Persistent near-tropical warmth on the Antarctic continent during the early Eocene epoch.

The warmest global climates of the past 65 million years occurred during the early Eocene epoch (about 55 to 48 million years ago), when the Equator-to-pole temperature gradients were much smaller than today and atmospheric carbon dioxide levels were in excess of one thousand parts per million by volume. Recently the early Eocene has received considerable interest because it may provide insight into the response of Earth's climate and biosphere to the high atmospheric carbon dioxide levels that are expected in the near future as a consequence of unabated anthropogenic carbon emissions. Climatic conditions of the early Eocene 'greenhouse world', however, are poorly constrained in critical regions, particularly Antarctica. Here we present a well-dated record of early Eocene climate on Antarctica from an ocean sediment core recovered off the Wilkes Land coast of East Antarctica. The information from biotic climate proxies (pollen and spores) and independent organic geochemical climate proxies (indices based on branched tetraether lipids) yields quantitative, seasonal temperature reconstructions for the early Eocene greenhouse world on Antarctica. We show that the climate in lowland settings along the Wilkes Land coast (at a palaeolatitude of about 70° south) supported the growth of highly diverse, near-tropical forests characterized by mesothermal to megathermal floral elements including palms and Bombacoideae. Notably, winters were extremely mild (warmer than 10 °C) and essentially frost-free despite polar darkness, which provides a critical new constraint for the validation of climate models and for understanding the response of high-latitude terrestrial ecosystems to increased carbon dioxide forcing.

Early evolution of the angiosperm clade Asteraceae in the Cretaceous of Antarctica.

The Asteraceae (sunflowers and daisies) are the most diverse family of flowering plants. Despite their prominent role in extant terrestrial ecosystems, the early evolutionary history of this family remains poorly understood. Here we report the discovery of a number of fossil pollen grains preserved in dinosaur-bearing deposits from the Late Cretaceous of Antarctica that drastically pushes back the timing of assumed origin of the family. Reliably dated to ∼76-66 Mya, these specimens are about 20 million years older than previously known records for the Asteraceae. Using a phylogenetic approach, we interpreted these fossil specimens as members of an extinct early diverging clade of the family, associated with subfamily Barnadesioideae. Based on a molecular phylogenetic tree calibrated using fossils, including the ones reported here, we estimated that the most recent common ancestor of the family lived at least 80 Mya in Gondwana, well before the thermal and biogeographical isolation of Antarctica. Most of the early diverging lineages of the family originated in a narrow time interval after the K/P boundary, 60-50 Mya, coinciding with a pronounced climatic warming during the Late Paleocene and Early Eocene, and the scene of a dramatic rise in flowering plant diversity. Our age estimates reduce earlier discrepancies between the age of the fossil record and previous molecular estimates for the origin of the family, bearing important implications in the evolution of flowering plants in general.

Parasitological Analysis and Gill Histopathology of Pearl Dace (Semotilus Margarita) and Brook Stickleback (Culaea Inconstans) Collected from the Athabasca Oil Sands Area (Canada).

Pearl dace (Semotilus margarita) and brook stickleback (Culaea inconstans) were collected from tributaries of the Athabasca River (Alberta, Canada), upstream (reference site) and downstream of oil sands deposits where fish were expected to be exposed to naturally occurring oil sands constituents. The objective was to determine if fish collected from these sites exhibited differences in the prevalence or intensity of infection by parasites or in gill histology. Dace did not display significant differences in these parameters. Alternately, upstream stickleback were predominantly infected by complex life history parasites, while downstream fish were primarily infected by parasites with simpler life histories. Moreover, downstream stickleback exhibited significantly more clubbing and aneurysms in secondary gill lamellae relative to upstream fish. This suggested a difference in habitat quality between upstream and downstream sites. However, based on basic body condition parameters of the fish, it would appear that any impacts upon the health of the fish due to the presence of naturally occurring oil sands associated chemical constituents would have been minor.

The role of the sonic hedgehog signalling pathway in patients with midline defects and congenital hypopituitarism.

