Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Linking environmental salinity to respiratory phenotypes and metabolic rate in fishes: a data mining and modelling approach.

The gill is the primary site of ionoregulation and gas exchange in adult teleost fishes. However, those characteristics that benefit diffusive gas exchange (large, thin gills) may also enhance the passive equilibration of ions and water that threaten osmotic homeostasis. Our literature review revealed that gill surface area and thickness were similar in freshwater (FW) and seawater (SW) species; however, the diffusive oxygen (O2) conductance (Gd) of the gill was lower in FW species. While a lower Gd may reduce ion losses, it also limits O2 uptake capacity and possibly aerobic performance in situations of high O2 demand (e.g. exercise) or low O2 availability (e.g. environmental hypoxia). We also found that FW fishes had significantly higher haemoglobin (Hb)-O2 binding affinities than SW species, which will increase the O2 diffusion gradient across the gills. Therefore, we hypothesized that the higher Hb-O2 affinity of FW fishes compensates, in part, for their lower Gd. Using a combined literature review and modelling approach, our results show that a higher Hb-O2 affinity in FW fishes increases the flux of O2 across their low-Gd gills. In addition, FW and SW teleosts can achieve similar maximal rates of O2 consumption (MO2,max) and hypoxia tolerance (Pcrit) through different combinations of Hb-O2 affinity and Gd. Our combined data identified novel patterns in gill and Hb characteristics between FW and SW fishes and our modelling approach provides mechanistic insight into the relationship between aerobic performance and species distribution ranges, generating novel hypotheses at the intersection of cardiorespiratory and ionoregulatory fish physiology.

How the gut and liver hibernate.

For hibernating mammals, the transition from summer active to winter hibernation seasons come with significant remodeling at cellular, organ and whole organism levels. This review summarizes and synthesizes what is known about hibernation-related remodeling in the gastrointestinal tract of the thirteen-lined ground squirrel, including intestinal and hepatic physiology and the gut microbiota. Hibernation alters intestinal epithelial, immune and cell survival pathways in ways that point to a protective phenotype in the face of prolonged fasting and major fluctuations in nutrient and oxygen delivery during torpor-arousal cycles. The prolonged fasting associated with hibernation alters lipid metabolism and systemic cholesterol dynamics, with both the gut and liver participating in these changes. Fasting also affects the gut microbiota, altering the abundance, composition and diversity of gut microbes and impacting the metabolites they produce in ways that may influence hibernation-related traits in the host. Finally, interventional studies have demonstrated that the hibernation phenotype confers resistance to experimental ischemia-reperfusion injury in both gut and liver, suggesting potential therapeutic roadmaps. We propose that the plasticity inherent to hibernation biology may contribute to this stress tolerance, and in the spirit of August Krogh, makes hibernators particularly valuable for study to identify solutions to certain problems.

A cost-effectiveness analysis of genomic sequencing in a prospective versus historical cohort of complex pediatric patients.

PURPOSE: Cost-effectiveness evaluations of first-line genomic sequencing (GS) in the diagnosis of children with genetic conditions are limited by the lack of well-defined comparative cohorts. We sought to evaluate the cost-effectiveness of early GS in pediatric patients with complex monogenic conditions compared with a matched historical cohort. METHODS: Data, including investigation costs, were collected in a prospective cohort of 92 pediatric patients undergoing singleton GS over an 18-month period (2016-2017) with two of the following: a condition with high mortality, multisystem disease involving three or more organs, or severe limitation of daily function. Comparative data were collected in a matched historical cohort who underwent traditional investigations in the years 2012-2013. RESULTS: GS yielded a diagnosis in 42% while traditional investigations yielded a diagnosis in 23% (p = 0.003). A change in management was experienced by 74% of patients diagnosed following GS, compared with 32% diagnosed following traditional investigations. Singleton GS at a cost of AU$3100 resulted in a mean saving per person of AU$3602 (95% confidence interval [CI] AU$2520-4685). Cost savings occurred across all investigation subtypes and were only minimally offset by clinical management costs. CONCLUSION: GS in complex pediatric patients saves significant costs and doubles the diagnostic yield of traditional approaches.

