The freeze-avoiding mountain pine beetle (Dendroctonus ponderosae) survives prolonged exposure to stressful cold by mitigating ionoregulatory collapse.

Insect performance is linked to environmental temperature, and surviving through winter represents a key challenge for temperate, alpine and polar species. To overwinter, insects have adapted a range of strategies to become truly cold hardy. However, although the mechanisms underlying the ability to avoid or tolerate freezing have been well studied, little attention has been given to the challenge of maintaining ion homeostasis at frigid temperatures in these species, despite this limiting cold tolerance for insects susceptible to mild chilling. Here, we investigated how prolonged exposure to temperatures just above the supercooling point affects ion balance in freeze-avoidant mountain pine beetle (Dendroctonus ponderosae) larvae in autumn, mid-winter and spring, and related it to organismal recovery times and survival. Hemolymph ion balance was gradually disrupted during the first day of exposure, characterized by hyperkalemia and hyponatremia, after which a plateau was reached and maintained for the rest of the 7-day experiment. The degree of ionoregulatory collapse correlated strongly with recovery times, which followed a similar asymptotical progression. Mortality increased slightly during extensive cold exposures, where hemolymph K+ concentration was highest, and a sigmoidal relationship was found between survival and hyperkalemia. Thus, the cold tolerance of the freeze-avoiding larvae of D. ponderosae appears limited by the ability to prevent ionoregulatory collapse in a manner similar to that of chill-susceptible insects, albeit at much lower temperatures. Based on these results, we propose that a prerequisite for the evolution of insect freeze avoidance may be a convergent or ancestral ability to maintain ion homeostasis during extreme cold stress.

Effects of a high cholesterol diet on chill tolerance are highly context-dependent in Drosophila.

Chill susceptible insects are thought to be injured through different mechanisms depending on the duration and severity of chilling. While chronic chilling causes "indirect" injury through disruption of metabolic and ion homeostasis, acute chilling is suspected to cause "direct" injury, in part through phase transitions of cell membrane lipids. Dietary supplementation of cholesterol can reduce acute chilling injury in Drosophila melanogaster (Shreve et al., 2007), but the generality of this effect and the mechanisms underlying it remain unclear. To better understand how and why cholesterol has this effect, we assessed how a high cholesterol diet and thermal acclimation independently and interactively impact several measures of chill tolerance. Cholesterol supplementation positively affected tolerance to acute chilling in warm-acclimated flies (as reported previously). Conversely, feeding on the high-cholesterol diet negatively affected tolerance to chronic chilling in both cold and warm acclimated flies, as well as tolerance to acute chilling in cold acclimated flies. Cholesterol had no effect on the ability of flies to remain active in the cold or recover movement after a cold stress. Our findings support the idea that dietary cholesterol reduces mechanical injury to membranes caused by direct chilling injury, and that acute and chronic chilling are associated with distinct mechanisms of injury. Feeding on a high-cholesterol diet may interfere with mechanisms involved in cold acclimation, leaving cholesterol augmented flies more susceptible to chilling injury under some conditions.

The Human Affectome.

Over the last decades, theoretical perspectives in the interdisciplinary field of the affective sciences have proliferated rather than converged due to differing assumptions about what human affective phenomena are and how they work. These metaphysical and mechanistic assumptions, shaped by academic context and values, have dictated affective constructs and operationalizations. However, an assumption about the purpose of affective phenomena can guide us to a common set of metaphysical and mechanistic assumptions. In this capstone paper, we home in on a nested teleological principle for human affective phenomena in order to synthesize metaphysical and mechanistic assumptions. Under this framework, human affective phenomena can collectively be considered algorithms that either adjust based on the human comfort zone (affective concerns) or monitor those adaptive processes (affective features). This teleologically-grounded framework offers a principled agenda and launchpad for both organizing existing perspectives and generating new ones. Ultimately, we hope the Human Affectome brings us a step closer to not only an integrated understanding of human affective phenomena, but an integrated field for affective research.

The Iowa Health Data Resource (IHDR): an innovative framework for transforming the clinical health data ecosystem.