INTRODUCTION: The Gli family of zinc finger (GLI) transcription factors mediates the sonic hedgehog signalling pathway (HH) essential for CNS, early pituitary and ventral forebrain development in mice. Human mutations in this pathway have been described in patients with holoprosencephaly (HPE), isolated congenital hypopituitarism (CH) and cranial/midline facial abnormalities. Mutations in Sonic hedgehog (SHH) have been associated with HPE but not CH, despite murine studies indicating involvement in pituitary development. OBJECTIVES/METHODS: We aimed to establish the role of the HH pathway in the aetiology of hypothalamo-pituitary disorders by screening our cohort of patients with midline defects and/or CH for mutations in SHH, GLI2, Shh brain enhancer 2 (SBE2) and growth-arrest specific 1 (GAS1). RESULTS: Two variants and a deletion of GLI2 were identified in three patients. A novel variant at a highly conserved residue in the zinc finger DNA-binding domain, c.1552G > A [pE518K], was identified in a patient with growth hormone deficiency and low normal free T4. A nonsynonymous variant, c.2159G > A [p.R720H], was identified in a patient with a short neck, cleft palate and hypogonadotrophic hypogonadism. A 26·6 Mb deletion, 2q12·3-q21·3, encompassing GLI2 and 77 other genes, was identified in a patient with short stature and impaired growth. Human embryonic expression studies and molecular characterisation of the GLI2 mutant p.E518K support the potential pathogenicity of GLI2 mutations. No mutations were identified in GAS1 or SBE2. A novel SHH variant, c.1295T>A [p.I432N], was identified in two siblings with variable midline defects but normal pituitary function. CONCLUSIONS: Our data suggest that mutations in SHH, GAS1 and SBE2 are not associated with hypopituitarism, although GLI2 is an important candidate for CH.

Supporting Autistic College Students: Examining the Mentoring, Organization and Social Support for Autism Inclusion on Campus (MOSSAIC) Program.

Recent trends suggest that autistic young adults are increasingly accessing postsecondary education than in years past. However, these students often face unique challenges that negatively impact their college experience resulting in high dropout. The Mentoring, Organization and Social Support for Autism Inclusion on Campus (MOSSAIC) Program is a peer-mentorship college transition program intended to support autistic students with executive functioning, social, and self-advocacy skills. This study investigated the experiences of 13 autistic mentees and 12 non-autistic mentors enrolled in the MOSSAIC program. Program feedback was collected using semi-structured interviews in order to understand student experiences, highlight benefits, and identify areas of improvement. Participants reported a general positive experience and improved skills in the domains of socialization, executive functioning, academic performance, and professional development. The most common suggestion for the program was the inclusion of autistic peer mentors. Mentees noted difficulty relating with non-autistic peers and feeling burdened with the need to educate their mentor on how to support autistic adults. These data provide valuable insight into how colleges can better improve support for autistic college students to ensure postsecondary success. Future peer mentorship programs should consider recruiting neurodiverse mentors from diverse backgrounds to improve congruence between mentor and mentee identities.

Discrimination of pollen of New Zealand manuka (Leptospermum scoparium agg.) and kanuka (Kunzea spp.) (Myrtaceae).

The very similar appearance of pollen of the New Zealand Myrtaceous taxa Leptospermum scoparium s.l. (manuka) and Kunzea spp. (kanuka) has led palynologists to combine them in paleoecological and melissopalynological studies. This is unfortunate, as differentiation of these taxa would improve understanding of past ecological change and has potential to add value to the New Zealand honey industry, where manuka honey attracts a premium price. Here, we examine in detail the pollen morphology of the 10 Kunzea species and a number of Leptospermum scoparium morphotypes collected from around New Zealand, using light microscopy, SEM, and Classifynder (an automated palynology system). Our results suggest that at a generic level the New Zealand Leptospermum and Kunzea pollen can be readily differentiated, but the differences between pollen from the morphotypes of Leptospermum or between the species of Kunzea are less discernible. While size is a determinant factor-equatorial diameter of Leptospermum scoparium pollen is 19.08 ± 1.28 µm, compared to 16.30 ± 0.95 µm for Kunzea spp.-other criteria such as surface texture and shape characteristics are also diagnostic. A support vector machine set up to differentiate Leptospermum from Kunzea pollen using images captured by the Classifynder system had a prediction accuracy of ~95%. This study is a step towards future melissopalynological differentiation of manuka honey using automated pollen image capture and classification approaches.

Mid-Miocene cooling and the extinction of tundra in continental Antarctica.