Shifts in metabolic fuel use coincide with maximal rates of ventilation and body surface rewarming in an arousing hibernator.

It is well established that hibernating mammals rely predominantly on lipid stores to fuel metabolism throughout the hibernation season. However, it is unclear if other endogenous fuels contribute to the rapid, ~400-fold increase in metabolic rate during the early phase of arousal from torpor. To investigate this issue, we used cavity ring-down spectroscopy, a technique that provides a real-time indication of fuel use by measuring the ratio of 13C to 12C in the exhaled CO2 of arousing 13-lined ground squirrels (Ictidomys tridecemlineatus). We used infrared thermography to simultaneously measure ventilation and surface temperature change in various body regions, and we interpreted these data in light of changing plasma metabolite abundances at multiple stages of arousal from torpor. We found that hibernating squirrels use a combination of lipids and, likely, carbohydrates to fuel the initial ~60 min of arousal before switching to predominantly lipid oxidation. This fuel switch coincided with times of maximal rates of ventilation and rewarming of different body surface regions, including brown adipose tissue. Infrared thermography revealed zonal rewarming, whereby the brown adipose tissue region was the first to warm, followed by the thoracic and head regions and, finally, the posterior half of the body. Consistent with the results from cavity ring-down spectroscopy, plasma metabolite dynamics during early arousal suggested a large reliance on fatty acids, with a contribution from carbohydrates and glycerol. Because of their high oxidative flux rates and efficient O2 use, carbohydrates might be an advantageous metabolic fuel during the early phase of arousal, when metabolic demands are high but ventilation rates and, thus, O2 supply are relatively low.

The utility and determination of Pcrit in fishes.

The critical O2 tension (Pcrit) is the lowest PO2  at which an animal can maintain some benchmark rate of O2 uptake (MO2 ). This PO2  has long served as a comparator of hypoxia tolerance in fishes and aquatic invertebrates, but its usefulness in this role, particularly when applied to fishes, has recently been questioned. We believe that Pcrit remains a useful comparator of hypoxia tolerance provided it is determined using the proper methods and hypoxia tolerance is clearly defined. Here, we review the available methods for each of the three steps of Pcrit determination: (1) measuring the most appropriate benchmark MO2  state for Pcrit determination (MO2,std, the MO2  required to support standard metabolic rate); (2) reducing water PO2 ; and (3) calculating Pcrit from the MO2  versus PO2  curve. We make suggestions on best practices for each step and for how to report Pcrit results to maximize their comparative value. We also discuss the concept of hypoxia tolerance and how Pcrit relates to a fish's overall hypoxia tolerance. When appropriate methods are used, Pcrit provides useful comparative physiological and ecological information about the aerobic contributions to a fish's hypoxic survival. When paired with other hypoxia-related physiological measurements (e.g. lactate accumulation, calorimetry-based measurements of metabolic depression, loss-of-equilibrium experiments), Pcrit contributes to a comprehensive understanding of how a fish combines aerobic metabolism, anaerobic metabolism and metabolic depression in an overall strategy for hypoxia tolerance.

Meeting the challenges of implementing rapid genomic testing in acute pediatric care.

