Photocaged Hoechst enables subnuclear visualization and cell selective staining of DNA in vivo

Selective targeting of DNA by means of fluorescent labelling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows for the visualization of nucleic acid by using DNA-targeting fluorescent fusion proteins in a cell-type and subcellular specific manner, it relies on the introduction of foreign genes. On the other hand, DNA-binding small fluorescent molecules can be used without genetic engineering but they are not spatially restricted. Here, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short-term cell tracking  in vivo  in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild-type organisms

COMAP Early Science: V. Constraints and Forecasts at z∼3z \sim 3

We present the current state of models for the $z\sim3$ carbon monoxide (CO) line-intensity signal targeted by the CO Mapping Array Project (COMAP) Pathfinder in the context of its early science results. Our fiducial model, relating dark matter halo properties to CO luminosities, informs parameter priors with empirical models of the galaxy-halo connection and previous CO(1-0) observations. The Pathfinder early science data spanning wavenumbers $k=0.051$-$0.62\,$Mpc$^{-1}$ represent the first direct 3D constraint on the clustering component of the CO(1-0) power spectrum. Our 95% upper limit on the redshift-space clustering amplitude $A_{\rm clust}\lesssim70\,\mu$K$^2$ greatly improves on the indirect upper limit of $420\,\mu$K$^2$ reported from the CO Power Spectrum Survey (COPSS) measurement at $k\sim1\,$Mpc$^{-1}$. The COMAP limit excludes a subset of models from previous literature, and constrains interpretation of the COPSS results, demonstrating the complementary nature of COMAP and interferometric CO surveys. Using line bias expectations from our priors, we also constrain the squared mean line intensity-bias product, $\langle{Tb}\rangle^2\lesssim50\,\mu$K$^2$, and the cosmic molecular gas density, $\rho_\text{H2}<2.5\times10^8\,M_\odot\,$Mpc$^{-3}$ (95% upper limits). Based on early instrument performance and our current CO signal estimates, we forecast that the five-year Pathfinder campaign will detect the CO power spectrum with overall signal-to-noise of 9-17. Between then and now, we also expect to detect the CO-galaxy cross-spectrum using overlapping galaxy survey data, enabling enhanced inferences of cosmic star-formation and galaxy-evolution history.Comment: Paper 5 of 7 in series. 17 pages + appendix and bibliography (30 pages total); 15 figures, 6 tables; accepted for publication in ApJ; v3 reflects the accepted version with minor changes and additions to tex

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis

AbstractThe formation of new blood vessel networks occurs via angiogenesis during development, tissue repair and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation and migration. Erk is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response however, involves a Vegfr-dependent mechanism that initiates concomitant with the wound inflammatory response. This work thus reveals a highly dynamic sequence in regenerative angiogenesis that was not previously appreciated. Altogether, this study demonstrates the utility of a unique biosensor strain for analysing dynamic endothelial Erk-signalling events and validates a new resource for the study of vascular signalling in real-time.</jats:p

The myth of a cancer-specific temperament: An analysis of affective temperament in cancer patients

ObjectiveWe investigate the prevalence of five affective temperaments (depressive, cyclothymic, hyperthymic, irritable, and anxious) in a large sample of cancer patients and associations of temperament with cancer site as well as the impact of temperament on overall survival of cancer patients.MethodsData for this prospective cohort study was collected in the outpatient clinic of a large cancer center. We used the Temperament Evaluation in Memphis, Pisa and San Diego – Münster Version (TEMPS-M) and recorded patient data. The sample consisted of 2531 patients with seven different cancer/disease-sites. Kruskal-Wallis tests and pairwise Wilcoxon rank sum test were applied to compare temperament scales across disease groups. For analyzing survival time, we used a Cox regression model and log-rank tests.ResultsThe five affective temperaments were similarly distributed across all disease groups. We found higher levels of depressive, cyclothymic, and anxious temperament in women and higher levels of hyperthymic and irritable temperament in men. Temperament was mostly not predictive of survival, with only two significant results in the regression models. Here, cyclothymic temperament was predictive of mortality in the full sample and hyperthymic temperament was predictive of the pancreatic cancer subsample.ConclusionsOur study provides evidence to debunk the myth of a cancer-specific temperament. Neither did we find a temperament profile that was different from studies with general population samples, nor were there any disease-specific profiles differentiating various types of cancer