7 Mutant RANTES Response (CCR5 DiscoveRx) IC 50 Overall Rank CHO-CCR2 xCelligence CCR5 DiscoveRx CCR2 DiscoveRx CCR7 DiscoveRx MΦ xCelligence % nM WT 35.1 90.0 6.6 ± 0.9 (3) 13.9 ± 1 (3) 12.2 ± 0.4 (2) 3 ± 0.8 (3) 3 Y80A 95.6 >1000 10 R89A 15.1 20.6 3.7 ± 1.7 (4) * 3.1 ± 0.4 (2) *** 4.1 ± 0.1 (2) ** 1 ± 0.4 (3) * 1 D141A 74.3 139.0 4 D141L 80.6 >750 8 D141R 79.4 >700 6 E143A 71.7 5 E143K 93.9 >5000 N.D. (3) N.D. (2) N.D. (3) 11 E143R 92.5 11 V185A 26.5 50.0 2 Y217A 66.5 >5000 7 Y217N 67.5 >1400 6 MR-Fc 91.2 >1400 N.D. (3) N.D. (2) N.D. (3) 9 Open in a separate window N.D., IC 50 could not be determined (i.e., a curve could not be fitted).

Techniques: Mutagenesis, Screening Assay

Journal: Molecular pharmacology

Article Title: Site-Directed Mutagenesis of the CC Chemokine Binding Protein 35K-Fc Reveals Residues Essential for Activity and Mutations That Increase the Potency of CC Chemokine Blockade

doi: 10.1124/mol.111.071985

Figure Lengend Snippet: Comparison of 35K-Fc WT, R89A, and E143K proteins by DiscoveRx β arrestin assay. A, dose-response curve of beta arrestin recruitment in response to CCR2 activation by MCP-1. B, MCP-1 (3 nM) was preincubated with the indicated concentration of WT 35K-Fc (●), R89A 35K-Fc (■) or E143K 35K-Fc (○) for 2 h then applied to the DiscoveRx assay. C and D, as for A and B, using CCR5 DiscoveRx cells and RANTES as the chemokine ligand. E and F, as for A and B, using CXCR2 DiscoveRx cells and IL-8 as the chemokine ligand. A and B show the mean ± S.E.M. of two technical replicates and are representative of two to three independent experiments. C and D show the mean ± S.E.M. of two technical replicates and are representative of three to four independent experiments. E and F show the mean ± S.E.M. of two technical replicates and are representative of two independent experiments.

Article Snippet: Data for all mutants are summarized in . table ft1 table-wrap mode="anchored" t5 caption a7 Mutant RANTES Response (CCR5 DiscoveRx) IC 50 Overall Rank CHO-CCR2 xCelligence CCR5 DiscoveRx CCR2 DiscoveRx CCR7 DiscoveRx MΦ xCelligence % nM WT 35.1 90.0 6.6 ± 0.9 (3) 13.9 ± 1 (3) 12.2 ± 0.4 (2) 3 ± 0.8 (3) 3 Y80A 95.6 >1000 10 R89A 15.1 20.6 3.7 ± 1.7 (4) * 3.1 ± 0.4 (2) *** 4.1 ± 0.1 (2) ** 1 ± 0.4 (3) * 1 D141A 74.3 139.0 4 D141L 80.6 >750 8 D141R 79.4 >700 6 E143A 71.7 5 E143K 93.9 >5000 N.D. (3) N.D. (2) N.D. (3) 11 E143R 92.5 11 V185A 26.5 50.0 2 Y217A 66.5 >5000 7 Y217N 67.5 >1400 6 MR-Fc 91.2 >1400 N.D. (3) N.D. (2) N.D. (3) 9 Open in a separate window N.D., IC 50 could not be determined (i.e., a curve could not be fitted).

