Range-wide sources of variation in reproductive rates of northern spotted owls.

We conducted a range-wide investigation of the dynamics of site-level reproductive rate of northern spotted owls using survey data from 11 study areas across the subspecies geographic range collected during 1993-2018. Our analytical approach accounted for imperfect detection of owl pairs and misclassification of successful reproduction (i.e., at least one young fledged) and contributed further insights into northern spotted owl population ecology and dynamics. Both nondetection and state misclassification were important, especially because factors affecting these sources of error also affected focal ecological parameters. Annual probabilities of site occupancy were greatest at sites with successful reproduction in the previous year and lowest for sites not occupied by a pair in the previous year. Site-specific occupancy transition probabilities declined over time and were negatively affected by barred owl presence. Overall, the site-specific probability of successful reproduction showed substantial year-to-year fluctuations and was similar for occupied sites that did or did not experience successful reproduction the previous year. Site-specific probabilities for successful reproduction were very small for sites that were unoccupied the previous year. Barred owl presence negatively affected the probability of successful reproduction by northern spotted owls in Washington and California, as predicted, but the effect in Oregon was mixed. The proportions of sites occupied by northern spotted owl pairs showed steep, near-monotonic declines over the study period, with all study areas showing the lowest observed levels of occupancy to date. If trends continue it is likely that northern spotted owls will become extirpated throughout large portions of their range in the coming decades.

Conspecific and congeneric interactions shape increasing rates of breeding dispersal of northern spotted owls.

Breeding dispersal, the movement from one breeding territory to another, is rare for philopatric species that evolved within relatively stable environments, such as the old-growth coniferous forests of the Pacific Northwest. Although dispersal is not inherently maladaptive, the consequences of increased dispersal on population dynamics in populations whose historical dispersal rates are low could be significant, particularly for a declining species. We examined rates and possible causes of breeding dispersal based on a sample of 4,118 northern spotted owls (Strix occidentalis caurina) monitored in seven study areas over 28 yr, 1990-2017, in Oregon and Washington, USA. Using a multistate mark-resight analysis, we investigated the potential impacts of an emergent congeneric competitor (barred owl Strix varia) and forest alteration (extrinsic factors), and social and individual conditions (intrinsic factors) on 408 successive and 1,372 nonsuccessive dispersal events between years. The annual probability of breeding dispersal increased for individual owls that had also dispersed in the previous year and decreased for owls on territories with historically high levels of reproduction. Intrinsic factors including pair status, prior reproductive success, and experience at a site, were also associated with breeding dispersal movements. The percent of monitored owls dispersing each year increased from ˜7% early in the study to ˜25% at the end of the study, which coincided with a rapid increase in numbers of invasive and competitively dominant barred owls. We suggest that the results presented here can inform spotted owl conservation efforts as we identify factors contributing to changing rates of demographic parameters including site fidelity and breeding dispersal. Our study further shows that increasing rates of breeding dispersal associated with population declines contribute to population instability and vulnerability of northern spotted owls to extinction, and the prognosis is unlikely to change unless active management interventions are undertaken.

A need for speed in Bayesian population models: a practical guide to marginalizing and recovering discrete latent states.

