Journal: Nature Communications

Article Title: Structural and mechanistic insights into a lysosomal membrane enzyme HGSNAT involved in Sanfilippo syndrome

doi: 10.1038/s41467-024-49614-1

Figure Lengend Snippet: a HGSNAT catalyzes the transmembrane acetylation of HS in the lysosome. The disaccharide repeating unit is represented by orange and magenta hexagons, while the terminal GlcN is shown as a green hexagon. Acetyl-CoA is represented by a yellow triangle (acetyl group) connected with a red circle (CoA). b Cryo-EM structure of human HGSNAT bound with acetyl-CoA. The two monomers within the dimer are colored in blue and orange respectively. Acetyl-CoA is colored in green. c Electrostatic potential of HGSNAT dimer. d Structure of HSGNAT dimer. e Topology map of the HGSNAT monomer colored in discrete colors corresponding to ( d ).

Article Snippet: The cDNA encoding the open reading frame of human HGSNAT (Uniprot: Q68CP4-2) was ordered from GenScript and cloned into the pEG-BacMam expression plasmid .

Techniques: Cryo-EM Sample Prep

Journal: Microbial Cell Factories

Article Title: Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris ( Komagataella phaffii ) P AOX1 -driven recombinant producer strains with different target gene dosage

doi: 10.1186/s12934-019-1240-8

Figure Lengend Snippet: Pichia pastoris physiological response to an increase in CRL1 gene dosage in fed-batch (FB) cultivations. a Specific methanol consumption rate ( q s ), overall biomass-to-substrate yield ( Y X / S * ). b Specific oxygen uptake rate ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$q_{{O_{2} }}$$\end{document} q O 2 ), specific carbon dioxide production rate ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$q_{{CO_{2} }}$$\end{document} q C O 2 ) and respiratory quotient (RQ)

Article Snippet: Recombinant strains of P. pastoris expressing CRL1 gene under the regulation of P AOX1 were constructed by using the pPICZαA plasmid (Invitrogen, Carlsbad, CA, US) assembled with the codon-optimized synthetic open reading frame (ORF) encoding the CRL1 gene sequence (GeneScript, Piscataway, NJ, USA).

Techniques:

Journal: Microbial Cell Factories

Article Title: Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris ( Komagataella phaffii ) P AOX1 -driven recombinant producer strains with different target gene dosage

doi: 10.1186/s12934-019-1240-8

Figure Lengend Snippet: Influence of the dilution rate on relative gene transcription levels (RTLs) in chemostat cultivations. a Genes AOX1 and CRL1 . b Gene MIT1 . MTH1 was used as housekeeping gene for the analysis. Error bars represent the standard deviation of two biological replicates

Article Snippet: Recombinant strains of P. pastoris expressing CRL1 gene under the regulation of P AOX1 were constructed by using the pPICZαA plasmid (Invitrogen, Carlsbad, CA, US) assembled with the codon-optimized synthetic open reading frame (ORF) encoding the CRL1 gene sequence (GeneScript, Piscataway, NJ, USA).

Techniques: Standard Deviation

Journal: Microbial Cell Factories

Article Title: Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris ( Komagataella phaffii ) P AOX1 -driven recombinant producer strains with different target gene dosage

doi: 10.1186/s12934-019-1240-8

Figure Lengend Snippet: Comparison of SCC and MCC Crl1 production kinetics and its relationship with CRL1 relative transcription levels. The specific Crl1 production rate ( q p ) was calculated for chemostat ( a ) and fed-batch cultivations ( b ). CRL1 transcriptional analyses were also done on chemostat cultivations ( a ). MTH1 was used as housekeeping gene for CRL1 RTL calculations. Error bars represent the standard deviation of two biological replicates

Article Snippet: Recombinant strains of P. pastoris expressing CRL1 gene under the regulation of P AOX1 were constructed by using the pPICZαA plasmid (Invitrogen, Carlsbad, CA, US) assembled with the codon-optimized synthetic open reading frame (ORF) encoding the CRL1 gene sequence (GeneScript, Piscataway, NJ, USA).

