Wave damping by giant kelp, Macrocystis pyrifera.

BACKGROUND AND AIMS: The increased likelihood and severity of storm events has brought into focus the role of coastal ecosystems in provision of shoreline protection by attenuating wave energy. Canopy-forming kelps, including giant kelp (Macrocystis pyrifera), are thought to provide this ecosystem service, but supporting data are extremely limited. Previous in situ examinations relied mostly on comparisons between nominally similar sites with and without kelp. Given that other factors (especially seafloor bathymetry and topographic features) often differ across sites, efforts to isolate the effects of kelp on wave energy propagation confront challenges. In particular, it can be difficult to distinguish wave energy dissipation attributable to kelp from frictional processes at the seabed that often covary with the presence of kelp. Here, we use an ecological transition from no kelp to a full forest, at a single site with static bathymetry, to resolve unambiguously the capacity of giant kelp to damp waves. METHODS: We measured waves within and outside rocky reef habitat, in both the absence and the presence of giant kelp, at Marguerite Reef, Palos Verdes, CA, USA. Nested within a broader kelp restoration project, this site transitioned from a bare state to one supporting a fully formed forest (density of 8 stipes m-2). We quantified, as a function of incident wave conditions, the decline in wave energy flux attributable to the presence of kelp, as waves propagated from outside and into reef habitat. KEY RESULTS: The kelp forest damped wave energy detectably, but to a modest extent. Interactions with the seabed alone reduced wave energy flux, on average, by 12 ±â€…1.4 % over 180 m of travel. The kelp forest induced an additional 7 ±â€…1.2 % decrease. Kelp-associated declines in wave energy flux were slightly greater for waves of longer periods and smaller wave heights. CONCLUSIONS: Macrocystis pyrifera forests have a limited, albeit measurable, capacity to enhance shoreline protection from nearshore waves. Expectations that giant kelp forests, whether extant or enhanced through restoration, have substantial impacts on wave-induced coastal erosion might require re-evaluation.

Rapid Range Expansion of a Marine Ectotherm Reveals the Demographic and Ecological Consequences of Short-Term Variability in Seawater Temperature and Dissolved Oxygen.

AbstractThe distributions of marine ectotherms are governed by physiological sensitivities to long-term trends in seawater temperature and dissolved oxygen. Short-term variability in these parameters has the potential to facilitate rapid range expansions, and the resulting ecological and socioeconomic consequences may portend those of future marine communities. Here, we combine physiological experiments with ecological and demographic surveys to assess the causes and consequences of sudden but temporary poleward range expansions of a marine ectotherm with considerable life history plasticity (California market squid, Doryteuthis opalescens). We show that sequential factors related to resource accessibility in the core range-the buildup of large populations as a result of competitive release and climate-associated temperature increase and oxygen loss that constrain aerobic activity-may drive these expansions. We also reveal that poleward range expansion alters the body size-and therefore trophic role-of invading populations, with potential negative implications for socioeconomically valuable resident species. To help forecast rapid range expansions of marine ectotherms, we advocate that research efforts focus on factors impacting resource accessibility in core ranges. Determining how environmental conditions in receiving ecosystems affect body size and how body size is related to trophic role will help refine estimates of the impacts of future marine communities.

Effects of heat acclimation on cardiac function in the intertidal mussel Mytilus californianus: can laboratory-based indices predict survival in the field?

Thermal performance curves are commonly used to investigate the effects of heat acclimation on thermal tolerance and physiological performance. However, recent work indicates that the metrics of these curves heavily depend on experimental design and may be poor predictors of animal survival during heat events in the field. In intertidal mussels, cardiac thermal performance (CTP) tests have been widely used as indicators of animals' acclimation or acclimatization state, providing two indices of thermal responses: critical temperature (Tcrit; the temperature above which heart rate abruptly declines) and flatline temperature (Tflat; the temperature where heart rate ceases). Despite the wide use of CTP tests, it remains largely unknown how Tcrit and Tflat change within a single individual after heat acclimation, and whether changes in these indices can predict altered survival in the field. Here, we addressed these issues by evaluating changes in CTP indices in the same individuals before and after heat acclimation. For control mussels, merely reaching Tcrit was not lethal, whereas remaining at Tcrit for ≥10 min was lethal. Heat acclimation significantly increased Tcrit only in mussels with an initially low Tcrit (<35°C), but improved their survival time above Tcrit by 20 min on average. Tflat increased by ∼1.6°C with heat acclimation, but it is unlikely that increased Tflat improves survival in the field. In summary, Tcrit and Tflat per se may fall short of providing quantitative indices of thermal tolerance in mussels; instead, a combination of Tcrit and tolerance time at temperatures ≥Tcrit better defines changes in thermal tolerance with heat acclimation.

