Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance

The phosphorylation of nucleic acid with 5′-OH termini catalyzed by polynucleotide kinase (PNK) involves several significant cellular events. Here a paper-based fluorescence assay with λ exonuclease assistance was reported for facile detection of PNK activity through monitoring the change of fluorescence intensity on paper surface. Cy5-labeled ssDNA was first immobilized on the surface of aldehyde group modified paper, and BHQ-labeled ssDNA was then employed to quench the fluorescence of the immobilized Cy5-labeled ssDNA with the help of an adaptor ssDNA. When PNK and λ exonuclease cleavage reaction were introduced, the fluorescence quenching effect on the paper surface was blocked because of the digestion of phosphorylated dsDNA by the coupled enzymes. By using this paper-based assay, PNK activity both in pure reaction buffer and in practical biosample have been successfully measured. Highly sensitive detection of PNK activity down to 0.0001 U mL^–1 and lysate of about 50 cells is achieved. The inhibition of PNK activity has also been investigated and a satisfactory result is obtained

Tunable Catalytic Performance of Single Pt Atom on Doped Graphene in Direct Dehydrogenation of Propane by Rational Doping: A Density Functional Theory Study

The catalytic reaction pathways and performance of supported single Pt atom on nitrogen- and boron-doped graphene in the direct dehydrogenation of propane (PDH) are investigated by using first principles calculations. The different dopants on graphene have distinct effects on the electronic structure of the supported Pt atom. The nitrogen on the support withdraws electrons from Pt, but boron donates electrons to Pt. Consequently, the d-band center of Pt atom is modified by either nitrogen or boron doping. The nitrogen doping shifts the d-band center of Pt atom closer to the Fermi level compared with the boron doping and the pristine ones. On the other hand, the d-band center has a significant influence on the C<i>–</i>H bond dissociation energy and reaction barrier. Therefore, better reactivity of Pt is found on the support with more nitrogen dopants as the d-band center is closer to the Fermi level. Also the calculated dissociation energy and the first C<i>–</i>H bond activation barrier obey the BEP rule. The different ratios between nitrogen and boron on the codoped graphene can continuously adjust the electronic structure of supported Pt and deliver the dissociation energy and reaction barrier in between the pure nitrogen- and boron-doped cases. Among various investigated supports, the graphene doped by pyridine nitrogen is predicted to be the most effective for enhancing Pt catalytic performance. The current work shows the promising catalytic performance of supported single Pt atom in PDH. More importantly, the tunable properties of the supported metal catalysts on the carbon materials are achieved by the rational doping, which provides a practical strategy for the catalyst optimization

Computational Design of a CeO₂‑Supported Pd-Based Bimetallic Nanorod for CO Oxidation

Engineering a bimetallic system with complementary chemical properties can be an effective way of tuning catalytic activity. In this work, CO oxidation on CeO₂(111)-supported Pd-based bimetallic nanorods was investigated using density functional theory calculations corrected by on-site Coulomb interactions. We studied a series of CeO₂(111)-supported Pd-based bimetallic nanorods (Pd–X, where X = Ag, Au, Cu, Pt, Rh, Ru) and found that Pd–Ag/CeO₂ and Pd–Cu/CeO₂ are the two systems where the binding sites of CO and O₂ are distinct; that is, in these two systems, CO and O₂ do not compete for the same binding sites. An analysis of the CO oxidation mechanisms suggests that the Pd–Ag/CeO₂ system is more effective for catalyzing CO oxidation as compared to Pd–Cu/CeO₂ because both CeO₂ lattice oxygen atoms and adsorbed oxygen molecules at Ag sites can oxidize CO with low energy barriers. Both the Pd–Ag and Pd–CeO₂ interfaces in Pd–Ag/CeO₂ were found to play important roles in CO oxidation. The Pd–Ag interface, which combines the different chemical nature of the two metals, not only separates the binding sites of CO and O₂ but also opens up active reaction pathways for CO oxidation. The strong metal–support interaction at the Pd–CeO₂ interface facilitates CO oxidation by the Mars–van Krevelen mechanism. Our study provides theoretical guidance for designing highly active metal/oxide catalysts for CO oxidation

Regional early and progressive loss of brain pericytes but not vascular smooth muscle cells in adult mice with disrupted platelet-derived growth factor receptor-β signaling

