Journal: bioRxiv

Article Title: CIS calibrates GM-CSF signalling strength to regulate macrophage polarization via a STAT5-IRF8 axis

doi: 10.1101/2022.03.31.486495

Figure Lengend Snippet: ( A ) IL-12 production by WT and Cish −/− GM-MØs stimulated with GpG or LPS for 20 hrs. *P<0.05, **P<0.01 by multiple group comparison of ANOVA test. (B ) Production of IL-12 by WT (Ly5.1) and Cish −/− (Ly5.2) GM-MØs derived from co-cultures with BM cells from individual mice. Harvested cells were stimulated with CpG for 4 hrs. ***P<0.001 by t test. ( C ) IL-12 production by spleen cells from WT and Cish −/− mice. Mice were engrafted with B16-GM for 9 days. Spleen cells were stimulated with CpG or LPS for 20 hrs. Bar graphs show mean ± SD of IL-12 concentration in culture supernatants. *P<0.05, **P<0.01 by t test. ( D ) IL-12 production by spleen MØs of WT and Cish −/− mice. Mix bone marrow chimera mice reconstituted with both WT (Ly5.1) and Cish −/− (Ly5.2) BM cells were engrafted with B16-GM for 9 days. Sorted MØs were stimulated with CpG for 20 hrs. Line graphs show IL-12 production by paired spleen MØs of WT and Cish −/− mice from the same hosts. *P<0.05, **P<0.01 by t test. ( E&F ) IL-12 production by human GM-MØs with Cish deletion. Human cord blood CD34 + cells were transfected with Cas9 assemble RNP and Cish guide RNA. Transfected CD34 + cells were cultured with human GM-CSF (5 ng/ml) for 7 days. Cells were stimulated with CpG or LPS. FACS plots show resulted human GM-MØ population ( E ). Bar graphs show IL-12 production by human GM-MØs ( F ). *P<0.05, **P<0.01 by multiple group comparison of ANOVA test. Data are representative of three independent experiments.

Article Snippet: CIS RNPs were assembled by incubating 1ml of 30mM CIS sgRNA (5’-CTCACCAGATTCCCGAAGGT-3’; Synthego), 1.7ml of 67nM Cas9 (Integrated DNA Technologies #1081058), 1ml of electroporation enhancer (Integrated DNA Technologies #1075915) and 1.3ml PBS for 10min at room temperature.

Techniques: Comparison, Derivative Assay, Concentration Assay, Transfection, Cell Culture

Journal: G3: Genes|Genomes|Genetics

Article Title: Tissue-Specific Split sfGFP System for Streamlined Expression of GFP Tagged Proteins in the Caenorhabditis elegans Germline

doi: 10.1534/g3.119.400162

Figure Lengend Snippet: Sequences used for CRISPR/Cas9, MosSCI, cloning and diagnosis. Table of the DNA/RNA sequences used for construction of the strains in this manuscript

Article Snippet: Ultramer oligonucleotides, tracrRNA, and crRNAs were obtained from IDT and mixed in the following concentrations: 14.35 µM Cas9-NLS (Berkeley MacroLab), 17.6 µM tracrRNA (IDT), 1.5 µM dpy10 crRNA (IDT), 5 µM dpy10 ssODN (IDT), 16.2 µM of target crRNA (IDT), and 6 µM of target ssODN (IDT) (see ).

Techniques: CRISPR, Clone Assay, Sequencing

Journal: Scientific reports

Article Title: CRISPR-assisted test for Schistosoma haematobium.

doi: 10.1038/s41598-023-31238-y

Figure Lengend Snippet: Figure 1. CATSH design and optimisation. (a) Visualisation of the targeted S. haematobium Dra1 repeat region with RPA primers, amplicon and crRNAs. (b) Schematic of the proposed CATSH workflow. Preparation of urine samples with in-house CATSH-compatible protocol, followed by CATSH testing. Real-time fluorescence is recorded on a portable reader. (c) Optimisation of ssDNA-FQ concentration. (d) Comparison of three crRNAs. (e) Optimisation of RPA input. (f) Optimisation of Cas12a to crRNA ratio. All reactions were run in five repeats. Bars represent the average endpoint fluorescence after background subtraction and error bars represent the standard deviation between repeats.

Article Snippet: Cas12a was diluted to its final concentration in 10X NEBuffer r2.1. crRNAs, RPA primers, synthetic target DNA and ssDNA-FQ were synthesised by Integrated DNA Technologies.

