Journal: bioRxiv

Article Title: GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production

doi: 10.1101/2020.02.18.954438

Figure Lengend Snippet: Generation of constitutive GADL1 KO mice. a Targeting strategy for knocking out exon 7 of the mouse Gadl1 locus on chromosome 9. Gadl1 coding sequences (hatched rectangles), non-coding exon portions (blue rectangles) and chromosome sequences (orange rectangles) are represented. The neomycin positive selection cassette is indicated between loxP sites (blue triangles) and FRT sites (plum triangles). b, c Growth curves of Gadl1 +/+ (n=4­34) and Gadl1 -/- mice (n=4-40) from postnatal week 2 to 70 for (b) male and 90 weeks for (c) female. Presented as mean ± SD. Differences between genotypes were significant ( p =0.0008 (64 weeks) and p =0.0005 (70 weeks) for males and p =0.0084 (90 weeks) and p =0.0001 (94 weeks) for females, respectively). d Southern blot analysis of genomic DNA from Gadl1 +/+ and Gadl1 -/- . e Genotyping of the offspring from intercrosses of Gadl1 +/- mice by PCR. The DNA band at 166 bp is the KO allele (primer 3), while bands at 330 bp (primer 2 and 3) and 750 bp (primer 1) are the WT alleles. f Representative western blots of OB samples from Gadl1 +/+ and Gadl1 -/- mice (34 weeks old, female) using anti GADL1 antibody. Positive control was recombinant His-tagged GADL1 (2 ng, lane 1). g Western blot of recombinant His-tagged truncated Gadl1 +/+ and Gadl1 -/- . h Enzyme activity towards CSA of recombinant His-tagged truncated Gadl1 +/+ and Gadl1 -/- , p < 0.001.

Article Snippet: Total RNA was purified from different tissues from both Gadl1 +/+ and Gadl1 -/- mice using the RNeasy purification kit from Qiagen (#74104 and for muscle tissue #74704). cDNA was synthesized in triplicates from this RNA using a High-Capacity RNA-to-cDNA™ Kit (#4387406, Applied Biosystems™). qRT-PCR was performed using the TaqMan gene expression assay (TaqMan Gene Expression Master Mix #4369016, Applied Biosystems™) The TaqMan probes were: Mm00520087_m1 (CSAD), Mm01348767_m1 (GADL1, exon 13-14), Mm01341531_m1 (GADL1, exon 3-4), Mm01341534_m1 (GADL1, exon 6-7), Mm99999915_g1 (GAPDH) and Mm00607939_s1 (β­actin). mRNA expression for all genotypes was normalized against the housekeeping genes GAPDH and β-actin using a variant (2 ΔCt ) of the Livak Method (2 -ΔΔCt ) as described in the Real-Time PCR applications guide from Bio-Rad( ).

Techniques: Selection, Southern Blot, Western Blot, Positive Control, Recombinant, Activity Assay

Journal: bioRxiv

Article Title: GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production

doi: 10.1101/2020.02.18.954438

Figure Lengend Snippet: The deletion of Gadl1 has tissue-specific effects on metabolite levels. a-d Top significant features of metabolites based on VIP scores > 2 of component 1 of PLS-DA. Untargeted metabolic profiling of a OB, b cerebral cortex, c SKM, and d liver tissue samples from Gadl1 +/+ (n=20) and Gadl1 -/- (n=21) mice. e The relative levels of β­alanine and carnosine derivatives in Gadl1 +/+ (grey) and Gadl1 -/- (red) mouse tissue.

Article Snippet: Total RNA was purified from different tissues from both Gadl1 +/+ and Gadl1 -/- mice using the RNeasy purification kit from Qiagen (#74104 and for muscle tissue #74704). cDNA was synthesized in triplicates from this RNA using a High-Capacity RNA-to-cDNA™ Kit (#4387406, Applied Biosystems™). qRT-PCR was performed using the TaqMan gene expression assay (TaqMan Gene Expression Master Mix #4369016, Applied Biosystems™) The TaqMan probes were: Mm00520087_m1 (CSAD), Mm01348767_m1 (GADL1, exon 13-14), Mm01341531_m1 (GADL1, exon 3-4), Mm01341534_m1 (GADL1, exon 6-7), Mm99999915_g1 (GAPDH) and Mm00607939_s1 (β­actin). mRNA expression for all genotypes was normalized against the housekeeping genes GAPDH and β-actin using a variant (2 ΔCt ) of the Livak Method (2 -ΔΔCt ) as described in the Real-Time PCR applications guide from Bio-Rad( ).

Techniques:

Journal: bioRxiv

Article Title: GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production

doi: 10.1101/2020.02.18.954438

Figure Lengend Snippet: 1 H-NMR and MRI of mouse tissues. a-c Measurement of carnosine in intact OB tissue. a Chemical structure of carnosine. b MAS- 1 H-NMR spectra of OB tissue samples from Gadl1 +/+ , Gadl1 +/- and Gadl1 -/- male mice (12 weeks). The two hydrogens of the imidazole ring in carnosine are marked a and b. c Relative integral based on NMR results, presented as mean ± SD, d-f MRI of the brain in d Gadl1 +/+ , e Gadl1 +/- and f Gadl1 -/- mice. The arrow indicates the OB.

