Live-cell super-resolution microscopy reveals a primary role for diffusion in polyglutamine-driven aggresome assembly.

The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed in vitro Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that the increasing surface area of the expanding aggresome increases the rate of accretion caused by diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies.

Secondary nucleation of monomers on fibril surface dominates α-synuclein aggregation and provides autocatalytic amyloid amplification.

Parkinson's disease (PD) is characterized by proteinaceous aggregates named Lewy Bodies and Lewy Neurites containing α-synuclein fibrils. The underlying aggregation mechanism of this protein is dominated by a secondary process at mildly acidic pH, as in endosomes and other organelles. This effect manifests as a strong acceleration of the aggregation in the presence of seeds and a weak dependence of the aggregation rate on monomer concentration. The molecular mechanism underlying this process could be nucleation of monomers on fibril surfaces or fibril fragmentation. Here, we aim to distinguish between these mechanisms. The nature of the secondary processes was investigated using differential sedimentation analysis, trap and seed experiments, quartz crystal microbalance experiments and super-resolution microscopy. The results identify secondary nucleation of monomers on the fibril surface as the dominant secondary process leading to rapid generation of new aggregates, while no significant contribution from fragmentation was found. The newly generated oligomeric species quickly elongate to further serve as templates for secondary nucleation and this may have important implications in the spreading of PD.

Human manual control precision depends on vestibular sensory precision and gravitational magnitude.

Precise motion control is critical to human survival on Earth and in space. Motion sensation is inherently imprecise, and the functional implications of this imprecision are not well understood. We studied a "vestibular" manual control task in which subjects attempted to keep themselves upright with a rotational hand controller (i.e., joystick) to null out pseudorandom, roll-tilt motion disturbances of their chair in the dark. Our first objective was to study the relationship between intersubject differences in manual control performance and sensory precision, determined by measuring vestibular perceptual thresholds. Our second objective was to examine the influence of altered gravity on manual control performance. Subjects performed the manual control task while supine during short-radius centrifugation, with roll tilts occurring relative to centripetal accelerations of 0.5, 1.0, and 1.33 GC (1 GC = 9.81 m/s2). Roll-tilt vestibular precision was quantified with roll-tilt vestibular direction-recognition perceptual thresholds, the minimum movement that one can reliably distinguish as leftward vs. rightward. A significant intersubject correlation was found between manual control performance (defined as the standard deviation of chair tilt) and thresholds, consistent with sensory imprecision negatively affecting functional precision. Furthermore, compared with 1.0 GC manual control was more precise in 1.33 GC (-18.3%, P = 0.005) and less precise in 0.5 GC (+39.6%, P < 0.001). The decrement in manual control performance observed in 0.5 GC and in subjects with high thresholds suggests potential risk factors for piloting and locomotion, both on Earth and during human exploration missions to the moon (0.16 G) and Mars (0.38 G). NEW & NOTEWORTHY The functional implications of imprecise motion sensation are not well understood. We found a significant correlation between subjects' vestibular perceptual thresholds and performance in a manual control task (using a joystick to keep their chair upright), consistent with sensory imprecision negatively affecting functional precision. Furthermore, using an altered-gravity centrifuge configuration, we found that manual control precision was improved in "hypergravity" and degraded in "hypogravity." These results have potential relevance for postural control, aviation, and spaceflight.

Human perception of whole body roll-tilt orientation in a hypogravity analog: underestimation and adaptation.

Overestimation of roll tilt in hypergravity ("G-excess" illusion) has been demonstrated, but corresponding sustained hypogravic conditions are impossible to create in ground laboratories. In this article we describe the first systematic experimental evidence that in a hypogravity analog, humans underestimate roll tilt. We studied perception of self-roll tilt in nine subjects, who were supine while spun on a centrifuge to create a hypogravity analog. By varying the centrifuge rotation rate, we modulated the centripetal acceleration (GC) at the subject's head location (0.5 or 1 GC) along the body axis. We measured orientation perception using a subjective visual vertical task in which subjects aligned an illuminated bar with their perceived centripetal acceleration direction during tilts (±11.5-28.5°). As hypothesized, based on the reduced utricular otolith shearing, subjects initially underestimated roll tilts in the 0.5 GC condition compared with the 1 GC condition (mean perceptual gain change = -0.27, P = 0.01). When visual feedback was given after each trial in 0.5 GC, subjects' perceptual gain increased in approximately exponential fashion over time (time constant = 16 tilts or 13 min), and after 45 min, the perceptual gain was not significantly different from the 1 GC baseline (mean gain difference between 1 GC initial and 0.5 GC final = 0.16, P = 0.3). Thus humans modified their interpretation of sensory cues to more correctly report orientation during this hypogravity analog. Quantifying the acute orientation perceptual learning in such an altered gravity environment may have implications for human space exploration on the moon or Mars. NEW & NOTEWORTHY Humans systematically overestimate roll tilt in hypergravity. However, human perception of orientation in hypogravity has not been quantified across a range of tilt angles. Using a centrifuge to create a hypogravity centripetal acceleration environment, we found initial underestimation of roll tilt. Providing static visual feedback, perceptual learning reduced underestimation during the hypogravity analog. These altered gravity orientation perceptual errors and adaptation may have implications for astronauts.