IMPORTANCE: This manuscript will be of interest to most Clinical and Translational Science Awards (CTSA) as they retool for the increasing emphasis on translational science from translational research. This effort is an extension of the EDW4R work that most CTSAs have done to deploy infrastructure and tools for researchers to access clinical data. OBJECTIVES: The Iowa Health Data Resource (IHDR) is a strategic investment made by the University of Iowa to improve access to real-world health data. The goals of IHDR are to improve the speed of translational health research, to boost interdisciplinary collaboration, and to improve literacy about health data. The first objective toward this larger goal was to address gaps in data access, data literacy, lack of computational environments for processing Personal Health Information (PHI) and the lack of processes and expertise for creating transformative datasets. METHODS: A three-pronged approach was taken to address the objective. The approach involves integration of an intercollegiate team of non-informatics faculty and staff, a data enclave for secure patient data analyses, and novel comprehensive datasets. RESULTS: To date, all five of the health science colleges (dentistry, medicine, nursing, pharmacy, and public health) have had at least one staff and one faculty member complete the two-month experiential learning curriculum. Over the first two years of this project, nine cohorts totaling 36 data liaisons have been trained, including 18 faculty and 18 staff. IHDR data enclave eliminated the need to duplicate computational infrastructure inside the hospital firewall which reduced infrastructure, hardware and human resource costs while leveraging the existing expertise embedded in the university research computing team. The creation of a process to develop and implement transformative datasets has resulted in the creation of seven domain specific datasets to date. CONCLUSION: The combination of people, process, and technology facilitates collaboration and interdisciplinary research in a secure environment using curated data sets. While other organizations have implemented individual components to address EDW4R operational demands, the IHDR combines multiple resources into a novel, comprehensive ecosystem IHDR enables scientists to use analysis tools with electronic patient data to accelerate time to science.

The digestive system of a cricket pulverizes polyethylene microplastics down to the nanoplastic scale.

Microplastics (MPs; <5 mm) are a growing concern and a poorly understood threat to biota. We used a generalist insect (a cricket; Gryllodes sigillatus) to examine whether individuals would ingest and physically degrade MPs in their food. We fed crickets a range of concentrations (0, 2.5, 5, and 10% w/w) of fluorescent polyethylene MPs mixed into a standard diet and dissected the gut regions to isolate the MPs within. Comparing plastic content and fragment size within gut regions, we sought to identify whether and where crickets can fragment ingested MP particles. Given the digestive tract morphology of this species, we expected that the crickets would both ingest and egest the MPs. We also predicted that the MPs would be fragmented into smaller pieces during this digestive process. We found that G. sigillatus egested much smaller pieces than they ingested, and this fragmentation occurs early in the digestive process of this insect. We found this for both sexes as well as across the range of concentrations of MPs. The degree of plastic breakdown relative to plastic feeding time suggests that the ability to fragment MPs is intrinsic and not altered by how much time crickets have spent eating the plastics. The amount of plastics found in each region of the gut in relation to feeding time also suggests that this size and shape of PE microplastic does not cause any physical blockage in the gut. This lack of evidence for blockage is likely due to plastic breakdown. We found a ∼1000-fold reduction in plastic size occurs during passage through the digestive system, yielding particles very near nanoplastics (NPs; <1 µm), and likely smaller, that are then excreted back into the environment. These findings suggest that generalist insects can act as agents of plastic transformation in their environment if/when encountering MPs.

A neurophysiological limit and its biogeographic correlations: cold-induced spreading depolarization in tropical butterflies.

The physiology of insects is directly influenced by environmental temperature, and thermal tolerance is therefore intrinsically linked to their thermal niche and distribution. Understanding the mechanisms that limit insect thermal tolerance is crucial to predicting biogeography and range shifts. Recent studies on locusts and flies suggest that the critical thermal minimum (CTmin) follows a loss of CNS function via a spreading depolarization. We hypothesized that other insect taxa share this phenomenon. Here, we investigate whether spreading depolarization events occur in butterflies exposed to cold. Supporting our hypothesis, we found that exposure to stressful cold induced spreading depolarization in all 12 species tested. This reinforces the idea that spreading depolarization is a common mechanism underlying the insect CTmin. Furthermore, our results highlight how CNS function is tuned to match the environment of a species. Further research into the physiology underlying spreading depolarization will likely elucidate key mechanisms determining insect thermal tolerance and ecology.

Quantifying microplastic ingestion, degradation and excretion in insects using fluorescent plastics.