Lung Deposition Using the Respimat® Soft Mist™ Inhaler Mono and Fixed-Dose Combination Therapies: An In Vitro/In Silico Analysis.

Tiotropium and olodaterol are mainstay treatments for chronic obstructive pulmonary disease (COPD) and yield important clinical improvements, especially when used in fixed-dose combination. Whilst previous studies have shown consistent delivery of tiotropium to the lungs with the Respimat® inhaler, no such study has been carried out for olodaterol or the components of their fixed-dose combination (TIO/OLO). Combining in vitro and in silico models, we measured the amount of drug retained in the mouth-throat area, entering the trachea and reaching the lung periphery. We applied a hybrid deposition model that considered the experimentally determined output of an Alberta throat model (in vitro - dose to lung) combined with a computational fluid dynamic model of the lungs (in silico). Regardless of the COPD breathing pattern, ≥50% of the nominal dose of either tiotropium, olodaterol, or TIO and OLO in the fixed-dose combination reached the lung. Of the dose reaching the lungs, greater than 50% is deposited in the lung periphery (from generation 8 onwards). Our study demonstrated that aerosol delivery via the Respimat inhaler achieved high deposition deep into the lung periphery with all formulations evaluated.

Transcutaneous immunization with CD40 ligation boosts cytotoxic T lymphocyte mediated antitumor immunity independent of CD4 helper cells in mice.

Transcutaneous immunization (TCI) is a novel vaccination strategy that utilizes skin-associated lymphatic tissue to induce immune responses. Employing T-cell epitopes and the TLR7 agonist imiquimod onto intact skin mounts strong primary, but limited memory CTL responses. To overcome this limitation, we developed a novel imiquimod-containing vaccination platform (IMI-Sol) rendering superior primary CD8+ and CD4+ T-cell responses. However, it has been unclear whether IMI-Sol per se is restricted in terms of memory formation and tumor protection. In our present work, we demonstrate that the combined administration of IMI-Sol and CD40 ligation unleashes fullblown specific T-cell responses in the priming and memory phase, strongly enhancing antitumor protection in mice. Interestingly, these effects were entirely CD4+ T cell independent, bypassing the necessity of helper T cells. Moreover, blockade of CD70 in vivo abrogated the boosting effect of CD40 ligation, indicating that the adjuvant effect of CD40 in TCI is mediated via CD70 on professional APCs. Furthermore, this work highlights the so far underappreciated importance of the CD70/CD27 interaction as a promising adjuvant target in TCI. Summing up, we demonstrate that the novel formulation IMI-Sol represents a powerful vaccination platform when applied in combination with sufficient adjuvant thereby overcoming current limitations of TCI.

A series of unfortunate events: characterizing the contingent nature of physiological extremes using long-term environmental records.

Accelerating shifts in global climate have focused the attention of ecologists and physiologists on extreme environmental events. However, the dynamic process of physiological acclimatization complicates study of these events' consequences. Depending on the range of plasticity and the amplitude and speed of environmental variation, physiology can be either in tune with the surroundings or dangerously out of synch. We implement a modified quantitative approach to identifying extreme events in environmental records, proposing that organisms are stressed by deviations of the environment from the current level of acclimatization, rather than by the environment's absolute state. This approach facilitates an unambiguous null model for the consequences of environmental variation, identifying a unique subset of events as 'extremes'. Specifically, it allows one to examine how both the temporal extent (the acclimatization window) and type of an environmental signal affect the magnitude and timing of extreme environmental events. For example, if physiology responds to the moving average of past conditions, a longer acclimatization window generally results in greater imposed stress. If instead physiology responds to historical maxima, longer acclimatization windows reduce imposed stress, albeit perhaps at greater constitutive cost. This approach should be further informed and tested with empirical experiments addressing the history-dependent nature of acclimatization.