PURPOSE: The purpose of the study was to implement and prospectively evaluate the outcomes of a rapid genomic diagnosis program at two pediatric tertiary centers. METHODS: Rapid singleton whole-exome sequencing (rWES) was performed in acutely unwell pediatric patients with suspected monogenic disorders. Laboratory and clinical barriers to implementation were addressed through continuous multidisciplinary review of process parameters. Diagnostic and clinical utility and cost-effectiveness of rWES were assessed. RESULTS: Of 40 enrolled patients, 21 (52.5%) received a diagnosis, with median time to report of 16 days (range 9-109 days). A result was provided during the first hospital admission in 28 of 36 inpatients (78%). Clinical management changed in 12 of the 21 diagnosed patients (57%), including the provision of lifesaving treatment, avoidance of invasive biopsies, and palliative care guidance. The cost per diagnosis was AU$13,388 (US$10,453). Additional cost savings from avoidance of planned tests and procedures and reduced length of stay are estimated to be around AU$543,178 (US$424,101). The clear relative advantage of rWES, joint clinical and laboratory leadership, and the creation of a multidisciplinary "rapid team" were key to successful implementation. CONCLUSION: Rapid genomic testing in acute pediatrics is not only feasible but also cost-effective, and has high diagnostic and clinical utility. It requires a whole-of-system approach for successful implementation.

Can variation among hypoxic environments explain why different fish species use different hypoxic survival strategies?

In aquatic environments, hypoxia is a multi-dimensional stressor that can vary in O2 level (partial pressure of O2 in water, PwO2 ), rate of induction and duration. Natural hypoxic environments can therefore be very different from one another. For the many fish species that have evolved to cope with these different hypoxic environments, survival requires adjusting energy supply and demand pathways to maintain energy balance. The literature describes innumerable ways that fishes combine aerobic metabolism, anaerobic metabolism and metabolic rate depression (MRD) to accomplish this, but it is unknown whether the evolutionary paths leading to these different strategies are determined primarily by species' phylogenetic histories, genetic constraint or their native hypoxic environments. We explored this idea by devising a four-quadrant matrix that bins different aquatic hypoxic environments according to their duration and PwO2  characteristics. We then systematically mined the literature for well-studied species native to environments within each quadrant, and, for each of 10 case studies, described the species' total hypoxic response (THR), defined as its hypoxia-induced combination of sustained aerobic metabolism, enhanced anaerobic metabolism and MRD, encompassing also the mechanisms underlying these metabolic modes. Our analysis revealed that fishes use a wide range of THRs, but that distantly related species from environments within the same matrix quadrant have converged on similar THRs. For example, environments of moderately hypoxic PwO2  favoured predominantly aerobic THRs, whereas environments of severely hypoxic PwO2  favoured MRD. Capacity for aerial emergence as well as predation pressure (aquatic and aerial) also contributed to these responses, in addition to other biotic and abiotic factors. Generally, it appears that the particular type of hypoxia experienced by a fish plays a major role in shaping its particular THR.

Rates of hypoxia induction alter mechanisms of O2 uptake and the critical O2 tension of goldfish.

The rate of hypoxia induction (RHI) is an important but overlooked dimension of environmental hypoxia that may affect an organism's survival. We hypothesized that, compared with rapid RHI, gradual RHI will afford an organism more time to alter plastic phenotypes associated with O2 uptake and subsequently reduce the critical O2 tension (Pcrit) of the rate of O2 uptake (MO2 ). We investigated this by determining Pcrit values for goldfish exposed to short (∼24 min), typical (∼84 min) and long (∼480 min) duration Pcrit trials to represent different RHIs. Consistent with our predictions, long duration Pcrit trials yielded significantly lower Pcrit values (1.0-1.4 kPa) than short and typical duration trials, which did not differ (2.6±0.3 and 2.5±0.2 kPa, respectively). Parallel experiments revealed these time-related shifts in Pcrit were associated with changes to aspects of the O2 transport cascade that took place over the hypoxia exposures: gill surface areas and haemoglobin-O2 binding affinities were significantly higher in fish exposed to gradual RHIs over 480 min than fish exposed to rapid RHIs over 60 min. Our results also revealed that the choice of respirometric technique (i.e. closed versus intermittent) does not affect Pcrit or routine MO2 , despite the significantly reduced water pH and elevated CO2 and ammonia levels measured following closed-circuit Pcrit trials of ∼90 min. Together, our results demonstrate that gradual RHIs result in alterations to physiological parameters that enhance O2 uptake in hypoxic environments. An organism's innate Pcrit is therefore most accurately determined using rapid RHIs (<90 min) so as to avoid the confounding effects of hypoxic acclimation.