Techniques: Amplification, Fluorescence, Concentration Assay, Comparison, Standard Deviation

Journal: Vaccine

Article Title: Intradermal fractional-dose inactivated polio vaccine (fIPV) adjuvanted with double mutant Enterotoxigenic Escherichia coli heat labile toxin (dmLT) is well-tolerated and augments a systemic immune response to all three poliovirus serotypes in a randomized placebo-controlled trial

doi: 10.1016/j.vaccine.2022.03.056

Figure Lengend Snippet: Adverse events (AE) possibly, probably, or definitely related to vaccine, through Day 28 post-vaccination.

Article Snippet: Additionally, preclinical and clinical work assessing the use of dmLT as an injectable adjuvant for Enterotoxigenic Escherichia coli (ETEC) vaccines has similarly found doses of 0.5 μg to be immunogenic with limited local reactogenicity , . dmLT was produced according to good manufacture practice (GMP) specification by IDT Biologika Corporation and supplied by PATH in the form of 500 μg lyophilized cake in 3 mL vials (lot 001-0816) and maintained at −20 °C during transport and storage at the clinical site for up to 12 months prior to use. dmLT was rehydrated with 0.5 mL of sterile water for injection to achieve a final concentration of 1 mg/ml dmLT.

Techniques: Injection

Journal: Cell chemical biology

Article Title: Site-Specific Incorporation of Genetically Encoded Photo-Crosslinkers Locates the Heteromeric Interface of a GPCR Complex in Living Cells

doi: 10.1016/j.chembiol.2020.07.006

Figure Lengend Snippet: (A) Cells co-transfected with constructs encoding suppressor tRNA and azF aaRS, along with HA-mGluR3-TAG4.44-mCitrine, were loaded with Fura-2 and monitored for intracellular calcium concentration after sequential administration of LY341495 (LY34) and/or LY379268 (LY37), or vehicle. Controls included cells transfected with the wild-type HA-mGluR3-mCitrine construct, and cells untransfected with Gαi9 (n = 4 independent experiments per experimental condition). Mean ± SEM. ***p < 0.001 by Student’s t test (mGluR3) or Dunnett’s post hoc test of one-way ANOVA (TAG4.44).

Article Snippet: Cat#06162 (1 R ,4 R ,5 S ,6 R )-4-Amino-2-oxabicyclo[3.1.0] hexane-4,6-dicarboxylic acid disodium salt ( LY379268 ) Tocris Bioscience Cat#5064 (2S)-2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid disodium salt ( LY341495 ) Tocris Bioscience Cat#4062 l -glutamic acid Tocris Bioscience Cat#0218 [ 3 H]] LY341495 American Radiolabeled Chemicals ART-1439 250 uCI Experimental Models: Cell Lines HEK293 cells (Female) ATCC Cat# CRL-1573 Oligonucleotides See Table S3 for sequences of primers used Integrated DNA technologies (IDT) N/A Recombinant DNA Plasmid: pSVBpUC carrying the amber suppressor tRNA gene were donated by Thomas P. Sakmar ( Ye et al., 2008 ) N/A Plasmid: pcDNA3.1 carrying the azF aminoacyl-tRNA synthetase gene Thomas P. Sakmar ( Ye et al., 2008 ) N/A Plasmid: chimeric Gα subunit Gα qi9 was donated by Philippe Rondard ( Xue et al., 2015 ) N/A Plasmid: pcDNA3.1-c-Myc-5-HT 2A -mCherry and Javier González-Maeso ( Moreno et al., 2016 ) N/A Plasmid: pcDNA3.1-HA-mGluR2-mCitrine Javier González-Maeso ( Moreno et al., 2016 ) N/A Plasmid: pcDNA3.1-HA-mGluR3-mCitrine Javier Gonzáez-Maeso ( Moreno et al., 2016 ) N/A Plasmid: HA-mGluR2C121A-mCitrine This paper N/A Plasmid: HA-mGluR2-TAG 1.37 -mCitrine This paper N/A Plasmid: HA-mGluR2-TAG 4.44 -mCitrine This paper N/A Plasmid: HA-mGluR2-TAG 4.56 -mCitrine This paper N/A Plasmid: HA-mGluR3-TAG 4.44 -mCitrine This paper N/A Software and Algorithms Softmax Pro Molecular Devices https://www.moleculardevices.com/products/microplate-readers/acquisition-and-analysis-software/softmax-pro-software#gref Prism software version 8 GraphPad https://www.graphpad.com/scientific-software/prism/ Excel Microsoft https://www.microsoft.com/en-us/microsoft-365/excel ImageJ ImageJ developers https://imagej.nih.gov/ij/ Other UV-A lamp Spectronics Corporation ML-3500S MAXIM Open in a separate window KEY RESOURCES TABLE Photoactivatable unnatural amino acids inform the structural interface of 5-HT 2A R-mGluR2 TAG mGluR2 constructs were co-expressed with 5-HT 2A R for photo-crosslinking UV-induced crosslinking only in cells co-expressing 5-HT 2A R and mGluR2-TAG 4.44 5-HT 2A R interacts with mGluR2 via the intracellular end of mGluR2’s TM4 SIGNIFICANCE GPCR homo/heteromerization is a highly controversial topic and the existence of GPCR oligomers has been questioned in multiple studies.

