The relative proportion of different types of rearrangements resulting from interstitial and telomeric DSBs.

<p>The percentage of NHEJ, small deletions, GCRs and large deletions is shown for clones EDS-7F2 (interstitial pEJ5-GFP and interstitial pDsRed-ISceI) and EDS-6J8 (telomeric pEJ5-GFP and interstitial pDsRed-ISceI) following expression of I-<i>Sce</i>I for 14 days. Untreated control cultures are compared with cultures treated with KU55933, shRNA-mediated knockdown of ATM, or combined KU55933 and shRNA-mediated knockdown of ATM. The category of large deletions also includes chromosome healing and inversions; however, these events are very rare. The percentage of GCRs as measured by DsRed⁺ cells (those with small deletions) is too low to be visible in this figure.</p

The effect of ATM deficiency on small deletions at interstitial and telomeric DSBs.

<p>The frequency of small deletions in genomic DNA from clones EDS-7F2 and EDS-6J8 that were infected with pQCXIH-ISceI and selected with hygromycin for 14 days was determined by first performing PCR using oligonucleotide primers spanning one of the I-<i>Sce</i>I sites in the pEJ5-GFP plasmid (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003386#pgen-1003386-g001" target="_blank">Figure 1</a>). The fraction of cells in the population that contain small deletions at the I-<i>Sce</i>I site was then determined from the fraction of the PCR product that was not digested with I-<i>Sce</i>I (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003386#s4" target="_blank">Materials and Methods</a>). All samples were analyzed in triplicate. Error bars represent standard deviation of three separate experiments.</p

The effect of ATM deficiency on large deletions at interstitial and telomeric DSBs.

<p>The frequency of GFP-negative cells (large deletions) was determined in clones GFP-7F1 (with interstitial pGFP-ISceI) and GFP-6D1 (with telomeric pGFP-ISceI) following the infection with either the pQCXIH control retrovirus vector or the pQCXIH-ISceI retrovirus vector and selection in hygromycin for 14 days. All samples were analyzed in triplicate. Error bars represent standard deviation of three separate experiments.</p

The frequency of NHEJ and GCRs at interstitial and telomeric I-<i>Sce</i>I-induced DSBs.

<p>(A) FACs analysis was used to determine the frequency of GFP⁺ (NHEJ) and DsRed⁺ (GCRs) cells in clone EDS-7F2 (with interstitial pEJ5-GFP and interstitial pDsRed-ISceI) and EDS-6J8 (with telomeric pEJ5-GFP and interstitial pDsRed-ISceI). (B) The frequency of DsRed⁺ cells was determined following infection with the pQCXIH-ISceI retrovirus and selection with hygromycin for 14 days. (C) The ratio of GFP⁺ (NHEJ) and DsRed⁺ (GCRs) cells was determined in clones that contain a single copy of the pEJ5-GFP plasmid integrated at an interstitial (EDS-7F2, EDS-7F4, EDS-7F5, EDS-7F6) or telomeric (EDS-6J8, EDS-6J29 EDS-6J31, EDS-6J35, EDS-6J46, EDS-6J49) site, and a single copy of the pDsRed-ISceI plasmid integrated at an interstitial site. Clones EDS-6J7 and EDS-6J10 contain a single copy of the pEJ5-GFP plasmid integrated at a telomeric site and 3 tandem copies of the pDsRed-ISceI plasmid integrated at an interstitial site. All samples were analyzed in triplicate. Error bars represent standard deviation of three separate experiments.</p

DNA sequence analysis of recombination junctions involved in the formation of GCRs in DsRed⁺ subclones.

<p>Genomic DNA was isolated from individual DsRed⁺ subclones following expression of I-<i>Sce</i>I in clones EDS-6J7 and EDS-6J8 (with telomeric pEJ5-GFP and interstitial pDsRed-ISceI). The sites containing the recombination junctions were amplified by PCR using one primer in the pEJ5-GFP plasmid and one primer in the pDsRed-ISceI plasmid (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003386#pgen-1003386-g001" target="_blank">Figure 1</a>). DNA sequence analysis was then performed on the PCR fragments to determine the structure of the recombination junction involved in the formation of the GCR. The location of the 4-nucleotide overhang generated by I-<i>Sce</i>I endonuclease (bold), deletions at the site of the DSB (dashes), and insertions (nucleotides between dashed lines) are shown.</p

The structure of the plasmids used to monitor NHEJ, GCRs, and large deletions.

