Journal: Science Advances

Article Title: Dense hydroxyl polyethylene glycol dendrimer targets activated glia in multiple CNS disorders

doi: 10.1126/sciadv.aay8514

Figure Lengend Snippet: ( A ) Key steps in synthetic pathway of PEGOL-60 ( 8 ). Reagents and conditions: (i) NaH, propargyl bromide, 0°C to room temperature (RT), 6 hours; (ii) NaH, allyl bromide, 0°C to RT, 2 hours; (iii) NaH, 2-(2-(2-azidoethoxy)ethoxy)ethyl 4-methylbenzenesulfonate ( 4 ), 0°C to RT, 3 to 4 hours; (iv) CuSO 4 ·5H 2 O, sodium ascorbate, 11,11,15,15-tetrakis((allyloxy)methyl)-1-azido-3,6,9,13,17-pentaoxaicos-19-ene ( 5 ), N , N′ -dimethylformamide (DMF), tetrahydrofuran (THF), H 2 O, microwave, 50°C, 6 hours, 76%; (v) 2,2-dimethoxy-2-phenylacetophenone, 1-thioglycerol ( 7 ), DMF, 12 hours. ( B ) 1 H NMR comparison of hexa-propargylated core ( 2 ), D1-allyl-30 ( 6 ), PEGOL-60 ( 8 ), and fluorescently labeled dendrimer PEGOL-60-Cy5 ( 11 ) (from bottom to top) showing the appearance/disappearance of characteristic proton signals. TEG, triethylene glycol. ( C ) Comparative HPLC chromatogram of D1-allyl-30 ( 6 ), PEGOL-60 ( 8 ), D-GABA-NHBOC ( 9 ), and PEGOL-60-Cy5 ( 11 ) showing evident shifts in retention times at each stage (absorption at 210 nm for 6 , 8 , and 9 and at 650 nm for 11 ). ( D ) MALDI-TOF spectrum of PEGOL-60 ( 8 ) showing a peak at mass of 7.4 kDa. ( E ) Synthesis of fluorescently labeled PEGOL-60-Cy5 ( 11 ): (i) N -(3-dimethylaminopropyl)- N ′-ethylcarbodiimide hydrochloride (EDC.HCl), 4-(dimethylamino)pyridine (DMAP), DMF, γ-(boc-amino)butyric acid, 12 hours; (ii) dichloromethane (DCM)/trifluoroacetic acid (3:1), 8 hours; (iii) N , N ′-diisopropylethylamine (DIPEA), Cy5 N -hydroxysuccinimide (NHS) ester, pH 7 to 7.4, DMF, 12 hours. ( F ) Stability analysis of fluorescently labeled PEGOL-60-Cy5 ( 11 ) by HPLC: HPLC chromatogram of PEGOL-60-Cy5 in water (left), extracted from human plasma in vitro after incubation for 24 hours at 37°C (middle), and urine obtained from rabbit bladder in vivo 24 hours after injection of PEGOL-60-Cy5 (right).

Article Snippet: PEGOL-60 ( 8 ) was coupled with γ-(boc-amino)butyric acid using N -(3-dimethylaminopropyl)- N ′-ethylcarbodiimide (EDC) and 4-(dimethylamino)pyridine (DMAP) to obtain dendrimer ( 9 , D-GABA-NHBOC), after which the BOC group was deprotected under mild acidic conditions [trifluoroacetic acid (TFA)/dichloromethane (DCM), 1/5] to get dendrimer ( 10 ).

Techniques: Labeling, In Vitro, Incubation, In Vivo, Injection

Journal: PLoS Pathogens

Article Title: Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

doi: 10.1371/journal.ppat.1009965

Figure Lengend Snippet: A ATCC 19977, D436H mutant, and D436H:leuS D complement strains were grown in Sauton media with 59 μg/mL norvaline (top left), 66 μg/mL leucine (top right), 59 μg/mL valine (lower left), or 66 μg/mL isoleucine (lower right). Data shown is from three biological replicates, one-way ANOVA with Tukey’s multiple comparisons test. ****, p < 0.0001. B D436H mutant grown in Sauton media with 59 μg/mL norvaline and a range of leucine (left), isoleucine (middle), or valine (right). D436H mutant grown in 0 μg/mL norvaline and 0 μg/mL BCAA represents growth control. D436H mutant grown in 59 μg/mL norvaline and 0 μg/mL BCAA represents inhibited growth control. BCAAs added to cultures from 33 μg/mL to 262 μg/mL (Leu/Ile) or 29 μg/mL to 234 μg/mL (Val). Data shown is mean ± SD from three biological replicates. Groups compared to inhibited growth control with one-way ANOVA with Dunnett’s multiple comparisons test. ***, p = 0.0009; ****, p ≤ 0.0001.

