Journal: Cell

Article Title: A conserved family of immune effectors cleaves cellular ATP upon viral infection.

doi: 10.1016/j.cell.2023.07.020

Figure Lengend Snippet: Figure 2. A clade of prokaryotic nucleosidases is associated with anti-phage systems (A) Phylogenetic tree of prokaryotic PNP domains. Prokaryotic proteins were clustered based on sequence homology, and clusters with homology to the PNP_UDP_1 domain (pfam PF01048) were identified. A representative sequence from each cluster was used to build the tree. The tree is based on the PNP domain only (see STAR Methods). The total number of proteins represented by the representative sequences is shown for each clade. Ultrafast bootstrap values are shown for major branches.34 Known housekeeping proteins are shown in gray. The p value in the Cap17 clade represents the false discovery rate of a binomial test assessing the association of proteins from this clade with known anti-phage systems (see STAR Methods and Table S3). (B) A detailed phylogenetic tree of the Cap17 PNP clade (marked blue in A). The PNP domain of E. coli MtnN was used as an outgroup. The colored ring represents known or predicted anti-phage systems harboring a PNP domain, with example systems shown as gene cassettes. Abbreviations: pAgo, prokaryotic argonaute; CD-NTase, cGAS/DncV-like nucleotidyltransferase; SAVED, SMODS-associated and fused to various effector domains; NLR, nucleotide-binding leucine-rich repeat; FGE-S, sulfatase-modifying factor enzyme 1; TPR, tetratricopeptide repeat; DRT8, defensive-associated reverse transcriptase type 8.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies primary mouse anti-FLAG antibody Sigma Cat#F1804; RRID:AB_262044 HRP-conjugated goat anti-rabbit secondary antibody Thermo Scientific Cat#31460: RRID:AB_228341 Bacterial and virus strains E. coli K-12 MG1655 American Type Culture Collection (ATCC) ATCC 47076;Genbank: NC_000913 NEB 5-alpha Competent E. coli New England BioLabs Cat#C2987H SECphi27 Doron et al.36 Genbank: LT961732.1 T5 Udi Qimron Genbank: AY543070.1 T4 Udi Qimron Genbank: AF158101.6 T6 German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) DSM 4622; Genbank: MH550421.1 T2 German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) DSM 16352; Genbank: LC348380.1 P1 Udi Qimron Genbank: NC_005856.1 Chemicals, peptides, and recombinant proteins Isopropyl b-d-1-thiogalactopyranoside (IPTG) Inalco Cat#1758-1400 L-Arabinose GoldBio Cat#A300 ATP Merck Cat#A1852-1VL ADP Merck Cat#D6000 AMP Sigma Cat#01930 Adenosine Sigma Cat#A9251 dATP Thermo Scientific Cat#R0141 dADP Alfa Aesar Cat#J65307 dAMP Sigma Cat#D6375 Deoxyadenosine Sigma Cat#D7400 Adenine Sigma Cat#A2786 NTPs (ATP, GTP, CTP, UTP) (MAXIscript SP6/T7 Transcription Kit) Ambion Cat#AM1322 dNTPs (dATP, dGTP, dCTP, dTTP) New England BioLabs Cat#N0447L Xanthine Oxidase Roche Cat#10110434001 iodonitrotetrazolium chloride Sigma Cat#I8377 20X Bolt MES SDS Running Buffer ThermoFisher Cat#B0002 20X Bolt transfer buffer Thermo Scientific Cat#BT00061 Critical commercial assays KLD enzyme mix New England Biolabs Cat#M0554S NEBuilder HiFi DNA Assembly Master Mix New England Biolabs Cat#E2621L NEBExpress Ni-NTA Magnetic Beads New England Biolabs Cat#S1423L BacTiter-Glo Microbial Cell Viability Assay Promega Cat#G8230 Oligonucleotides Primers, see Table S7 This paper N/A Recombinant DNA pSG1 Doron et al.36 N/A pBAD-GFP Bernheim et al.13 N/A (Continued on next page) e1 Cell 186, 3619–3631.e1–e5, August 17, 2023

Techniques: Sequencing, Binding Assay, Reverse Transcription

Journal: eLife

Article Title: High-resolution and high-accuracy topographic and transcriptional maps of the nucleosome barrier

doi: 10.7554/eLife.48281

Figure Lengend Snippet:

Article Snippet: Strain , BL21(DE3) competent cells , NEB , Cat# c2527H , .

