A Comprehensive Assessment of All-Oleate Polysorbate 80: Free Fatty Acid Particle Formation, Interfacial Protection and Oxidative Degradation.

PURPOSE: Enzymatic polysorbate (PS) degradation and resulting free fatty acid (FFA) particles are detrimental to biopharmaceutical drug product (DP) stability. Different types and grades of polysorbate have varying propensity to form FFA particles. This work evaluates the homogenous all-oleate (AO) PS80 alongside heterogeneous PS20 and PS80 grades in terms its propensity to form FFA particles and other important attributes like interfacial protection and oxidation susceptibility. METHODS: FFA particle formation rates were compared by degrading PS using non-immobilized hydrolases and fast degrading DP formulations. Interfacial protection of monoclonal antibodies (mAbs) was assessed by agitation studies in saline using non-degraded and degraded PS. Several antioxidants were assessed for their ability to mitigate AO PS80 oxidation and subsequent mAb oxidation by a 40°C placebo stability study and a 2, 2'-Azobis (2-amidinopropane) dihydrochloride stress model, respectively. RESULTS: Visible and subvisible particles were significantly delayed in AO PS80 formulations compared with heterogeneous PS20 and PS80 formulations. Non-degraded AO PS80 was less protective of mAbs against the air-water interface compared with heterogeneous PS20. Interfacial protection by AO PS80 improved upon degradation owing to high surface activity of FFAs. Diethylenetriaminepentaacetic acid (DTPA) completely mitigated AO PS80 oxidation unlike L-methionine and N-Acetyl-DL-Tryptophan. However, DTPA did not mitigate radical mediated mAb oxidation. CONCLUSION: AO PS80 is a promising alternative to reduce FFA particle formation compared with other PS types and grades. However, limitations observed here---such as lower protection against interfacial stresses and higher propensity for oxidation---need to be considered in assessing the risk/benefit ratio in using AO PS80.

Evaluating a Modified High Purity Polysorbate 20 Designed to Reduce the Risk of Free Fatty Acid Particle Formation.

PURPOSE: To evaluate a modified high purity polysorbate 20 (RO HP PS20)-with lower levels of stearate, palmitate and myristate esters than the non-modified HP PS20-as a surfactant in biopharmaceutical drug products (DP). RO HP PS20 was designed to provide functional equivalence as a surfactant while delaying the onset of free fatty acid (FFA) particle formation upon hydrolytic degradation relative to HP PS20. METHODS: Analytical characterization of RO HP PS20 raw material included fatty acid ester (FAE) distribution, higher order ester (HOE) fraction, FFA levels and trace metals. Functional assessments included 1) vial and intravenous bag agitation; 2) oxidation via a placebo and methionine surrogate study; and 3) hydrolytic PS20 degradation studies to evaluate FFA particle formation with and without metal nucleation. RESULTS: Interfacial protection and oxidation propensity were comparable between the two polysorbates. Upon hydrolytic degradation, FFA particle onset was delayed in RO HP PS20. The delay was more pronounced when HOEs of PS20 were preferentially degraded. Furthermore, the hydrolytic degradants of RO HP PS20 formed fewer particles in the presence of spiked aluminum. CONCLUSION: This work highlights the criticality of having tighter control on long chain FAE levels of PS20 to reduce the occurrence of FFA particle formation upon hydrolytic degradation and lower the variability in its onset. By simultaneously meeting compendial PS20 specifications while narrowing the allowable range for each FAE and shifting its composition towards the shorter carbon chain species, RO HP PS20 provides a promising alternative to HP PS20 for biopharmaceutical DPs.

A Chemical Chaperone Decouples TDP-43 Disordered Domain Phase Separation from Fibrillation.

Ribonucleoprotein (RNP) condensations through liquid-liquid phase separation play vital roles in the dynamic formation-dissolution of stress granules (SGs). These condensations are, however, usually assumed to be linked to pathologic fibrillation. Here, we show that physiologic condensation and pathologic fibrillation of RNPs are independent processes that can be unlinked with the chemical chaperone trimethylamine N-oxide (TMAO). Using the low-complexity disordered domain of the archetypical SG-protein TDP-43 as a model system, we show that TMAO enhances RNP liquid condensation yet inhibits protein fibrillation. Our results demonstrate effective decoupling of physiologic condensation from pathologic aggregation and suggest that selective targeting of protein fibrillation (without altering condensation) can be employed as a therapeutic strategy for RNP aggregation-associated degenerative disorders.