Techniques: Beta-Arrestin Assay, Activation Assay, Concentration Assay

Journal: Journal of Bacteriology

Article Title: Three Paralogous LysR-Type Transcriptional Regulators Control Sulfur Amino Acid Supply in Streptococcus mutans ▿ †

doi: 10.1128/JB.00119-10

Figure Lengend Snippet: Bacterial strains and plasmids

Article Snippet: When required, 5-bromo-4-chloro-3-indolyl-β- d -galactoside (0.04 g/liter), isopropyl 1-thio-β- d -galactopyranoside (IPTG; 0.04 g/liter), ampicillin (100 μg/ml for E. coli ), erythromycin (8 μg/ml for S. mutans and 100 μg/ml for E. coli ), and tetracycline (3 μg/ml for S. mutans ) were added to the culture medium. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristics Reference or source E. coli strain TG1 supE Δ thi(lac-proAB) hsdD5 (F′ + traD36 proAB lacI q Z ΔM15) 25 S. mutans strains UA159 S. mutans wild-type strain (ATCC 700610) ATCC JIM8877 UA159 Δ cysR This work JIM8878 UA159 Δ homR , Em r This work JIM8879 UA159 Δ gshT , Em r This work JIM8880 UA159 Δ cysK , Em r This work JIM8882 UA159 Δ tcyA , Em r This work JIM8884 UA159 Δ tcytE , Em r This work JIM8886 UA159 Δ tcyA Δ tcytE , Em r Tet r This work JIM8890 UA159 containing pJIM5788 integrated at the P homR locus, Em r This work JIM8892 UA159 containing pJIM5790 integrated at the P tcyD locus, Em r This work JIM8887 UA159 containing pJIM5786 integrated at the P cysK locus, Em r This work JIM8888 UA159 containing pJIM5787 integrated at the P cysK locus, Em r This work JIM8889 JIM8877 containing pJIM5786 integrated at the P cysK locus, Em r This work Plasmids pGEM-T easy Ap r , M13 ori pBR322 ori , linear T-overhang vector Promega pGhost 9 Em r , ori + Δ repA , integrative promoter probe vector 43 pJIM4900 Em r , ori + Δ repA , integrative promoter probe vector containing luxAB genes 57 pJIM5785 SalI fusion of pGhost 9 and pGEM-T Easy containing 818-bp fragment upstream and 850-bp fragment downstream cysR gene ligated in BamHI This work pJIM5788 P HomR - lux -SpeI site fusion of pJIM4900 and pGEM-T Easy containing P homR on a 551-bp fragment This work pJIM5786 P cysK - lux -BclI/BamHI site fusion of pJIM4900 and pGEM-T Easy containing P cysk on a 591-bp fragment This work pJIM5787 P cysK - lux -BclI/BamHI site fusion of pJIM4900 and pGEM-T Easy containing P cysK carrying nucleotide substitution on the CysR box on a 591-bp fragment This work pJIM5790 P tcyD - lux -SpeI site fusion of pJIM4900 and pGEM-T Easy containing P tcyD on a 591-bp fragment This work Open in a separate window Bacterial strains and plasmids DNA manipulation procedures.

Techniques: Plasmid Preparation

Journal:

Article Title: Internalization of Swine Vesicular Disease Virus into Cultured Cells: a Comparative Study with Foot-and-Mouth Disease Virus ▿ †

doi: 10.1128/JVI.02436-08

Figure Lengend Snippet: Dynamin and cell signaling requirements for virus infection. The effects of dynasore (A), Bis (B), OV (C), genistein (D), PP2 (E), and wortmannin (F) on VSV, SVDV, and FMDV infection were studied by plaque assay. The following treatments induced measurable reported effects: dynasore, inhibition of TF internalization (49); wortmannin, endosomal vacuolation (46); and OV, tyrosine phosphorylation (73). Statistically significant differences between control and drug treatments are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.005).

Article Snippet: Another differential factor is that NH 4 Cl blockage of endosomal acidification is produced by neutralization of endosomal pH, whereas concanamycin inhibits the proton flux into the endosome but does not neutralize remaining acid pH within the endosomes. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Cellular target Drug Virus b SVDV FMDV VSV Dynamin Dynasore + + +PI Endosomal pH NH 4 Cl + + + Microtubules Nocodazole + − +PI Actin microfilaments Cyt D − − − Cell signaling machinery OV +PI + + Genistein +PI − +PI PP2 + + + Bis − − − Wortmannin − − − Cholesterol MβCD + + − Filipin − − − Lovastatin − − − Nystatin − − − Open in a separate window a Only drugs for which results with the three viruses compared were obtained are included. b +, Statistically significant inhibitory effect of the drug on virus infection only when added preinfection; +PI, the drug also had a statistically significant inhibitory effect when added postinfection; −, no statistically significant effect when added pre- or postinfection.

