acarbose, alpha-glucosidase inhibitor  (Advisains)

Journal: bioRxiv

Article Title: Spatial transcriptomics and single-nucleus RNA sequencing reveal a transcriptomic atlas of adult human spinal cord

doi: 10.1101/2023.09.26.559582

Figure Lengend Snippet: ( A ) UMAP plot showing the coclustering of mouse and human neurons. Dots, individual cells. Colors, species. ( B ) UMAP plot showing the distribution of putative homologous neuronal clusters of humans and mice. Dots, individual cells. Colors, clusters. ( C ) Heatmap of conserved cell-type-specific gene expression (columns) in human and mouse cell types (rows; m, mouse; h, human). Genes in each species are included in the heatmap if they are significantly enriched in a cluster compared to all other clusters (FDR < 0.01, top 50 genes by log 2 -fold change [FC] per cell type). ( D-E ) Dot plot showing the expression of classical marker genes in the human (D) and mouse (E) spinal cord. ( F-G ) Heatmap showing the expression of classical marker genes in the human (F) and mouse (G) spinal cord. ( H ) Representative image showing the distribution of NPY-positive spots in the human spinal cord (left) and representative immunofluorescence image of NPY in a coronal cryosection of the human lumber spinal cord. ( I ) Representative RNAscope in situ hybridization images of Npy and Rbfox3 in a coronal cryosection of mouse lumber spinal cord. ( J ) Representative image showing the distribution of TAC1-positive spots in the human spinal cord and representative immunofluorescence image of TAC1 in a coronal cryosection of the human lumber spinal cord. ( K ) Representative RNAscope in situ hybridization images of Tac1 and Rbfox3 in a coronal cryosection of mouse lumber spinal cord. UMAP, uniform manifold approximation and projection.

Article Snippet: The lumber spinal cord and L3-5 DRGs were extracted and stored in 4% paraformaldehyde overnight, followed by incubation in 30% sucrose for 48 h. Cryosections from human and mouse spinal cord or DRGs were cut at 12 μm using a freezing microtome (CM1850; Leica, Buffalo Grove, IL, USA) and incubated at 4°C overnight with primary antibodies including: NeuN (1:500, mouse, Millipore, Cat#:MAB377; 1:500, rabbit, Abcam, Cat#: ab104225), MBP (1:300, rabbit, Abcam, Cat#: ab218011), GFAP (1:500, guinea pig, Synaptic System, Cat#:173004), Iba1 (1:300, rabbit, Wako, Cat#:019-19741), VGLUT2 (1:500, mouse, Millipore, Cat#: MAB5504), GAD67 (1:300, mouse, Santa Cruz Biotechnology, Cat#: sc-28376), CHAT (1:300, rabbit, Abcam, Cat#: ab181023), PDYN (1:200, rabbit, GeneTex, Cat#: GTX113515), NPY (1:200, rabbit, Abcam, Cat#: ab22145), TAC1 (1:300, mouse, Abcam, Cat#: ab14184), SNA10A (1:200, mouse, Abcam, Cat#: AB93616), SST (1:300, rabbit, Abclonal, Cat#: A9274), CCK (1:200, rabbit, Thermo Fisher, Cat#: PA5-103116), and FOXP2 (1:200, rabbit, Abcam, Cat#: ab16046).

Techniques: Expressing, Marker, Immunofluorescence, In Situ Hybridization

Journal: bioRxiv

Article Title: Spatial transcriptomics and single-nucleus RNA sequencing reveal a transcriptomic atlas of adult human spinal cord

doi: 10.1101/2023.09.26.559582

Figure Lengend Snippet: ( A ) Putative ligand‒receptor interactions of neuronal clusters between the human DRG and spinal cord. The thickness of connecting lines is proportional to the number of total ligand‒receptor interactions between the two connecting cell types. ( B ) DRG CALCA-spinal CALCRL interactions (left). The spatial location of the CALCRL-cluster (right). ( C ) DRG NGF-spinal NGFR interactions (left). The spatial location of the NGFR-cluster (right). ( D ) DRG SST-spinal SSTR1 interactions (left). The spatial location of the SSTR1-cluster (right). ( E ) DRG APOE-spinal LRP1 interactions. ( F ) DRG SLIT2-spinal ROBO2 interactions. ( G ) DRG TAC1-spinal TACR1 interactions. ( H ) DRG NRG1-spinal ERBB4 interactions. Dot size denotes relative expression of a gene in each cell type, and colors indicate cell type. Arrows between cell types denote the 10 highest ligand‒receptor scores. NF, neurofilament neurons; PEP, peptidergic neurons; NP, nonpeptidergic neurons.