Article Snippet: Total RNA was purified from different tissues from both Gadl1 +/+ and Gadl1 -/- mice using the RNeasy purification kit from Qiagen (#74104 and for muscle tissue #74704). cDNA was synthesized in triplicates from this RNA using a High-Capacity RNA-to-cDNA™ Kit (#4387406, Applied Biosystems™). qRT-PCR was performed using the TaqMan gene expression assay (TaqMan Gene Expression Master Mix #4369016, Applied Biosystems™) The TaqMan probes were: Mm00520087_m1 (CSAD), Mm01348767_m1 (GADL1, exon 13-14), Mm01341531_m1 (GADL1, exon 3-4), Mm01341534_m1 (GADL1, exon 6-7), Mm99999915_g1 (GAPDH) and Mm00607939_s1 (β­actin). mRNA expression for all genotypes was normalized against the housekeeping genes GAPDH and β-actin using a variant (2 ΔCt ) of the Livak Method (2 -ΔΔCt ) as described in the Real-Time PCR applications guide from Bio-Rad( ).

Techniques:

Journal: bioRxiv

Article Title: GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production

doi: 10.1101/2020.02.18.954438

Figure Lengend Snippet: RNA sequence and qRT-PCR analysis of mouse tissues. a-c Relative RNA expression levels (volcano plots) in OB tissue. a Gadl1 +/+ to Gadl1 +/- ratio, b Gadl1 +/+ to Gadl1 -/- ratio, c Gadl1 -/- to Gadl1 +/- ratio. d-f qRT-PCR analysis of normalized mRNA expression in OB, brain, SKM, and liver tissues from 35 week old females Gadl1 +/+ (grey) , Gadl1 +/- (blue) and Gadl1 -/- (red) mice for d Gadl1 exon 7 and 8 ,e Gadl1 exon 10 and 11, and f CSAD. n=3 for each genotype. Presented on a Ln y-scale as mean of 2 ΔCt and upper limit (95%).

Article Snippet: Total RNA was purified from different tissues from both Gadl1 +/+ and Gadl1 -/- mice using the RNeasy purification kit from Qiagen (#74104 and for muscle tissue #74704). cDNA was synthesized in triplicates from this RNA using a High-Capacity RNA-to-cDNA™ Kit (#4387406, Applied Biosystems™). qRT-PCR was performed using the TaqMan gene expression assay (TaqMan Gene Expression Master Mix #4369016, Applied Biosystems™) The TaqMan probes were: Mm00520087_m1 (CSAD), Mm01348767_m1 (GADL1, exon 13-14), Mm01341531_m1 (GADL1, exon 3-4), Mm01341534_m1 (GADL1, exon 6-7), Mm99999915_g1 (GAPDH) and Mm00607939_s1 (β­actin). mRNA expression for all genotypes was normalized against the housekeeping genes GAPDH and β-actin using a variant (2 ΔCt ) of the Livak Method (2 -ΔΔCt ) as described in the Real-Time PCR applications guide from Bio-Rad( ).

Techniques: Sequencing, Quantitative RT-PCR, RNA Expression, Expressing

Journal: bioRxiv

Article Title: GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production

doi: 10.1101/2020.02.18.954438

Figure Lengend Snippet: Behavioral phenotypes associated with Gadl1 -/- . a 3-chamber task: time spent at each cylinder was not different between the genotypes. Both Gadl1 +/+ and Gadl1 -/- mice prefer the social cylinder indicating sociability remains similar. b Open Field task: the cumulative time spent in the center was not different between the genotypes. On this measure, no anti-anxiety effect was observed. However, on the latency to enter the center (another anxiety measure), Gadl1 -/- were quicker to enter the center which may suggest some anxiolytic effects of the Gadl1 -/­ phenotype which would require confirmation in additional studies. c Open Field task: the exploration velocity was not different between the genotypes indicating that no effect on motor function. Similar observations were made with the total distance moved data. Taken together, this suggests no effect of genotype on activity metrics. d Elevated plus maze: the ratio of the time spent (s) on the open and closed arms was found not to be different between the genotypes. Both genotypes prefer the closed (sheltered arm) suggesting no difference in anxiety on this measure. e Resident Intruder Paradigm: the attack latency against an intruder from the 1 st to the 5 th day (Test 1-5) was not different between the genotypes. Both genotypes attack faster on the second day compared to the first day after which the attack latency remains constant. This suggests no effect of genotype on aggression.

