Cell wall and organelle modifications during nitrogen starvation in Nannochloropsis oceanica F&M-M24

AbstractNannochloropsis oceanica F&M-M24 is able to increase its lipid content during nitrogen starvation to more than 50% of the total biomass. We investigated the ultrastructural changes and the variation in the content of main cell biomolecules that accompany the final phase of lipid accumulation. Nitrogen starvation induced a first phase of thylakoid disruption followed by chloroplast macroautophagy and formation of lipid droplets. During this phase, the total amount of proteins decreased by one-third, while carbohydrates decreased by 12–13%, suggesting that lipid droplets were formed by remodelling of chloroplast membranes and synthesis of fatty acids from carbohydrates and amino acids. The change in mitochondrial ultrastructure suggests also that these organelles were involved in the process. The cell wall increased its thickness and changed its structure during starvation, indicating that a disruption process could be partially affected by the increase in wall thickness for biomolecules recovery from starved cells. The wall thickness in strain F&M-M24 was much lower than that observed in other strains of N. oceanica, showing a possible advantage of this strain for the purpose of biomolecules extraction. The modifications following starvation were interpreted as a response to reduction of availability of a key nutrient (nitrogen). The result is a prolonged survival in quiescence until an improvement of the environmental conditions (nutrient availability) allows the rebuilding of the photosynthetic apparatus and the full recovery of cell functions

Evolution of Intra-specific Regulatory Networks in a Multipartite Bacterial Genome

<div><p>Reconstruction of the regulatory network is an important step in understanding how organisms control the expression of gene products and therefore phenotypes. Recent studies have pointed out the importance of regulatory network plasticity in bacterial adaptation and evolution. The evolution of such networks within and outside the species boundary is however still obscure. <i>Sinorhizobium meliloti</i> is an ideal species for such study, having three large replicons, many genomes available and a significant knowledge of its transcription factors (TF). Each replicon has a specific functional and evolutionary mark; which might also emerge from the analysis of their regulatory signatures. Here we have studied the plasticity of the regulatory network within and outside the <i>S. meliloti</i> species, looking for the presence of 41 TFs binding motifs in 51 strains and 5 related rhizobial species. We have detected a preference of several TFs for one of the three replicons, and the function of regulated genes was found to be in accordance with the overall replicon functional signature: house-keeping functions for the chromosome, metabolism for the chromid, symbiosis for the megaplasmid. This therefore suggests a replicon-specific wiring of the regulatory network in the <i>S. meliloti</i> species. At the same time a significant part of the predicted regulatory network is shared between the chromosome and the chromid, thus adding an additional layer by which the chromid integrates itself in the core genome. Furthermore, the regulatory network distance was found to be correlated with both promoter regions and accessory genome evolution inside the species, indicating that both pangenome compartments are involved in the regulatory network evolution. We also observed that genes which are not included in the species regulatory network are more likely to belong to the accessory genome, indicating that regulatory interactions should also be considered to predict gene conservation in bacterial pangenomes.</p></div

Regulon downstream genes.

<p>Regulatory network general statistics over the strains used in this study.</p><p>^<i>a</i> Position according to the Rm1021 reference strain;</p><p>^<i>b</i> Mean Absolute Deviation;</p><p>NA: not defined.</p><p>Regulon downstream genes.</p

Correlations between pangenome diversity and regulatory network distances.

<p>R and S indicate the Pearson’s and Spearman’s correlation coefficients between the regulatory network and each pangenome partition distances (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004478#sec008" target="_blank">Materials and Methods</a> for the definition of the distances metrics used here). Outliers have been defined using a Z-score threshold of 3.5 on the mean absolute deviation of the distances. a) correlations within the <i>S. meliloti</i> species for the accessory genome; b) correlations within the <i>S. meliloti</i> species for coding and upstream regions; and c) correlation between the outgroups.</p

Regulon conservation.

<p>Regulatory network conservation in <i>S. meliloti</i> and near rhizobial species. For each regulator the number of conserved downstream genes over the average regulon size is reported.</p><p>^<i>a</i><i>S. meliloti</i> strain Rm1021 is also considered.</p><p>NA: not defined.</p><p>Regulon conservation.</p

Variability in regulon size.

<p>Color intensity indicates the number of downstream regulated genes in each strain; gray squares indicate the TF absence in the genome of that particular strain. Blue squares indicate that there are more than 64 genes predicted to be under the control of the TF. TFs are colored according to the replicon they belong to: red for chromosome, green for the pSymA megaplasmid and blue for the pSymB chromid.</p

TFs preferentially associated with a replicon.

<p>a) K-means clustering of the normalized proportion of genes regulated in each of the three main replicons of <i>S. meliloti</i>, visualized in a two-dimensional PCA. The dark blue and cyan clusters contain TFs with no clear replicon preference; b) Variability in the number of regulatory links in the same replicon and between replicons. All differences are significant (t-test p-value < 0.05).</p

Regulatory network dynamics.

<p>a) Graphical representation of the six states in which each regulatory link (a gene found with a TFBS in at least one genome) can be found in the <i>S. meliloti</i> species and between the outgroup species; b) states probabilities and states transitions probabilities inside the <i>S. meliloti</i> species: nodes and edges sizes are proportional to the probability in the model. For each state, the sum of transition probabilities is one; transition probabilities below 0.1 are not shown; c) states probabilities and states transitions probabilities between the outgroup species.</p

Tocilizumab for patients with COVID-19 pneumonia. The single-arm TOCIVID-19 prospective trial

BackgroundTocilizumab blocks pro-inflammatory activity of interleukin-6 (IL-6), involved in pathogenesis of pneumonia the most frequent cause of death in COVID-19 patients.MethodsA multicenter, single-arm, hypothesis-driven trial was planned, according to a phase 2 design, to study the effect of tocilizumab on lethality rates at 14 and 30 days (co-primary endpoints, a priori expected rates being 20 and 35%, respectively). A further prospective cohort of patients, consecutively enrolled after the first cohort was accomplished, was used as a secondary validation dataset. The two cohorts were evaluated jointly in an exploratory multivariable logistic regression model to assess prognostic variables on survival.ResultsIn the primary intention-to-treat (ITT) phase 2 population, 180/301 (59.8%) subjects received tocilizumab, and 67 deaths were observed overall. Lethality rates were equal to 18.4% (97.5% CI: 13.6-24.0, P=0.52) and 22.4% (97.5% CI: 17.2-28.3, P&lt;0.001) at 14 and 30 days, respectively. Lethality rates were lower in the validation dataset, that included 920 patients. No signal of specific drug toxicity was reported. In the exploratory multivariable logistic regression analysis, older age and lower PaO2/FiO2 ratio negatively affected survival, while the concurrent use of steroids was associated with greater survival. A statistically significant interaction was found between tocilizumab and respiratory support, suggesting that tocilizumab might be more effective in patients not requiring mechanical respiratory support at baseline.ConclusionsTocilizumab reduced lethality rate at 30 days compared with null hypothesis, without significant toxicity. Possibly, this effect could be limited to patients not requiring mechanical respiratory support at baseline.Registration EudraCT (2020-001110-38); clinicaltrials.gov (NCT04317092)