Fluorescence Self-Quenching from Reporter Dyes Informs on the Structural Properties of Amyloid Clusters Formed in Vitro and in Cells.

The characterization of the aggregation kinetics of protein amyloids and the structural properties of the ensuing aggregates are vital in the study of the pathogenesis of many neurodegenerative diseases and the discovery of therapeutic targets. In this article, we show that the fluorescence lifetime of synthetic dyes covalently attached to amyloid proteins informs on the structural properties of amyloid clusters formed both in vitro and in cells. We demonstrate that the mechanism behind such a "lifetime sensor" of protein aggregation is based on fluorescence self-quenching and that it offers a good dynamic range to report on various stages of aggregation without significantly perturbing the process under investigation. We show that the sensor informs on the structural density of amyloid clusters in a high-throughput and quantitative manner and in these aspects the sensor outperforms super-resolution imaging techniques. We demonstrate the power and speed of the method, offering capabilities, for example, in therapeutic screenings that monitor biological self-assembly. We investigate the mechanism and advantages of the lifetime sensor in studies of the K18 protein fragment of the Alzheimer's disease related protein tau and its amyloid aggregates formed in vitro. Finally, we demonstrate the sensor in the study of aggregates of polyglutamine protein, a model used in studies related to Huntington's disease, by performing correlative fluorescence lifetime imaging microscopy and structured-illumination microscopy experiments in cells.

Temperature Treatment of Highly Porous Zirconium-Containing Metal-Organic Frameworks Extends Drug Delivery Release.

Utilizing metal-organic frameworks (MOFs) as a biological carrier can lower the amount of the active pharmaceutical ingredient (API) required in cancer treatments to provide a more efficacious therapy. In this work, we have developed a temperature treatment process for delaying the release of a model drug compound from the pores of NU-1000 and NU-901, while taking care to utilize these MOFs' large pore volume and size to achieve exceptional model drug loading percentages over 35 wt %. Video-rate super-resolution microscopy reveals movement of MOF particles when located outside of the cell boundary, and their subsequent immobilization when taken up by the cell. Through the use of optical sectioning structured illumination microscopy (SIM), we have captured high-resolution 3D images showing MOF uptake by HeLa cells over a 24 h period. We found that addition of a model drug compound into the MOF and the subsequent temperature treatment process does not affect the rate of MOF uptake by the cell. Endocytosis analysis revealed that MOFs are internalized by active transport and that inhibiting the caveolae-mediated pathway significantly reduced cellular uptake of MOFs. Encapsulation of an anticancer therapeutic, alpha-cyano-4-hydroxycinnamic acid (α-CHC), and subsequent temperature treatment produced loadings of up to 81 wt % and demonstrated efficacy at killing cells beyond the burst release effect.

Imaging Aß(1-42) fibril elongation reveals strongly polarised growth and growth incompetent states.

The major hallmark of Alzheimer's disease is the deposition of plaques of amyloid fibrils formed from amyloid-ß (Aß) peptides. Kinetic studies have contributed significantly towards a mechanistic understanding of amyloid fibril self-assembly, however dynamic features of the aggregation process cannot be captured using ensemble methods. Here we present an assay for imaging Aß42 aggregation dynamics at the single fibril level, allowing for the quantitative extraction of concentration and temperature dependent kinetic parameters. From direct observation of elongation using TIRF and super-resolution optical microscopy, we find that Aß42 fibril growth is strongly polarized, with fast and slow growing ends arising from different elongation rates, but also from a growth incompetent state, which dominates the process at the slow growing end. Our findings reveal the surprising complexity of the Aß42 fibril elongation reaction at the microscopic level.

Nanoscale click-reactive scaffolds from peptide self-assembly.