Techniques: Transfection, Construct, Concentration Assay

Journal: Cell chemical biology

Article Title: Site-Specific Incorporation of Genetically Encoded Photo-Crosslinkers Locates the Heteromeric Interface of a GPCR Complex in Living Cells

doi: 10.1016/j.chembiol.2020.07.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cat#06162 (1 R ,4 R ,5 S ,6 R )-4-Amino-2-oxabicyclo[3.1.0] hexane-4,6-dicarboxylic acid disodium salt ( LY379268 ) Tocris Bioscience Cat#5064 (2S)-2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid disodium salt ( LY341495 ) Tocris Bioscience Cat#4062 l -glutamic acid Tocris Bioscience Cat#0218 [ 3 H]] LY341495 American Radiolabeled Chemicals ART-1439 250 uCI Experimental Models: Cell Lines HEK293 cells (Female) ATCC Cat# CRL-1573 Oligonucleotides See Table S3 for sequences of primers used Integrated DNA technologies (IDT) N/A Recombinant DNA Plasmid: pSVBpUC carrying the amber suppressor tRNA gene were donated by Thomas P. Sakmar ( Ye et al., 2008 ) N/A Plasmid: pcDNA3.1 carrying the azF aminoacyl-tRNA synthetase gene Thomas P. Sakmar ( Ye et al., 2008 ) N/A Plasmid: chimeric Gα subunit Gα qi9 was donated by Philippe Rondard ( Xue et al., 2015 ) N/A Plasmid: pcDNA3.1-c-Myc-5-HT 2A -mCherry and Javier González-Maeso ( Moreno et al., 2016 ) N/A Plasmid: pcDNA3.1-HA-mGluR2-mCitrine Javier González-Maeso ( Moreno et al., 2016 ) N/A Plasmid: pcDNA3.1-HA-mGluR3-mCitrine Javier Gonzáez-Maeso ( Moreno et al., 2016 ) N/A Plasmid: HA-mGluR2C121A-mCitrine This paper N/A Plasmid: HA-mGluR2-TAG 1.37 -mCitrine This paper N/A Plasmid: HA-mGluR2-TAG 4.44 -mCitrine This paper N/A Plasmid: HA-mGluR2-TAG 4.56 -mCitrine This paper N/A Plasmid: HA-mGluR3-TAG 4.44 -mCitrine This paper N/A Software and Algorithms Softmax Pro Molecular Devices https://www.moleculardevices.com/products/microplate-readers/acquisition-and-analysis-software/softmax-pro-software#gref Prism software version 8 GraphPad https://www.graphpad.com/scientific-software/prism/ Excel Microsoft https://www.microsoft.com/en-us/microsoft-365/excel ImageJ ImageJ developers https://imagej.nih.gov/ij/ Other UV-A lamp Spectronics Corporation ML-3500S MAXIM Open in a separate window KEY RESOURCES TABLE Photoactivatable unnatural amino acids inform the structural interface of 5-HT 2A R-mGluR2 TAG mGluR2 constructs were co-expressed with 5-HT 2A R for photo-crosslinking UV-induced crosslinking only in cells co-expressing 5-HT 2A R and mGluR2-TAG 4.44 5-HT 2A R interacts with mGluR2 via the intracellular end of mGluR2’s TM4 SIGNIFICANCE GPCR homo/heteromerization is a highly controversial topic and the existence of GPCR oligomers has been questioned in multiple studies.

Techniques: Recombinant, Plasmid Preparation, Software