Article Snippet: The lumber spinal cord and L3-5 DRGs were extracted and stored in 4% paraformaldehyde overnight, followed by incubation in 30% sucrose for 48 h. Cryosections from human and mouse spinal cord or DRGs were cut at 12 μm using a freezing microtome (CM1850; Leica, Buffalo Grove, IL, USA) and incubated at 4°C overnight with primary antibodies including: NeuN (1:500, mouse, Millipore, Cat#:MAB377; 1:500, rabbit, Abcam, Cat#: ab104225), MBP (1:300, rabbit, Abcam, Cat#: ab218011), GFAP (1:500, guinea pig, Synaptic System, Cat#:173004), Iba1 (1:300, rabbit, Wako, Cat#:019-19741), VGLUT2 (1:500, mouse, Millipore, Cat#: MAB5504), GAD67 (1:300, mouse, Santa Cruz Biotechnology, Cat#: sc-28376), CHAT (1:300, rabbit, Abcam, Cat#: ab181023), PDYN (1:200, rabbit, GeneTex, Cat#: GTX113515), NPY (1:200, rabbit, Abcam, Cat#: ab22145), TAC1 (1:300, mouse, Abcam, Cat#: ab14184), SNA10A (1:200, mouse, Abcam, Cat#: AB93616), SST (1:300, rabbit, Abclonal, Cat#: A9274), CCK (1:200, rabbit, Thermo Fisher, Cat#: PA5-103116), and FOXP2 (1:200, rabbit, Abcam, Cat#: ab16046).

Techniques: Expressing

Journal: Cancer Research

Article Title: Oxidative Phosphorylation Fueled by Fatty Acid Oxidation Sensitizes Leukemic Stem Cells to Cold

doi: 10.1158/0008-5472.CAN-23-1006

Figure Lengend Snippet: OxPhos leukemic cells have a 4°C-sensitive–specific lipidome dependent on FA metabolism. A, Maximal respiratory capacity, ATP-linked respiration, mtDNAc, median fluorescence intensity (MFI) for the mitochondrial potential probe TMRE, and the mitochondrial calcium probe Rhod-2 as a function of Q1V − Time at 4°C for primary AML. Respiration was measured with Seahorse assays under basal and maximal conditions. The relative mtDNA copy number was calculated relative to a reference DNA sample (healthy donor) set to 1. Nonparametric Spearman correlation tests were applied for n = 10 to 18 pairs. Thin black line shows experimentally derived linear regression trend line with 95% confidence band. B, Glycolytic-forced cells submitted to CKC4. Mitochondrial respiration was impaired by the complex I inhibitor rotenone. C, Kinetic of viability of pre-rotenone or untreated control AML cells at 4°C. D, OxPhos-forced cells submitted to CKC4. K562 cells were incubated at 37°C in glucose-free medium with galactose for 72 hours. E, Representative FACS dot plot of side scatter against DAPI for untreated control or galactose pretreated K562 cells after 72 hours at 4°C. F, Principal component analysis score plot of the total lipidome by MS (including species and subspecies analysis) of three OxPhos (Kasumi-1, THP-1, MV4-11) and three glycolytic leukemic cells (KG-1a, NB-4, U937), triplicate per cell type. G, Fold change of ether-phosphatidylcholine (PC), phosphatidylserine (PS), and sphingomyelin (SM) for OxPhos (O), and glycolytic cells (G) as indicated, normalized to the mean percentage for OxPhos cells (see also ; Supplementary Fig. S2I). H, Fold change of PC, PS, and SM for untreated OxPhos MV4-11 and glycolytic reprogrammed rotenone-treated MV4-11 cells. I, Venn diagram of significantly different proportion of lipid subspecies for OxPhos and glycolytic cells cohorts and for MV4-11 and MV4-11 rotenone-treated cells (see also ). J, Repartition of PE-C-30 to PE-C-44 subspecies within the PE family for OxPhos and glycolytic cells cohorts and for MV4-11 and MV4-11 rotenone-treated cells. K, Diagram showing the hypothesis tested in L and M . L and M, Indicated cells were pretreated with nontoxic dose of mitochondrial β-oxidation inhibitor etomoxir ( L ) or with lipid synthesis inhibitor Triacsin C ( M ) for 72 hours before undergoing a CKC4. N, Total proteome by MS for 10 patient samples. Pearson correlations were calculated for all quantified proteins versus Q1V − Time determined, and a ranked list of Pearson coefficients was used to perform GSEAs (see also Supplementary Table S2). GSEA of FA β-oxidation in CKC4-resistant versus CKC4-sensitive primary AML samples. O, Overview of study findings. OxPhos-reliant blast and LSCs cells have a FA-specific metabolism, shaping the composition of their membranes and accounting for their faster permeabilization compared with glycolysis-dependent leukemic blasts and progenitors. Mann–Whitney test was applied. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: In some experiments, cells were treated for 48 to 72 hours with etomoxir (#HY-50202, MedChem express) or Triacsin C (#ab141888, Abcam) with IC25 dose specific of each cell tested and predetermined by luciferase assay.