Techniques: Avidin-Biotin Assay, Sequencing, Lambda DNA Preparation, Recombinant, Construct, Software, Magnetic Beads, Plasmid Preparation

Journal: PLOS One

Article Title: Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices

doi: 10.1371/journal.pone.0320393

Figure Lengend Snippet: (A) Overall operation of the cDDLAMP assay for the detection of STEC strains. Culture free enrichment: E. coli was enriched from milk samples and lettuce surface rinse water using magnetic beads functionalized with anti- E. coli (α- E. coli ) antibodies. cDDLAMP: gDNA was extracted and amplified by LAMP in heat block to produce DNAzyme-containing LAMP amplicons. Colorimetric detection: The colorimetric signal produced by DNAzyme reaction was captured for analysis by using SpotXel Reader app on a smartphone. A micro-USB-powered light pad and a light-proof enclosure were used to eliminate the impact of ambient light. (B) cDDLAMP: Top panel shows a schematic illustration of target stx1 , stx2 , and eae genes of E. coli O157:H7. LAMP Dumbbell: The forward inner primer (FIP) and backward inner primer (BIP) were modified with reverse-complement sequence of DNAzyme (rcDNAzyme), rcEAD2 or rcDz-00. LAMP product: LAMP reaction generates amplicons containing two types of DNAzymes (EAD2, and Dz-00) in each amplification unit. Upon adding hemin, the DNAzymes produce a colorimetric signal in the presence of H 2 O 2 and TMB. The color change is captured by smartphone camera, and analyzed with SpotXel, a smartphone-based microplate reader application.

Article Snippet: For the specificity evaluation of cDDLAMP system, gDNA of O157:H7 EDL932 (ATCC 43894), six clinical STEC strains (O111:H8, O26:H11, O103:H11, O45:H2, O145:NT, and O121:H19), three non-pathogenic E. coli strains (ATCC 43745, 25922,and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ) were tested.

Techniques: Magnetic Beads, Amplification, Blocking Assay, Produced, Modification, Sequencing

Journal: PLOS One

Article Title: Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices

doi: 10.1371/journal.pone.0320393

Figure Lengend Snippet: The following bacterial species were tested: O157:H7 EDL932 (ATCC 43894), six clinical STEC strains (O111:H8, O26:H11, O103:H11, O45:H2, O145:NT, and O121:H19), three non-pathogenic E. coli strains(ATCC 43745, 25922,and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ). Means from three independent experiments are shown with error bars representing standard error of the means. a–c; Asterisks above means denote significant differences and means carrying different superscripts are significantly different at p < 0.05 (****P < 0.0001). The cut-off value of stx1 indicated as dotted line = 1.45. The stx2 and eae genes were 1.37and 1.18, respectively.

Article Snippet: For the specificity evaluation of cDDLAMP system, gDNA of O157:H7 EDL932 (ATCC 43894), six clinical STEC strains (O111:H8, O26:H11, O103:H11, O45:H2, O145:NT, and O121:H19), three non-pathogenic E. coli strains (ATCC 43745, 25922,and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ) were tested.

Techniques: Bacteria

Journal: PLOS One

Article Title: Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices

doi: 10.1371/journal.pone.0320393

Figure Lengend Snippet: ClustalW alignment of the stx1 (A), stx2 (B), and eae (C) genes from STEC strains is presented. Target sequences for stx1 , stx2 , and eae from E. coli O157:H7 (strain EDL933*) were retrieved from the GenBank database (accession number CP008957.1). Annotation labels indicate the binding regions for the primers, including outer primers (F3, B3), loop primers (LF, LB), forward inner primer (FIP; F1c, F2), and backward inner primer (BIP; B1c, B2). Shading indicates sequence similarity as follows: black (100%), dark gray (80–100%), light gray (60–80%), and white (<60%). Sequence logos, generated using Geneious 2019.2.1 software, display the consensus across the sequences.

Article Snippet: For the specificity evaluation of cDDLAMP system, gDNA of O157:H7 EDL932 (ATCC 43894), six clinical STEC strains (O111:H8, O26:H11, O103:H11, O45:H2, O145:NT, and O121:H19), three non-pathogenic E. coli strains (ATCC 43745, 25922,and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ) were tested.

Techniques: Binding Assay, Sequencing, Generated, Software

Journal: Cell Reports Medicine

Article Title: Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic

doi: 10.1016/j.xcrm.2021.100319

Figure Lengend Snippet:

Article Snippet: Human respiratory syncytial virus , ATCC , Cat# VR-1580DQ.

