Journal: PLoS ONE

Article Title: Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans

doi: 10.1371/journal.pone.0012570

Figure Lengend Snippet: ( A ) Cytosolic protease activities in E. coli and D. radiodurans . ( B ) Cytosolic distribution and concentration of amino acids in D. radiodurans . No-IR, non-irradiated control cells held in 25 mM phosphate buffer, pH 7.4 on ice, then washed and held in 25 mM phosphate buffer, pH 7.4 (32°C) for 0 or 30 min. +IR, cells irradiated to 7 kGy in 25 mM phosphate buffer, pH 7.4 on ice, then washed and held in 25 mM phosphate buffer, pH 7.4 (32°C) for 0 or 30 min. Cells were harvested, resuspended in 20% TCA, and broken open. Aliquots of neutralized supernatant were analyzed for free amino acid and peptide-derived amino acid content. ( C ) Radioprotection of Bam HI by amino acids. PiB, potassium phosphate buffer, pH 7.4. ( D ) Radioprotection of Bam HI by the decapeptide (H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH; 1,261 Da). Ns/Nb, nucleosides and bases (1 mM; see for the Ns/Nb added). ( E ) Radioprotection of glutamine synthetase (GS) by Mn 2+ and leucine (Leu), uridine (U), or the decapeptide (DP) in potassium phosphate buffer (PiB), pH 7.4 or sodium bicarbonate buffer (HCO 3 ), pH 7.4. Adenosine could not be evaluated because it is an allosteric inhibitor of glutamine synthetase.

Article Snippet: The synthetic decapeptide (H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH) was obtained from Elim Biopharmaceuticals, Inc. Hayward, California, USA, and was authenticated at NHLBI by high performance liquid chromatography-mass spectrometry (HPLC-MS).

Techniques: Concentration Assay, Irradiation, Control, Derivative Assay

Journal: PLoS ONE

Article Title: Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans

doi: 10.1371/journal.pone.0012570

Figure Lengend Snippet: Structural forms of the plasmid (pUC19): OC, open circular; L, linear; and SC, super-coiled. SSB, single-strand break; DSB, double-strand break. M, DNA size marker; PiB, phosphate buffer, pH 7.4. In the absence of HO • -scavenging agents, approximately 80% of ionizing radiation-induced damage to purified DNA in aqueous solution is caused by HO • , where one SSB in a SC circular plasmid molecule yields an OC form . In contrast to DNA damage, 3 mM decapeptide, 25 mM phosphate buffer, pH 7.4, and 1 mM Mn 2+ preserved the activity of enzymes exposed to 50 kGy .

Article Snippet: The synthetic decapeptide (H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH) was obtained from Elim Biopharmaceuticals, Inc. Hayward, California, USA, and was authenticated at NHLBI by high performance liquid chromatography-mass spectrometry (HPLC-MS).

Techniques: Plasmid Preparation, Marker, Purification, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Experimental approach used to conduct the microarray analysis from TRPV1 neurons, 72 hours after i.pl. CFA. ( B ) FACS isolation of TRPV1-pHluorin neurons. Representative FACS plot of GFP + population in WT (top) and TRPV1-pHluorin (bottom) mice. SSC-A side scatter area; FSCA- forward scatter area. ( C ) Scatter plot representation of genes regulated in CFA conditions. Genes that passed a threshold of log 2 fold change in differential expression analysis are represented as green when downregulated and red when upregulated. All genes are listed in . ( D ) qRT-PCR assessment of ALKAL2 upregulation in the DRG ipsilateral to the CFA injection (Ipsi) ( n = 9), compared with the contralateral side (Contra) ( n = 9) and naive control ( n = 8). Statistical analysis was performed using Kruskal-Wallis followed by Dunn’s post hoc test. * P < 0.05; *** P < 0.001. ( E ) Representative Western blot of ALKAL2 in the DRG ipsilateral to the CFA injection compared with the contralateral side. ( F ) Quantification of ALKAL2 protein level from Western blot experiments. Each dot represents a sample collected from a different animal ( n = 6 per group). Statistical analysis was performed by unpaired t test ( F ). **** P < 0.0001. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Microarray, Isolation, Expressing, Quantitative RT-PCR, Injection, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Heatmap of the expression of ALKAL2 and selected population markers on the 17 populations of sensory neurons from DRG described in Zeisel et al. . ( B ) Representative confocal images of coimmunostaining for ALKAL2 and TRPV1, IB4, GFRα3, and NF200 in DRG neurons. Scale bars: 50 μm. ( C ) Dot plot summarizing the results in B : 77.64% ± 2.95% of TRPV1, 77.64% ± 3.06% of GFRα3, 41.65% ± 7.08% of IB4, and 36.46% ± 2.64% of NF200-positive neurons express ALKAL2 (each symbol represents a DRG section from n = 4 individual animals). Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. **** P < 0.001. ( D ) Representative confocal images of coimmunostaining for ALKAL2 and NF200, IB4, and GFRα3 in the sciatic nerve. Scale bars: 50 μm. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Representative RNAScope image showing expression of ALKAL2 (light blue) in human DRG neurons coexpressing Nav1.8 (pink). ( B ) Bar graph summarizing the results (each symbol represents an individual patient, n = 3). Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Schematic illustrating the coculture system used to chronically expose DRG neurons to ALKAL2. HEK cells were plated into the upper chamber of a Transwell and then transfected with ALKAL2 plasmid for 16 hours. Cells were washed, and DRG neurons were plated in the lower chamber of the Transwell for another 16 hours of coculture and then immunostained for Tuj1. Scale bars: 50 μm. ALKAL2 induces a significant increase of total neurites ( B ), number of branch points per neuron ( C ), and total neurite length per neuron ( D ) (<20 μm). Control, n = 19; ALKAL2, n = 12; ALKAL2+lorlatinib, n = 18. Statistical analysis was performed using Kruskal-Wallis followed by Dunn’s post hoc test. * P < 0.05; ** P < 0.01; **** P < 0.001. ( E ) Representative confocal images illustrating the TRPV1-GFP innervation of the skin paw following i.pl. injection of CFA (3 days). Scale bars: 50 μm. ( F ) CFA-induced sprouting is reversed by daily administration of lorlatinib (1 mg/kg). Control, n = 5; CFA+vehicle, n = 5; CFA+lorlatinib, n = 5. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. * P < 0.05; ** P < 0.01. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Transfection, Plasmid Preparation, Injection