Techniques: Comparison, Standard Deviation

Journal: Microbial Cell Factories

Article Title: Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris ( Komagataella phaffii ) P AOX1 -driven recombinant producer strains with different target gene dosage

doi: 10.1186/s12934-019-1240-8

Figure Lengend Snippet: Comparison of SCC and MCC strain Crl1-related yields. Overall product-to-biomass yield ( Y P / X ) and product-to-substrate yield ( Y P / S ). a Chemostat cultivations. b Fed-batch cultivations. In a , error bars represent the standard deviation of two biological replicates

Article Snippet: Recombinant strains of P. pastoris expressing CRL1 gene under the regulation of P AOX1 were constructed by using the pPICZαA plasmid (Invitrogen, Carlsbad, CA, US) assembled with the codon-optimized synthetic open reading frame (ORF) encoding the CRL1 gene sequence (GeneScript, Piscataway, NJ, USA).

Techniques: Comparison, Standard Deviation

Journal: Microbial Cell Factories

Article Title: Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris ( Komagataella phaffii ) P AOX1 -driven recombinant producer strains with different target gene dosage

doi: 10.1186/s12934-019-1240-8

Figure Lengend Snippet: Crl1 production time evolution expressed as total activity units in fed-batch cultivations at different µ : (filled circle, open circle), 0.028 h −1 ; (filled square, open square), 0.047 h −1 ; (filled triangle), 0.063 h −1 ; (open triangle), 0.084 h −1

Article Snippet: Recombinant strains of P. pastoris expressing CRL1 gene under the regulation of P AOX1 were constructed by using the pPICZαA plasmid (Invitrogen, Carlsbad, CA, US) assembled with the codon-optimized synthetic open reading frame (ORF) encoding the CRL1 gene sequence (GeneScript, Piscataway, NJ, USA).

Techniques: Activity Assay

Journal: Microbial Cell Factories

Article Title: Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris ( Komagataella phaffii ) P AOX1 -driven recombinant producer strains with different target gene dosage

doi: 10.1186/s12934-019-1240-8

Figure Lengend Snippet: Effect of dilution rate on the CRL1 relative transcription level and specific production rate ratios between MCC and SCC. Error bars represent the standard deviation of q p and RTL ratios

Article Snippet: Recombinant strains of P. pastoris expressing CRL1 gene under the regulation of P AOX1 were constructed by using the pPICZαA plasmid (Invitrogen, Carlsbad, CA, US) assembled with the codon-optimized synthetic open reading frame (ORF) encoding the CRL1 gene sequence (GeneScript, Piscataway, NJ, USA).

Techniques: Standard Deviation

Journal: Microbial Cell Factories

Article Title: Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris ( Komagataella phaffii ) P AOX1 -driven recombinant producer strains with different target gene dosage

doi: 10.1186/s12934-019-1240-8

Figure Lengend Snippet: Comparison of Crl1 production-related parameters for chemostat cultivations

Article Snippet: Recombinant strains of P. pastoris expressing CRL1 gene under the regulation of P AOX1 were constructed by using the pPICZαA plasmid (Invitrogen, Carlsbad, CA, US) assembled with the codon-optimized synthetic open reading frame (ORF) encoding the CRL1 gene sequence (GeneScript, Piscataway, NJ, USA).

Techniques: Comparison

Journal: Microbial Cell Factories

Article Title: Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris ( Komagataella phaffii ) P AOX1 -driven recombinant producer strains with different target gene dosage

doi: 10.1186/s12934-019-1240-8

Figure Lengend Snippet: Comparison of Crl1 production-related parameters for fed-batch cultivations

Article Snippet: Recombinant strains of P. pastoris expressing CRL1 gene under the regulation of P AOX1 were constructed by using the pPICZαA plasmid (Invitrogen, Carlsbad, CA, US) assembled with the codon-optimized synthetic open reading frame (ORF) encoding the CRL1 gene sequence (GeneScript, Piscataway, NJ, USA).

Techniques: Comparison

Journal: Microbial Cell Factories

Article Title: Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris ( Komagataella phaffii ) P AOX1 -driven recombinant producer strains with different target gene dosage

doi: 10.1186/s12934-019-1240-8

Figure Lengend Snippet: Effect of dilution rate on the AOX1 – CRL1 relative transcription level ratio between MCC and SCC Error bars represent the standard deviation of RTL ratios

Article Snippet: Recombinant strains of P. pastoris expressing CRL1 gene under the regulation of P AOX1 were constructed by using the pPICZαA plasmid (Invitrogen, Carlsbad, CA, US) assembled with the codon-optimized synthetic open reading frame (ORF) encoding the CRL1 gene sequence (GeneScript, Piscataway, NJ, USA).