Techniques: Fluorescence, Derivative Assay, Incubation, MANN-WHITNEY

Journal: Biomedicines

Article Title: Apabetalone Downregulates Fibrotic, Inflammatory and Calcific Processes in Renal Mesangial Cells and Patients with Renal Impairment

doi: 10.3390/biomedicines11061663

Figure Lengend Snippet: Apabetalone blocks TGF-β1-induced HRMC activation. ( A ): HRMCs were treated with TGF-β1 ± BETi or TGFBRi for 24 h followed by gene expression analysis by real-time PCR ( n = 5). ( B ): Representative images of HRMCs treated with TGF-β1 ± apabetalone for 48 h, followed by immunofluorescence microscopy for α-SMA (green); nuclei were stained with DAPI (blue); apa = apabetalone. ( C ): Fluorescence intensity of α-SMA was quantified as percent of the image area. ( D ): Collagen gel contraction was evaluated after 4 days of treatment ( n = 6). ( E ) Collagen deposition was evaluated by picrosirius red staining after 5 days of treatment ( n = 4). Data in bar graphs are the mean ± SD. Statistical analysis by one-way ANOVA followed by Dunnett’s Multiple Comparison Test. *** p < 0.001, ** p < 0.01, NS not significant. ACTA2: α-SMA gene. α-SMA: alpha smooth muscle actin. TGF-β1: Transforming growth factor β1. Apabetalone: BD2-selective BET inhibitor. JQ1: pan-BET inhibitor. MZ1: PROTAC that directs BET proteins for degradation. TGFBRi: small molecule inhibitor of the TGF-β receptor.

Article Snippet: Recombinant, receptor active TGF-β1 was purchased from StemCell Technologies (Vancouver, BC, Canada) or Abcam (Cambridge, UK), a small molecule inhibitor of TGF-β receptors (TGFBRi) was purchased from Abcam (catalog # ab141890), and LPS (E. coli O111:B4) was purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Microscopy, Staining, Fluorescence