Techniques: Virus, Infection, Plaque Assay, Inhibition, Phospho-proteomics, Control

Journal:

Article Title: Internalization of Swine Vesicular Disease Virus into Cultured Cells: a Comparative Study with Foot-and-Mouth Disease Virus ▿ †

doi: 10.1128/JVI.02436-08

Figure Lengend Snippet: Summary of effects on SVDV, FMDV, and VSV entry of the drugs tested a

Article Snippet: Another differential factor is that NH 4 Cl blockage of endosomal acidification is produced by neutralization of endosomal pH, whereas concanamycin inhibits the proton flux into the endosome but does not neutralize remaining acid pH within the endosomes. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Cellular target Drug Virus b SVDV FMDV VSV Dynamin Dynasore + + +PI Endosomal pH NH 4 Cl + + + Microtubules Nocodazole + − +PI Actin microfilaments Cyt D − − − Cell signaling machinery OV +PI + + Genistein +PI − +PI PP2 + + + Bis − − − Wortmannin − − − Cholesterol MβCD + + − Filipin − − − Lovastatin − − − Nystatin − − − Open in a separate window a Only drugs for which results with the three viruses compared were obtained are included. b +, Statistically significant inhibitory effect of the drug on virus infection only when added preinfection; +PI, the drug also had a statistically significant inhibitory effect when added postinfection; −, no statistically significant effect when added pre- or postinfection.

Techniques: Virus

Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Crystal structure of the aromatic-amino-acid aminotransferase from Streptococcus mutans

doi: 10.1107/S2053230X18018472

Figure Lengend Snippet: Macromolecule-production information

Article Snippet: Macromolecule-production information is summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Source organism S. mutans DNA source S. mutans strain UA159 (ATCC 700610) Forward primer 5′-CGC GGATCC ATGGATTTGAGTAAACGTTTTA-3′ Reverse primer 5′-CCG CTCGAG TTAGTCTGCATATTGCTCC-3′ Cloning vector pGEX-6P-1 Expression vector pGEX-6P-1 Expression host E. coli strain BL21 (DE3) Complete amino-acid sequence of the construct produced GPMDLSKRFNKNLNKIEVSMIRQFDQSISDIPDVLKLTLGEPDFATPKHIKEAAKRAIDADESHYTGMAGLLALRQAASAFVKEKYHLTYNPDNEILVTIGATEALSASLTAILEPGDKVLLPAPAYPGYEPVVNLVGAEVVEIDTRSNDFVLTPEMLEEAILKEGEALKAVILNYPTNPTGVTYSRQQIKNLAEVLKKYPIFVISDEVYAELTYTGESHVSIAEYLPDQTILISGLSKSHAMTGWRLGLIFAPAVLTAQLIKSHQYLVTAATTSVQFAAIEALTNGKDDALPMKEEYIKRRDYIIEKMEAMKFKIIKPDGAFYIFAKIPVAQGQDSFKFLQDFAKEKAVAFIPGVAFGKYGEGYLRISYAASMETIKEAMKRLKEFMEQYAD Open in a separate window caption a8 Macromolecule-production information

Techniques: Cloning, Plasmid Preparation, Expressing, Sequencing, Construct, Produced