<div><p>Pericytes regulate key neurovascular functions of the brain. Studies in pericyte-deficient transgenic mice with aberrant signaling between endothelial-derived platelet-derived growth factor BB (PDGF-BB) and platelet-derived growth factor receptor β (PDGFRβ) in pericytes have contributed to better understanding of the role of pericytes in the brain. Here, we studied <i>Pdgfrβ</i>^<i>F7/F7</i> mice, which carry seven point mutations that disrupt PDGFRβ signaling causing loss of pericytes and vascular smooth muscle cells (VSMCs) in the developing brain. We asked whether these mice have a stable or progressive vascular phenotype after birth, and whether both pericyte and VSMCs populations are affected in the adult brain. We found an early and progressive region-dependent loss of brain pericytes, microvascular reductions and blood-brain barrier (BBB) breakdown, which were more pronounced in the cortex, hippocampus and striatum than in the thalamus, whereas VSMCs population remained unaffected at the time when pericyte loss was already established. For example, compared to age-matched controls, <i>Pdgfrβ</i>^<i>F7/F7</i> mice between 4–6 and 36–48 weeks of age developed a region-dependent loss in pericyte coverage (22–46, 24–44 and 4–31%) and cell numbers (36–49, 34–64 and 11–36%), reduction in capillary length (20–39, 13–46 and 1–30%), and an increase in extravascular fibrinogen-derived deposits (3.4–5.2, 2.8–4.1 and 0–3.6-fold) demonstrating BBB breakdown in the cortex, hippocampus and thalamus, respectively. Capillary reductions and BBB breakdown correlated with loss of pericyte coverage. Our data suggest that <i>Pdgfrβ</i>^<i>F7/F7</i> mice develop an aggressive and rapid vascular phenotype without appreciable early involvement of VSMCs, therefore providing a valuable model to study regional effects of pericyte loss on brain vascular and neuronal functions. This model could be a useful tool for future studies directed at understanding the role of pericytes in the pathogenesis of neurological disorders associated with pericyte loss such as vascular dementia, Alzheimer’s disease, amyotrophic lateral sclerosis, stroke and human immunodeficiency virus-associated neurocognitive disorder.</p></div

Normal thickness of vascular smooth muscle cells (VSMCs)-covered arteriolar wall, VSMCs cell numbers and functional response to adenosine in adult <i>Pdgfrβ</i>^<i>F7/F7</i> (<i>F7/F7</i>) mice.

<p>(A) Representative high magnification confocal images (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176225#sec002" target="_blank">Methods</a> for details) of α-smooth muscle actin (SMA)-positive VSMCs (green, left panels) and lectin-positive endothelial profiles (red, middle panels), and merged (left panels) in penetrating arterioles of the somatosensory cortex S1 layer 1 cortex (S1Cx) of 12-week old F7/F7 and age-matched littermate control mice (+/+). Interrupted lines indicate the edge of fluorescent signal. Da, SMA-positive arteriolar diameter; Dv, lectin-positive endothelial diameter. Scale bar = 50 μm. (B-C) Quantification of the arteriolar wall thickness (B) and VSMCs numbers (C) in 4-6-, 12-16- and 36-48-week old F7/F7 mice compared to the respective age-matched littermate controls (+/+). In B and C, individual points represent 10 vessels per animal from 5 animals per group. Mean ± S.E.M. from 50 arterioles per group. ns, not significant by one-way ANOVA. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176225#sec002" target="_blank">Methods</a> for details. (D) Laser Doppler flowmetry measurements of cerebral blood flow (CBF) response to endothelium-independent VSMCs relaxant adenosine (400 μM) in 4-6-, 12-16-, and 36-48-week old F7/F7 mice compared to age-matched littermate controls (+/+). Mean ± S.E.M.; n = 3 mice per group; ns, not significant by one-way ANOVA.</p

Loss of pericyte coverage correlates with capillary reductions in the cortex and hippocampus of adult <i>Pdgfrβ</i>^<i>F7/F7</i> (<i>F7/F7</i>) mice.

<p>(<b>A-C</b>) Correlations between age-dependent reduction in capillary length and loss of pericyte coverage in the somatosensory cortex S1 region (S1Cx) (<b>A</b>), the CA1 region of the hippocampus (Hp) (<b>B</b>), and posterior thalamus (Thal) (<b>C</b>) in <i>F7/F7</i> mice. Single data points were from 4-6- (grey), 12–16 (red), and 36–48 (blue) week-old <i>F7/F7</i> mice (n = 15 individual points; each point represents the mean value per mouse calculated as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176225#pone.0176225.g001" target="_blank">Fig 1</a> legend and Methods). r = Pearson’s coefficient. <i>P</i>, significance.</p

Early and progressive loss of pericyte coverage and capillary reductions in the somatosensory cortex of adult Pdgfrβ^<i>F7/F7</i> (<i>F7/F7</i>) mice.