Journal: Biomedicines

Article Title: Apabetalone Downregulates Fibrotic, Inflammatory and Calcific Processes in Renal Mesangial Cells and Patients with Renal Impairment

doi: 10.3390/biomedicines11061663

Figure Lengend Snippet: BET proteins regulate expression of key drivers of fibrosis in HRMCs. HRMCs were treated with TGF-β1 ± BETi or TGFBRi for 24 h, followed by gene expression analysis by real-time PCR (left column ( A , C , E , G ); n = 4 or 5). For secreted proteins (right column ( B , D , F , H )), HRMCs were treated for 48 h. Cell culture media were clarified of debris by centrifugation, and the indicated proteins quantified by ELISA ( n = 3). Data are presented as mean ± SD. Statistical analysis by one-way ANOVA followed by Dunnett’s Multiple Comparison Test. * p < 0.05, ** p < 0.01, *** p < 0.001, NS not significant. THBS1 : thrombospondin 1 gene. FN1 : fibronectin gene. POSTN : periostin gene. SPARC gene and SPARC protein: secreted protein acidic and rich in cysteine. TGF-β1: Transforming growth factor β1. Apabetalone: BD2-selective BET inhibitor. JQ1: pan-BET inhibitor. MZ1: PROTAC that directs BET proteins for degradation.

Article Snippet: Recombinant, receptor active TGF-β1 was purchased from StemCell Technologies (Vancouver, BC, Canada) or Abcam (Cambridge, UK), a small molecule inhibitor of TGF-β receptors (TGFBRi) was purchased from Abcam (catalog # ab141890), and LPS (E. coli O111:B4) was purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Centrifugation, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Apabetalone Downregulates Fibrotic, Inflammatory and Calcific Processes in Renal Mesangial Cells and Patients with Renal Impairment

doi: 10.3390/biomedicines11061663

Figure Lengend Snippet: BET proteins regulate expression of inflammatory mediators. HRMCs were stimulated with TGF-β1 ± BETi or TGFBRi for 48 h ( A , B ) or with LPS ± BETi for 24 h ( C – E ). Gene expression was analyzed by real-time PCR ( n = 4 or 5) and secreted IL-6 levels by ELISA ( n = 3). Data are presented as mean ± SD. Statistical analysis by one-way ANOVA followed by Dunnett’s Multiple Comparison Test. *** p < 0.001, NS not significant. IL: interleukin. PTGS2: Prostaglandin-endoperoxide synthase 2. COX2: Cyclooxygenase 2. TGF-β1: Transforming growth factor β1. LPS: Lipopolysaccharide. Apabetalone: BD2-selective BET inhibitor. JQ1: pan-BET inhibitor. MZ1: PROTAC that directs BET proteins for degradation.

Article Snippet: Recombinant, receptor active TGF-β1 was purchased from StemCell Technologies (Vancouver, BC, Canada) or Abcam (Cambridge, UK), a small molecule inhibitor of TGF-β receptors (TGFBRi) was purchased from Abcam (catalog # ab141890), and LPS (E. coli O111:B4) was purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Apabetalone Downregulates Fibrotic, Inflammatory and Calcific Processes in Renal Mesangial Cells and Patients with Renal Impairment

doi: 10.3390/biomedicines11061663

Figure Lengend Snippet: Apabetalone suppresses TGF-β1 stimulated phosphatase expression associated with calcification. HRMCs were treated with TGF-β1 ± BETi for 48 h, followed by ALPL gene expression analysis by real-time PCR ( A ) or cell-associated alkaline phosphatase activity using the artificial fluorescent substrate 4-methylumbelliferyl phosphate (4-MUP) ( B ). Data are presented as the mean ± SD ( n = 5). Statistical analysis one-way ANOVA followed by Dunnett’s Multiple Comparison Test. *** p < 0.001. ALPL : Tissue-nonspecific alkaline phosphatase gene. TGF-β1: Transforming growth factor β1. Apabetalone: BD2-selective BET inhibitor. JQ1: pan-BET inhibitor. MZ1: PROTAC that directs BET proteins for degradation.