BACKGROUND: Due to their natural tendency to self-assemble, proteins and peptides are important components for organic nanotechnology. One particular class of peptides of recent interest is those that form amyloid fibrils, as this self-assembly results in extremely strong, stable quasi-one-dimensional structures which can be used to organise a wide range of cargo species including proteins and oligonucleotides. However, assembly of peptides already conjugated to proteins is limited to cargo species that do not interfere sterically with the assembly process or misfold under the harsh conditions often used for assembly. Therefore, a general method is needed to conjugate proteins and other molecules to amyloid fibrils after the fibrils have self-assembled. RESULTS: Here we have designed an amyloidogenic peptide based on the TTR105-115 fragment of transthyretin to form fibrils that display an alkyne functionality, important for bioorthogonal chemical reactions, on their surface. The fibrils were formed and reacted both with an azide-containing amino acid and with an azide-functionalised dye by the Huisgen cycloaddition, one of the class of "click" reactions. Mass spectrometry and total internal reflection fluorescence optical microscopy were used to show that peptides incorporated into the fibrils reacted with the azide while maintaining the structure of the fibril. These click-functionalised amyloid fibrils have a variety of potential uses in materials and as scaffolds for bionanotechnology. DISCUSSION: Although previous studies have produced peptides that can both form amyloid fibrils and undergo "click"-type reactions, this is the first example of amyloid fibrils that can undergo such a reaction after they have been formed. Our approach has the advantage that self-assembly takes place before click functionalization rather than pre-functionalised building blocks self-assembling. Therefore, the molecules used to functionalise the fibril do not themselves have to be exposed to harsh, amyloid-forming conditions. This means that a wider range of proteins can be used as ligands in this process. For instance, the fibrils can be functionalised with a green fluorescent protein that retains its fluorescence after it is attached to the fibrils, whereas this protein loses its fluorescence if it is exposed to the conditions used for aggregation.

Human perceptual overestimation of whole body roll tilt in hypergravity.

Hypergravity provides a unique environment to study human perception of orientation. We utilized a long-radius centrifuge to study perception of both static and dynamic whole body roll tilt in hypergravity, across a range of angles, frequencies, and net gravito-inertial levels (referred to as G levels). While studies of static tilt perception in hypergravity have been published, this is the first to measure dynamic tilt perception (i.e., with time-varying canal stimulation) in hypergravity using a continuous matching task. In complete darkness, subjects reported their orientation perception using a haptic task, whereby they attempted to align a hand-held bar with their perceived horizontal. Static roll tilt was overestimated in hypergravity, with more overestimation at larger angles and higher G levels, across the conditions tested (overestimated by ∼35% per additional G level, P < 0.001). As our primary contribution, we show that dynamic roll tilt was also consistently overestimated in hypergravity (P < 0.001) at all angles and frequencies tested, again with more overestimation at higher G levels. The overestimation was similar to that for static tilts at low angular velocities but decreased at higher angular velocities (P = 0.006), consistent with semicircular canal sensory integration. To match our findings, we propose a modification to a previous Observer-type canal-otolith interaction model. Specifically, our data were better modeled by including the hypothesis that the central nervous system treats otolith stimulation in the utricular plane differently than stimulation out of the utricular plane. This modified model was able to simulate quantitatively both the static and the dynamic roll tilt overestimation in hypergravity measured experimentally.

Human manual control performance in hyper-gravity.

Hyper-gravity provides a unique environment to study how misperceptions impact control of orientation relative to gravity. Previous studies have found that static and dynamic roll tilts are perceptually overestimated in hyper-gravity. The current investigation quantifies how this influences control of orientation. We utilized a long-radius centrifuge to study manual control performance in hyper-gravity. In the dark, subjects were tasked with nulling out a pseudo-random roll disturbance on the cab of the centrifuge using a rotational hand controller to command their roll rate in order to remain perceptually upright. The task was performed in 1, 1.5, and 2 G's of net gravito-inertial acceleration. Initial performance, in terms of root-mean-square deviation from upright, degraded in hyper-gravity relative to 1 G performance levels. In 1.5 G, initial performance degraded by 26 % and in 2 G, by 45 %. With practice, however, performance in hyper-gravity improved to near the 1 G performance level over several minutes. Finally, pre-exposure to one hyper-gravity level reduced initial performance decrements in a different, novel, hyper-gravity level. Perceptual overestimation of roll tilts in hyper-gravity leads to manual control performance errors, which are reduced both with practice and with pre-exposure to alternate hyper-gravity stimuli.