A single heat-stress bout induces rapid and prolonged heat acclimation in the California mussel, Mytilus californianus.

Climate change is not only causing steady increases in average global temperatures but also increasing the frequency with which extreme heating events occur. These extreme events may be pivotal in determining the ability of organisms to persist in their current habitats. Thus, it is important to understand how quickly an organism's heat tolerance can be gained and lost relative to the frequency with which extreme heating events occur in the field. We show that the California mussel, Mytilus californianus-a sessile intertidal species that experiences extreme temperature fluctuations and cannot behaviourally thermoregulate-can quickly (in 24-48 h) acquire improved heat tolerance after exposure to a single sublethal heat-stress bout (2 h at 30 or 35°C) and then maintain this improved tolerance for up to three weeks without further exposure to elevated temperatures. This adaptive response improved survival rates by approximately 75% under extreme heat-stress bouts (2 h at 40°C). To interpret these laboratory findings in an ecological context, we evaluated 4 years of mussel body temperatures recorded in the field. The majority (approx. 64%) of consecutive heat-stress bouts were separated by 24-48 h, but several consecutive heat bouts were separated by as much as 22 days. Thus, the ability of M. californianus to maintain improved heat tolerance for up to three weeks after a single sublethal heat-stress bout significantly improves their probability of survival, as approximately 33% of consecutive heat events are separated by 3-22 days. As a sessile animal, mussels likely evolved the capability to rapidly gain and slowly lose heat tolerance to survive the intermittent, and often unpredictable, heat events in the intertidal zone. This adaptive strategy will likely prove beneficial under the extreme heat events predicted with climate change.

Mussel acclimatization to high, variable temperatures is lost slowly upon transfer to benign conditions.

Climate change is increasing the temperature variability animals face, and thermal acclimatization allows animals to adjust adaptively to this variability. Although the rate of heat acclimatization has received some study, little is known about how long these adaptive changes remain without continuing exposure to heat stress. This study explored the rate at which field acclimatization states are lost when temperature variability is minimized during constant submersion. California mussels (Mytilus californianus) with different acclimatization states were collected from high- and low-zone sites (∼12 versus ∼5°C daily temperature ranges, respectively) and then kept submerged at 15°C for 8 weeks. Each week, the cardiac thermal performance of mussels was measured as a metric of acclimatization state: critical (Tcrit) and flatline (Tflat) temperatures were recorded. Over 8 weeks of constant submersion, the mean Tcrit of high-zone mussels decreased by 1.07°C from baseline, but low-zone mussels' mean Tcrit was unchanged. High- and low-zone mussels' mean maximum heart rate (HR) and resting HR decreased ∼12 and 35%, respectively. Tflat was unchanged in both groups. These data suggest that Tcrit and HR are more physiologically plastic in response to the narrowing of an animal's daily temperature range than Tflat is, and that an animal's prior acclimatization state (high versus low) influences the acclimatory capacity of Tcrit Approximately 2 months were required for the cardiac thermal performance of the high-zone mussels to reach that of the low-zone mussels, suggesting that acclimatization to high and variable temperatures may persist long enough to enable these animals to cope with intermittent bouts of heat stress.

Establishing typical values for hemocyte mortality in individual California mussels, Mytilus californianus.