A major obstacle in understanding the evolution of Cenozoic climate has been the lack of well dated terrestrial evidence from high-latitude, glaciated regions. Here, we report the discovery of exceptionally well preserved fossils of lacustrine and terrestrial organisms from the McMurdo Dry Valleys sector of the Transantarctic Mountains for which we have established a precise radiometric chronology. The fossils, which include diatoms, palynomorphs, mosses, ostracodes, and insects, represent the last vestige of a tundra community that inhabited the mountains before stepped cooling that first brought a full polar climate to Antarctica. Paleoecological analyses, (40)Ar/(39)Ar analyses of associated ash fall, and climate inferences from glaciological modeling together suggest that mean summer temperatures in the region cooled by at least 8 degrees C between 14.07 +/- 0.05 Ma and 13.85 +/- 0.03 Ma. These results provide novel constraints for the timing and amplitude of middle-Miocene cooling in Antarctica and reveal the ecological legacy of this global climate transition.

Prefertilization Exposure of Rainbow Trout Eggs to Per- and Polyfluoroalkyl Substances to Simulate Accumulation During Oogenesis.

Aqueous film-forming foams (AFFFs) are used in firefighting and are sources of per- and polyfluoroalkyl substances (PFAS) to the environment through surface runoff and groundwater contamination at defense and transportation sites. Little is known regarding the toxicity and bioaccumulation of newer AFFF formulations containing novel PFAS. To mimic maternal transfer of PFAS, prefertilization rainbow trout eggs were exposed to three PFAS using novel methodologies. Batches of unfertilized oocytes were exposed for 3 h to 0, 0.01, 0.1, 1, or 10 µg/ml separately to perfluorooctanoic acid, perfluorohexanoic acid, or perfluorooctanesulfonic acid in either coelomic fluid or Cortland's solution. After exposure, the gametes were fertilized and rinsed with dechlorinated water. Egg yolk was aspirated from a subset of fertilized eggs for PFAS quantification. Each PFAS was detected in yolks of eggs exposed to the respective PFAS, and yolk concentrations were directly proportional to concentrations in aqueous media to which they were exposed. Exposure in coelomic fluid or Cortland's solution resulted in similar concentrations of PFAS in egg yolks. Ratios of PFAS concentrations in oocytes to concentrations in exposure media (oocyte fluid ratios) were <0.99 when exposed from 0.01 to 10 µg/ml and <0.45 when exposed from 0.1 to 10 µg/ml for both media and all three PFAS, demonstrating that the water solubility of the chemicals was relatively great. Prefertilization exposure of eggs effectively introduced PFAS into unfertilized egg yolk. This method provided a means of mimicking maternal transfer to evaluate toxicity to developing embryos from an early stage. This method is more rapid and efficient than injection of individual fertilized eggs and avoids trauma from inserting needles into eggs. Environ Toxicol Chem 2021;40:3159-3165. © 2021 SETAC.

Systemic thyroid hormone is necessary and sufficient to induce ultraviolet-sensitive cone loss in the juvenile rainbow trout retina.

Rainbow trout possess ultraviolet-sensitive (UVS) cones in their retina that degenerate naturally during development. This phenomenon can be induced with exogenous thyroxine [T4, a thyroid hormone (TH)] treatment. However, the previous T4 exposure experiments employed static water immersion; a method that could introduce confounding stress effects on the fish. Because of this, it was uncertain if T4 alone was sufficient to induce retinal changes or if stress-related hormones were also necessary to initiate this process. Furthermore, it was unclear whether endogenous T4 was the factor responsible for initiating natural UVS cone loss during development. The current study examined the role of systemic T4 on the juvenile rainbow trout retina using a slow-release implant. Exogenous T4 treatment resulted in SWS1 opsin downregulation and UVS cone loss after four weeks of exposure, signifying that T4 is sufficient to induce this process. Blocking endogenous T4 production with propylthiouracil (PTU, an anti-thyroid agent) attenuated SWS1 downregulation and UVS cone loss in the retina of naturally developing rainbow trout, suggesting that endogenous T4 is necessary to initiate retinal remodelling during development. Quantitative real-time RT-PCR analysis demonstrated that several TH-regulating components are expressed in the trout retina, and that expression levels of the TH receptor isoform TRbeta and the type 2 deiodinase (D2) change with T4 treatment. This suggests that T4 may act directly on the retina to induce UVS cone loss. Taken together, these results demonstrate that systemic TH is necessary and sufficient to induce SWS1 opsin downregulation and UVS cone loss in the retina of juvenile rainbow trout.

Female inheritance of malarial lap genes is essential for mosquito transmission.