Techniques: Standard Deviation

Journal: PLoS ONE

Article Title: Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

doi: 10.1371/journal.pone.0015553

Figure Lengend Snippet: A . RT-PCR analysis of pancreatic islets and the whole brain of WT and CDKAL1 KO mice. The CDKAL1 transcript was not detected in CDKAL1 KO mice. B . Immunoblot analysis. Homogenates of mouse pancreatic islets and whole brain (30 µg) were subjected to SDS-PAGE and immunoblotted with anti-CDKAL1 antibody. The protein band below the CDKAL1 protein (*) is a nonspecific protein band detected by the anti-CDKAL1 antibody.

Article Snippet: EGFP–CDKAL1 was generated using a cDNA fragment encoding a full open reading frame of murine CDKAL1 (Dnaform, Kanagawa, Japan), N-terminally tagged with EGFP, and subcloned into the HindIII/KpnI site of a pcDNA3 vector (Invitrogen).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, SDS Page

Journal: PLoS ONE

Article Title: Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

doi: 10.1371/journal.pone.0015553

Figure Lengend Snippet: A . CDKAL1 KO mice have normal islet architecture. Pancreatic sections were peroxidase stained for insulin. Scale bar: 100 µm. B . Relative area occupied by β cells (percentage of total pancreatic area). Random sections of the entire pancreas from WT and CDKAL1 KO mice were immunostained (as shown in A ) and analyzed (60 sections from each of three mice per group). C–F . Electron micrographs of pancreatic tissue sections. C Representative sections (scale bar: 5 µm), D β cell size, E total number of granules per cell section, and F mean granule diameter in ultra-thin sections (100 nm) (n = 20 cells per group) of CDKAL1 KO and WT β cells. G–I . Insulin content in CDKAL1 KO mice. G Pancreatic insulin content measured in acid-ethanol extracts from WT and KO mice by ELISA (n = 6 per group). H DNA content per islet and I islet insulin content per DNA from WT and KO mice (n = 6 per group). Results are means±SEM.

Article Snippet: EGFP–CDKAL1 was generated using a cDNA fragment encoding a full open reading frame of murine CDKAL1 (Dnaform, Kanagawa, Japan), N-terminally tagged with EGFP, and subcloned into the HindIII/KpnI site of a pcDNA3 vector (Invitrogen).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

doi: 10.1371/journal.pone.0015553

Figure Lengend Snippet: A . Insulin release (for 30 min) in batch-incubated WT and CDKAL1 KO β cells in the presence of 2.2 mM or 16.7 mM glucose (n = 8 per group). B . Histogram showing the number of fusion events from GFP-tagged granules in wild-type (WT) and CDKAL1 KO β cells (per 200 µm 2 ) at 1-min intervals after stimulation with 22 mM glucose and measured by TIRF microscopy. Data are mean±SEM (WT, n = 16 cells; KO, n = 14 cells). Time 0 indicates the addition of 22 mM glucose. The red column shows fusion events from previously docked granules, and the green column shows those from newcomers. C . Histogram showing the number of fusion events in WT and KO β cells (per 200 µm 2 ) at 1-min intervals after 40 mM high K + stimulation measured by TIRF microscopy (n = 8 cells per group).

Article Snippet: EGFP–CDKAL1 was generated using a cDNA fragment encoding a full open reading frame of murine CDKAL1 (Dnaform, Kanagawa, Japan), N-terminally tagged with EGFP, and subcloned into the HindIII/KpnI site of a pcDNA3 vector (Invitrogen).

Techniques: Incubation, Microscopy

Journal: PLoS ONE

Article Title: Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

doi: 10.1371/journal.pone.0015553

Figure Lengend Snippet: A . Total internal reflection fluorescence (TIRF) microscopy of insulin granules morphologically docked to the plasma membrane. (top) Typical TIRF images of docked insulin granules in WT and CDKAL1 KO β cells. The surrounding lines represent the outline of cells attached to the cover glass. Scale bar: 5 µm. Pancreatic β cells were prepared from WT and KO mice, fixed, and immunostained for insulin. (bottom) Number of insulin granules morphologically docked to the plasma membrane. Individual fluorescent spots shown in the TIRF images were manually counted per 200 µm 2 in 15 cells per group. B . Electron micrograph of β cell sections. (top) Typical images of the plasma membrane area facing the blood capillary (C) of WT and KO β cells (B). Bar: 500 nm. (bottom) Number of morphologically docked insulin granules per 10 µm of the plasma membrane. Granules at their shortest distance of <10 nm from the plasma membrane were defined as morphologically docked granules (red arrowheads). Results are means±SEM. C . Expression of SNARE proteins in wild-type (WT) and KO islets by immunoblotting. Equal amounts of islet protein (30 µg) were separated by SDS-PAGE and immunoblotted. β-actin was used as a loading control.