Bayesian population models can be exceedingly slow due, in part, to the choice to simulate discrete latent states. Here, we discuss an alternative approach to discrete latent states, marginalization, that forms the basis of maximum likelihood population models and is much faster. Our manuscript has two goals: (1) to introduce readers unfamiliar with marginalization to the concept and provide worked examples and (2) to address topics associated with marginalization that have not been previously synthesized and are relevant to both Bayesian and maximum likelihood models. We begin by explaining marginalization using a Cormack-Jolly-Seber model. Next, we apply marginalization to multistate capture-recapture, community occupancy, and integrated population models and briefly discuss random effects, priors, and pseudo-R2 . Then, we focus on recovery of discrete latent states, defining different types of conditional probabilities and showing how quantities such as population abundance or species richness can be estimated in marginalized code. Last, we show that occupancy and site-abundance models with auto-covariates can be fit with marginalized code with minimal impact on parameter estimates. Marginalized code was anywhere from five to >1,000 times faster than discrete code and differences in inferences were minimal. Discrete latent states and fully conditional approaches provide the best estimates of conditional probabilities for a given site or individual. However, estimates for parameters and derived quantities such as species richness and abundance are minimally affected by marginalization. In the case of abundance, marginalized code is both quicker and has lower bias than an N-augmentation approach. Understanding how marginalization works shrinks the divide between Bayesian and maximum likelihood approaches to population models. Some models that have only been presented in a Bayesian framework can easily be fit in maximum likelihood. On the other hand, factors such as informative priors, random effects, or pseudo-R2 values may motivate a Bayesian approach in some applications. An understanding of marginalization allows users to minimize the speed that is sacrificed when switching from a maximum likelihood approach. Widespread application of marginalization in Bayesian population models will facilitate more thorough simulation studies, comparisons of alternative model structures, and faster learning.

The past and future roles of competition and habitat in the range-wide occupancy dynamics of Northern Spotted Owls.

Slow ecological processes challenge conservation. Short-term variability can obscure the importance of slower processes that may ultimately determine the state of a system. Furthermore, management actions with slow responses can be hard to justify. One response to slow processes is to explicitly concentrate analysis on state dynamics. Here, we focus on identifying drivers of Northern Spotted Owl (Strix occidentalis caurina) territorial occupancy dynamics across 11 study areas spanning their geographic range and forecasting response to potential management actions. Competition with Barred Owls (Strix varia) has increased Spotted Owl territory extinction probabilities across all study areas and driven recent declines in Spotted Owl populations. Without management intervention, the Northern Spotted Owl subspecies will be extirpated from parts of its current range within decades. In the short term, Barred Owl removal can be effective. Over longer time spans, however, maintaining or improving habitat conditions can help promote the persistence of northern spotted owl populations. In most study areas, habitat effects on expected Northern Spotted Owl territorial occupancy are actually greater than the effects of competition from Barred Owls. This study suggests how intensive management actions (removal of a competitor) with rapid results can complement a slower management action (i.e., promoting forest succession).

Dynamic N-occupancy models: estimating demographic rates and local abundance from detection-nondetection data.

Occupancy modeling is a widely used analytical technique for assessing species distributions and range dynamics. However, occupancy analyses frequently ignore variation in abundance of occupied sites, even though site abundances affect many of the parameters being estimated (e.g., extinction, colonization, detection probability). We introduce a new model ("dynamic N-occupancy") capable of providing accurate estimates of local abundance, population gains (reproduction/immigration), and apparent survival probabilities while accounting for imperfect detection using only detection/nondetection data. Our model utilizes heterogeneity in detection based on variations in site abundances to estimate latent demographic rates via a dynamic N-mixture modeling framework. We validate our model using simulations across a wide range of values and examine the data requirements, including the number of years and survey sites needed, for unbiased and precise estimation of parameters. We apply our model to estimate spatiotemporal heterogeneity in abundances of barred owls (Strix varia) within a recently invaded region in Oregon (USA). Estimates of apparent survival and population gains are consistent with those from a nearby radio-tracking study and elucidate how barred owl abundances have increased dramatically over time. The dynamic N-occupancy model greatly improves inferences on individual-level population processes from occupancy data by explicitly modeling the latent population structure.

The roles of competition and habitat in the dynamics of populations and species distributions.