Journal: Cell metabolism

Article Title: Homeostatic responses regulate selfish mitochondrial genome dynamics in C. elegans

doi: 10.1016/j.cmet.2016.06.008

Figure Lengend Snippet: (A) Schematic of C. elegans mtDNA showing the uaDf5 and mptDf1 deletions (long and short red bars, respectively). Grey arrows show protein and rRNA-encoding genes and their orientation. White boxes show genes encoding tRNAs. (B) Schematic illustrating the selection strategy to force loss of uaDf5 mtDNA from a heteroplasmic C. elegans line. Each generation, the progeny of individuals with the lowest uaDf5 levels were selected for subsequent propagation. (C) Single worm PCR of wildtype and uaDf5 mtDNA. Successive propagation of individual worms with low uaDf5 levels (red boxes) results in complete loss of uaDf5 mtDNA from the population over multiple generations. (D) ddPCR data from single worms confirming complete loss of uaDf5. Positive droplets containing uaDf5-specific PCR product exhibit increased fluorescence intensity (blue) compared to negative droplets that contain no uaDf5 mtDNA (gray). For each droplet, the droplet reader detects droplet size, shape, and fluorescence intensity, and automatically distinguishes positive from negative droplets on the basis of these criteria. Sample 1, control containing uaDf5.

Article Snippet: Moreover, uaDf5 levels steadily increase in individuals that inherit it at a low frequency ( Tsang and Lemire, 2002 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 Mutant mtDNA uaDf5 can be forced out from a stably persisting heteroplasmy in C. elegans (A) Schematic of C. elegans mtDNA showing the uaDf5 and mptDf1 deletions (long and short red bars, respectively).

Techniques: Selection, Fluorescence, Control

Journal: Cell metabolism

Article Title: Homeostatic responses regulate selfish mitochondrial genome dynamics in C. elegans

doi: 10.1016/j.cmet.2016.06.008

Figure Lengend Snippet: (A) Histogram showing uaDf5 frequency (%) distribution in individuals from a population stably maintaining uaDf5 mtDNA. Heteroplasmy frequency was determined using ddPCR to quantify wildtype and uaDf5 mtDNA copy number in single individuals. (B) mtDNA levels in individual day 4 adult worms, normalized to actin and rank-ordered by uaDf5 mtDNA copy number. (C) Wider variation in uaDf5 relative to wildtype copy number (p<0.05) suggests that wildtype mtDNA, but not uaDf5 mtDNA, is subject to homeostatic copy number control. Grey data points show mtDNA copy number from single individuals. Box and whisker plot shows the median, lower and upper quartile (boxes), and minimum and maximum (error bars) mtDNA copy number. (D) mptDf1 frequency distribution obtained from single individuals from a population stably maintaining mptDf1 heteroplasmy. (E) mtDNA levels in individual L4 worms, normalized to actin and rank-ordered by mptDf1 copy number. (F) Similar to uaDf5, wider variation in mptDf1 relative to wildtype copy number (p<0.05) suggests that wildtype mtDNA, but not mptDf1 mtDNA, is subject to homeostatic copy number control. AU, arbitrary units.

Article Snippet: Moreover, uaDf5 levels steadily increase in individuals that inherit it at a low frequency ( Tsang and Lemire, 2002 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 Mutant mtDNA uaDf5 can be forced out from a stably persisting heteroplasmy in C. elegans (A) Schematic of C. elegans mtDNA showing the uaDf5 and mptDf1 deletions (long and short red bars, respectively).

Techniques: Stable Transfection, Control, Whisker Assay

Journal: Cell metabolism

Article Title: Homeostatic responses regulate selfish mitochondrial genome dynamics in C. elegans