Plastic pollution is a growing threat to our natural environment. Plastic waste/pollution results from high emissions of both macro (>5 mm) and microplastics (MPs; <5 mm) as well as environmental fractioning of macroplastics into MPs. MPs have been shown to have a range of negative impacts on biota. Harmonized methods to accurately measure and count MPs from animal samples are limited, but what methods exist are not ideal for a controlled laboratory environment where plastic ingestion, degradation and elimination can be quantified and related to molecular, physiological and organismal traits. Here, we propose a complete method for isolating and quantifying fluorescent MPs by combining several previously reported approaches into one comprehensive workflow. We combine tissue dissection, organic material digestion, sample filtering and automated imaging techniques to show how fluorescently labelled MPs provided to insects (e.g. in their diet) in a laboratory setting can be isolated, identified and quantified. As a proof of concept, we fed crickets (Gryllodes sigillatus) a diet of 2.5% (w/w) fluorescently labelled plastics and isolated and quantified plastic particles within the gut and frass.

Locust gut epithelia do not become more permeable to fluorescent dextran and bacteria in the cold.

The insect gut, which plays a role in ion and water balance, has been shown to leak solutes in the cold. Cold stress can also activate insect immune systems, but it is unknown whether the leak of the gut microbiome is a possible immune trigger in the cold. We developed a novel feeding protocol to load the gut of locusts (Locusta migratoria) with fluorescent bacteria before exposing them to -2°C for up to 48 h. No bacteria were recovered from the hemolymph of cold-exposed locusts, regardless of exposure duration. To examine this further, we used an ex vivo gut sac preparation to re-test cold-induced fluorescent FITC-dextran leak across the gut and found no increased rate of leak. These results question not only the validity of FITC-dextran as a marker of paracellular barrier permeability in the gut, but also to what extent the insect gut becomes leaky in the cold.

A cold and quiet brain: mechanisms of insect CNS arrest at low temperatures.

Exposure to cold causes insects to enter a chill coma at species-specific temperatures and such temperature sensitivity contributes to geographic distribution and phenology. Coma results from abrupt spreading depolarization (SD) of neural tissue in the integrative centers of the central nervous system (CNS). SD abolishes neuronal signaling and the operation of neural circuits, like an off switch for the CNS. Turning off the CNS by allowing ion gradients to collapse will conserve energy and may offset negative consequences of temporary immobility. SD is modified by prior experience via rapid cold hardening (RCH) or cold acclimation that alter properties of Kv channels, Na+/K+-ATPase, and Na+/K+/2Cl- cotransporter. The stress hormone octopamine mediates RCH. Future progress depends on developing a more complete understanding of ion homeostasis in and of the insect CNS.

Cold-induced immune activation in chill-susceptible insects.

Chilling injuries in chill-susceptible insects, such as the model dipteran Drosophila melanogaster, have been well-documented as a consequence of stressful low-temperature exposures. Cold stress also causes upregulation of genes in the insect immune pathways, some of which are also upregulated following other forms of sterile stress. The adaptive significance and underlying mechanisms surrounding cold-induced immune activation, however, are still unclear. Here, we review recent work on the roles of reactive oxygen species, damage-associated molecular patterns, and antimicrobial peptides in insect immune signaling or function. Using this emerging knowledge, we propose a conceptual model linking biochemical and molecular causes of immune activation to its consequences during and following cold stress.

Metabolically defined body size and body shape phenotypes and risk of postmenopausal breast cancer in the European Prospective Investigation into Cancer and Nutrition.