The role of competition in structuring biotic communities at fine spatial scales is well known from detailed process-based studies. Our understanding of competition's importance at broader scales is less resolved and mainly based on static species distribution maps. Here, we bridge this gap by examining the joint occupancy dynamics of an invading species (Barred Owl, Strix varia) and a resident species (Northern Spotted Owl, Strix occidentalis caurina) in a 1000-km study area over a 22-year period. Past studies of these competitors have focused on the dynamics of one species at a time, hindering efforts to parse out the roles of habitat and competition and to forecast the future of the resident species. In addition, while these studies accounted for the imperfect detection of the focal species, no multi-season analysis of these species has accounted for the imperfect detection of the secondary species, potentially biasing inference. We analyzed survey data using models that combine the general multistate-multi-season occupancy modeling framework with autologistic modeling, allowing us to account for important aspects of our study system. We found that local extinction probability increases for each species when the other is present; however, the effect of the invader on the resident is greater. Although the species prefer different habitats, these habitats are highly correlated at the patch scale, and the impacts of invader on the resident are greatest in patches that would otherwise be optimal. As a consequence, competition leads to a weaker relationship between habitat and Northern Spotted Owl occupancy. Colonization and extinction rates of the invader are closely related to neighborhood occupancy, and over the first half of the study the availability of colonists limited the rate of population growth. Competition is likely to exclude the resident species, both through its immediate effects on local extinction and by indirectly lowering colonization rates as Northern Spotted Owl occupancy declines. Our analysis suggests that dispersal limitation affects both the invasion dynamics and the scale at which the effects of competition are observed. We also provide predictions regarding the potential costs and benefits of managing Barred Owl populations at different target levels.

A miniaturized culture platform for control of the metabolic environment.

The heart is a metabolic "omnivore" and adjusts its energy source depending on the circulating metabolites. Human cardiac organoids, a three-dimensional in vitro model of the heart wall, are a useful tool to study cardiac physiology and pathology. However, cardiac tissue naturally experiences shear stress and nutrient fluctuations via blood flow in vivo, whilst in vitro models are conventionally cultivated in a static medium. This necessitates the regular refreshing of culture media, which creates acute cellular disturbances and large metabolic fluxes. To culture human cardiac organoids in a more physiological manner, we have developed a perfused bioreactor for cultures in a 96-well plate format. The designed bioreactor is easy to fabricate using a common culture plate and a 3D printer. Its open system allows for the use of traditional molecular biology techniques, prevents flow blockage issues, and provides easy access for sampling and cell assays. We hypothesized that a perfused culture would create more stable environment improving cardiac function and maturation. We found that lactate is rapidly produced by human cardiac organoids, resulting in large fluctuations in this metabolite under static culture. Despite this, neither medium perfusion in bioreactor culture nor lactate supplementation improved cardiac function or maturation. In fact, RNA sequencing revealed little change across the transcriptome. This demonstrates that cardiac organoids are robust in response to fluctuating environmental conditions under normal physiological conditions. Together, we provide a framework for establishing an easily accessible perfusion system that can be adapted to a range of miniaturized cell culture systems.

The Kidneys Are Not All Normal: Transplanted Kidneys and Their Speckle Distributions.

OBJECTIVE: Modelling ultrasound speckle to characterise tissue properties has generated considerable interest. As speckle is dependent on the underlying tissue architecture, modelling it may aid in tasks such as segmentation or disease detection. For the transplanted kidney, where ultrasound is used to investigate dysfunction, it is unknown which statistical distribution best characterises such speckle. This applies to the regions of the transplanted kidney: the cortex, the medulla and the central echogenic complex. Furthermore, it is unclear how these distributions vary by patient variables such as age, sex, body mass index, primary disease or donor type. These traits may influence speckle modelling given their influence on kidney anatomy. We investigate these two aims. METHODS: B-mode images from n = 821 kidney transplant recipients (one image per recipient) were automatically segmented into the cortex, medulla and central echogenic complex using a neural network. Seven distinct probability distributions were fitted to each region's histogram, and statistical analysis was performed. DISCUSSION: The Rayleigh and Nakagami distributions had model parameters that differed significantly between the three regions (p ≤ 0.05). Although both had excellent goodness of fit, the Nakagami had higher Kullbeck-Leibler divergence. Recipient age correlated weakly with scale in the cortex (Ω: ρ = 0.11, p = 0.004), while body mass index correlated weakly with shape in the medulla (m: ρ = 0.08, p = 0.04). Neither sex, primary disease nor donor type exhibited any correlation. CONCLUSION: We propose the Nakagami distribution be used to characterize transplanted kidneys regionally independent of disease etiology and most patient characteristics.

