Additional file 1: of Methylxanthines induce structural and functional alterations of the cardiac system in zebrafish embryos

Heart rate of BMP transgenic zebrafish embryos [tg(Bmp:EGFP)] at 48, 72, 96 and 120 hpf after microinjection with one dose of each drug. The doses selected were: aminophylline 2.5 ng, caffeine: 0.75 ng, diprophylline 5 ng, etophylline 3 ng, theophylline 2 ng, IBMX 0.5 ng, pentoxifylline 1 ng, theophylline 1 ng. For each drug, ten normal embryos were randomly selected for counting the heart beat. One way ANOVA with Dunnett’s test was used to test the significance. Asterisk indicates that the p-value differ significantly from control. (JPEG 1268 kb

Importance and Difficulties in the Use of Chiroptical Methods to Assign the Absolute Configuration of Natural Products: The Case of Phytotoxic Pyrones and Furanones Produced by <i>Diplodia corticola</i>

α-Pyrones and furanones are metabolites produced by <i>Diplodia corticola</i>, a pathogen of cork oak. Previously, the absolute configuration (AC) of diplopyrone was defined by chiroptical methods and Mosher’s method. Using X-ray and chiroptical methods, the AC of sapinofuranone C was assigned, while that of the (4<i>S,</i>5<i>S</i>)-enantiomer of sapinofuranone B was established by enantioselective total synthesis. Diplofuranone A and diplobifuranylones A–C ACs are still unassigned. Here electronic and vibrational circular dichroism (ECD and VCD) and optical rotatory dispersion (ORD) spectra are reported and compared with density functional theory computations. The AC of the (4<i>S</i>,5<i>S</i>)-enantiomer of sapinofuranone B and sapinofuranone C is checked for completeness. The AC of diplobifuranylones A–C is assigned as (2<i>S</i>,2′<i>S</i>,5′<i>S</i>,6′<i>S</i>), (2<i>S</i>,2′<i>R</i>,5′<i>S</i>,6′<i>R</i>), and (2<i>S</i>,2′<i>S</i>,5′<i>R</i>,6′<i>R</i>), respectively, with the Mosher’s method applied to define the absolute configuration of the carbinol stereogenic carbon. The AC assignment of sapinofuranones is problematic: while diplofuranone A is (4<i>S</i>,9<i>R</i>), sapinofuranones B and C are (4<i>S</i>,5<i>S</i>) according to ORD and VCD, but not to ECD. To eliminate these ambiguities, ECD and VCD spectra of a di-<i>p</i>-bromobenzoate derivative of sapinofuranone C are measured and calculated. For phytotoxicity studies, it is relevant that all six compounds share the <i>S</i> configuration for the stereogenic carbon atom of the lactone moiety

Additional file 1 of Social isolation consequences: lessons from COVID-19 pandemic in a context of dynamic lock-down in Chile

Supplementary Material 1

Endogenous products of APP metabolism negatively affect HIPK2 DNA-binding activity.

<p>(<b>a</b>) ChIP experiments were performed with anti-HIPK2 antibody on HEK-293 and HEK-APP cells that were also treated with β-secretase inhibitor at 1 µmol/L for 48 h and on HEK-293 treated with conditioned medium from HEK-APP cells for 48 h in the absence or presence of β-secretase inhibitor; PCR analyses were performed on the immunoprecipitated DNA samples using specific primers for the human HIF-1α promoter as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010171#pone-0010171-g001" target="_blank">Figure 1</a>. (<b>b</b>) ChIP experiments were performed with anti-HIPK2 antibody on HEK-293 and HEK-APP cells that were also treated with β-secretase inhibitor at 1 µmol/L for 4 8h and on HEK-293 treated with conditioned medium from HEK-APP cells for 48 h in the absence or presence of β-secretase inhibitor; PCR analyses were performed on the immunoprecipitated DNA samples using specific primers for the MT2A promoter. (<b>c</b>) HEK-293 and HEK-APP cells were transfected with MT2A-luc and HIF-1α-luc reporter construct and luciferase activity was measured 36 h after transfection. Results normalized to β-galactosidase activity are presented as fold of induction of luciferase activity ±S.D. At least three independent experiments performed in duplicate. * <i>p</i><0.01 (Student <i>t</i>-test). (<b>d</b>) MT2A and HIF-1α mRNA expression was determined in HEK-APP compared to HEK-293 cells by reverse-transcriptase (RT)-PCR. GAPDH was used as loading control.</p

Levels of Aβ 1-40 and Aβ 1-42 after treatment with β secretase inhibitor.