BACKGROUND: Excess body fatness and hyperinsulinemia are both associated with an increased risk of postmenopausal breast cancer. However, whether women with high body fatness but normal insulin levels or those with normal body fatness and high levels of insulin are at elevated risk of breast cancer is not known. We investigated the associations of metabolically defined body size and shape phenotypes with the risk of postmenopausal breast cancer in a nested case-control study within the European Prospective Investigation into Cancer and Nutrition. METHODS: Concentrations of C-peptide-a marker for insulin secretion-were measured at inclusion prior to cancer diagnosis in serum from 610 incident postmenopausal breast cancer cases and 1130 matched controls. C-peptide concentrations among the control participants were used to define metabolically healthy (MH; in first tertile) and metabolically unhealthy (MU; >1st tertile) status. We created four metabolic health/body size phenotype categories by combining the metabolic health definitions with normal weight (NW; BMI < 25 kg/m2 , or WC < 80 cm, or WHR < 0.8) and overweight or obese (OW/OB; BMI ≥ 25 kg/m2 , or WC ≥ 80 cm, or WHR ≥ 0.8) status for each of the three anthropometric measures separately: (1) MHNW, (2) MHOW/OB, (3) MUNW, and (4) MUOW/OB. Conditional logistic regression was used to compute odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: Women classified as MUOW/OB were at higher risk of postmenopausal breast cancer compared to MHNW women considering BMI (OR = 1.58, 95% CI = 1.14-2.19) and WC (OR = 1.51, 95% CI = 1.09-2.08) cut points and there was also a suggestive increased risk for the WHR (OR = 1.29, 95% CI = 0.94-1.77) definition. Conversely, women with the MHOW/OB and MUNW were not at statistically significant elevated risk of postmenopausal breast cancer risk compared to MHNW women. CONCLUSION: These findings suggest that being overweight or obese and metabolically unhealthy raises risk of postmenopausal breast cancer while overweight or obese women with normal insulin levels are not at higher risk. Additional research should consider the combined utility of anthropometric measures with metabolic parameters in predicting breast cancer risk.

Dietary fatty acids and endometrial cancer risk within the European Prospective Investigation into Cancer and Nutrition.

BACKGROUND: Diet may impact important risk factors for endometrial cancer such as obesity and inflammation. However, evidence on the role of specific dietary factors is limited. We investigated associations between dietary fatty acids and endometrial cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC). METHODS: This analysis includes 1,886 incident endometrial cancer cases and 297,432 non-cases. All participants were followed up for a mean of 8.8 years. Multivariable Cox proportional hazard models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) of endometrial cancer across quintiles of individual fatty acids estimated from various food sources quantified through food frequency questionnaires in the entire EPIC cohort. The false discovery rate (q-values) was computed to control for multiple comparisons. RESULTS: Consumption of n-6 γ-linolenic acid was inversely associated with endometrial cancer risk (HR comparing 5th with 1st quintileQ5-Q1=0.77, 95% CI = 0.64; 0.92, ptrend=0.01, q-value = 0.15). This association was mainly driven by γ-linolenic acid derived from plant sources (HRper unit increment=0.94, 95%CI= (0.90;0.98), p = 0.01) but not from animal sources (HRper unit increment= 1.00, 95%CI = (0.92; 1.07), p = 0.92). In addition, an inverse association was found between consumption of n-3 α-linolenic acid from vegetable sources and endometrial cancer risk (HRper unit increment= 0.93, 95%CI = (0.87; 0.99), p = 0.04). No significant association was found between any other fatty acids (individual or grouped) and endometrial cancer risk. CONCLUSION: Our results suggest that higher consumption of γ-linolenic acid and α-linoleic acid from plant sources may be associated with lower risk of endometrial cancer.

Plasticity in Na+/K+-ATPase thermal kinetics drives variation in the temperature of cold-induced neural shutdown of adult Drosophila melanogaster.

Most insects can acclimate to changes in their thermal environment and counteract temperature effects on neuromuscular function. At the critical thermal minimum, a spreading depolarization (SD) event silences central neurons, but the temperature at which this event occurs can be altered through acclimation. SD is triggered by an inability to maintain ion homeostasis in the extracellular space in the brain and is characterized by a rapid surge in extracellular K+ concentration, implicating ion pump and channel function. Here, we focused on the role of the Na+/K+-ATPase specifically in lowering the SD temperature in cold-acclimated Drosophila melanogaster. After first confirming cold acclimation altered SD onset, we investigated the dependency of the SD event on Na+/K+-ATPase activity by injecting the inhibitor ouabain into the head of the flies to induce SD over a range of temperatures. Latency to SD followed the pattern of a thermal performance curve, but cold acclimation resulted in a left-shift of the curve to an extent similar to its effect on the SD temperature. With Na+/K+-ATPase activity assays and immunoblots, we found that cold-acclimated flies have ion pumps that are less sensitive to temperature, but do not differ in their overall abundance in the brain. Combined, these findings suggest a key role for plasticity in Na+/K+-ATPase thermal sensitivity in maintaining central nervous system function in the cold, and more broadly highlight that a single ion pump can be an important determinant of whether insects can respond to their environment to remain active at low temperatures.