Automatic measurement of kidney dimensions in two-dimensional ultrasonography is comparable to expert sonographers.

Purpose: Length and width measurements of the kidneys aid in the detection and monitoring of structural abnormalities and organ disease. Manual measurement results in intra- and inter-rater variability, is complex and time-consuming, and is fraught with error. We propose an automated approach based on machine learning for quantifying kidney dimensions from two-dimensional (2D) ultrasound images in both native and transplanted kidneys. Approach: An nnU-net machine learning model was trained on 514 images to segment the kidney capsule in standard longitudinal and transverse views. Two expert sonographers and three medical students manually measured the maximal kidney length and width in 132 ultrasound cines. The segmentation algorithm was then applied to the same cines, region fitting was performed, and the maximum kidney length and width were measured. Additionally, single kidney volume for 16 patients was estimated using either manual or automatic measurements. Results: The experts resulted in length of 84.8±26.4 mm [95% CI: 80.0, 89.6] and a width of 51.8±10.5 mm [49.9, 53.7]. The algorithm resulted a length of 86.3±24.4 [81.5, 91.1] and a width of 47.1±12.8 [43.6, 50.6]. Experts, novices, and the algorithm did not statistically significant differ from one another (p>0.05). Bland-Altman analysis showed the algorithm produced a mean difference of 2.6 mm (SD = 1.2) from experts, compared to novices who had a mean difference of 3.7 mm (SD = 2.9 mm). For volumes, mean absolute difference was 47 mL (31%) consistent with ∼1 mm error in all three dimensions. Conclusions: This pilot study demonstrates the feasibility of an automatic tool to measure in vivo kidney biometrics of length, width, and volume from standard 2D ultrasound views with comparable accuracy and reproducibility to expert sonographers. Such a tool may enhance workplace efficiency, assist novices, and aid in tracking disease progression.

Pharmacological or genetic inhibition of LTCC promotes cardiomyocyte proliferation through inhibition of calcineurin activity.

Cardiomyocytes (CMs) lost during ischemic cardiac injury cannot be replaced due to their limited proliferative capacity, which leads to progressive heart failure. Calcium (Ca2+) is an important signal transducer that regulates key cellular processes, but its role in regulating CM proliferation is incompletely understood. A drug screen targeting proteins involved in CM calcium cycling in human embryonic stem cell-derived cardiac organoids (hCOs) revealed that only the inhibition of L-Type Calcium Channel (LTCC), but not other Ca2+ regulatory proteins (SERCA or RYR), induced the CM cell cycle. Furthermore, overexpression of Ras-related associated with Diabetes (RRAD), an endogenous inhibitor of LTCC, induced CM cell cycle activity in vitro, in human cardiac slices, and in vivo. Mechanistically, LTCC inhibition by RRAD induces the cell cycle in CMs by modulating calcineurin activity and translocating Hoxb13 to the CM nucleus. Together, this represents a robust pathway for regenerative strategies.

Effect of flow change on brain injury during an experimental model of differential hypoxaemia in cardiogenic shock supported by extracorporeal membrane oxygenation.

Differential hypoxaemia (DH) is common in patients supported by femoral veno-arterial extracorporeal membrane oxygenation (V-A ECMO) and can cause cerebral hypoxaemia. To date, no models have studied the direct impact of flow on cerebral damage. We investigated the impact of V-A ECMO flow on brain injury in an ovine model of DH. After inducing severe cardiorespiratory failure and providing ECMO support, we randomised six sheep into two groups: low flow (LF) in which ECMO was set at 2.5 L min-1 ensuring that the brain was entirely perfused by the native heart and lungs, and high flow (HF) in which ECMO was set at 4.5 L min-1 ensuring that the brain was at least partially perfused by ECMO. We used invasive (oxygenation tension-PbTO2, and cerebral microdialysis) and non-invasive (near infrared spectroscopy-NIRS) neuromonitoring, and euthanised animals after five hours for histological analysis. Cerebral oxygenation was significantly improved in the HF group as shown by higher PbTO2 levels (+ 215% vs - 58%, p = 0.043) and NIRS (67 ± 5% vs 49 ± 4%, p = 0.003). The HF group showed significantly less severe brain injury than the LF group in terms of neuronal shrinkage, congestion and perivascular oedema (p < 0.0001). Cerebral microdialysis values in the LF group all reached the pathological thresholds, even though no statistical difference was found between the two groups. Differential hypoxaemia can lead to cerebral damage after only a few hours and mandates a thorough neuromonitoring of patients. An increase in ECMO flow was an effective strategy to reduce such damages.