<p>Levels of Aβ 1-40 and Aβ 1-42 peptides were measured with a commercial ELISA kit in the cellular extracts and conditioned media of HEK-APP cells untreated or treated with β secretase inhibitor at 1 µmol/L for 48 hours. Results are representative of at least three independent experiments ± S.E.M.</p><p>* p<0.001 βSI treatment vs corresponding control.</p><p># p<0.01 βSI treatment vs corresponding control.</p

Zinc supplementation to HEK-APP restores p53 pro-apoptotic transcriptional activity.

<p>(<b>a</b>) HEK-293 and HEK-APP cells were transfected with p53AIP1-luc reporter construct and 24 h after transfection treated with doxorubicin (3.4 µM) and zinc (100 µM) for 24 h before luciferase activity was assayed. Results normalized to β-galactosidase activity are shown as relative luciferase activity ±S.D. At least three independent experiments performed in duplicate. * <i>p</i><0.05 vs HEK-293 or HEK-APP; ** <i>p</i><0.01 vs HEK-APP (Bonferroni Multiple Comparison test). (<b>b</b>) Bax mRNA expression was determined in HEK-APP compared to HEK-293 cells by reverse-transcriptase (RT)-PCR after treatment with doxorubicin (3.4 µM) and zinc (100 µM) for 24 h. GAPDH was used as loading control. (<b>c</b>) Total cell extracts of HEK-APP cells treated with doxorubicin (3.4 µM) and zinc (100 µM) for 24 h were analysed for Bax and p53 expression. Protein loading control was shown as Ponceau staining.</p

Effects of peroxynitrite compound SIN-1 on p53 conformation.

<p>Lymphocytes derive from a healthy subject were exposed to 500 µM SIN-1 in the presence or absence of uric acid at the same concentration. In particular SIN-1 was added 30 min after uric acid addition and incubated for the next 4 hours. Cells were then processed for (<b>A</b>) RNS generation study by FACS analysis measuring DCF fluorescence; (<b>B</b>) Pab 240 positive p53 isoform (unfolded p53) measured by ELISA assay, and (<b>C</b>) the degree of p53 nitration on tyrosine residues investigated by immunoprecipitation experiment with the two conformational specific antibodies (PAb 1620 and pab 240) followed by immunoblottin, with anti-rabbit-anti-3NT or anti-goat anti-p53 (R19).</p

Expression of SOD1 and SOD2 protein levels and activity in ADmut, EOSAD and control lymphocytes.

<p>Protein extracts derived from immortalized lymphocytes of ADmut, EOSAD and healthy individuals were prepared as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029789#s4" target="_blank">method</a> section. <b>A</b>) Western blot analysis carried out with monoclonal anti-SOD1 and anti-SOD2 antibodies on protein extracts derived from 2 controls, 2 ADmut, and 2 EOSAD. Tubulin expression was used to normalize the samples. <b>B</b>) and <b>C</b>) SOD1 and SOD2 levels of 9 controls, 9 ADmut and 9 EOSAD were measured using Scion Image program: quantitative analysis was expressed as intensity (optical density) of SOD1 or SOD2 bands over tubulin levels. <b>D</b>) enzymatic activity of superoxide dismutase (SOD) measured in controls (n = 9), EOSAD (n = 9) and ADmut (n = 9) lymphocytes using specific enzymatic assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029789#s4" target="_blank">method</a> section).</p

Demographic and clinical characteristic of the subjects enrolled in the study.

<p>Abbreviations: EOSAD, early onset sporadic AD; ADmut, familial AD; CTR, control; M, male; F, female; LOI, length of illness; MMSE, Mini Mental State Examination; N, number of individuals.</p><p>Values are expressed as mean ± SD.</p

The enzymatic activity of glutathione peroxidase (GPX) and glutathione reductase (GRD).

<p>A) glutathione peroxidase (GPX) was measured in controls (n = 9), EOSAD (n = 9) and ADmut (n = 9) lymphocytes using specific assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029789#s4" target="_blank">method</a> section). B) glutathione reductase (GRD) was measured in controls (n = 9), EOSAD (n = 9) and ADmut (n = 9) lymphocytes using specific assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029789#s4" target="_blank">method</a> section).</p