Article Snippet: Total RNA was purified from different tissues from both Gadl1 +/+ and Gadl1 -/- mice using the RNeasy purification kit from Qiagen (#74104 and for muscle tissue #74704). cDNA was synthesized in triplicates from this RNA using a High-Capacity RNA-to-cDNA™ Kit (#4387406, Applied Biosystems™). qRT-PCR was performed using the TaqMan gene expression assay (TaqMan Gene Expression Master Mix #4369016, Applied Biosystems™) The TaqMan probes were: Mm00520087_m1 (CSAD), Mm01348767_m1 (GADL1, exon 13-14), Mm01341531_m1 (GADL1, exon 3-4), Mm01341534_m1 (GADL1, exon 6-7), Mm99999915_g1 (GAPDH) and Mm00607939_s1 (β­actin). mRNA expression for all genotypes was normalized against the housekeeping genes GAPDH and β-actin using a variant (2 ΔCt ) of the Livak Method (2 -ΔΔCt ) as described in the Real-Time PCR applications guide from Bio-Rad( ).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production

doi: 10.1101/2020.02.18.954438

Figure Lengend Snippet: Substrate specificity. a Chemical structure comparison of GADL1 substrates CSA, Asp, and Glu. b 3D substrate structures to show the size and shape differences of Glu compared to CSA and Asp. c Active sites of GADL1, GAD, and CSAD and the predicted mode of binding of Glu to GAD. The prediction is based on the complex between GAD and the inhibitor chelidonic acid.

Article Snippet: Total RNA was purified from different tissues from both Gadl1 +/+ and Gadl1 -/- mice using the RNeasy purification kit from Qiagen (#74104 and for muscle tissue #74704). cDNA was synthesized in triplicates from this RNA using a High-Capacity RNA-to-cDNA™ Kit (#4387406, Applied Biosystems™). qRT-PCR was performed using the TaqMan gene expression assay (TaqMan Gene Expression Master Mix #4369016, Applied Biosystems™) The TaqMan probes were: Mm00520087_m1 (CSAD), Mm01348767_m1 (GADL1, exon 13-14), Mm01341531_m1 (GADL1, exon 3-4), Mm01341534_m1 (GADL1, exon 6-7), Mm99999915_g1 (GAPDH) and Mm00607939_s1 (β­actin). mRNA expression for all genotypes was normalized against the housekeeping genes GAPDH and β-actin using a variant (2 ΔCt ) of the Livak Method (2 -ΔΔCt ) as described in the Real-Time PCR applications guide from Bio-Rad( ).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production

doi: 10.1101/2020.02.18.954438

Figure Lengend Snippet: Gadl1 -/- mice have altered levels of antioxidant enzymes. a-e Representative western blots (b, d, f) and normalized protein expression (a, c, e) for glutathione reductase (GSR), superoxide dismutase 1 (CuZnSOD), and SOD2 (Mn) in (a, b) OB, (c, d) cerebral cortex and (e, f) skeletal muscle tissue from female Gadl1 +/+ and Gadl1 -/­ mice.

Article Snippet: Total RNA was purified from different tissues from both Gadl1 +/+ and Gadl1 -/- mice using the RNeasy purification kit from Qiagen (#74104 and for muscle tissue #74704). cDNA was synthesized in triplicates from this RNA using a High-Capacity RNA-to-cDNA™ Kit (#4387406, Applied Biosystems™). qRT-PCR was performed using the TaqMan gene expression assay (TaqMan Gene Expression Master Mix #4369016, Applied Biosystems™) The TaqMan probes were: Mm00520087_m1 (CSAD), Mm01348767_m1 (GADL1, exon 13-14), Mm01341531_m1 (GADL1, exon 3-4), Mm01341534_m1 (GADL1, exon 6-7), Mm99999915_g1 (GAPDH) and Mm00607939_s1 (β­actin). mRNA expression for all genotypes was normalized against the housekeeping genes GAPDH and β-actin using a variant (2 ΔCt ) of the Livak Method (2 -ΔΔCt ) as described in the Real-Time PCR applications guide from Bio-Rad( ).

Techniques: Western Blot, Expressing

Journal: Bioengineering

Article Title: Stabilization of Poly (β-Amino Ester) Nanoparticles for the Efficient Intracellular Delivery of PiggyBac Transposon

doi: 10.3390/bioengineering8020016

Figure Lengend Snippet: PEG-PDHA NPs uptake by U87MG cells. ( a ) Flow cytometry and ( b ) confocal images of intact U87MG cells. ( c ) Flow cytometry and ( d ) confocal images of U878MG cells co-cultured for 1 h with PEG-PDHA NPs. TRITC-conjugated phalloidin was used to stain for actin cytoskeleton (red), Eosin isothiocyanate was used to label PEG-PDHA NPs (green) and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei (blue). Scale bar = 50 μm.

Article Snippet: Rhodamine B Isothiocyanate was purchased from CHEM-IMPEX International Inc. Eosin-5-Isothiocyanate was obtained from Chemodex.

Techniques: Flow Cytometry, Cell Culture, Staining