The Virtuous Technology Cycle Concept and its Application to Next Generation Sequencing

External access to scientific technology plays an increasingly important role in pharmaceuticals. One advantage of accessing technology externally is to avoid the costs associated with purchase and the time required for the development of new methods; in addition, access to external scientific expertise can be beneficial. However, few conceptual frameworks exist for achieving an optimal mix of internal and external technology access. Here we describe the Virtuous Technology Cycle (VTC) concept and exemplify its application to Next-Generation Sequencing (NGS), which is seen as one of the most exciting and transformative technologies in molecular biology. Based on selected examples, we show that the VTC concept can greatly enhance the number of technologies accessed and thus significantly increase flexibility and efficiency in drug discovery. We also discuss the challenges of externally accessing NGS technologies

Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters

International audienceOlder age is one of the strongest risk factors for severe COVID-19. In this study, we determined whether age-associated cellular senescence contributes to the severity of experimental COVID-19. Aged golden hamsters accumulate senescent cells in the lungs, and the senolytic drug ABT-263, a BCL-2 inhibitor, depletes these cells at baseline and during SARS-CoV-2 infection. Relative to young hamsters, aged hamsters had a greater viral load during the acute phase of infection and displayed higher levels of sequelae during the post-acute phase. Early treatment with ABT-263 lowered pulmonary viral load in aged (but not young) animals, an effect associated with lower expression of ACE2, the receptor for SARS-CoV-2. ABT-263 treatment also led to lower pulmonary and systemic levels of senescence-associated secretory phenotype factors and to amelioration of early and late lung disease. These data demonstrate the causative role of age-associated pre-existing senescent cells on COVID-19 severity and have clear clinical relevance

Chronic DOX treatment (3 mg/kg/week) alters levels of 25 microRNAs from week 2 onwards.

<p>Variation of cardiac microRNA levels versus vehicle are reported for animals treated with DOX 3 mg/kg/week for 2 and 4 weeks. Values were calculated via the relative quantification (ΔΔCt) method by using the mammalian U6 snRNA as a normalizer. MicroRNAs showing same trend at 2 and 4 weeks are italicized. # indicates microRNAs selected for further analysis. Significant P values (<0.05) are in bold. FC  =  fold change.</p

Study design and representative micrograph showing DOX-related vacuolation in the myocardium.

<p>(A) Six adult male rats were injected with the indicated doses of vehicle, doxorubicin (DOX), dexrazoxane (DZR), etoposide (EPS) or a combination of DOX and DZR for 2, 4 or 6 weeks. Cardiac tissue was excised and deep frozen for gene expression and microRNA profiling experiments. A representative micrograph of a toluidine blue stained myocardial section of a control (B) and of a DOX treated animal (C). Black arrows indicate sarcoplasmic micro- and macro- vacuolation of cardiomyocytes.</p

DOX 3 mg/kg/week altered levels of genomic cardiomyopathy indicators (Ankrd/Carp, Nppb, Myh7 and Myh6).

<p>Expression fold change relative to vehicle were represented for DOX 3 mg/kg/week at 2, 4 and 6 weeks time point (n = 6) for (A) Ankrd/Carp, (B) Nppb, (C) Myh7 and (D) Myh6. For each time point and each probe set, vehicle values were averaged and normalized to 1. The same correction was applied to the DOX treated values. Affymetrix probe-set number is indicated in brackets. Error bars represent standard deviation. T-test was performed for vehicle- vs. DOX-treated at each time point. *P<0.05, **P<0.01, ***P<0.005, NS = Non-Significant. (No t-test for #, as n = 2).</p

Ambra1 expression was induced by DOX treatment <i>in vivo</i> and miR-34c could control its endogenous expression levels in H9c2 cardiac myoblasts.

<p>(A) Fold change of Ambra1 probe set in rat heart tissue treated with DOX. Fold change and statistical significance were assessed <i>vs.</i> each vehicle group. n = 4 to 6 (except #, n = 2) (B) Endogenous levels of Ambra1 were measured after miR-34c over-expression (miR-34c mimic) or inhibition (miR-34c HI) in absence of presence of DOX 0.1. Fold change value were normalized <i>vs.</i> the respective negative transfection controls in the untreated condition (n = 3). *P<0.05, **P<0.01, ***P<0.005. FC  =  fold change.</p

miR-34c directly controls DOX-induced Sipa1 mRNA decrease.

<p>(A) Sipa1 mRNA raw expression values decreased in the heart of rats treated with DOX. #, n = 2. (B) DOX treatment for 24 h caused a decrease of Spa1 mRNA and an increase of miR-34c in cardiac myoblast cells (H9c2). (C) H9c2 endogenous Sipa1 mRNA was decreased by transfection of miR-34c mimic, and increased using a miR-34c hairpin inhibitor (HI). Transfection with miR-34c mimic and inhibitor respectively exacerbated and rescued Sipa1 mRNA levels in H9c2 treated with DOX 0.1 and 1 µM overnight in comparison to negative controls. (D) Alignment of mammalian miR-34 family. Capital letters indicate mismatch in the sequence. Sipa1 3′-UTR wt and MUT construct: 12 nt surrounding the predicted seed are shown. HEK 293 cells were co-transfected with pmiR-GLO-Sipa1 and the indicated miRNA mimic or a <i>C. elegans</i> negative control mimic. Averaged and normalized Renilla luciferase signal was obtained from 3 independent experiments, each run in quadruplicate. Y axis represents percentual residual luciferase activity in the indicated conditions. Mutant 3′ UTR restores luciferase activity in Sipa1/miR-34c. *P<0.05, **P<0.01, ***P<0.005.</p

Relative quantification of DOX-responder microRNAs in rat heart across all groups.

<p>Relative quantification of (A) miR-208b, (B) miR-215, (C) miR-216b, (D) miR-367 and (E) miR-34c in DOX, DOX + DZR, EPS groups, normalized versus vehicle treated animals. Expression levels were measured by single assay qPCR (n = 3, except #, n = 2). DOX: Doxorubicin, DZR: dexrazoxane, EPS: etoposide; numbers indicate the weekly dose of each compound in mg/kg/week. Empty spaces represent non-sampled animals. The vehicle treated is the first column of each time-point. The animals used in this experiment were distinct from the ones represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040395#pone-0040395-t001" target="_blank">Table 1</a>. Error bars represent SD. T-test results are indicated by asterisks for significant DOX-treated groups vs. their own vehicle-treated, unless otherwise specified by horizontal range bars; *P<0.05, **P<0.01, ***P<0.005, NS = Non-Significant).</p