Journal:

Article Title: Inward rectifier potassium conductance regulates membrane potential of canine colonic smooth muscle

doi: 10.1111/j.1469-7793.1999.0247r.x

Figure Lengend Snippet: A representative gel of Q-PCR for Kir2.1 in the submucosal layer of canine colon is shown in A; 2-fold serial dilutions of mimic DNA were included in the PCR reactions while Kir2.1 cDNA remained constant. The concentration of Kir2.1 cRNA in different regions of the GI tract expressed relative to β-actin cDNA is illustrated in B. The amount of Kir2.1 cDNA in SCM, ICM and MyCM preparations is depicted in C. Results are expressed as means ±s.e.m.; * significant difference in the level of Kir2.1 transcript in ICM and MyCM compared with SCM preparations (P < 0.05).

Article Snippet: Quantitative PCR (Q-PCR) Q-PCR was performed by use of the PCR MIMIC Construction Kit (Clontech, CA, USA), which is based upon a competitive PCR approach: non-homologous engineered DNA standards (referred to as PCR MIMICs) compete with target DNA for the same gene-specific primers.

Techniques: Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies

doi: 10.3389/fmicb.2022.1038120

Figure Lengend Snippet: Overview of the experiments. (A) Eight protocols were initially tested with Mock community samples (Zymo, D6300) to evaluate loss of DNA during processing. Two protocols were excluded prior to processing clinical samples. (B) Three pools (A, B, and C) were created by blindly pooling six 2 ml samples from premature infants for each pool. Six aliquots (2 ml each) from each pool were processed according to the remaining six protocols. (C) After reviewing results, four protocols were excluded and an additional pool was created using six samples from premature infants, obtained right after birth (Pool D). Six aliquots (2 ml) from this pool were processed according to the remaining two protocols. Two of the aliquots were spiked with mock community. (D) The most promising protocol (Mol_MasterPure) was further tested on individual patient samples.

Article Snippet: Additionally, Femto™ Quantification kits for host and bacterial DNA (Zymo, E2005 and E2006) were used according to the manufacturer’s instructions for four individual patient samples.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies

doi: 10.3389/fmicb.2022.1038120

Figure Lengend Snippet: Pooled patient samples. (A) DNA yield extracted from pools A, B, and C according to different protocols. Each bar represents DNA yield of one sample. Samples that proceeded to WMS are marked with black dots (library concentration > 10 nM). (B) Comparison of relative reduction in total DNA yield with protocols for DNA depletion. MasterPure (no depletion) was used as a reference. (C) Relative change in the proportion of microbial DNA/host DNA, evaluated with real-time qPCR. (D) Host DNA content evaluated with WMS in samples from patients’ pools A, B, and C. MasterPure protocol served as a reference. (E) Increase in number of bacterial reads in depleted samples, relative to non-depleted reference samples. (B–E) Values for individual samples are presented as dots. Bars correspond to mean values from samples from different patient pools, processed with the same protocol.

Article Snippet: Additionally, Femto™ Quantification kits for host and bacterial DNA (Zymo, E2005 and E2006) were used according to the manufacturer’s instructions for four individual patient samples.

Techniques: Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies

doi: 10.3389/fmicb.2022.1038120

Figure Lengend Snippet: Information regarding library preparation, host DNA content and number of bacterial, and ARG associated reads.

Article Snippet: Additionally, Femto™ Quantification kits for host and bacterial DNA (Zymo, E2005 and E2006) were used according to the manufacturer’s instructions for four individual patient samples.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies

doi: 10.3389/fmicb.2022.1038120

Figure Lengend Snippet: Rarefaction analysis. Rarefaction analysis. Rarefaction curves at species level for samples at various sequencing depth (% clean reads) for pools A (A) , B (B) , C (C) , and D (D) . Protocols including MolYsis for host DNA depletion retrieved highest species richness across all pools. Number of bacterial reads obtained at 100% clean reads are listed next to samples rarefaction curve.

Article Snippet: Additionally, Femto™ Quantification kits for host and bacterial DNA (Zymo, E2005 and E2006) were used according to the manufacturer’s instructions for four individual patient samples.

Techniques: Sequencing

Journal: Frontiers in Microbiology

Article Title: Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies

doi: 10.3389/fmicb.2022.1038120

Figure Lengend Snippet: Detailed information on individual patient samples.

Article Snippet: Additionally, Femto™ Quantification kits for host and bacterial DNA (Zymo, E2005 and E2006) were used according to the manufacturer’s instructions for four individual patient samples.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies

doi: 10.3389/fmicb.2022.1038120

Figure Lengend Snippet: Individual patient samples. (A) . Total microbial load relative to the abundance of I. haloterans (log scale). (B) Bacterial/Host DNA ratio for individual patient samples evaluated with real time qPCR (red) and WGS (blue).