Hemocytes are immune cells in the hemolymph of invertebrates that play multiple roles in response to stressors; hemocyte mortality can thus serve as an indicator of overall animal health. However, previous research has often analyzed hemolymph samples pooled from several individuals, which precludes tracking individual responses to stressors over time. The ability to track individuals is important, however, because large inter-individual variation in response to stressors can confound the interpretation of pooled samples. Here, we describe protocols for analysis of inter- and intra-individual variability in hemocyte mortality across repeated hemolymph samples of California mussels, Mytilus californianus, free from typical abiotic stressors. To assess individual variability in hemocyte mortality with serial sampling, we created four groups of 15 mussels each that were repeatedly sampled four times: at baseline (time zero) and three subsequent times separated by either 24, 48, 72, or 168 h. Hemocyte mortality was assessed by fluorescence-activated cell sorting (FACS) of cells stained with propidium iodide. Our study demonstrates that hemolymph can be repeatedly sampled from individual mussels without mortality; however, there is substantial inter- and intra-individual variability in hemocyte mortality through time that is partially dependent on the sampling interval. Across repeated samples, individual mussels' hemocyte mortality had, on average, a range of ~6% and a standard deviation of ~3%, which was minimized with sampling periods ≥72 h apart. Due to this intra-individual variability, obtaining ≥2 samples from a specimen will more accurately establish an individual's baseline. Pooled-sample means were similar to individual-sample means; however, pooled samples masked the individual variation in each group. Overall, these data lay the foundation for future work exploring individual mussels' temporal responses to various stressors on a cellular level.

Impact of heating rate on cardiac thermal tolerance in the California mussel, Mytilus californianus.

Intertidal communities of wave-swept rocky shores have served as a powerful model system for experiments in ecology, and mussels (the dominant competitor for space in the mid-intertidal zone) play a central role in determining community structure in this physically stressful habitat. Consequently, the ability to account for mussels' physiological responses to thermal stress affects ecologists' capacity to predict the impacts of a warming climate on this ecosystem. Here, we examined the effect of heating rate on cardiac thermal tolerance in the ribbed mussel, Mytilus californianus, comparing populations from high and low sites in the intertidal zone where emersion duration leads to different mean daily heating rates. Two temperature-related cardiac variables were examined: (1) the critical temperature (Tcrit) at which heart rate (HR) precipitously declines, and (2) flatline temperature (FLT) where HR reaches zero. Mussels were heated in air at slow, moderate and fast rates, and HR was measured via an infrared sensor affixed to the shell. Faster heating rates significantly increased Tcrit in high- but not low-zone mussels, and Tcrit was higher in high- versus low-zone mussels, especially at the fastest heating rate. By contrast, FLT did not differ between zones, and was minimally affected by heating rate. As heating rate significantly impacted high- but not low-zone mussels' cardiac thermal tolerance, realistic zone-specific heating rates must be used in laboratory tests if those tests are to provide accurate information for ecological models attempting to predict the effects of increasing temperature on intertidal communities.

The fallacy of the average: on the ubiquity, utility and continuing novelty of Jensen's inequality.

Biologists often cope with variation in physiological, environmental and ecological processes by measuring how living systems perform under average conditions. However, performance at average conditions is seldom equal to average performance across a range of conditions. This basic property of nonlinear averaging - known as 'Jensen's inequality' or 'the fallacy of the average' - has important implications for all of biology. For instance, a burgeoning awareness of Jensen's inequality has improved our ability to predict how plants and animals will respond to a warmer and more variable future climate. But for many biologists, the fallacy of the average is still a novel concept. Here, I highlight the importance of Jensen's inequality, provide a simple graphical approach to understanding its effects, and explore its consequences at atomic, molecular, organismal and ecological levels.

The extraordinary joint material of an articulated coralline alga. I. Mechanical characterization of a key adaptation.

Flexibility is key to survival for seaweeds exposed to the extreme hydrodynamic environment of wave-washed rocky shores. This poses a problem for coralline algae, whose calcified cell walls make them rigid. Through the course of evolution, erect coralline algae have solved this problem by incorporating joints (genicula) into their morphology, allowing their fronds to be as flexible as those of uncalcified seaweeds. To provide the flexibility required by this structural innovation, the joint material of Calliarthron cheilosporioides, a representative articulated coralline alga, relies on an extraordinary tissue that is stronger, more extensible and more fatigue resistant than the tissue of other algal fronds. Here, we report on experiments that reveal the viscoelastic properties of this material. On the one hand, its compliance is independent of the rate of deformation across a wide range of deformation rates, a characteristic of elastic solids. This deformation rate independence allows joints to maintain their flexibility when loaded by the unpredictable - and often rapidly imposed - hydrodynamic force of breaking waves. On the other hand, the genicular material has viscous characteristics that similarly augment its function. The genicular material dissipates much of the energy absorbed as a joint is deformed during cyclic wave loading, which potentially reduces the chance of failure by fatigue, and the material accrues a limited amount of deformation through time. This limited creep increases the flexibility of the joints while preventing them from gradually stretching to the point of failure. These new findings provide the basis for understanding how the microscale architecture of genicular cell walls results in the adaptive mechanical properties of coralline algal joints.

