Genomic approaches to understand the pathogenesis of the acute repiratory distress syndrome

The acute respiratory distress syndrome (ARDS) is an acute lung inflammatory process that commonly develops as a consequence of severe infections, being sepsis its main cause of development. Despite the fatality of the syndrome, there is a lack of specific therapeutic options and effective prognostic methods for patients. Since many studies support the influence of genetic factors and microbiome shifts in the origin and evolution of ARDS, here we have aimed to address its pathophysiology using different genomic approaches. We have performed a genome-wide association study in European patients with sepsis, revealing a novel gene associated with ARDS susceptibility. Additionally, we have sequenced the bacterial DNA extracted from lung aspirates from a subset of the individuals with sepsis, reporting the association of the reduction of bacterial diversity with intensive care unit mortality during the first 8 h of sepsis diagnosis. Finally, the exploration of the genomic variation of a recently admixed population has pointed out genomic regions related to the ethnicity and harboring novel genes associated with response to infections and with a form of ARDS, among many other traits. All these findings have allowed us to further understand the pathogenesis of the syndrome and of main risk factors, as well as i) to propose VEGFR-1 as a potential therapeutic target, ii) to suggest the bacterial diversity as an early prognostic biomarker in critical patients, and iii) to lay the foundations for designing fine and admixture mapping studies in Canary Islanders to identify novel risk genes for complex traits such as sepsis and ARDS

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Relaxase MobM Induces a Molecular Switch at Its Cognate Origin of Transfer

The MOBV1 family of relaxases is broadly distributed in plasmids and other mobile genetic elements isolated from staphylococci, enterococci, and streptococci. The prototype of this family is protein MobM encoded by the streptococcal promiscuous plasmid pMV158. MobM cleaves the phosphodiester bond of a specific dinucleotide within the origin of transfer (oriT) to initiate conjugative transfer. Differently from other relaxases, MobM and probably other members of the family, cleaves its target single-stranded DNA through a histidine residue rather than the commonly used tyrosine. The oriT of the MOBV1 family differs from other well-known conjugative systems since it has sequences with three inverted repeats, which were predicted to generate three mutually-exclusive hairpins on supercoiled DNA. In this work, such hypothesis was evaluated through footprinting experiments on supercoiled plasmid DNA. We have found a change in hairpin extrusion mediated by protein MobM. This conformational change involves a shift from the main hairpin generated on “naked” DNA to a different hairpin in which the nick site is positioned in a single-stranded configuration. Our results indicate that the oriTpMV158 acts as a molecular switch in which, depending on the inverted repeat recognized by MobM, pMV158 mobilization could be turned “on” or “off.

Apoptotic efficacy of etomoxir in human acute myeloid leukemia cells. Cooperation with arsenic trioxide and glycolytic inhibitors, and regulation by oxidative stress and protein kinase activities

26 p.-9 fig.Fatty acid synthesis and oxidation are frequently exacerbated in leukemia cells, and may therefore represent a target for therapeutic intervention. In this work we analyzed the apoptotic and chemo-sensitizing action of the fatty acid oxidation inhibitor etomoxir in human acute myeloid leukemia cells. Etomoxir caused negligible lethality at concentrations up to 100 mM, but efficaciously cooperated to cause apoptosis with the anti-leukemic agent arsenic trioxide (ATO, Trisenox), and with lower efficacy with other anti-tumour drugs(etoposide,cisplatin), in HL60 cells. Etomoxir-ATO cooperation was also observed in NB4 human acute promyelocytic cells, but not in normal (non-tumour) mitogen-stimulated human peripheral blood lymphocytes. Biochemical determinations in HL60 cells indicated that etomoxir (25–200 mM) dose-dependently inhibited mitochondrial respiration while slightly stimulating glycolysis, and only caused marginal alterations in total ATP content and adenine nucleotide pool distribution. In addition, etomoxir caused oxidative stress (increase in intracellular reactive oxygen species accumulation, decrease in reduced glutathione content), as well as pro-apoptotic LKB-1/AMPK pathway activation, all of which may in part explain the chemo-sensitizing capacity of the drug. Etomoxir also cooperated with glycolytic inhibitors (2-deoxy-D-glucose, lonidamine) to induce apoptosis in HL60 cells, but not in NB4 cells. The combined etomoxir plus 2-deoxy-D-glucose treatment did not increase oxidative stress,caused moderate decrease in net ATP content, increased the AMP/ATP ratio with concomitant drop in energy charge, and caused defensive Akt and ERK kinase activation. Apoptosis generation by etomoxir plus 2-deoxy-D-glucose was further increased by co-incubation with ATO, which is apparently explained by the capacity of ATO to attenuate Akt and ERK activation. In summary, co-treatment with etomoxir may represent an interesting strategy to increase the apoptotic efficacy of ATO and (with some limitations) 2-deoxy-D-glucose which, although clinically important anti-tumour agents, exhibit low efficacy in monotherapy.This work was supported by grant SAF2010-20256 (to P.A.) from the Spanish Ministerio de Ciencia e Innovación, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, Dirección General de Investigación(http://www.mineco.gob.es/portal/site/mineco/idi), and by Grant S2010/BMD-2402 (to E.R.) from the Comunidad de Madrid (https://www.madrimasd.org).Peer reviewe