Fluorescence Lifetime Imaging Microscopy of Biomolecular Condensates.

Biomolecular condensates of ribonucleoproteins (RNPs) such as the transactivation response element (TAR) DNA-binding protein 43 (TDP-43) arise from liquid-liquid phase separation (LLPS) and play vital roles in various biological processes including the formation-dissolution of stress granules (SGs). These condensates are thought to be directly linked to neurodegenerative diseases, providing a depot of aggregation-prone proteins and serving as a cauldron of protein aggregation and fibrillation. Despite recent research efforts, biochemical processes and rearrangements within biomolecular condensates that trigger subsequent protein misfolding and aggregation remain to be elucidated. Fluorescence lifetime imaging microscopy (FLIM) provides a minimally intrusive high-sensitivity and high-resolution imaging method to monitor in-droplet spatiotemporal changes that initiate and lead to protein aggregation. In this chapter, we describe a FLIM application for characterizing chemical chaperone-assisted decoupling of TDP-43 liquid-liquid phase separation and aggregation/fibrillation, highlighting potential therapeutic strategies to combat pathological RNP-associated aggregates without compromising cellular stress responses.

Single actomyosin motor interactions in skeletal muscle.

We present a study of intramuscular motion during contraction of skeletal muscle myofibrils. Myofibrillar actin was labeled with fluorescent dye so that the ratio of fluorescently labeled to unlabeled protein was 1:10(5). Such sparse labeling assured that there was on average only one actin-marker present in the focus at a given time. From the intensity signal in the two orthogonal detection channels, significant fluctuations, similar to fluorescent burst in diffusion-based single-molecule detection schemes, were identified via a threshold algorithm and analyzed with respect to their intensity and polarization. When only rigor complexes were formed, the fluctuations of polarized intensity were characterized by unimodal Gaussian photon distributions. During contraction, in contrast, bimodal Gaussian photon distributions were observed above the rigor background threshold. This suggests that the bimodal Gaussian photon distributions represent pre- and post-power stroke conformations. Clusters of polarized photons indicated an anisotropy decay of single actomyosin motors of ~9s during muscle contraction. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

NANOG prion-like assembly mediates DNA bridging to facilitate chromatin reorganization and activation of pluripotency.

Human NANOG expression resets stem cells to ground-state pluripotency. Here we identify the unique features of human NANOG that relate to its dose-sensitive function as a master transcription factor. NANOG is largely disordered, with a C-terminal prion-like domain that phase-transitions to gel-like condensates. Full-length NANOG readily forms higher-order oligomers at low nanomolar concentrations, orders of magnitude lower than typical amyloids. Using single-molecule Förster resonance energy transfer and fluorescence cross-correlation techniques, we show that NANOG oligomerization is essential for bridging DNA elements in vitro. Using chromatin immunoprecipitation sequencing and Hi-C 3.0 in cells, we validate that NANOG prion-like domain assembly is essential for specific DNA recognition and distant chromatin interactions. Our results provide a physical basis for the indispensable role of NANOG in shaping the pluripotent genome. NANOG's unique ability to form prion-like assemblies could provide a cooperative and concerted DNA bridging mechanism that is essential for chromatin reorganization and dose-sensitive activation of ground-state pluripotency.

Degenerin channel activation causes caspase-mediated protein degradation and mitochondrial dysfunction in adult C. elegans muscle.

BACKGROUND: Declines in skeletal muscle structure and function are found in various clinical populations, but the intramuscular proteolytic pathways that govern declines in these individuals remain relatively poorly understood. The nematode Caenorhabditis elegans has been developed into a model for identifying and understanding these pathways. Recently, it was reported that UNC-105/degenerin channel activation produced muscle protein degradation via an unknown mechanism. METHODS: Generation of transgenic and double mutant C. elegans, RNAi, and drug treatments were utilized to assess molecular events governing protein degradation. Western blots were used to measure protein content. Cationic dyes and adenosine triphosphate (ATP) production assays were utilized to measure mitochondrial function. RESULTS: unc-105 gain-of-function mutants display aberrant muscle protein degradation and a movement defect; both are reduced in intragenic revertants and in let-2 mutants that gate the hyperactive UNC-105 channel. Degradation is not suppressed by interventions suppressing proteasome-mediated, autophagy-mediated, or calpain-mediated degradation nor by suppressors of degenerin-induced neurodegeneration. Protein degradation, but not the movement defect, is decreased by treatment with caspase inhibitors or RNAi against ced-3 or ced-4. Adult unc-105 muscles display a time-dependent fragmentation of the mitochondrial reticulum that is associated with impaired mitochondrial membrane potential and that correlates with decreased rates of maximal ATP production. Reduced levels of CED-4, which is sufficient to activate CED-3 in vitro, are observed in unc-105 mitochondrial isolations. CONCLUSIONS: Constitutive cationic influx into muscle appears to cause caspase degradation of cytosolic proteins as the result of mitochondrial dysfunction, which may be relevant to ageing and sarcopenia.