<p>(A) Representative confocal microscopy analysis of coronal sections showing CD13-positive pericyte coverage (magenta, left panels), lectin-positive endothelial vascular profiles (blue, middle panels) and merged (right panels) in the S1 region of the somatosensory cortex (S1Cx) layers II-IV of 6-, 12- and 38-week old F7/F7 and 38-week old control (+/+ 38w) mice. Bar = 40 μm. (B-C) Quantification of pericyte coverage (B) and total capillary length (C) in 4–6, 12–16, and 36-48-week old F7/F7 mice compared to age-matched littermate controls (+/+). Pericyte coverage was determined as a percentage (%) of CD13-positive pericyte surface area covering lectin-positive endothelial surface (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176225#sec002" target="_blank">Methods</a> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176225#pone.0176225.s001" target="_blank">S1 Fig</a></b>). Total capillary length was determined in mm of lectin-positive endothelial profiles of vessels ≤ 6 μm in diameter, and expressed per mm3 of cortical tissue. In each animal, 4–6 randomly selected fields were analyzed in 4 non-adjacent sections (~100 μm apart), and averaged per mouse to obtain individual values that were taken for statistical comparisons. Mean ± S.E.M., n = 5 animals per group. In B and C, one-way ANOVA and Bonferroni’s post hoc tests were used to compare data in F7/F7 mutants versus age-matched littermate controls and/or between different age groups of F7/F7 mutants only. P < 0.05 indicates statistically significant differences between groups.</p

Accumulation of fibrinogen and fibrin perivascular deposits in the brain of adult <i>Pdgfrβ</i>^<i>F7/F7</i> (<i>F7/F7</i>) mice.

<p><b>(A-C)</b> Representative confocal images of lectin-positive endothelial profiles and extravascular fibrinogen and fibrin deposits in the somatosensory cortex S1 region (S1Cx) layer IV (<b>A</b>), the CA1 region stratum pyrmidale of the hippocampus (Hp) (<b>B</b>) and posterior thalamus (<b>C</b>) of 6-, 16- and 46-week old <i>F7/F7</i> mice and 16-week old control (<i>+/+</i>) mice. Bar = 20 μm. (<b>D-F</b>) Quantification of fibrinogen and fibrin-positive extravascular deposits in the S1Cx (<b>D</b>), Hp (<b>E</b>) and Thal (<b>F</b>) of 4–6, 12–16, and 36-48-week old <i>F7/F7</i> mice and age-matched littermate controls (<i>+/+</i>). Mean ± SEM, n = 5 mice per group. In each animal, 4–6 randomly selected fields in the cortex, hippocampus and thalamus were analyzed in 4 non-adjacent sections (~100 μm apart) and averaged to calculate individual values per mouse. In <b>D-F</b>, one-way ANOVA and Bonferroni’s post hoc tests were used to compare data in mutants versus age-matched littermate controls, and/or between different age groups of <i>F7/F7</i> mutants. <i>P</i> < 0.05 indicates statistically significant differences between groups. (<b>G-I</b>) Correlations between age-dependent fibrinogen and fibrin perivascular accumulation and loss of pericyte coverage in the S1Cx (<b>G</b>), Hp (<b>H</b>), and Thal (<b>I</b>) regions in <i>F7/F7</i> mice. Single data points were from 4-6- (grey), 12-16- (red), and 36-48- (blue) week old <i>F7/F7</i> mice (n = 15 individual points per mouse; r = Pearson’s coefficient; <i>P</i>, significance.</p

Seasonal Disparities in Airborne Bacteria and Associated Antibiotic Resistance Genes in PM_2.5 between Urban and Rural Sites

The atmosphere represents an unappreciated compartment for the environmental dissemination of antibiotic resistance genes (ARGs), particularly via airborne fine particles (PM_2.5), with strong implications for the inhalational exposure of the general population. We examined the seasonal variations in airborne bacteria and several ARGs in PM_2.5 across an industrial–urban–rural transect in a megacity of China over an annual cycle. Seasonality was most apparent at the rural site with a remarkable wintertime reduction in the total level of bacteria and an enrichment of certain ARGs in winter but dilution in spring. This contrasted with the relative consistency across seasons at urban and industrial sites. The statistical correlation between ARGs and the mobile genetic element (MGE), <i>int</i>I1, weakened from rural to urban and industrial sites, which hints at the diluting role of <i>int</i>I1 in horizontal gene transfers across the land use gradient. Differing mechanisms may regulate site-specific population exposure to transferable ARGs, and the identification of additional MGEs is warranted. Compared to drinking water and the accidental ingestion of agricultural soil, airborne PM_2.5 contributes to a similar extent to the human daily intake of certain ARGs and <i>int</i>I1. Collectively, this study highlights the importance of PM_2.5 in the dissemination of, and pathways of human exposure to, common environmental ARGs

Additional file 1 of Identification and expression analysis of ATP-binding cassette (ABC) transporters revealed its role in regulating stress response in pear (Pyrus bretchneideri)

Additional file 1: Table S1 Detailed information of the ABC transporter gene family in pea