Table1.DOC

<p>The MOB_V1 family of relaxases is broadly distributed in plasmids and other mobile genetic elements isolated from staphylococci, enterococci, and streptococci. The prototype of this family is protein MobM encoded by the streptococcal promiscuous plasmid pMV158. MobM cleaves the phosphodiester bond of a specific dinucleotide within the origin of transfer (oriT) to initiate conjugative transfer. Differently from other relaxases, MobM and probably other members of the family, cleaves its target single-stranded DNA through a histidine residue rather than the commonly used tyrosine. The oriT of the MOB_V1 family differs from other well-known conjugative systems since it has sequences with three inverted repeats, which were predicted to generate three mutually-exclusive hairpins on supercoiled DNA. In this work, such hypothesis was evaluated through footprinting experiments on supercoiled plasmid DNA. We have found a change in hairpin extrusion mediated by protein MobM. This conformational change involves a shift from the main hairpin generated on “naked” DNA to a different hairpin in which the nick site is positioned in a single-stranded configuration. Our results indicate that the oriT_pMV158 acts as a molecular switch in which, depending on the inverted repeat recognized by MobM, pMV158 mobilization could be turned “on” or “off.”</p

Effect of etomoxir, etoposide and cisplatin on cell proliferation, cycle phase distribution and apoptosis generation in different cell models.

<p>Cells were incubated for 24 h with the indicated concentrations of etomoxir (Eto), ATO, etoposide (ETP) or cisplatin (CDDP), alone or in combination. When nothing is indicated, etomoxir was used at 100 µM. (A) Apoptosis generation, measured by flow cytometry, in NB4 cells. (B) The same, in mitogen-stimulated human PBLs. (C, D) Changes in proliferation activity, measured by cell counting (C), and apoptosis generation, measured by flow cytometry (D), in HL60 cells. Changes in proliferation are expressed in relation to untreated (Cont) cultures. (E) Flow cytometry histograms showing changes in cycle distribution in HL60 cell cultures treated with etoposide and cisplatin. The bar charts in (A–D) represent the mean ± S.D. of at least three determinations. Symbols and n.s. indicate significant and non-significant differences, respectively, between the indicated pairs of values (C), or between the combined treatment and the sum of values in the corresponding individual treatments (A, B, D). For other conditions see legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115250#pone-0115250-g001" target="_blank">Fig. 1</a>.</p

Scheme summarizing the main results in this work.

<p>Etomoxir decreases respiration while increasing glycolytic activity. In addition, it causes oxidative stress (ROS over-production, GSH depletion) which, together with AMPK activation, may explain the potentiation of ATO-provoked apoptosis. Etomoxir may also cooperate in some cell models with glycolytic inhibitors (2-DG, lonidamine) to cause apoptosis. This response is further enhanced by co-incubation with ATO, due to the capacity of this agent to attenuate the 2-DG/etomoxir-provoked Akt and ERK activation.</p

Effect of etomoxir and ATO on cell viability, cycle phase distribution and apoptosis generation in HL60 cells.

<p>Cells were incubated for 24 h with the indicated concentrations of etomoxir (Eto) and ATO, alone and in combination. When nothing is indicated, ATO was used at 2 µM. For convenience, the drug concentrations are sometimes indicated as subheadings. When indicated, the cells were co-incubated with the pan-caspase inhibitor z-VAD-fmk (50 µM). (A) Changes in cell viability, as evidenced by the MTT assay. Absorption values are indicated in relation to untreated (Cont) cultures. (B) Frequency of cells at the different phases of the growth cycle, namely G₁, S and G₂/M, and with sub-G₁ DNA content (apoptotic). Examples of flow cytometry histograms are presented in (E). (C) Frequency of apoptotic cells, as determined by flow cytometry. (D) Caspase-3 cleavage/activation, determined by immunoblot. β-actin is included as a loading control. The bar charts in (A–C) represent the mean ± S.D. of at least three determinations. Symbols mean: (*) significant differences between the indicated pair values; (^#) significant differences between the combined treatment and the sum of values in the corresponding individual treatments (e.g., co-incubation with 100 µM Eto and 2 µM ATO, in relation to the sum of 100 µM Eto alone plus 2 µM ATO alone) (n.s., non-significant). To better discern differences, in this case the sum of values in individual treatments is indicated by a horizontal white line within the bar corresponding to the combined treatment. Single symbol, <i>p</i><0.05; double symbol, <i>p</i><0.01; triple symbol, <i>p</i><0.001.</p