Calorespirometry reveals that goldfish prioritize aerobic metabolism over metabolic rate depression in all but near-anoxic environments.

Metabolic rate depression (MRD) has long been proposed as the key metabolic strategy of hypoxic survival, but surprisingly, the effects of changes in hypoxic O2 tensions (PwO2 ) on MRD are largely unexplored. We simultaneously measured the O2 consumption rate (MO2 ) and metabolic heat of goldfish using calorespirometry to test the hypothesis that MRD is employed at hypoxic PwO2  values and initiated just below Pcrit, the PwO2 below which MO2  is forced to progressively decline as the fish oxyconforms to decreasing PwO2 Specifically, we used closed-chamber and flow-through calorespirometry together with terminal sampling experiments to examine the effects of PwO2  and time on MO2 , metabolic heat and anaerobic metabolism (lactate and ethanol production). The closed-chamber and flow-through experiments yielded slightly different results. Under closed-chamber conditions with a continually decreasing PwO2 , goldfish showed a Pcrit of 3.0±0.3 kPa and metabolic heat production was only depressed at PwO2  between 0 and 0.67 kPa. Under flow-through conditions with PwO2  held at a variety of oxygen tensions for 1 and 4 h, goldfish also initiated MRD between 0 and 0.67 kPa but maintained MO2  to 0.67 kPa, indicating that Pcrit is at or below this PwO2 Anaerobic metabolism was strongly activated at PwO2 ≤1.3 kPa, but only used within the first hour at 1.3 and 0.67 kPa, as anaerobic end-products did not accumulate between 1 and 4 h exposure. Taken together, it appears that goldfish reserve MRD for near-anoxia, supporting routine metabolic rate at sub-PcritPwO2  values with the help of anaerobic glycolysis in the closed-chamber experiments, and aerobically after an initial (<1 h) activation of anaerobic metabolism in the flow-through experiments, even at 0.67 kPa PwO2.

Metabolic depression and the evolution of hypoxia tolerance in threespine stickleback, Gasterosteus aculeatus.

Anthropogenic increases in global temperature and agricultural runoff are increasing the prevalence of aquatic hypoxia throughout the world. We investigated the potential for a relatively rapid evolution of hypoxia tolerance using two isolated (for less than 11 000 years) populations of threespine stickleback: one from a lake that experiences long-term hypoxia (Alta Lake, British Columbia) and one from a lake that does not (Trout Lake, British Columbia). Loss-of-equilibrium (LOE) experiments revealed that the Alta Lake stickleback were significantly more tolerant of hypoxia than the Trout Lake stickleback, and calorimetry experiments revealed that the enhanced tolerance of Alta Lake stickleback may be associated with their ability to depress metabolic rate (as indicated by metabolic heat production) by 33% in hypoxia. The two populations showed little variation in their capacities for O2 extraction and anaerobic metabolism. These results reveal that intraspecific variation in hypoxia tolerance can develop over relatively short geological timescales, as can metabolic rate depression, a complex biochemical response that may be favoured in long-term hypoxic environments.

Ambient CO2, fish behaviour and altered GABAergic neurotransmission: exploring the mechanism of CO2-altered behaviour by taking a hypercapnia dweller down to low CO2 levels.