Techniques: Virus, Recombinant, Protease Inhibitor, Reverse Transcription, Ligation, Sequencing, Plasmid Preparation, Software, Magnetic Beads

Journal: The EMBO Journal

Article Title: CROP: a retromer‐PROPPIN complex mediating membrane fission in the endo‐lysosomal system

doi: 10.15252/embj.2021109646

Figure Lengend Snippet: Sequence alignment of various Atg18 and WIPI1/2 orthologs showing a conserved stretch (in red) of residues in blade 2. The stretch containing T56 is mapped on the structure of Atg18 from S. cerevisiae (pdb #6KYB) (Lei et al , ), the LFSTSL motif in from S. cerevisiae is shown in orange. Arrows show the Thr56 residue. Pull‐down. Cells ( SEY6210 atg18∆ , atg21∆ ) expressing genomically tagged Vps26 yomCherry and the indicated Atg18 HA3‐yEGFP variants were logarithmically grown in SC ‐URA media. Vps26 yomCherry was pulled down from whole‐cell extracts with RFP‐trap magnetic beads and analyzed by SDS–PAGE and Western blotting against the indicated proteins. Bands were quantified on a LICOR fluorescence imager. Signals of Atg18 HA3yEGFP were normalized relative to those of Vps26 yomCherry . Vps1 served as a loading control. n = 3 independent replicates. Data were subjected to an unpaired t ‐test. Bars represent the mean and errors bars the SEM, *** P < 0.001). Influence of substitutions on Atg18 localization. The cells from c were stained with calcofluor white to mark the cell walls and analyzed by confocal microscopy. The calcofluor signal (blue) is only shown in the merge. Scale bar: 5 µm.

Article Snippet: The QExactive data were searched with Mascot 2.6 (Matrix Science, London, UK) software using a Saccharomyces cerevisiae (strain ATCC 204508 / S288c) reference proteome from UniProt ( www.uniprot.org , February 2017 version: 6,049 sequences) as database.

Techniques: Sequencing, Residue, Expressing, Magnetic Beads, SDS Page, Western Blot, Fluorescence, Control, Staining, Confocal Microscopy

Journal:

Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity

doi:

Figure Lengend Snippet: Immunization with AdMDC/DCs induces protective immunity specific to the pathogen used for pulsing DCs. (a) P. aeruginosa challenge. C57BL/6 mice were immunized with AdMDC-modified DCs pulsed with heat-killed P. aeruginosa or E. coli (25922 strain). After 3 weeks, immunized animals were infected intratracheally with 2 × 105 CFU P. aeruginosa PAO1 enmeshed in agar beads. (b) E. coli challenge. Immunization of mice was identical to that in a, but 108 CFU E. coli (25922 strain) were used for the intratracheal infection. For both parts, controls included mice without any immunization. Survival was recorded as the percentage of surviving animals (n = 10 mice per group).

Article Snippet: DCs were incubated with AdMDC, AdNull, or PBS at an moi of 100 for 4 hours at 37°C in the presence of heat-killed (56°C, 30 minutes) P. aeruginosa or (as a control) Escherichia coli (25922 strain; ATCC) at a ratio of ten bacteria per one DC.

Techniques: Modification, Infection

Journal:

Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity

doi:

Figure Lengend Snippet: Responses of CD4+ T cells from immunized mice in a microbe-specific manner. (a–d) Proliferation. C57BL/6 mice were immunized with AdMDC-modified DCs pulsed with P. aeruginosa PAO1 strain or E. coli. Three weeks after immunization, splenic CD4+ T cells were isolated from immunized or nonimmunized mice using the magnetic beads. In 96-well culture plates, 5 × 105 CD4+ T cells were stimulated with 5 × 104 DCs that had been pulsed with heat-killed P. aeruginosa strain PAO1 (a), heat-killed P. aeruginosa strain PA103 (b), heat-killed E. coli (c), or without any microbes (d), and irradiated. In (a–d), controls included DC culture without CD4+ T cells. The number of viable cells was determined using the MTS assay, as described in Methods. The data are presented as the mean percentage increase over baseline of duplicate wells. (e) IL-4 secretion. The culture medium was collected 4 days after the initiation of the coculture described above, and the levels of murine IL-4 were assayed by ELISA. The data are presented as means ± SE (n = 3 per data point).

Article Snippet: DCs were incubated with AdMDC, AdNull, or PBS at an moi of 100 for 4 hours at 37°C in the presence of heat-killed (56°C, 30 minutes) P. aeruginosa or (as a control) Escherichia coli (25922 strain; ATCC) at a ratio of ten bacteria per one DC.

Techniques: Modification, Isolation, Magnetic Beads, Irradiation, MTS Assay, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: The evolution of a counter-defense mechanism in a virus constrains its host range

doi: 10.7554/eLife.79549

Figure Lengend Snippet:

Article Snippet: Strain, strain background (T6) , T6 , ATCC , Cat# 11303-B6; phML-40 , N/A.

Techniques: Cloning, Control, Magnetic Beads, Recombinant, Concentration Assay, Plasmid Preparation, Amplification, Software, Sequencing, DNA Sequencing, Shear, Illumina Sequencing