Donor heart ischemic time can be extended beyond 9 hours using hypothermic machine perfusion in sheep.

BACKGROUND: The global shortage of donor hearts available for transplantation is a major problem for the treatment of end-stage heart failure. The ischemic time for donor hearts using traditional preservation by standard static cold storage (SCS) is limited to approximately 4 hours, beyond which the risk for primary graft dysfunction (PGD) significantly increases. Hypothermic machine perfusion (HMP) of donor hearts has been proposed to safely extend ischemic time without increasing the risk of PGD. METHODS: Using our sheep model of 24 hours brain death (BD) followed by orthotopic heart transplantation (HTx), we examined post-transplant outcomes in recipients following donor heart preservation by HMP for 8 hours, compared to donor heart preservation for 2 hours by either SCS or HMP. RESULTS: Following HTx, all HMP recipients (both 2 hours and 8 hours groups) survived to the end of the study (6 hours after transplantation and successful weaning from cardiopulmonary bypass), required less vasoactive support for hemodynamic stability, and exhibited superior metabolic, fluid status and inflammatory profiles compared to SCS recipients. Contractile function and cardiac damage (troponin I release and histological assessment) was comparable between groups. CONCLUSIONS: Overall, compared to current clinical SCS, recipient outcomes following transplantation are not adversely impacted by extending HMP to 8 hours. These results have important implications for clinical transplantation where longer ischemic times may be required (e.g., complex surgical cases, transport across long distances). Additionally, HMP may allow safe preservation of "marginal" donor hearts that are more susceptible to myocardial injury and facilitate increased utilization of these hearts for transplantation.

Vascular cells improve functionality of human cardiac organoids.

Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor ß (PDGFRß) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models.

Neighborhood and habitat effects on vital rates: expansion of the Barred Owl in the Oregon coast ranges.

In this paper, we modify dynamic occupancy models developed for detection-nondetection data to allow for the dependence of local vital rates on neighborhood occupancy, where neighborhood is defined very flexibly. Such dependence of occupancy dynamics on the status of a relevant neighborhood is pervasive, yet frequently ignored. Our framework permits joint inference about the importance of neighborhood effects and habitat covariates in determining colonization and extinction rates. Our specific motivation is the recent expansion of the Barred Owl (Strix varia) in western Oregon, USA, over the period 1990-2010. Because the focal period was one of dramatic range expansion and local population increase, the use of models that incorporate regional occupancy (sources of colonists) as determinants of dynamic rate parameters is especially appropriate. We began our analysis of 21 years of Barred Owl presence/nondetection data in the Tyee Density Study Area (TDSA) by testing a suite of six models that varied only in the covariates included in the modeling of detection probability. We then tested whether models that used regional occupancy as a covariate for colonization and extinction outperformed models with constant or year-specific colonization or extinction rates. Finally we tested whether habitat covariates improved the AIC of our models, focusing on which habitat covariates performed best, and whether the signs of habitat effects are consistent with a priori hypotheses. We conclude that all covariates used to model detection probability lead to improved AIC, that regional occupancy influences colonization and extinction rates, and that habitat plays an important role in determining extinction and colonization rates. As occupancy increases from low levels toward equilibrium, colonization increases and extinction decreases, presumably because there are more and more dispersing juveniles. While both rates are affected, colonization increases more than extinction decreases. Colonization is higher and extinction is lower in survey polygons with more riparian forest. The effects of riparian forest on extinction rates are greater than on colonization rates. Model results have implications for management of the invading Barred Owl, both through habitat alteration and removal.