Recent studies suggest that projected rises of aquatic CO2 levels cause acid-base regulatory responses in fishes that lead to altered GABAergic neurotransmission and disrupted behaviour, threatening fitness and population survival. It is thought that changes in Cl(-) and HCO3 (-) gradients across neural membranes interfere with the function of GABA-gated anion channels (GABAA receptors). So far, such alterations have been revealed experimentally by exposing species living in low-CO2 environments, like many oceanic habitats, to high levels of CO2 (hypercapnia). To examine the generality of this phenomenon, we set out to study the opposite situation, hypothesizing that fishes living in typically hypercapnic environments also display behavioural alterations if exposed to low CO2 levels. This would indicate that ion regulation in the fish brain is fine-tuned to the prevailing CO2 conditions. We quantified pH regulatory variables and behavioural responses of Pangasianodon hypophthalmus, a fish native to the hypercapnic Mekong River, acclimated to high-CO2 (3.1 kPa) or low-CO2 (0.04 kPa) water. We found that brain and blood pH was actively regulated and that the low-CO2 fish displayed significantly higher activity levels, which were reduced after treatment with gabazine, a GABAA receptor blocker. This indicates an involvement of the GABAA receptor and altered Cl(-) and HCO3 (-) ion gradients. Indeed, Goldman calculations suggest that low levels of environmental CO2 may cause significant changes in neural ion gradients in P. hypophthalmus. Taken together, the results suggest that brain ion regulation in fishes is fine-tuned to the prevailing ambient CO2 conditions and is prone to disruption if these conditions change.

Characterizing the metabolic capacity of the anoxic hagfish heart.

Pacific hagfish, Eptatretus stoutii, can recover from 36 h of anoxia at 10°C. Such anoxia tolerance demands the mobilization of anaerobic fuels and the removal of metabolic wastes--processes that require a functional heart. The purpose of this study was to measure the metabolic response of the excised, cannulated hagfish heart to anoxia using direct calorimetry. These experiments were coupled with measurements of cardiac pH and metabolite concentrations, at multiple time points, to monitor acid-base balance and anaerobic ATP production. We also exposed hagfish to anoxia to compare the in vitro responses of the excised hearts with the in vivo responses. The calorimetry results revealed a significant reduction in the rate of metabolic heat production over the first hour of anoxia exposure, and a recovery over the subsequent 6 h. This response is likely attributable to a rapid anoxia-induced depression of aerobic ATP-production pathways followed by an upregulation of anaerobic ATP-production pathways such that the ATP production rate was restored to that measured in normoxia. Glycogen-depletion measurements suggest that metabolic processes were initially supported by glycolysis but that an alternative fuel source was used to support the sustained rates of ATP production. The maintenance of intracellular pH during anoxia indicates a remarkable ability of the myocytes to buffer/regulate protons and thus protect cardiac function. Altogether, these results illustrate that the low metabolic demand of the hagfish heart allows for near-routine levels of cardiac metabolism to be supported anaerobically. This is probably a significant contributor to the hagfish's exceptional anoxia tolerance.

Sex and Age Differences in Atypical Chief Complaints for Acute Decompensated Heart Failure in Emergency Department Visits.

Objectives: Around one million United States emergency department (ED) visits annually are due to acute decompensated heart failure (ADHF) symptoms. Characterizing ED symptom presentation of ADHF patients may improve clinical care, yet sex and age differences in ED chief complaints have not been thoroughly investigated. This paper aims to describe differences in chief complaints and comorbid conditions for ED patients with a ADHF diagnosis, stratified by sex and age. Methods: Retrospective analysis of adults presenting to North Carolina EDs in NC DETECT, a statewide syndromic surveillance system, between 2010 and 2016 with a diagnosis of ADHF. Frequencies of chief complaint categories for ED visits and comorbid conditions, stratified by sex and age, were evaluated and standardized differences computed. Results: Top chief complaints were dyspnea (19.1%), chest pain (13.5%), and other respiratory complaints (13.4%). In the 18-44 age group, females when compared to males reported more nausea/vomiting (6.7% versus 4.1%) and headache (4.2% versus 2.0%). In those 45-64 and 65+ years old, complaints were similar by sex. When stratified by age group alone, the 18-44 and 45-64 age groups had more complaints of chest pain, whereas balance issues, weakness, and confusion were more common in the 65+ age group. Conclusion: Sex and age differences in atypical ADHF symptoms were seen in in ED patients with ADHF. Characterizing variation of ADHF symptoms in ED patients can inform the identification of ED patients with ADHF and the management of ADHF-related symptoms.