Squat exercise biomechanics during short-radius centrifugation.

INTRODUCTION: Centrifuge-induced artificial gravity (AG) with exercise is a promising comprehensive countermeasure against the physiological de-conditioning that results from exposure to weightlessness. However, body movements onboard a rotating centrifuge are affected by both the gravity gradient and Coriolis accelerations. The effect of centrifugation on squat exercise biomechanics was investigated, and differences between AG and upright squat biomechanics were quantified. METHODS: There were 28 subjects (16 male) who participated in two separate experiments. Knee position, foot reaction forces, and motion sickness were recorded during the squats in a 1-G field while standing upright and while supine on a horizontally rotating 2 m radius centrifuge at 0, 23, or 30 rpm. RESULTS: No participants terminated the experiment due to motion sickness symptoms. Total mediolateral knee deflection increased by 1.0 to 2.0 cm during centrifugation, and did not result in any injuries. There was no evidence of an increased mediolateral knee travel "after-effect" during postrotation supine squats. Peak foot reaction forces increased with rotation rate up to approximately 200% bodyweight (iRED on ISS provides approximately 210% bodyweight resistance). The ratio of left-to-right foot force throughout the squat cycle on the centrifuge was nonconstant and approximately sinusoidal. Total foot reaction force versus knee flexion-extension angles differed between upright and AG squats due to centripetal acceleration on the centrifuge. DISCUSSION: A brief exercise protocol during centrifugation can be safely completed without significant after-effects in mediolateral knee position or motion sickness. Several recommendations are made for the design of future centrifuge-based exercise protocols for in-space applications.

Optimal estimator models for spatial orientation and vestibular nystagmus.

Mathematical models have played an important role in research on the vestibular system over the past century, from the torsion pendulum analogies of the semicircular canal to the optimal estimator "observer" models of multisensory interaction and adaptation. This short review is limited to our own contributions in bringing the technology of feedback control theory to bear on the understanding of human spatial orientation, eye movements, and nystagmus, both on Earth and in space. It points to the importance of the "internal model" concept for treatment of the manner in which the brain constantly makes predictions about future sensory feedback, adjusts the weightings of sensors according to their signal-to-noise ratios, and adapts control according to the motion environment, and availability of sensory cues.

Asymmetry in vestibular responses to cross-coupled stimulus.

Head turns performed while rotating about another axis result in a cross-coupled stimulus (CCS) to the vestibular system. The CCS causes a tumbling sensation, and the magnitude of the tumbling sensation is dependent on the type of head turn (HT) that is performed. Asymmetric CCS responses to different rotational directions are widely acknowledged, yet poorly understood. The objective of this study was to: 1) correctly describe the asymmetries in responses to different configurations of CCS stimulation and 2) test two previously proposed hypotheses for explaining the asymmetries, dominant direction, and dominant end position. The dominant direction hypothesis states that the tumbling sensations evoked by the CCS will be more intense for certain directions of the tumbling sensation than for others. The dominant end position hypothesis states that head turns ending in the nose-up position result in more intense sensations than those ending on the side positions. Subjects performed four types of 60-degree yaw head turns while lying horizontally on a centrifuge. Subjects were either supine or prone, while rotating clockwise or counterclockwise. Three experimental conditions were tested: clockwise supine (n = 33); counterclockwise supine (n = 10); and clockwise prone (n = 10). Subjective tumbling intensity scores were recorded for each head turn. Head turns to the left are dominant for clockwise supine centrifugation (P < 0.0001) and head turns to the right are dominant for counterclockwise supine centrifugation (P = 0.0020), matching what is expected from previous studies. However, for prone centrifugation, head turns to the left are more intense than head turns to the right (P = 0.0078), refuting the dominant direction hypothesis. The dominant end position effect is small in magnitude and cannot by itself explain the asymmetries. For every test condition, there is a dominant direction, but the dominant direction is not just a function of the HT and centrifuge rotation directions, instead it is also dependent on the subject's orientation on the centrifuge. An alternative perceived danger hypothesis that matches the data from all three experiments is proposed.

Use of short-radius centrifugation to augment ankle-brachial indices.