The extraordinary joint material of an articulated coralline alga. II. Modeling the structural basis of its mechanical properties.

By incorporating joints into their otherwise rigid fronds, erect coralline algae have evolved to be as flexible as other seaweeds, which allows them to thrive - and even dominate space - on wave-washed shores around the globe. However, to provide the required flexibility, the joint tissue of Calliarthron cheilosporioides, a representative articulated coralline alga, relies on an extraordinary tissue that is stronger, more extensible and more fatigue resistant than that of other algae. Here, we used the results from recent experiments to parameterize a conceptual model that links the microscale architecture of cell walls to the adaptive mechanical properties of joint tissue. Our analysis suggests that the theory of discontinuous fiber-wound composite materials (with cellulose fibrils as the fibers and galactan gel as the matrix) can explain key aspects of the material's mechanics. In particular, its adaptive viscoelastic behavior can be characterized by two, widely separated time constants. We speculate that the short time constant (∼14 s) results from the viscous response of the matrix to the change in cell-wall shape as a joint is stretched, a response that allows the material both to remain flexible and to dissipate energy as a frond is lashed by waves. We propose that the long time constant (∼35 h), is governed by the shearing of the matrix between cellulose fibrils. The resulting high apparent viscosity ensures that joints avoid accumulating lethal deformation in the course of a frond's lifetime. Our synthesis of experimental measurements allows us to draw a chain of mechanistic inference from molecules to cell walls to fronds and community ecology.

Thermal variation, thermal extremes and the physiological performance of individuals.

In this review we consider how small-scale temporal and spatial variation in body temperature, and biochemical/physiological variation among individuals, affect the prediction of organisms' performance in nature. For 'normal' body temperatures - benign temperatures near the species' mean - thermal biology traditionally uses performance curves to describe how physiological capabilities vary with temperature. However, these curves, which are typically measured under static laboratory conditions, can yield incomplete or inaccurate predictions of how organisms respond to natural patterns of temperature variation. For example, scale transition theory predicts that, in a variable environment, peak average performance is lower and occurs at a lower mean temperature than the peak of statically measured performance. We also demonstrate that temporal variation in performance is minimized near this new 'optimal' temperature. These factors add complexity to predictions of the consequences of climate change. We then move beyond the performance curve approach to consider the effects of rare, extreme temperatures. A statistical procedure (the environmental bootstrap) allows for long-term simulations that capture the temporal pattern of extremes (a Poisson interval distribution), which is characterized by clusters of events interspersed with long intervals of benign conditions. The bootstrap can be combined with biophysical models to incorporate temporal, spatial and physiological variation into evolutionary models of thermal tolerance. We conclude with several challenges that must be overcome to more fully develop our understanding of thermal performance in the context of a changing climate by explicitly considering different forms of small-scale variation. These challenges highlight the need to empirically and rigorously test existing theories.

Aperture effects in squid jet propulsion.

Squid are the largest jet propellers in nature as adults, but as paralarvae they are some of the smallest, faced with the inherent inefficiency of jet propulsion at a low Reynolds number. In this study we describe the behavior and kinematics of locomotion in 1 mm paralarvae of Dosidicus gigas, the smallest squid yet studied. They swim with hop-and-sink behavior and can engage in fast jets by reducing the size of the mantle aperture during the contraction phase of a jetting cycle. We go on to explore the general effects of a variable mantle and funnel aperture in a theoretical model of jet propulsion scaled from the smallest (1 mm mantle length) to the largest (3 m) squid. Aperture reduction during mantle contraction increases propulsive efficiency at all squid sizes, although 1 mm squid still suffer from low efficiency (20%) because of a limited speed of contraction. Efficiency increases to a peak of 40% for 1 cm squid, then slowly declines. Squid larger than 6 cm must either reduce contraction speed or increase aperture size to maintain stress within maximal muscle tolerance. Ecological pressure to maintain maximum velocity may lead them to increase aperture size, which reduces efficiency. This effect might be ameliorated by nonaxial flow during the refill phase of the cycle. Our model's predictions highlight areas for future empirical work, and emphasize the existence of complex behavioral options for maximizing efficiency at both very small and large sizes.