doi: 10.1016/j.cmet.2016.06.008

Figure Lengend Snippet: (A) Schematic showing expected expression of mtDNA-encoded transcripts. The presence of uaDf5 mtDNA is expected to result in stoichiometric imbalance of gene expression, as the expression of uaDf5 and wildtype mtDNA copies (red and blue lines, respectively) combine to generate total expression (black line) at elevated levels for genes located outside the deletion but at wildtype levels for genes missing from the uaDf5 mtDNA. (B) Animals heteroplasmic for uaDf5 exhibit expression levels similar to that of wildtype animals for mtDNA-encoded genes affected by the deletion (CYTB and ND1), as well as a nuclear-encoded mitochondrial gene (NUO2) and actin. However, uaDf5 heteroplasmy results in overexpression for mtDNA-encoded genes located outside the uaDf5 deletion (COXI, COXII, COXIII, ND4, and ND5). All transcript levels are normalized to wildtype. Error bars represent standard deviation. (C) Mitochondrially targeted GFP (GFPmt), but not cytosolic GFP (cGFPcyt), is significantly reduced in uaDf5 heteroplasmic individuals. (D) Western blot analysis of wildtype and uaDf5 heteroplasmic animals expressing GFPmt reveals reduced levels in uaDf5 heteroplasmic individuals relative to actin. Data are shown from two biological replicates each for wildtype and uaDf5 strain. (E) Fluorescence increase in uaDf5 animals stained with mitochondrial membrane potential independent dye MitoTracker Green FM and (F) membrane potential dependent dye TMRE. Error bars represent standard deviation. AU, arbitrary units.

Article Snippet: Moreover, uaDf5 levels steadily increase in individuals that inherit it at a low frequency ( Tsang and Lemire, 2002 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 Mutant mtDNA uaDf5 can be forced out from a stably persisting heteroplasmy in C. elegans (A) Schematic of C. elegans mtDNA showing the uaDf5 and mptDf1 deletions (long and short red bars, respectively).

Techniques: Expressing, Gene Expression, Over Expression, Standard Deviation, Western Blot, Fluorescence, Staining, Membrane

Journal: Cell metabolism

Article Title: Homeostatic responses regulate selfish mitochondrial genome dynamics in C. elegans

doi: 10.1016/j.cmet.2016.06.008

Figure Lengend Snippet: (A) Transcription of two UPRmt-activated molecular chaperones (hsp-60 and hsp-6) is increased in individuals with uaDf5 compared to wildtype individuals. (B) Quantification of fluorescence between wildtype homoplasmic and uaDf5 heteroplasmic animals shows increased activation of the UPRmt marker hsp-60∷GFP in the presence of uaDf5 mtDNA. Each data point is from a single individual picked randomly from a population. (C) Visual comparison of GFP fluorescence between uaDf5 and wildtype animals, each expressing hsp-60∷GFP. Wildtype animals were picked at random from a population but only uaDf5 animals with apparent fluorescence were picked to show UPRmt activation. (D) Positive relationship between uaDf5 frequency and hsp-60∷GFP fluorescence (trendline) indicates that UPRmt activation increases at higher uaDf5 frequency. Each data point corresponds to a single individual. Error bars represent standard deviation. AU, arbitrary units.

Article Snippet: Moreover, uaDf5 levels steadily increase in individuals that inherit it at a low frequency ( Tsang and Lemire, 2002 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 Mutant mtDNA uaDf5 can be forced out from a stably persisting heteroplasmy in C. elegans (A) Schematic of C. elegans mtDNA showing the uaDf5 and mptDf1 deletions (long and short red bars, respectively).

Techniques: Fluorescence, Activation Assay, Marker, Comparison, Expressing, Standard Deviation

Journal: Cell metabolism

Article Title: Homeostatic responses regulate selfish mitochondrial genome dynamics in C. elegans

doi: 10.1016/j.cmet.2016.06.008

Figure Lengend Snippet: (A) Growth under RNAi-mediated knockdown of atfs-1, required for UPRmt activation, results in a shift to lower uaDf5 frequency relative to growth under control conditions (p<0.05). (B) uaDf5 frequency decreases in heteroplasmic animals homozygous for the atfs-1(tm4525) loss-of-function allele compared to heteroplasmic animals that express wildtype atfs-1, in which high uaDf5 levels are stably maintained. uaDf5 frequency decreases further in the atfs-1 null animals after multiple generations but is not lost completely. (C) Quantification of mtDNA copy number in individual day 4 adult animals homozygous for the atfs-1 loss-of-function allele, normalized to actin and rank-ordered by uaDf5 mtDNA copy number. (D) Wider variation in uaDf5 relative to wildtype copy number (p<0.05) in atfs-1 null animals suggests that mtDNA copy number control persists in absence of UPRmt. (E) PCR of single heteroplasmic individuals against the atfs-1 wildtype or atfs-1 null nuclear background shows that uaDf5 is retained in both lines but is at lower levels in the null animals after about 30 generations. Note that because mutant and wildtype templates compete for amplification, the wildtype band appears fainter when uaDf5 levels are high but does not actually reflect reduced wildtype mtDNA levels (see Fig. 2B). Error bars represent standard deviation. AU, arbitrary units.