BACKGROUND: Peripheral arterial disease is mainly caused by atherosclerosis and is characterized by decreased circulation, lower blood pressure, and insufficient tissue perfusion in the lower extremities. The hemodynamics of standing and altered gravity environments have been well studied relative to arm blood pressures but are less well understood for ankle pressures. METHODS: Because regional blood pressure depends, in part, on the gravitational pressure gradient, we hypothesized that artificial gravity exposure on a short-arm centrifuge with the center of rotation above the head would increase blood pressure in the lower extremities. Cardiovascular parameters for 12 healthy subjects were measured during exposure to supine short-arm centrifugation at 20, 25, and 30 revolutions per minute (rpm), corresponding to centripetal accelerations of 0.94, 1.47, and 2.11 Gz at the foot level, respectively. RESULTS: Systolic ankle blood pressure significantly increased at all levels of centrifugation. Ankle-brachial indices (the ratio of systolic ankle to arm blood pressures) increased significantly from 1.17 +/- 0.03 to 1.58 +/- 0.03 at 0.94 Gz (P < 0.005), 1.74 +/- 0.02 at 1.47 Gz (P < 0.005), and 1.89 +/- 0.06 at 2.11 Gz (P < 0.005). Systolic arm blood pressure significantly increased at 2.11 Gz, but heart rate did not change significantly. All parameters returned to normal after cessation of centrifugation. CONCLUSIONS: We demonstrated that short-radius centrifugation leads to an increase in ankle-brachial indices. This could have potential implications for the treatment of peripheral arterial disease.

Computational model of cardiovascular response to centrifugation and lower body cycling exercise.

Short-radius centrifugation combined with exercise has been suggested as a potential countermeasure against spaceflight deconditioning. Both the long-term and acute physiological responses to such a combination are incompletely understood. We developed and validated a computational model to study the acute cardiovascular response to centrifugation combined with lower body ergometer exercise. The model consisted of 21 compartments, including the upper body, renal, splanchnic, and leg circulation, as well as a four-chamber heart and pulmonary circulation. It also included the effects of gravity gradient and ergometer exercise. Centrifugation and exercise profiles were simulated and compared with experimental data gathered on 12 subjects exposed to a range of gravitational levels (1 and 1.4G measured at the feet) and workload intensities (25-100 W). The model was capable of reproducing cardiovascular changes (within ± 1 SD from the group-averaged behavior) due to both centrifugation and exercise, including dynamic responses during transitions between the different phases of the protocol. The model was then used to simulate the hemodynamic response of hypovolemic subjects (blood volume reduced by 5-15%) subjected to similar gravitational stress and exercise profiles, providing insights into the physiological responses of experimental conditions not tested before. Hypovolemic results are in agreement with the limited available data and the expected responses based on physiological principles, although additional experimental data are warranted to further validate our predictions, especially during the exercise phases. The model captures the cardiovascular response for a range of centrifugation and exercise profiles, and it shows promise in simulating additional conditions where data collection is difficult, expensive, or infeasible.NEW & NOTEWORTHY Artificial gravity combined with exercise is a potential countermeasure for spaceflight deconditioning, but the long-term and acute cardiovascular response to such gravitational stress is still largely unknown. We provide a novel mathematical model of the cardiovascular system that incorporates gravitational stress generated by centrifugation and lower body cycling exercise, and we validate it with experimental measurements from human subjects. Simulations of experimental conditions not used for model development corroborate the model's predictive capabilities.

Incremental adaptation to yaw head turns during 30 RPM centrifugation.

A 3-day incremental protocol was conducted with the aim of adapting human subjects to make head movements comfortably during 30 RPM centrifugation. With motion sickness as a potentially limiting factor, the protocol was designed using a quantitative motion sickness model based upon the neural mismatch sensory conflict theory. Centrifuge velocity was incremented from 14 RPM on day 1, to 23 RPM on day 2, to 30 RPM on day 3, with subjects making a total of 42 head movements on each day. Twenty-four subjects completed the experiment with average motion sickness levels below five (out of 20). Four subjects aborted due to motion sickness. Adaptation of non-compensatory vertical nystagmus was observed through an 18% decrease in the vertical aVOR time constant over the 3 days. Subjective intensity ratings for the head movements decreased by approximately 40% over the 3 days, while illusory motion duration decreased by 18%. Feasibility of head movements during 30 RPM rotation was demonstrated with only 3 days of incremental training.

The effect of head turn velocity on cross-coupled stimulation during centrifugation.