Article Snippet: Recombinant, receptor active TGF-β1 was purchased from StemCell Technologies (Vancouver, BC, Canada) or Abcam (Cambridge, UK), a small molecule inhibitor of TGF-β receptors (TGFBRi) was purchased from Abcam (catalog # ab141890), and LPS (E. coli O111:B4) was purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Biomedicines

Article Title: Apabetalone Downregulates Fibrotic, Inflammatory and Calcific Processes in Renal Mesangial Cells and Patients with Renal Impairment

doi: 10.3390/biomedicines11061663

Figure Lengend Snippet: Transcriptomics show BET inhibitors affect pathways of ECM reorganization. HRMCs were treated with TGF-β1 alone ( A ) or cotreated with TGF-β1 and the indicated BETi ( B – E ) for 24 h, followed by transcriptomic assessment by RNA-seq. Reactome Enrichment analysis of differentially expressed genes (DEGs) identified gene sets affected by treatments. Shown are the top six most significantly enriched gene sets for each treatment. The name of the gene set is followed by the number of DEGs from RNA-seq/number of genes in that Reactome gene set (gene ratio). The size of each circle is proportional to the percent overlap between DEGs and the Reactome gene set. Statistical significance was determined using the Mann–Whitney U Test and shown as −log10(padj). TGF-β1: Transforming growth factor β1. Apabetalone: BD2-selective BET inhibitor. JQ1: pan-BET inhibitor. MZ1: PROTAC that directs BET proteins for degradation.

Article Snippet: Recombinant, receptor active TGF-β1 was purchased from StemCell Technologies (Vancouver, BC, Canada) or Abcam (Cambridge, UK), a small molecule inhibitor of TGF-β receptors (TGFBRi) was purchased from Abcam (catalog # ab141890), and LPS (E. coli O111:B4) was purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: RNA Sequencing Assay, MANN-WHITNEY

Journal: Biomedicines

Article Title: Apabetalone Downregulates Fibrotic, Inflammatory and Calcific Processes in Renal Mesangial Cells and Patients with Renal Impairment

doi: 10.3390/biomedicines11061663

Figure Lengend Snippet: The effect of BETi on expression of genes in the “ECM Organization” gene set from the Reactome database. ECM: extracellular matrix. DEGs: differentially expressed genes. TGF-β1: Transforming growth factor β1. Apa: apabetalone, a BD2-selective BET inhibitor. JQ1: pan-BET inhibitor. MZ1: PROTAC that directs BET proteins for degradation.

Article Snippet: Recombinant, receptor active TGF-β1 was purchased from StemCell Technologies (Vancouver, BC, Canada) or Abcam (Cambridge, UK), a small molecule inhibitor of TGF-β receptors (TGFBRi) was purchased from Abcam (catalog # ab141890), and LPS (E. coli O111:B4) was purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Expressing

Journal: Biomedicines

Article Title: Apabetalone Downregulates Fibrotic, Inflammatory and Calcific Processes in Renal Mesangial Cells and Patients with Renal Impairment

doi: 10.3390/biomedicines11061663

Figure Lengend Snippet: IPA upstream regulator analysis of TGF-β1 via plasma proteomics. Plasma levels of 1305 proteins were determined in subjects with renal impairment (CKD; n = 8) and controls without renal impairment matched for age, weight, and sex ( n = 8) following a single dose of apabetalone (100 mg). Orange indicates predicted activation (IPA z-score > 2, and Benjamini–Hochberg adjusted p < 0.05), while blue indicates predicted inhibition (IPA z-score < −2, p < 0.05). TGF-β1: Transforming growth factor β1. Apabetalone: BD2-selective BET inhibitor.

Article Snippet: Recombinant, receptor active TGF-β1 was purchased from StemCell Technologies (Vancouver, BC, Canada) or Abcam (Cambridge, UK), a small molecule inhibitor of TGF-β receptors (TGFBRi) was purchased from Abcam (catalog # ab141890), and LPS (E. coli O111:B4) was purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Activation Assay, Inhibition