Indefatigable: an erect coralline alga is highly resistant to fatigue.

Intertidal organisms are subjected to intense hydrodynamic forces as waves break on the shore. These repeated insults can cause a plant or animal's structural materials to fatigue and fail, even though no single force would be sufficient to break the organism. Indeed, the survivorship and maximum size of at least one species of seaweed is set by the accumulated effects of small forces rather than the catastrophic imposition of a single lethal force. One might suppose that fatigue would be especially potent in articulated coralline algae, in which the strain of the entire structure is concentrated in localized joints, the genicula. However, previous studies of joint morphology suggest an alternative hypothesis. Each geniculum is composed of a single tier of cells, which are attached at their ends to the calcified segments of the plant (the intergenicula) but have minimal connection to each other along their lengths. This lack of neighborly attachment potentially allows the weak interfaces between cells to act as 'crack stoppers', inhibiting the growth of fatigue cracks. We tested this possibility by repeatedly loading fronds of Calliarthron cheilosporioides, a coralline alga common on wave-washed shores in California. When repeatedly loaded to 50-80% of its breaking strength, C. cheilosporioides commonly survives more than a million stress cycles, with a record of 51 million. We show how this extraordinary fatigue resistance interacts with the distribution of wave-induced water velocities to set the limits to size in this species.

A bendable biological ceramic.

A fatigue-resistant mollusk hinge could in spire the development of new materials.

Quality by Design (QbD) Approach for a Nanoparticulate Imiquimod Formulation as an Investigational Medicinal Product.

The present article exemplifies the application of the concept of quality by design (QbD) for the systematic development of a nanoparticulate imiquimod (IMQ) emulsion gel formulation as an investigational medicinal product (IMP) for evaluation in an academic phase-I/II clinical trial for the treatment of actinic keratosis (AK) against the comparator Aldara (EudraCT: 2015-002203-28). The design of the QbD elements of a quality target product profile (QTPP) enables the identification of the critical quality attributes (CQAs) of the drug product as the content of IMQ, the particle-size distribution, the pH, the rheological properties, the permeation rate and the chemical, physical and microbiological stability. Critical material attributes (CMAs) and critical process parameters (CPPs) are identified by using a risk-based approach in an Ishikawa diagram and in a risk-estimation matrix. In this study, the identified CPPs of the wet media ball-milling process's milling time and milling speed are evaluated in a central composite design of experiments (DoEs) approach, revealing criticality for both factors for the resulting mean particle size, while only the milling time is significantly affecting the polydispersity. To achieve a mean particle size in the range of 300-400 nm with a minimal PdI, the optimal process conditions are found to be 650 rpm for 135 min. Validating the model reveals a good correlation between the predicted and observed values. Adequate control strategies were implemented for intermediate products as in-process controls (IPCs) and quality control (QC) tests of the identified CQAs. The IPC and QC data from 13 "IMI-Gel" batches manufactured in adherence to good manufacturing practice (GMP) reveal consistent quality with minimal batch-to-batch variability.

The fine art of surfacing: its efficacy in broadcast spawning.

Many organisms reproduce by releasing gametes into the surrounding fluid. For some such broadcast spawners, gametes are positively or negatively buoyant, and, as a result, fertilization occurs on a two-dimensional surface rather than in the bulk of the air or water. We here rationalize this behaviour by considering the encounter rates of gametes on the surface and in the fluid bulk. The advantage of surfacing is quantified by considering an infinitely wide body of water of constant depth. Differential loss rates at the surface and in the bulk are considered and their influence on the robustness of surface search assessed. For small and moderate differential loss rates, the advantage of surfacing is very robust and significant; only for large loss rate differences can the advantage of surfacing be nullified.