<p>(A) NHEJ was monitored using the pEJ5-GFP plasmid integrated at interstitial (not shown) or telomeric sites (shown). pEJ5-GFP contains a GFP gene that is initially inactive due to the presence of a puromycin-resistance (puro) gene located between the GFP gene and its promoter. NHEJ occurring between the distal ends of the I-<i>Sce</i>I-induced DSBs at either end of the puro gene results in the activation of the GFP gene. A PCR product generated with oligonucleotide primers spanning one of the I-<i>Sce</i>I sites in the pEJ5-GFP plasmid (red arrows) was digested with I-<i>Sce</i>I endonuclease to determine the frequency of small deletions at a single I-<i>Sce</i>I-induced DSB. Cell clones containing the pGFP-ISceI plasmid integrated at interstitial sites (not shown) or telomeric sites (shown) were used for analysis of large deletions that inactivate the GFP gene. (B) GCRs were monitored using cell clones that contain the pEJ5-GFP plasmid integrated at an interstitial (not shown) or telomeric (shown) site and the pDsRed-ISceI plasmid integrated at an interstitial site at a different location in the genome. The DsRed gene in the pDsRed-ISceI plasmid is initially inactive due to the lack of a promoter, but becomes activated following NHEJ between the I-<i>Sce</i>I-induced DSBs in the pEJ5-GFP and pDsRed-ISceI plasmids. The location of oligonucleotide primers used for PCR to analyze the junctions between the pEJ5-GFP and pDsRed-ISceI plasmids are shown (red arrows). The location of the ampicillin gene and plasmid origin of replication (Amp/ori), chicken β-actin promoter (promoter), puro gene (Puro), GFP coding sequence (GFP), and telomere are shown. Also show is the genomic DNA (solid line), directions of transcription (black arrows), and I-<i>Sce</i>I recognition sites (I-<i>Sce</i>I).</p

A Systematic Review and Meta-Analysis of MicroRNA as Predictive Biomarkers of Acute Kidney Injury

Acute kidney injury (AKI) affects 10–15% of hospitalised patients and arises after severe infections, major surgeries, or exposure to nephrotoxic drugs. AKI diagnosis based on creatinine level changes lacks specificity and may be delayed. MicroRNAs are short non-coding RNA secreted by all cells. This review of studies measuring miRNAs in AKI aimed to verify miRNAs as diagnostic markers. The study included data from patients diagnosed with AKI due to sepsis, ischaemia, nephrotoxins, radiocontrast, shock, trauma, and cardiopulmonary bypass. Out of 71 studies, the majority focused on AKI in sepsis patients, followed by cardiac surgery patients, ICU patients, and individuals receiving nephrotoxic agents or experiencing ischaemia. Studies that used untargeted assays found 856 differentially regulated miRNAs, although none of these were confirmed by more than one study. Moreover, 68 studies measured miRNAs by qRT-PCR, and 2 studies reported downregulation of miR-495-3p and miR-370-3p in AKI patients with sepsis after the AKI diagnosis. In three studies, upregulation of miR-21 was reported at the time of the AKI diagnosis with a significant pooled effect of 0.56. MiR-21 was also measured 19–24 h after cardiac surgery in three studies. However, the pooled effect was not significant. Despite the considerable research into miRNA in AKI, there is a knowledge gap in their applicability as diagnostic markers of AKI in humans.</p

Investigating Catalase Activity Through Hydrogen Peroxide Decomposition by Bacteria Biofilms in Real Time Using Scanning Electrochemical Microscopy

Catalase activity through hydrogen peroxide decomposition in a 1 mM bulk solution above <i>Vibrio fischeri</i> (γ-<i>Protebacteria-Vibrionaceae</i>) bacterial biofilms of either symbiotic or free-living strains was studied in real time by scanning electrochemical microscopy (SECM). The catalase activity, in units of micromoles hydrogen peroxide decomposed per minute over a period of 348 s, was found to vary with incubation time of each biofilm in correlation with the corresponding growth curve of bacteria in liquid culture. Average catalase activity for the same incubation times ranging from 1 to 12 h was found to be 0.28 ± 0.07 μmol H₂O₂/min for the symbiotic biofilms and 0.31 ± 0.07 μmol H₂O₂/min for the free-living biofilms, suggesting similar catalase activity. Calculations based on Comsol Multiphysics simulations in fitting experimental biofilm data indicated that approximately (3 ± 1) × 10⁶ molecules of hydrogen peroxide were decomposed by a single bacterium per second, signifying the presence of a highly active catalase. A 2-fold enhancement in catalase activity was found for both free-living and symbiotic biofilms in response to external hydrogen peroxide concentrations as low as 1 nM in the growth media, implying a similar mechanism in responding to oxidative stress

MOESM1 of Airway autoimmune responses in severe eosinophilic asthma following low-dose Mepolizumab therapy

Additional file 1. Figures and immunological methods