Journal: Nature

Article Title: The human microbiome encodes resistance to the antidiabetic drug acarbose

doi: 10.1038/s41586-021-04091-0

Figure Lengend Snippet: a. Phosphorylation rates (relative to the acarbose phosphorylation rate, which is set as 100%) at which Mak1 and AcbK phosphorylate a diverse panel of carbohydrates and aminoglycosides with structural similarities to acarbose. No phosphorylation was detected for all but one (validamycin) of the substrates under the same experimental conditions (see Methods). Experiments were done in duplicates with the average value used for rate comparisons; raw data is available in Supplementary Table 3. b. Michaelis-Menten saturation curves for AcbK (grey) and Mak1 (blue) performed at 1 μm enzyme concentration. Km and kcat values are indicated in their respective colours and individual kobs measurement replicates are shown on the graph for both enzymes. Raw data is available in Supplementary Table 3. c. kobs of both AcbK (grey) and Mak1 (blue) across different temperatures from 25–40 °C in 3 °C steps. The difference in kobs between the two enzymes can be seen across different temperatures. d, e. Hill plot (logarithm of kobs on the the y axis and logarithm of protein concentration on the x axis) of Mak1 (d) and AcbK (e). The Hill coefficient (slope) is greater than 1 for Mak1 (d, n=1.71 ± 0.06, n=1 is shown in grey dashed line for reference) but not for AcbK (e, n=1.13 ± 0.02), suggesting that only Mak1 is a cooperative enzyme. f. Relative change in kobs (y axis) for the single mutants D160A and D247A as well as the double mutant (D160A, H162A) of Mak1 as compared to wild type protein (N = 2). g. Extracted Ion Chromatograms (EICs) for acarbose (left, m/z = 646.4, [M+H]+) and acarbose-O6A-phosphate (right, m/z = 726.4, [M+H]+), showing that the addition of EDTA (blue traces) abolishes the activity of Mak1, while the addition of excess MgCl2 (red traces) restores it.

Article Snippet: Analyses of acarbose phosphorylation was carried out at 37 °C using 1 mM acarbose (Abcam, ab141891), 2 mM ATP (Sigma-Aldrich) and 10 μM recombinant protein, unless otherwise noted.

Techniques: Concentration Assay, Protein Concentration, Mutagenesis, Activity Assay

Journal: Nature

Article Title: The human microbiome encodes resistance to the antidiabetic drug acarbose

doi: 10.1038/s41586-021-04091-0

Figure Lengend Snippet: a, Side-by-side comparison of the extremely similar structures of Mak1 and AcbK with the acarbose substrate and AMP-PNP bound, as well as the coordinated metal (Mn+2, purple sphere). Both proteins form a canonical ribokinase fold where the substrate binds in a small cleft between the two main domains: a core α/β domain and a smaller β-clasp domain that forms the dimer interface. b, Left, Mak1 forms homodimers with extensive interactions between the two monomers, most notably β3 of one monomer and β8 of the other (yellow highlighted boxes; β-strands from each monomer are shaded differently for clarity). A molecular surface view is shown (right) with one Mak1 monomer coloured red and the other coloured purple, highlighting the extensive surface area (1,298 Å2) that is involved in forming the dimer through the β-clasp domains. c, Magnified view of the Mak1 substrate-binding pocket where an extensive network of hydrogen bonds form with all the hydroxyl groups in the acarbose A ring and hold it in place. Distances are shown for each of the hydrogen bonds. d, Magnified view of the Mak1 active site with important residues shown (Asp160, His162 and Asp247) that are all involved in priming the O6A hydroxyl of acarbose for nucleophilic attack and in facilitating the transfer of the phosphate from an ATP (AMP-PNP shown) to the O6A hydroxyl of acarbose. The distance shown is from the O6A hydroxyl to the γ-phosphate. A corresponding set of figures for AcbK is provided in Extended Data Fig. 7.

Article Snippet: Analyses of acarbose phosphorylation was carried out at 37 °C using 1 mM acarbose (Abcam, ab141891), 2 mM ATP (Sigma-Aldrich) and 10 μM recombinant protein, unless otherwise noted.

