Innate Structure of DNA Foci Restricts the Mixing of DNA from Different Chromosome Territories

The distribution of chromatin within the mammalian nucleus is constrained by its organization into chromosome territories (CTs). However, recent studies have suggested that promiscuous intra- and inter-chromosomal interactions play fundamental roles in regulating chromatin function and so might define the spatial integrity of CTs. In order to test the extent of DNA mixing between CTs, DNA foci of individual CTs were labeled in living cells following incorporation of Alexa-488 and Cy-3 conjugated replication precursor analogues during consecutive cell cycles. Uniquely labeled chromatin domains, resolved following random mitotic segregation, were visualized as discrete structures with defined borders. At the level of resolution analysed, evidence for mixing of chromatin from adjacent domains was only apparent within the surface volumes where neighboring CTs touched. However, while less than 1% of the nuclear volume represented domains of inter-chromosomal mixing, the dynamic plasticity of DNA foci within individual CTs allows continual transformation of CT structure so that different domains of chromatin mixing evolve over time. Notably, chromatin mixing at the boundaries of adjacent CTs had little impact on the innate structural properties of DNA foci. However, when TSA was used to alter the extent of histone acetylation changes in chromatin correlated with increased chromatin mixing. We propose that DNA foci maintain a structural integrity that restricts widespread mixing of DNA and discuss how the potential to dynamically remodel genome organization might alter during cell differentiation

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Extending the Schizosaccharomyces pombe molecular genetic toolbox.

Targeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70+ and pku80+ genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1+ to around 75-80% (a 16-fold increase). We describe how a natMX6/rpl42+ cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system

A cartoon indicating the approach used by all three integration vector systems.

<p>A cartoon indicating the approach used by all three integration vector systems.</p

Manipulating native loci with an <i>rpl42⁺/natMX6</i> cassette.

<p>A) Approaches used for targeted mutagenesis. B) The structure of the pFA6arpl42natMX6 plasmid. C) The phenotype switches arising from the progression through the indicated genotypes.</p

Inclusion of <i>pku70.Δ</i> and <i>pku80.Δ</i> in host strain radically enhances targeting at the <i>fin1</i>⁺ locus.

<p>A) Cartoons depicting the structure of the DNA fragments used to direct the integration of a <i>natMX6</i> cassette 3′ to the Fin1 coding sequences at the <i>fin1</i>⁺ locus and the integration of sequences encoding three GFP molecules, a stop codon and the <i>kan^RMX6</i> marker at the end of the <i>fin1</i>⁺ locus. B) PCR amplification reactions with the oligonucleotides indicated by arrows in panel A to monitor the structure of the genomic regions at the <i>fin1</i>⁺ locus. For the “<i>fin1 ORF</i>” transformation amplification gives an 850 bp fragment (red cross next to each panel), whereas with successful integration generates an 2050 bp fragment (red tick next to each panel). For the “<i>fin1.3GFP</i>” transformation amplification with the same primers used to screen “ <i>fin1 ORF”</i> transformants generated an 850 bp fragment in the recipient host (red cross next to each panel) and an 4650 bp fragment in the correct transformant (red tick next to each panel). C) A table showing the frequency of correct integration events in the indicated strains with the indicated concentrations of each DNA fragment as determined by PCR analysis of 48 candidate transformants in each case.</p

The pINTH series of vectors for the expression of a gene of interest from the <i>hph.171k</i> locus on chromosome III.

<p>Cartoons depicting the structure of the indicated pINTH vectors.</p

Strains used in this study.

<p>Strains used in this study.</p

The pREPN series of vectors for the expression of a gene of interest from an ectopic plasmid.

<p>Cartoons depicting the structure of the indicated pREPN vectors.</p

The pINTK series of vectors for the expression of a gene of interest from the <i>lys1</i> locus.

<p>Cartoons depicting the structure of the indicated pINTK vectors.</p