Article Snippet: EGFP–CDKAL1 was generated using a cDNA fragment encoding a full open reading frame of murine CDKAL1 (Dnaform, Kanagawa, Japan), N-terminally tagged with EGFP, and subcloned into the HindIII/KpnI site of a pcDNA3 vector (Invitrogen).

Techniques: Fluorescence, Microscopy, Clinical Proteomics, Membrane, Expressing, Western Blot, SDS Page, Control

Journal: PLoS ONE

Article Title: Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

doi: 10.1371/journal.pone.0015553

Figure Lengend Snippet: A 22 mM glucose- and B 40 mM high K + -induced changes in [Ca 2+ ] i in WT and CDKAL1 KO β cells. Changes in [Ca 2+ ] i were measured by Fura-2 acetoxymethyl (2 µM). Time 0 indicates when the stimulants were added. The fluorescence ratio (340/360) at time 0 was taken as 1. Results are means±SEM (n = 12 cells per group).

Article Snippet: EGFP–CDKAL1 was generated using a cDNA fragment encoding a full open reading frame of murine CDKAL1 (Dnaform, Kanagawa, Japan), N-terminally tagged with EGFP, and subcloned into the HindIII/KpnI site of a pcDNA3 vector (Invitrogen).

Techniques: Fluorescence

Journal: PLoS ONE

Article Title: Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

doi: 10.1371/journal.pone.0015553

Figure Lengend Snippet: A . ATP content in WT and CDKAL1 KO islets. HPLC was used to measure ATP content in islets from WT and KO mice after incubation with 2 or 22 mM glucose. Results are means±SEM (n = 7 per group). B , C . Changes in mitochondrial membrane potential in response to 22 mM glucose ( B ) and 10 mM KIC ( C ) in WT and KO β cells. Changes in mitochondrial membrane potential were measured by a mitochondrial potential sensitive dye TMRE (10 nM). Time 0 indicates when the stimulants were added. The fluorescence intensity at time 0 was taken as 100%. Results are means±SEM (n = 12 cells per group).

Article Snippet: EGFP–CDKAL1 was generated using a cDNA fragment encoding a full open reading frame of murine CDKAL1 (Dnaform, Kanagawa, Japan), N-terminally tagged with EGFP, and subcloned into the HindIII/KpnI site of a pcDNA3 vector (Invitrogen).

Techniques: Incubation, Membrane, Fluorescence

Journal: PLoS ONE

Article Title: Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

doi: 10.1371/journal.pone.0015553

Figure Lengend Snippet: A . Subcellular fractionation for CDKAL1 localization. The postnuclear fraction (S1) from MIN6 β cells homogenates was fractionated by differential centrifugation (see ). Equal amounts (2 µg protein) of the fractions obtained were analyzed by immunoblotting with the indicated antibodies. S2, soluble cytosolic/microsomal fraction; Pellet, membrane fraction; S3, soluble cytosolic fraction. The protein band below the CDKAL1 protein (*) is a nonspecific protein band detected by the anti-CDKAL1 antibody. B . Immunocytochemical localization of CDKAL1 in MIN6 β cells. MIN6 β cells were transfected with EGFP–CDKAL1 and intracellular localization was examined using a confocal microscope with MitoTracker Red staining or immunostaining with anti-calnexin IgG (ER marker), as indicated.

Article Snippet: EGFP–CDKAL1 was generated using a cDNA fragment encoding a full open reading frame of murine CDKAL1 (Dnaform, Kanagawa, Japan), N-terminally tagged with EGFP, and subcloned into the HindIII/KpnI site of a pcDNA3 vector (Invitrogen).

Techniques: Fractionation, Centrifugation, Western Blot, Membrane, Transfection, Microscopy, Staining, Immunostaining, Marker