Techniques: Binding Assay

Journal: Nature

Article Title: The human microbiome encodes resistance to the antidiabetic drug acarbose

doi: 10.1038/s41586-021-04091-0

Figure Lengend Snippet: a. AcbK forms homodimers with extensive interactions between the two monomers. These interactions include multiple β strands in the β-clasp domain, most notably the β3 of one monomer and the β8 of the other (magenta highlighted box, with β strands from each monomer shaded differently for clarity). A molecular surface view is shown on the right with one AcbK monomer coloured green and the other coloured blue, highlighting the extensive surface area (1,298 Å2) involved in forming the dimer via the β-clasp domains. b. Zoomed in view of the AcbK substrate binding pocket where an extensive network of hydrogen bonds (from residues Asp16, Asn99, Ser109, and Asp248) form with all the hydroxyl groups in the acarbose A ring and hold it in place. Distances are shown for each of the hydrogen bonds mentioned above. c. Zoomed in view of the AcbK active site with important residues shown (Asp162, His164, Asp248), all involved in priming the O6A hydroxyl of acarbose for nucleophilic attack and in facilitating the transfer of the phosphate from an ATP (AMP-PNP shown) to the O6A hydroxyl of acarbose. Distance shown is from the O6A hydroxyl to the γ-phosphate. d. Full-length amino acid sequence alignment of experimentally tested Mcks/Maks, as well as AcbK and PfkB. The bars on the top of the alignment denote the average pairwise percent identity at each residue. e. Selected segments of the amino acid sequence alignment of experimentally tested Mcks/Maks, as well as AcbK and PfkB. Blue colours highlight amino acid residues that are deemed important for hydrogen bonding with the A ring of acarbose, yellow colours highlight those involved in the transfer of the phosphate group to acarbose and green indicates a residue involved in both processes. Grey colours indicate other conserved residues in the alignment. The bars on the top of the alignment denote the average pairwise percent identity at each residue.

Article Snippet: Analyses of acarbose phosphorylation was carried out at 37 °C using 1 mM acarbose (Abcam, ab141891), 2 mM ATP (Sigma-Aldrich) and 10 μM recombinant protein, unless otherwise noted.

Techniques: Binding Assay, Sequencing

Journal: Frontiers in Physiology

Article Title: Adventitial macrophage accumulation impairs perivascular nerve function in mesenteric arteries with inflammatory bowel disease

doi: 10.3389/fphys.2023.1198066

Figure Lengend Snippet: Primary antibody information.

Article Snippet: Substance P (SP) , mouse , Abcam , ab14184 , 1:250 , AB_300971.

Techniques: Concentration Assay, Activity Assay

Journal: Frontiers in Physiology

Article Title: Adventitial macrophage accumulation impairs perivascular nerve function in mesenteric arteries with inflammatory bowel disease

doi: 10.3389/fphys.2023.1198066

Figure Lengend Snippet: Primary antibody information.

Article Snippet: Substance P (SP) , mouse , Abcam , ab14184 , 1:250 , AB_300971.

Techniques: Concentration Assay, Activity Assay

Journal: Frontiers in Physiology

Article Title: Adventitial macrophage accumulation impairs perivascular nerve function in mesenteric arteries with inflammatory bowel disease

doi: 10.3389/fphys.2023.1198066

Figure Lengend Snippet: Macrophages depletion increases fluorescence area of sensory neurotransmitters but not the total perivascular nerves or perivascular sympathetic nerves. (A) Representative max z-projections of perivascular nerves labels from two sets of immunolabeling: perivascular sympathetic nerves (tyrosine hydroxylase, TH, green) + all perivascular nerves (PGP 9.5, red) and sensory neurotransmitters calcitonin gene related peptide (CGRP, green) + substance P (SP, red). Both sets are shown in (top-to-bottom) Control-PBS, Control-CLOD, IBD-PBS, and IBD-CLOD groups. Scale bar = 100 µm). (B–E) Quantitation of nerve density. Data are mean ± SE fluorescence area (B) all perivascular nerves (PGP 9.5), (C) perivascular sympathetic nerves (TH), and (D, E) sensory neurotransmitters CGRP and SP of confocal z-projections through the mesenteric artery adventitia of vessels from Control (blue, circles markers) and IBD (red, squares) mice treated with PBS (open bars), or clodronate (striped bars) liposomes. N = 10–25 images from 3-4 mice per group.* = p < 0.05 vs. CONT-PBS.

Article Snippet: Substance P (SP) , mouse , Abcam , ab14184 , 1:250 , AB_300971.

Techniques: Fluorescence, Immunolabeling, Quantitation Assay