Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage

Abstract Cytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5–14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54–1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns

Toward quantitative super-resolution methods for cryo-CLEM

Cryogenic ultrastructural imaging techniques such as cryo-electron tomography have produced a revolution in how the structure of biological systems is investigated by enabling the determination of structures of protein complexes immersed in a complex biological matrix within vitrified cell and model organisms. However, so far, the portfolio of successes has been mostly limited to highly abundant complexes or to structures that are relatively unambiguous and easy to identify through electron microscopy. In order to realize the full potential of this revolution, researchers would have to be able to pinpoint lower abundance species and obtain functional annotations on the state of objects of interest which would then be correlated to ultrastructural information to build a complete picture of the structure-function relationships underpinning biological processes. Fluorescence imaging at cryogenic conditions has the potential to be able to meet these demands. However, wide-field images acquired at low numeric aperture (NA) using air immersion objective have a low resolving power and cannot provide accurate enough three-dimensional (3D) localization to enable the assignment of functional annotations to individual objects of interest or target sample debulking to ensure the preservation of the structures of interest. It is therefore necessary to develop super-resolved cryo-fluorescence workflows capable of fulfilling this role and enabling new biological discoveries. In this chapter, we present the current state of development of two super-resolution cryogenic fluorescence techniques, superSIL-STORM and astigmatism-based 3D STORM, show their application to a variety of biological systems and discuss their advantages and limitations. We further discuss the future applicability to cryo-CLEM workflows though examples of practical application to the study of membrane protein complexes both in mammalian cells and in Escherichia coli

Regional Practice Variation and Outcomes in the Standard Versus Accelerated Initiation of Renal Replacement Therapy in Acute Kidney Injury (STARRT-AKI) Trial: A Post Hoc Secondary Analysis.

ObjectivesAmong patients with severe acute kidney injury (AKI) admitted to the ICU in high-income countries, regional practice variations for fluid balance (FB) management, timing, and choice of renal replacement therapy (RRT) modality may be significant.DesignSecondary post hoc analysis of the STandard vs. Accelerated initiation of Renal Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial (ClinicalTrials.gov number NCT02568722).SettingOne hundred-fifty-three ICUs in 13 countries.PatientsAltogether 2693 critically ill patients with AKI, of whom 994 were North American, 1143 European, and 556 from Australia and New Zealand (ANZ).InterventionsNone.Measurements and main resultsTotal mean FB to a maximum of 14 days was +7199 mL in North America, +5641 mL in Europe, and +2211 mL in ANZ (p p p p p p p p = 0.007).ConclusionsAmong STARRT-AKI trial centers, significant regional practice variation exists regarding FB, timing of initiation of RRT, and initial use of continuous RRT. After adjustment, such practice variation was associated with lower ICU and hospital stay and 90-day mortality among ANZ patients compared with other regions

A Bayesian reanalysis of the Standard versus Accelerated Initiation of Renal-Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial

Background Timing of initiation of kidney-replacement therapy (KRT) in critically ill patients remains controversial. The Standard versus Accelerated Initiation of Renal-Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial compared two strategies of KRT initiation (accelerated versus standard) in critically ill patients with acute kidney injury and found neutral results for 90-day all-cause mortality. Probabilistic exploration of the trial endpoints may enable greater understanding of the trial findings. We aimed to perform a reanalysis using a Bayesian framework. Methods We performed a secondary analysis of all 2927 patients randomized in multi-national STARRT-AKI trial, performed at 168 centers in 15 countries. The primary endpoint, 90-day all-cause mortality, was evaluated using hierarchical Bayesian logistic regression. A spectrum of priors includes optimistic, neutral, and pessimistic priors, along with priors informed from earlier clinical trials. Secondary endpoints (KRT-free days and hospital-free days) were assessed using zero–one inflated beta regression. Results The posterior probability of benefit comparing an accelerated versus a standard KRT initiation strategy for the primary endpoint suggested no important difference, regardless of the prior used (absolute difference of 0.13% [95% credible interval [CrI] − 3.30%; 3.40%], − 0.39% [95% CrI − 3.46%; 3.00%], and 0.64% [95% CrI − 2.53%; 3.88%] for neutral, optimistic, and pessimistic priors, respectively). There was a very low probability that the effect size was equal or larger than a consensus-defined minimal clinically important difference. Patients allocated to the accelerated strategy had a lower number of KRT-free days (median absolute difference of − 3.55 days [95% CrI − 6.38; − 0.48]), with a probability that the accelerated strategy was associated with more KRT-free days of 0.008. Hospital-free days were similar between strategies, with the accelerated strategy having a median absolute difference of 0.48 more hospital-free days (95% CrI − 1.87; 2.72) compared with the standard strategy and the probability that the accelerated strategy had more hospital-free days was 0.66. Conclusions In a Bayesian reanalysis of the STARRT-AKI trial, we found very low probability that an accelerated strategy has clinically important benefits compared with the standard strategy. Patients receiving the accelerated strategy probably have fewer days alive and KRT-free. These findings do not support the adoption of an accelerated strategy of KRT initiation