Ingestion of Microplastic Fibres, But Not Microplastic Beads, Impacts Growth Rates in the Tropical House Cricket Gryllodes Sigillatus.

Microplastic is a growing concern as an environmental contaminant as it is ubiquitous in our ecosystems. Microplastics are present in terrestrial environments, yet the majority of studies have focused on the adverse effects of microplastics on aquatic biota. We hypothesized that microplastic ingestion by a terrestrial insect would have localized effects on gut health and nutrient absorption, such that prolonged dietary microplastic exposure would impact growth rate and adult body size. We further hypothesized that plastic form (fibres vs. beads) would influence these effects because of the nature of gut-plastic interactions. Freshly hatched tropical house crickets (Gryllodes sigillatus) were fed a standard diet containing different concentrations of either fluorescent polyethylene microplastic beads (75-105 µm), or untreated polyethylene terephthalate microfibers (< 5 mm) until they died or reached adulthood (approximately 8 weeks). Weight and body length were measured weekly and microplastic ingestion was confirmed through fluorescence microscopy and visual inspection of the frass. While, to our surprise, we found no effect of polyethylene bead ingestion on growth rate or final body size of G. sigillatus, females experienced a reduction in size and weight when fed high concentrations of polyethylene terephthalate microfibers. These results suggest that high concentrations of polyethylene beads of the 100 µm size range can pass through the cricket gut without a substantial negative effect on their growth and development time, but high concentrations of polyethylene terephthalate microfibers cannot. Although we report the negative effects of microplastic ingestion on the growth of G. sigillatus, it remains uncertain what threats microplastics pose to terrestrial insects.

Cytokine induced 3-D organotypic psoriasis skin model demonstrates distinct roles for NF-κB and JAK pathways in disease pathophysiology.

Psoriasis vulgaris is an inflammatory skin disease that affects 2%-3% of the population worldwide. One of the major challenges in discovering novel therapies is the poor translatability of animal models to human disease. Therefore, it is imperative to develop human preclinical models of psoriasis that are amenable to pharmacological intervention. Here, we report a 3-D reconstituted human epidermis (RHE) culture system treated with cytokines commonly associated with psoriasis (TNFα, IL-17A and IL-22) that reproduced some key features of the human disease. The effects on epidermal morphology, gene transcription and cytokine production, which are dysregulated in psoriasis were assessed. Certain morphological features of psoriatic epidermis were evident in cytokine-stimulated RHEs, including hypogranulosis and parakeratosis. In addition, RHEs responded to a cytokine mix in a dose-dependent manner by expressing genes and proteins associated with impaired keratinocyte differentiation (keratin 10/K10, loricrin), innate immune responses (S100A7, DEFB4, elafin) and inflammation (IL-1α, IL-6, IL-8, IL-10, IL-12/23p40, IL-36γ, GM-CSF and IFNγ) typical of psoriasis. These disease-relevant changes in morphology, gene transcription and cytokine production were robustly attenuated by pharmacologically blocking TNFα/IL-17A-induced NF-κB activation with IKK-2 inhibitor IV. Conversely, inhibition of IL-22-induced JAK1 signalling with ABT-317 strongly attenuated morphological features of the disease but had no effect on NFκB-dependent cytokine production, suggesting distinct mechanisms of action by the cytokines driving psoriasis. These data support the use of cytokine-induced RHE models for identifying and targeting keratinocyte signalling pathways important for disease progression and may provide translational insights into novel keratinocyte mechanisms for novel psoriasis therapies.

Pharmacological disruption of the MTDH-SND1 complex enhances tumor antigen presentation and synergizes with anti-PD-1 therapy in metastatic breast cancer.

Despite increased overall survival rates, curative options for metastatic breast cancer remain limited. We have previously shown that metadherin (MTDH) is frequently overexpressed in poor prognosis breast cancer, where it promotes metastasis and therapy resistance through its interaction with staphylococcal nuclease domain-containing 1 (SND1). Through genetic and pharmacological targeting of the MTDH-SND1 interaction, we reveal a key role for this complex in suppressing antitumor T cell responses in breast cancer. The MTDH-SND1 complex reduces tumor antigen presentation and inhibits T cell infiltration and activation by binding to and destabilizing Tap1/2 messenger RNAs, which encode key components of the antigen-presentation machinery. Following small-molecule compound C26-A6 treatment to disrupt the MTDH-SND1 complex, we showed enhanced immune surveillance and sensitivity to anti-programmed cell death protein 1 therapy in preclinical models of metastatic breast cancer, in support of this combination therapy as a viable approach to increase immune-checkpoint blockade therapy responses in metastatic breast cancer.