Article Snippet: Additionally, Femto™ Quantification kits for host and bacterial DNA (Zymo, E2005 and E2006) were used according to the manufacturer’s instructions for four individual patient samples.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies

doi: 10.3389/fmicb.2022.1038120

Figure Lengend Snippet: Mechanism of Host DNA depletion protocols. Graphical illustration of different host DNA depletion protocols according to their mechanism of action. LyPMA protocol is composed of osmotic lysis and DNA fragmentation with photolysis. MolYsis and QIAamp use chemical lysis of host cells followed by enzymatic degradation of extracellular DNA. In theory, all methods result in selective lysis of human cells, followed by removal of extracellular DNA, human and bacterial. In our study, only samples depleted with MolYsis™ showed a reduction in host DNA content. Parts of the figure were drawn by using pictures from Servier Medical Art by Servier (smart. servier.com ).

Article Snippet: Additionally, Femto™ Quantification kits for host and bacterial DNA (Zymo, E2005 and E2006) were used according to the manufacturer’s instructions for four individual patient samples.

Techniques: Lysis

Journal: Nucleic Acids Research

Article Title: Epstein–Barr Virus DNase (BGLF5) induces genomic instability in human epithelial cells

doi: 10.1093/nar/gkp1169

Figure Lengend Snippet: The nuclease activity of EBV DNase is important for the effect of DNA damage. ( a ) TW01 cells were transfected with vector, DNase, Mutant 1, Mutant 2 and Mutant 3, respectively. After 24 h transfection, cells were collected for detection of DNA damage. DNA damage was analyzed by comet assay, which is as described in ‘Materials and Methods’ section. ( b ) The results of comet assay were quantitated by visual scoring. Visual scores were presented as classes 0–4 which increase with the level of genomic DNA damage ( Supplementary Figure S1 ). All data presented represent the means and the standard deviations of at least three independent experiments. Two asterisks denote P < 0.01.

Article Snippet: A single-cell gel electrophoresis (comet assay) kit was employed for evaluating DNA damage (Trevigen, Inc.).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Single Cell Gel Electrophoresis

Journal: Frontiers in Genetics

Article Title: Expanded CUG Repeat RNA Induces Premature Senescence in Myotonic Dystrophy Model Cells

doi: 10.3389/fgene.2022.865811

Figure Lengend Snippet: Expanded CUG repeat RNA causes DNA damage in a telomere-independent manner. (A) Measurement of relative telomere length by qPCR. Telomere length was measured at three different stages (at days 43, 49, and 61) in cells not expressing CUG exp (CUG exp− ) (blue) and cells expressing CUG exp (CUG exp+ ) (red). Data are presented as means ± SD of three independent experiments. (B) Representative images of DNA damage in comet assays at day 53 (left). DNA damage was evaluated according to the percentage tail DNA and Olive tail moment (right). Data are presented as means ± SD of three independent experiments. * p < 0.01, ** p < 0.001. (C) Representative images of DNA damage in γ-H2AX assays at day 53 (left). Bar graph showing percentage of γ-H2AX positive cells (right). Data are presented as means ± SD of three independent experiments. * p < 0.05. (D) Quantification of DNA damage response by AP site measurement. The AP site lesions per 100,000 base pairs were measured in the cells before transfection (day 5) and at day 49 in cells not expressing CUG exp (CUG exp− ) and cells expressing CUG exp (CUG exp+ ). Data are presented as means ± SD of three independent experiments. * p < 0.01.

Article Snippet: The number of AP sites was assessed using a colorimetric DNA Damage Quantification Kit-AP Site Counting (Dojindo), according to the manufacturer’s instructions.

Techniques: Expressing, Transfection

Journal: Frontiers in Genetics

Article Title: Expanded CUG Repeat RNA Induces Premature Senescence in Myotonic Dystrophy Model Cells

doi: 10.3389/fgene.2022.865811

Figure Lengend Snippet: Proposed mechanism of premature senescence by CUG exp RNA expression. RNA toxicity by CUG exp causes mitochondrial dysfunction, which contributes to ROS overproduction, triggering DNA damage and DDR. Accumulated DNA damage activates cell cycle regulators, such as p53, p21, and p16. ROS-induced DNA damage upregulates the secreted mediators PAI-1 and IGFBP3 in a p53-dependent and -independent manner, which drive premature senescence. These multiple steps in the process of senescence interact with one another, inducing premature senescence.

Article Snippet: The number of AP sites was assessed using a colorimetric DNA Damage Quantification Kit-AP Site Counting (Dojindo), according to the manufacturer’s instructions.