Article Snippet: We observed a 23-fold reduction in escape mutant frequency when M . abscessus ATCC 19977 was plated on 7H10 plates containing 10X MIC 90 EPT and 590 μg/mL norvaline (1.89 x 10 −10 ) over EPT alone (4.28 x 10 −9 ) ( ); as a control, RFB did not benefit from norvaline supplementation.

Techniques: Mutagenesis, Control

Journal: PLoS Pathogens

Article Title: Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

doi: 10.1371/journal.ppat.1009965

Figure Lengend Snippet: A-B M . abscessus ATCC 19977 or D436H mutant was grown in 59 μg/mL valine or 59 μg/mL norvaline for 12 hours or 3 days. Valine was used as a specificity control. Total cell lysate was collected. (Nor)valinated protein medians from the proteomes were compared using Kruskal-Wallis with Dunn’s multiple comparisons test. ***, p = 0.0005; ****, p < 0.0001; ns, no statistical significance. C-F Protein fold abundance shown as D436H mutant relative to M . abscessus ATCC 19977. Dashed red lines indicate 0.5x and 2x fold abundance thresholds for biologically relevant changes.

Article Snippet: We observed a 23-fold reduction in escape mutant frequency when M . abscessus ATCC 19977 was plated on 7H10 plates containing 10X MIC 90 EPT and 590 μg/mL norvaline (1.89 x 10 −10 ) over EPT alone (4.28 x 10 −9 ) ( ); as a control, RFB did not benefit from norvaline supplementation.

Techniques: Mutagenesis, Control

Journal: PLoS Pathogens

Article Title: Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

doi: 10.1371/journal.ppat.1009965

Figure Lengend Snippet: A Protein-protein interaction network from the 130 most abundant proteins in D436H mutant when challenged with 59 μg/mL norvaline for 12 hours. 942 PPIs identified with PPI enrichment p-value < 1.0x10 -16 . B PPI network from 130 randomly selected proteins in D436H mutant when challenged with 59 μg/mL norvaline for 12 hours. 102 PPIs identified with PPI enrichment p-value of 0.0397. C Clp protease/chaperonin family local network cluster identified in a (false discovery rate: 0.00072). D Temporal signature of the heat shock response. Protein expression levels of D436H norvaline relative to WT norvaline were measured by spectral counting.

Article Snippet: We observed a 23-fold reduction in escape mutant frequency when M . abscessus ATCC 19977 was plated on 7H10 plates containing 10X MIC 90 EPT and 590 μg/mL norvaline (1.89 x 10 −10 ) over EPT alone (4.28 x 10 −9 ) ( ); as a control, RFB did not benefit from norvaline supplementation.

Techniques: Mutagenesis, Expressing

Journal: PLoS Pathogens

Article Title: Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

doi: 10.1371/journal.ppat.1009965

Figure Lengend Snippet: A Mutant frequency to 10X MIC 90 EPT (0.63 μg/mL) or 10X MIC 90 EPT + 10X MIC 90 norvaline (590 μg/mL) in M . abscessus ATCC 19977 and M . tuberculosis H37Rv. Data shown is mean ± SD from biological triplicates. Means compared using Poisson distribution. *, p < 0.0001; ns, no statistical significance. B M . abscessus susceptibility to EPT with norvaline as an adjuvant. Data shown is mean ± SD from one experiment representative of three. C EPT in vivo activity in SCID mouse model of M . abscessus lung infection with norvaline supplementation. Rifabutin (RFB) acts as positive control. Data shown is mean ± SD of three to five mice per treatment group. Means compared using one-way ANOVA with Tukey’s multiple comparisons test. **, p < 0.007; ***, p < 0.0005; ****; p < 0.0001; ns, no statistical significance.

Article Snippet: We observed a 23-fold reduction in escape mutant frequency when M . abscessus ATCC 19977 was plated on 7H10 plates containing 10X MIC 90 EPT and 590 μg/mL norvaline (1.89 x 10 −10 ) over EPT alone (4.28 x 10 −9 ) ( ); as a control, RFB did not benefit from norvaline supplementation.

Techniques: Mutagenesis, Adjuvant, In Vivo, Activity Assay, Infection, Positive Control