Short-radius centrifugation (SRC) provides a practical means of producing artificial gravity for long duration space flights, though perceptual side-effects could limit its operational feasibility. Head turns (HT) during SRC, other than those about the centrifugation axis, produce Cross-Coupled Stimulation (CCS), perceived as a tumbling sensation. CCS can be nauseagenic, though adaptation can minimize this detrimental effect over time. The force environment of CCS suggests that the head turn velocity plays a role in determining the stimulus magnitude, though its degree has not been characterized. Twenty-three subjects performed right quadrant head turns of 8 different velocities while spinning at 19 and 23 RPMs on the SRC over two consecutive days. The perceptual effects were characterized by subjective metrics, investigating the acute differences between velocities as well as the chronic effects on adaptation. It was found that the perceived CCS magnitude can be regulated by modulating HT velocity. Further, a threshold of HT velocity exists above which an asymptotic perceptual response is observed, and below which the perceptual response diminishes at an exponential rate relative to head turn velocity. Finally, the effects of HT velocity are independent of HT direction, though the differing head turn directions likely produce contextually specific stimuli. These results suggest that HT velocity modulation could provide a practical means of incremental adaptation to CCS during SRC.

Vestibular adaptation to centrifugation does not transfer across planes of head rotation.

Out-of-plane head movements performed during fast rotation produce non-compensatory nystagmus, sensations of illusory motion, and often motion sickness. Adaptation to this cross-coupled Coriolis stimulus has previously been demonstrated for head turns made in the yaw (transverse) plane of motion, during supine head-on-axis rotation. An open question, however, is if adaptation to head movements in one plane of motion transfers to head movements performed in a new, unpracticed plane of motion. Evidence of transfer would imply the brain builds up a generalized model of the vestibular sensory-motor system, instead of learning a variety of individual input/output relations separately. To investigate, over two days 9 subjects performed pitch head turns (sagittal plane) while rotating, before and after a series of yaw head turns while rotating. A Control Group of 10 subjects performed only the pitch movements. The vestibulo-ocular reflex (VOR) and sensations of illusory motion were recorded in the dark for all movements. Upon comparing the two groups we failed to find any evidence of transfer from the yaw plane to the pitch plane, suggesting that adaptation to cross-coupled stimuli is specific to the particular plane of head movement. The findings have applications for the use of centrifugation as a possible countermeasure for long duration spaceflight. Adapting astronauts to unconstrained head movements while rotating will likely require exposure to head movements in all planes and directions.

Short-Term Cardiovascular Response to Short-Radius Centrifugation With and Without Ergometer Exercise.

Artificial gravity (AG) has often been proposed as an integrated multi-system countermeasure to physiological deconditioning associated with extended exposure to reduced gravity levels, particularly if combined with exercise. Twelve subjects underwent short-radius centrifugation along with bicycle ergometry to quantify the short-term cardiovascular response to AG and exercise across three AG levels (0 G or no rotation, 1 G, and 1.4 G; referenced to the subject's feet and measured in the centripetal direction) and three exercise intensities (25, 50, and 100 W). Continuous cardiovascular measurements were collected during the centrifugation sessions using a non-invasive monitoring system. The cardiovascular responses were more prominent at higher levels of AG and exercise intensity. In particular, cardiac output, stroke volume, pulse pressure, and heart rate significantly increased with both AG level (in most of exercise group combinations, showing averaged increments across exercise conditions of 1.4 L/min/g, 7.6 mL/g, 5.22 mmHg/g, and 2.0 bpm/g, respectively), and workload intensity (averaged increments across AG conditions of 0.09 L/min/W, 0.17 mL/W, 0.22 mmHg/W, and 0.74 bpm/W respectively). These results suggest that the addition of AG to exercise can provide a greater cardiovascular benefit than exercise alone. Hierarchical regression models were fitted to the experimental data to determine dose-response curves of all cardiovascular variables as a function of AG-level and exercise intensity during short-radius centrifugation. These results can inform future studies, decisions, and trade-offs toward potential implementation of AG as a space countermeasure.

Structured illumination microscopy combined with machine learning enables the high throughput analysis and classification of virus structure.

Optical super-resolution microscopy techniques enable high molecular specificity with high spatial resolution and constitute a set of powerful tools in the investigation of the structure of supramolecular assemblies such as viruses. Here, we report on a new methodology which combines Structured Illumination Microscopy (SIM) with machine learning algorithms to image and classify the structure of large populations of biopharmaceutical viruses with high resolution. The method offers information on virus morphology that can ultimately be linked with functional performance. We demonstrate the approach on viruses produced for oncolytic viriotherapy (Newcastle Disease Virus) and vaccine development (Influenza). This unique tool enables the rapid assessment of the quality of viral production with high throughput obviating the need for traditional batch testing methods which are complex and time consuming. We show that our method also works on non-purified samples from pooled harvest fluids directly from the production line.