Small-molecule inhibitors that disrupt the MTDH-SND1 complex suppress breast cancer progression and metastasis.

Metastatic breast cancer is a leading health burden worldwide. Previous studies have shown that metadherin (MTDH) promotes breast cancer initiation, metastasis and therapy resistance; however, the therapeutic potential of targeting MTDH remains largely unexplored. Here, we used genetically modified mice and demonstrate that genetic ablation of Mtdh inhibits breast cancer development through disrupting the interaction with staphylococcal nuclease domain-containing 1 (SND1), which is required to sustain breast cancer progression in established tumors. We performed a small-molecule compound screening to identify a class of specific inhibitors that disrupts the protein-protein interaction (PPI) between MTDH and SND1 and show that our lead candidate compounds C26-A2 and C26-A6 suppressed tumor growth and metastasis and enhanced chemotherapy sensitivity in preclinical models of triple-negative breast cancer (TNBC). Our results demonstrate a significant therapeutic potential in targeting the MTDH-SND1 complex and identify a new class of therapeutic agents for metastatic breast cancer.

Improved object recognition memory using post-encoding repetitive transcranial magnetic stimulation.

BACKGROUND: Brain stimulation is known to affect canonical pathways and proteins involved in memory. However, there are conflicting results on the ability of brain stimulation to improve to memory, which may be due to variations in timing of stimulation. HYPOTHESIS: We hypothesized that repetitive transcranial magnetic stimulation (rTMS) given following a learning task and within the time period before retrieval could help improve memory. METHODS: We implanted male B6129SF2/J mice (n = 32) with a cranial attachment to secure the rTMS coil so that the mice could be given consistent stimulation to the frontal area whilst freely moving. Mice then underwent the object recognition test sampling phase and given treatment +3, +24, +48 h following the test. Treatment consisted of 10 min 10 Hz rTMS stimulation (TMS, n = 10), sham treatment (SHAM, n = 11) or a control group which did not do the behavior test or receive rTMS (CONTROL n = 11). At +72 h mice were tested for their exploration of the novel vs familiar object. RESULTS: At 72-h's, only the mice which received rTMS had greater exploration of the novel object than the familiar object. We further show that promoting synaptic GluR2 and maintaining synaptic connections in the perirhinal cortex and hippocampal CA1 are important for this effect. In addition, we found evidence that these changes were linked to CAMKII and CREB pathways in hippocampal neurons. CONCLUSION: By linking the known biological effects of rTMS to memory pathways we provide evidence that rTMS is effective in improving memory when given during the consolidation and maintenance phases.

A lack of repeatability creates the illusion of a trade-off between basal and plastic cold tolerance.

The thermotolerance-plasticity trade-off hypothesis predicts that ectotherms with greater basal thermal tolerance have a lower acclimation capacity. This hypothesis has been tested at both high and low temperatures but the results often conflict. If basal tolerance constrains plasticity (e.g. through shared mechanisms that create physiological constraints), it should be evident at the level of the individual, provided the trait measured is repeatable. Here, we used chill-coma onset temperature and chill-coma recovery time (CCO and CCRT; non-lethal thermal limits) to quantify cold tolerance of Drosophila melanogaster across two trials (pre- and post-acclimation). Cold acclimation improved cold tolerance, as expected, but individual measurements of CCO and CCRT in non-acclimated flies were not (or only slightly) repeatable. Surprisingly, however, there was still a strong correlation between basal tolerance and plasticity in cold-acclimated flies. We argue that this relationship is a statistical artefact (specifically, a manifestation of regression to the mean; RTM) and does not reflect a true trade-off or physiological constraint. Thermal tolerance trade-off patterns in previous studies that used similar methodology are thus likely to be impacted by RTM. Moving forward, controlling and/or correcting for RTM effects is critical to determining whether such a trade-off or physiological constraint exists.