Long-term outcomes of heart transplantation in adults with congenital heart disease: The impact of single-ventricle versus biventricular physiology.

Objective: Congenital heart disease is a risk factor for mortality after orthotopic heart transplantation; however, the impact of preoperative circulation type and primary congenital heart disease diagnosis remains poorly delineated. Methods: We retrospectively reviewed patients with adult congenital heart disease aged 16 years or more who underwent orthotopic heart transplantation at our institution between 2008 and 2022. Patients were categorized as having single-ventricle or biventricular circulation. The primary end point was 5-year post-transplant survival. Results: Sixty-one patients with adult congenital heart disease (single-ventricle: n = 26 [42.6%], biventricular: n = 35 [57.4%]) underwent orthotopic heart transplantation at 33.7 [interquartile range, 19.1-48.7] years. The most common congenital heart disease diagnosis was hypoplastic left heart syndrome (n = 11, 42.3%) in the single-ventricle group and congenitally corrected transposition of the great arteries (n = 7, 20.0%) in the biventricular group. Twenty-four patients previously underwent Fontan palliation. At transplant, patients in the single-ventricle group were younger (18.5 [interquartile range, 17.6-32.3] years vs 45.0 [interquartile range, 33.0-52.2] years, P < .001) and more likely to have biopsy-proven cirrhosis (46.2% vs 14.3%, P = .01) and protein-losing enteropathy (42.3% vs 2.9%, P < .001). Patients in the single-ventricle group also had longer bypass times (223.4 ± 65.3 minutes vs 187.4 ± 59.5 minutes, P = .03) and longer durations of mechanical ventilatory support (3.5 [interquartile range, 2.0-6.0] days vs 1.0 [interquartile range, 1.0-2.0] days, P < .001). Operative mortality was comparable (11.5% vs 8.6%, P = 1). Median follow-up was 6.0 [interquartile range, 2.4-10.0] years. Five-year survival was worse in the single-ventricle group (66.0% ± 10.0% vs 91.3% ± 4.8%, P = .03), as was freedom from major rejection (58.3% ± 10.2% vs 84.0% ± 6.6%, P = .02). In univariable analysis, hypoplastic left heart syndrome and Fontan circulation were risk factors for post-transplant mortality (hypoplastic left heart syndrome: hazard ratio, 5.0, P < .001; Fontan: hazard ratio, 3.5, P = .03). Conclusions: Adult patients with congenital heart disease undergoing heart transplant with single-ventricle physiology experienced a more complicated post-transplant course, with worse long-term survival and freedom from rejection. Multicenter studies are required to guide orthotopic heart transplantation decision-making in this complex cohort.

Clinical Utility of the Molecular Microscope Diagnostic System in a Real-World Transplant Cohort: Moving Towards a New Paradigm.