Techniques: RNA Expression

Journal: PLoS Genetics

Article Title: A Network of Conserved Damage Survival Pathways Revealed by a Genomic RNAi Screen

doi: 10.1371/journal.pgen.1000527

Figure Lengend Snippet: (A) A schematic representation of the experimental design used to screen for genes involved in survival of an MMS exposure using RNAi in Drosophila . (B) A schematic representation of the workflow from the initial RNAi screen, the normalization and validation of results, and the follow-up work conducted, with a summary of the number of dsRNA amplicons and genes (FBgn; FlyBase gene number) involved at each step in the process. Also provided are the final numbers of MMS survival proteins and MMS survival pathways (BER: base excision repair; NER: nucleotide excision repair; MMR: mismatch repair; HRR: homologous recombination repair; RECQ: RecQ helicases; DDR: DNA damage response; Proteasome; GSH: glutathione synthesis; TOR: TOR pathway; Transcription: basal transcription; Ribosome; ATPase; Notch: Notch signaling pathway).

Article Snippet: Apurinic or apyrmidinic sites (AP) that were generated following DNA damage were measured using a colorimetric assay kit for DNA damage quantification (Oxford Biomedical Science, Oxford, MI), following manufacturer's protocol.

Techniques: Biomarker Discovery, Homologous Recombination

Journal: PLoS Genetics

Article Title: A Network of Conserved Damage Survival Pathways Revealed by a Genomic RNAi Screen

doi: 10.1371/journal.pgen.1000527

Figure Lengend Snippet: CG numbers are given for each Drosophila pathway component, as well as the protein names or complex names for their human orthologues. Pathway entry points are noted with Roman numerals at the top, and end points are at the bottom. A key for the following symbols is provided. Symbols encircled with thick lines represent proteins that act together or in a complex, while symbols encircled with thin lines represent paralogues or proteins that may substitute for one another. Proteins found to affect MMS survival are noted as down (death) or up triangles (resistance). Statistically significant proteins are indicated with black triangles, while trend hits are indicated with grey triangles. Essential genes are noted with a thick bar and any with downwards or upwards pointing boxes were also validated as conferring death or resistance, respectively, to MMS upon knock-down. Shaded squares are proteins not found to be hits after validation, and open squares were not tested in our validation. Yeast orthologues previously found to be required for MMS survival are noted with a dot under the symbol. (A) Base Excision Repair. (B) DNA Damage Response. (C) Proteasome.

Article Snippet: Apurinic or apyrmidinic sites (AP) that were generated following DNA damage were measured using a colorimetric assay kit for DNA damage quantification (Oxford Biomedical Science, Oxford, MI), following manufacturer's protocol.

Techniques: Knockdown, Biomarker Discovery

Journal: PLoS Genetics

Article Title: A Network of Conserved Damage Survival Pathways Revealed by a Genomic RNAi Screen

doi: 10.1371/journal.pgen.1000527

Figure Lengend Snippet: (A) 150 of the validated MMS survival proteins (red nodes) are represented in the known Drosophila interactome, with protein:protein interactions represented by grey edges; 104 of these validated hits are “orphans,” not connected to other proteins. (B) Nodes and orphans from (A), including 11 proteins essential for viability (black nodes) that are connected to two or more validated hits, improves the connectivity of the DNA damage survival network. In this network, only 93 hits are orphans and a larger network of 36 proteins, containing 29 validated hits, is observed. (C) Nodes and orphans from (B), including additional pathway hits not found in screen (green nodes). In this network, 86 screen hits and an additional 54 pathway hits are orphans and a larger network of 63 proteins, containing 33 validated screen hits, 20 additional pathway hits and 10 essential proteins. (D) Nodes and orphans from (C), including all non-hit members of the 13 MMS survival pathways (open nodes). In this network, 60 screen hits and an additional 40 pathway hits are orphans and a larger network of 247 proteins, containing 79 validated screen hits (26 of which are within pathways), 35 additional pathway hits, 101 non-hit pathway proteins, and 32 essential proteins. (E) Network of proteins validated to be required after MMS treatment (red nodes), proteins essential for viability (black nodes), and 13 pathways (blue pathway nodes) validated to be involved in MMS survival. All proteins in each pathway, regardless of being a hit, are represented in its metanode, with node size representing protein number. After all pathway components are used to create the network, only 54 proteins are orphan, and 96 proteins are contained within the main interconnected network. Network connectivity coefficients are given at the lower right-hand corner for (A–D).

Article Snippet: Apurinic or apyrmidinic sites (AP) that were generated following DNA damage were measured using a colorimetric assay kit for DNA damage quantification (Oxford Biomedical Science, Oxford, MI), following manufacturer's protocol.

Techniques: Protein-Protein interactions