An innovative ovine model of severe cardiopulmonary failure supported by veno-arterial extracorporeal membrane oxygenation.

Refractory cardiogenic shock (CS) often requires veno-arterial extracorporeal membrane oxygenation (VA-ECMO) to sustain end-organ perfusion. Current animal models result in heterogenous cardiac injury and frequent episodes of refractory ventricular fibrillation. Thus, we aimed to develop an innovative, clinically relevant, and titratable model of severe cardiopulmonary failure. Six sheep (60 ± 6 kg) were anaesthetized and mechanically ventilated. VA-ECMO was commenced and CS was induced through intramyocardial injections of ethanol. Then, hypoxemic/hypercapnic pulmonary failure was achieved, through substantial decrease in ventilatory support. Echocardiography was used to compute left ventricular fractional area change (LVFAC) and cardiac Troponin I (cTnI) was quantified. After 5 h, the animals were euthanised and the heart was retrieved for histological evaluations. Ethanol (58 ± 23 mL) successfully induced CS in all animals. cTnI levels increased near 5000-fold. CS was confirmed by a drop in systolic blood pressure to 67 ± 14 mmHg, while lactate increased to 4.7 ± 0.9 mmol/L and LVFAC decreased to 16 ± 7%. Myocardial samples corroborated extensive cellular necrosis and inflammatory infiltrates. In conclusion, we present an innovative ovine model of severe cardiopulmonary failure in animals on VA-ECMO. This model could be essential to further characterize CS and develop future treatments.

A clinically relevant sheep model of orthotopic heart transplantation 24 h after donor brainstem death.

BACKGROUND: Heart transplantation (HTx) from brainstem dead (BSD) donors is the gold-standard therapy for severe/end-stage cardiac disease, but is limited by a global donor heart shortage. Consequently, innovative solutions to increase donor heart availability and utilisation are rapidly expanding. Clinically relevant preclinical models are essential for evaluating interventions for human translation, yet few exist that accurately mimic all key HTx components, incorporating injuries beginning in the donor, through to the recipient. To enable future assessment of novel perfusion technologies in our research program, we thus aimed to develop a clinically relevant sheep model of HTx following 24 h of donor BSD. METHODS: BSD donors (vs. sham neurological injury, 4/group) were hemodynamically supported and monitored for 24 h, followed by heart preservation with cold static storage. Bicaval orthotopic HTx was performed in matched recipients, who were weaned from cardiopulmonary bypass (CPB), and monitored for 6 h. Donor and recipient blood were assayed for inflammatory and cardiac injury markers, and cardiac function was assessed using echocardiography. Repeated measurements between the two different groups during the study observation period were assessed by mixed ANOVA for repeated measures. RESULTS: Brainstem death caused an immediate catecholaminergic hemodynamic response (mean arterial pressure, p = 0.09), systemic inflammation (IL-6 - p = 0.025, IL-8 - p = 0.002) and cardiac injury (cardiac troponin I, p = 0.048), requiring vasopressor support (vasopressor dependency index, VDI, p = 0.023), with normalisation of biomarkers and physiology over 24 h. All hearts were weaned from CPB and monitored for 6 h post-HTx, except one (sham) recipient that died 2 h post-HTx. Hemodynamic (VDI - p = 0.592, heart rate - p = 0.747) and metabolic (blood lactate, p = 0.546) parameters post-HTx were comparable between groups, despite the observed physiological perturbations that occurred during donor BSD. All p values denote interaction among groups and time in the ANOVA for repeated measures. CONCLUSIONS: We have successfully developed an ovine HTx model following 24 h of donor BSD. After 6 h of critical care management post-HTx, there were no differences between groups, despite evident hemodynamic perturbations, systemic inflammation, and cardiac injury observed during donor BSD. This preclinical model provides a platform for critical assessment of injury development pre- and post-HTx, and novel therapeutic evaluation.