Objectives: To evaluate the clinical implications of adjunctive molecular gene expression analysis (MMDx ) of biopsy specimens in heart transplant (HT ) recipients with suspected rejection. Introduction: Histopathological evaluation remains the standard method for rejection diagnosis in HT. However, the wide interobserver variability combined with a relatively common incidence of "biopsy-negative" rejection has raised concerns about the likelihood of false-negative results. MMDx, which uses gene expression to detect early signs of rejection, is a promising test to further refine the assessment of HT rejection. Methods: Single-center prospective study of 418 consecutive for-cause endomyocardial biopsies performed between November 2022 and May 2024. Each biopsy was graded based on histology and assessed for rejection patterns using MMDx. MMDx results were deemed positive if borderline or definitive rejection was present. The impact of MMDx results on clinical management was evaluated. Primary outcomes were 1-year survival and graft dysfunction following MMDx-guided clinical management. Secondary outcomes included changes in donor-specific antibodies, MMDx gene transcripts, and donor-derived cell-free DNA (dd-cfDNA) levels. Results: We analyzed 418 molecular samples from 237 unique patients. Histology identified rejection in 32 cases (7.7%), while MMDx identified rejection in 95 cases (22.7%). Notably, in 79 of the 95 cases where MMDx identified rejection, histology results were negative, with the majority of these cases being antibody-mediated rejection (62.1%). Samples with rejection on MMDx were more likely to show a combined elevation of dd-cfDNA and peripheral blood gene expression profiling than those with borderline or negative MMDx results (36.7% vs 28.0% vs 10.3%; p<0.001). MMDx results led to the implementation of specific antirejection protocols or changes in immunosuppression in 20.4% of cases, and in 73.4% of cases where histology was negative and MMDx showed rejection. 1-year survival was better in the positive MMDx group where clinical management was guided by MMDx results (87.0% vs 78.6%; log rank p=0.0017). Conclusions: In our cohort, MMDx results more frequently indicated rejection than histology, often leading to the initiation of antirejection treatment. Intervention guided by positive MMDx results was associated with improved outcomes.

The early Miocene onset of a ventilated circulation regime in the Arctic Ocean.

Deep-water formation in the northern North Atlantic Ocean and the Arctic Ocean is a key driver of the global thermohaline circulation and hence also of global climate. Deciphering the history of the circulation regime in the Arctic Ocean has long been prevented by the lack of data from cores of Cenozoic sediments from the Arctic's deep-sea floor. Similarly, the timing of the opening of a connection between the northern North Atlantic and the Arctic Ocean, permitting deep-water exchange, has been poorly constrained. This situation changed when the first drill cores were recovered from the central Arctic Ocean. Here we use these cores to show that the transition from poorly oxygenated to fully oxygenated ('ventilated') conditions in the Arctic Ocean occurred during the later part of early Miocene times. We attribute this pronounced change in ventilation regime to the opening of the Fram Strait. A palaeo-geographic and palaeo-bathymetric reconstruction of the Arctic Ocean, together with a physical oceanographic analysis of the evolving strait and sill conditions in the Fram Strait, suggests that the Arctic Ocean went from an oxygen-poor 'lake stage', to a transitional 'estuarine sea' phase with variable ventilation, and finally to the fully ventilated 'ocean' phase 17.5 Myr ago. The timing of this palaeo-oceanographic change coincides with the onset of the middle Miocene climatic optimum, although it remains unclear if there is a causal relationship between these two events.

The Cenozoic palaeoenvironment of the Arctic Ocean.

The history of the Arctic Ocean during the Cenozoic era (0-65 million years ago) is largely unknown from direct evidence. Here we present a Cenozoic palaeoceanographic record constructed from >400 m of sediment core from a recent drilling expedition to the Lomonosov ridge in the Arctic Ocean. Our record shows a palaeoenvironmental transition from a warm 'greenhouse' world, during the late Palaeocene and early Eocene epochs, to a colder 'icehouse' world influenced by sea ice and icebergs from the middle Eocene epoch to the present. For the most recent approximately 14 Myr, we find sedimentation rates of 1-2 cm per thousand years, in stark contrast to the substantially lower rates proposed in earlier studies; this record of the Neogene reveals cooling of the Arctic that was synchronous with the expansion of Greenland ice (approximately 3.2 Myr ago) and East Antarctic ice (approximately 14 Myr ago). We find evidence for the first occurrence of ice-rafted debris in the middle Eocene epoch (approximately 45 Myr ago), some 35 Myr earlier than previously thought; fresh surface waters were present at approximately 49 Myr ago, before the onset of ice-rafted debris. Also, the temperatures of surface waters during the Palaeocene/Eocene thermal maximum (approximately 55 Myr ago) appear to have been substantially warmer than previously estimated. The revised timing of the earliest Arctic cooling events coincides with those from Antarctica, supporting arguments for bipolar symmetry in climate change.