Article Snippet: Moreover, uaDf5 levels steadily increase in individuals that inherit it at a low frequency ( Tsang and Lemire, 2002 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 Mutant mtDNA uaDf5 can be forced out from a stably persisting heteroplasmy in C. elegans (A) Schematic of C. elegans mtDNA showing the uaDf5 and mptDf1 deletions (long and short red bars, respectively).

Techniques: Knockdown, Activation Assay, Control, Stable Transfection, Mutagenesis, Amplification, Standard Deviation

Journal: Cell metabolism

Article Title: Homeostatic responses regulate selfish mitochondrial genome dynamics in C. elegans

doi: 10.1016/j.cmet.2016.06.008

Figure Lengend Snippet: (A) Growth under RNAi-mediated knockdown of atfs-1 across seven generations reduces average uaDf5 frequency. However, restoration of atfs-1 expression by returning atfs-1 knockdown animals to control conditions results in recovery of elevated uaDf5 frequency in a single generation. (B) When starting uaDf5 frequency is high (75-80%), constitutive UPRmt activation in individuals heterozygous for an atfs-1 gain-of-function allele causes no further rise in average uaDf5 frequency; (C) however, uaDf5 frequency rises when the atfs-1 gain-of-function allele is crossed into a strain harboring lower uaDf5 levels (∼30%). Error bars represent standard deviation.

Article Snippet: Moreover, uaDf5 levels steadily increase in individuals that inherit it at a low frequency ( Tsang and Lemire, 2002 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 Mutant mtDNA uaDf5 can be forced out from a stably persisting heteroplasmy in C. elegans (A) Schematic of C. elegans mtDNA showing the uaDf5 and mptDf1 deletions (long and short red bars, respectively).

Techniques: Knockdown, Expressing, Control, Activation Assay, Standard Deviation

Journal: Cell metabolism

Article Title: Homeostatic responses regulate selfish mitochondrial genome dynamics in C. elegans

doi: 10.1016/j.cmet.2016.06.008

Figure Lengend Snippet: (A) Heteroplasmic individuals exhibit delayed growth: as 100% of progeny from wildtype parents reach adulthood in three days, approximately 10% of uaDf5 progeny remain in the larval stage. Knockdown of atfs-1 showed no effect on development in homoplasmic wildtype animals and did not further enhance developmental delay in uaDf5 heteroplasmic animals. (B) No significant difference was observed between uaDf5 and wildtype animals, or between atfs-1 knockdown and control conditions, on the percentage of embryos that remain unhatched after one day or (C) on the percentage of lethality among day 4 adults. (D) Quantification of Pink-1∷GFP fluorescence shows increased mitophagy in uaDf5 animals upon pdr-1;atfs-1 double knockdown compared to knockdown of pdr-1 alone. AU, arbitrary units. (E) Crossing scheme employed to isolate uaDf5 animals in wildtype, atfs-1 null, pdr-1 null, and atfs-1;pdr-1 double mutant backgrounds. (F) Quantification of uaDf5 levels shows recovery of uaDf5 levels in atfs-1;pdr-1 double mutants compared to atfs-1 single mutant animals. uaDf5 recovers to the highest levels in pdr-1 single mutants. Error bars represent standard deviation. AU, arbitrary units.

Article Snippet: Moreover, uaDf5 levels steadily increase in individuals that inherit it at a low frequency ( Tsang and Lemire, 2002 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 Mutant mtDNA uaDf5 can be forced out from a stably persisting heteroplasmy in C. elegans (A) Schematic of C. elegans mtDNA showing the uaDf5 and mptDf1 deletions (long and short red bars, respectively).

Techniques: Knockdown, Control, Fluorescence, Mutagenesis, Standard Deviation