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Resting membrane potential (RMP) of small DRG neurons in control (–56.06 ± 1.03 mV, n = 19), ALKAL2 (–55.15 ± 2.21 mV, n = 13), or ALKAL2+lorlatinib (–54.5 ± 1.53 mV, n = 13) groups. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. ( B ) AP threshold in control (–35.59 ± 2.26 mV, n = 16), ALKAL2 (–40.57 ± 2.88 mV, n = 11), and ALKAL2+lorlatinib (–31.59 ± 1.76 mV, n = 9) groups. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. ( C ) Representative AP discharge evoked by 100, 200, 300, and 400 pA current injections (1 s) in control, ALKAL2- (10 nM), and ALKAL2+lorlatinib-treated (10 nM+1 μM) DRG neurons. ( D ) Measure of AP frequency evoked by current injection in the different groups represented in E (control, n = 13; ALKAL2 [10 nM], n = 11; ALKAL2+lorlatinib treated [10 nM+1 μM], n = 9). Statistical analysis was performed using 2-way ANOVA followed by Tukey’s post hoc test. * P < 0.05; *** P < 0.001; **** P < 0.0001 versus control. $ P < 0.05; $$$$ P < 0.0001 versus ALKAL2+lorlatinib. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Injection

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Administration i.t. of ALKAL2 (1 μM) promotes thermal hyperalgesia, which is reversed by the administration of lorlatinib (ALKAL2, n = 9; ALKAL2+lorlatinib, n = 8). Statistical analysis was performed using 2-way ANOVA followed by Bonferroni’s post hoc test. * P < 0.05; *** P < 0.001. ( B ) Representative confocal images illustrating pALK induction in the lamina I and II of the spinal dorsal horn following i.t. infusion of ALKAL2. Activation of pALK is reversed by administration of lorlatinib prior to ALKAL2 administration. Scale bars: 100 μm. ( C ) Bar graph of the pALK signal intensity represented in B in the spinal dorsal horn (control, n = 7; ALKAL2, n = 8; ALKAL2+lorlatinib, n = 6; 1 hour). Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. * P < 0.05. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Schematic illustrating the experimental protocol of ALKAL2 oligodeoxynucleotide injection in the CFA pain model. ( B ) Representative confocal images of ALKAL2 immunostaining in DRG sections from control, ALKAL2, or scrambled ODN–treated animals. Scale bars: 50 μm. ( C ) Western blot of ALKAL2 in lumbar DRG lysates at D9 following injection of ALKAL2 or scrambled ODN, compared with naive control mice. ( D ) Bar graph illustrating the reduction in ALKAL2 protein expression in the ODN-treated animals ( n = 4–6 mice per group). Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. * P < 0.05; *** P < 0.001. ( E ) Measure of thermal withdrawal latency in contralateral and ipsilateral hind paws of CFA-injected animals that received saline control ( n = 8), scrambled ( n = 7), or ALKAL2 ODN ( n = 8). Statistical analysis was performed using 2-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001 ODN ipsi vs Control ipsi; $$P< 0.01, $$$P < 0.001, $$$$P < 0.0001 ODN ipsi vs sODN ipsi. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Injection, Immunostaining, Western Blot, Expressing