Intensive care unit scoring systems outperform emergency department scoring systems for mortality prediction in critically ill patients: a prospective cohort study.

BACKGROUND: Multiple scoring systems have been developed for both the intensive care unit (ICU) and the emergency department (ED) to risk stratify patients and predict mortality. However, it remains unclear whether the additional data needed to compute ICU scores improves mortality prediction for critically ill patients compared to the simpler ED scores. METHODS: We studied a prospective observational cohort of 227 critically ill patients admitted to the ICU directly from the ED at an academic, tertiary care medical center. We compared Acute Physiology and Chronic Health Evaluation (APACHE) II, APACHE III, Simplified Acute Physiology Score (SAPS) II, Modified Early Warning Score (MEWS), Rapid Emergency Medicine Score (REMS), Prince of Wales Emergency Department Score (PEDS), and a pre-hospital critical illness prediction score developed by Seymour et al. (JAMA 2010, 304(7):747-754). The primary endpoint was 60-day mortality. We compared the receiver operating characteristic (ROC) curves of the different scores and their calibration using the Hosmer-Lemeshow goodness-of-fit test and visual assessment. RESULTS: The ICU scores outperformed the ED scores with higher area under the curve (AUC) values (p = 0.01). There were no differences in discrimination among the ED-based scoring systems (AUC 0.698 to 0.742; p = 0.45) or among the ICU-based scoring systems (AUC 0.779 to 0.799; p = 0.60). With the exception of the Seymour score, the ED-based scoring systems did not discriminate as well as the best-performing ICU-based scoring system, APACHE III (p = 0.005 to 0.01 for comparison of ED scores to APACHE III). The Seymour score had a superior AUC to other ED scores and, despite a lower AUC than all the ICU scores, was not significantly different than APACHE III (p = 0.09). When data from the first 24 h in the ICU was used to calculate the ED scores, the AUC for the ED scores improved numerically, but this improvement was not statistically significant. All scores had acceptable calibration. CONCLUSIONS: In contrast to prior studies of patients based in the emergency department, ICU scores outperformed ED scores in critically ill patients admitted from the emergency department. This difference in performance seemed to be primarily due to the complexity of the scores rather than the time window from which the data was derived.

Confocal fluctuation spectroscopy and imaging.

Currently, work with subnanomolar concentrations is routine while femtomolar and even single-molecule studies are possible with some efforts getting high on single-molecule biophysics and biochemistry. Methodological breakthroughs, such as reducing the background light contribution in single-molecule studies, which has plagued many studies of molecular fluorescence in dilute solution, and particularly in live cells, have recently described by us. We first demonstrated how optimized time-gating of the fluorescence signal, together with time-correlated, single-photon counting, can be used to substantially boost the experimental signal-to-noise ratio about 140-fold, making it possible to measure analyte concentrations that are as low as 15 pM. By detection of femtomolar bulk concentrations, confocal microsopy has the potential to address the observation of one and the same molecule in dilute solution without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than currently available. We present relevant physics. The equations are derived using Einstein's approach showing how it fits with Fick's law and the autocorrelation function. An improved technology is being developed at ISS for femtomolar microscopy. The general concepts and provided experimental examples should help to compare our approach to those used in conventional confocal microscopy.

Reducing background contributions in fluorescence fluctuation time-traces for single-molecule measurements in solution.

We first report on the development of new microscope means that reduce background contributions in fluorescence fluctuation methods: i) excitation shutter, ii) electronic switches, and iii) early and late time-gating. The elements allow for measuring molecules at low analyte concentrations. We first found conditions of early and late time-gating with time-correlated single-photon counting that made the fluorescence signal as bright as possible compared with the fluctuations in the background count rate in a diffraction-limited optical set-up. We measured about a 140-fold increase in the amplitude of autocorrelated fluorescence fluctuations at the lowest analyte concentration of about 15 pM, which gave a signal-to-background advantage of more than two-orders of magnitude. The results of this original article pave the way for single-molecule detection in solution and in live cells without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than are currently available.