Members of the LCCL/lectin adhesive-like protein (LAP) family, a family of six putative secreted proteins with predicted adhesive extracellular domains, have all been detected in the sexual and sporogonic stages of Plasmodium and have previously been predicted to play a role in parasite-mosquito interactions and/or immunomodulation. In this study we have investigated the function of PbLAP1, 2, 4, and 6. Through phenotypic analysis of Plasmodium berghei loss-of-function mutants, we have demonstrated that PbLAP2, 4, and 6, as previously shown for PbLAP1, are critical for oocyst maturation and sporozoite formation, and essential for transmission from mosquitoes to mice. Sporozoite formation was rescued by a genetic cross with wild-type parasites, which results in the production of heterokaryotic polyploid ookinetes and oocysts, and ultimately infective Deltapblap sporozoites, but not if the individual Deltapblap parasite lines were crossed amongst each other. Genetic crosses with female-deficient (Deltapbs47) and male-deficient (Deltapbs48/45) parasites show that the lethal phenotype is only rescued when the wild-type pblap gene is inherited from a female gametocyte, thus explaining the failure to rescue in the crosses between different Deltapblap parasite lines. We conclude that the functions of PbLAPs1, 2, 4, and 6 are critical prior to the expression of the male-derived gene after microgametogenesis, fertilization, and meiosis, possibly in the gametocyte-to-ookinete period of differentiation. The phenotypes detectable by cytological methods in the oocyst some 10 d after the critical period of activity suggests key roles of the LAPs or LAP-dependent processes in the regulation of the cell cycle, possibly in the regulation of cytoplasm-to-nuclear ratio, and, importantly, in the events of cytokinesis at sporozoite formation. This phenotype is not seen in the other dividing forms of the mutant parasite lines in the liver and blood stages.

Indication of global deforestation at the Cretaceous-Tertiary boundary by New Zealand fern spike.

The devastating effect on terrestrial plant communities of a bolide impact at the Cretaceous-Tertiary boundary is shown in fossil pollen and spore assemblages by a diverse flora being abruptly replaced by one dominated by a few species of fern. Well documented in North America, this fern spike signals widespread deforestation due to an impact winter or massive wildfires. A Southern Hemisphere record of a fern spike, together with a large iridium anomaly, indicates that the devastation was truly global. Recovery of New Zealand plant communities followed a pattern consistent with major climatic perturbations occurring after an impact winter that was possibly preceded by global wildfires.

Changes in thyroid hormone reception precede SWS1 opsin downregulation in trout retina.

Rainbow trout undergo natural cone degeneration and thus are interesting models for examining mechanisms of neural degeneration. They have ultraviolet-sensitive (UVS) cones that are lost over most of the retina during development; only a small functional population remains in the dorsal retina. How this spatial distribution of UVS cones is maintained is unclear. Thyroxine (T4) induces UVS cone loss, and local thyroid hormone regulation was hypothesized to control UVS cone distribution. Thyroid hormone receptor alpha (TRalpha), thyroid hormone receptor beta (TRbeta) and Type 2 deiodinase (D2) regulate thyroid hormone exposure to target cells. Regional retinal expression of these genes was investigated during exogenous T4 treatment and natural smoltification of rainbow trout. Each retina from dark-adapted parr, T4-treated parr and natural smolts was divided into four quadrants, and total RNA was isolated. Quantitative real-time RT-PCR analysis demonstrated that all retinal quadrants had increased accumulation of TRbeta transcripts 2 days post-T4 treatment, corresponding to initiation of SWS1 opsin downregulation. Smolts exhibited decreased accumulation of TRalpha and TRbeta transcripts in all quadrants, but this effect was most pronounced in the dorso-temporal (DT) retinal quadrant where UVS cones persist. By contrast, in 2 day T4-treated parr, the DT quadrant showed increased expression of TRalpha and TRbeta. Furthermore, D2 transcripts decreased in the DT quadrant of T4-treated parr but increased in the DT quadrant of smolts. These results suggest that T4 upregulates TRbeta expression to initiate SWS1 opsin downregulation, while TRalpha and TRbeta downregulation occurs to prevent natural loss of UVS cones from the DT retina.

Chronic toxicity of oil sands tailings pond sediments to early life stages of fathead minnow (Pimephales promelas).

In this study fathead minnow (Pimephales promelas) embryo-larval stages were exposed to two oil sands tailings pond sediments which had previously been shown to decrease the survival of embryo-larval larval stages of walleye (Sander vitreus) and northern pike (Esox lucius). Fathead minnow are standard test species and we wanted to compare their sensitivity to the other two species. Fathead minnow larvae were exposed for 20 days (5 days in the egg stage and 15 days in the larval stage) with daily renewal of sediments and waters. Sediments contained polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs (APAHs). Results from an earlier study showed that Sediment 1 contained 173 µg/g total PAHs + APAHs (97 % alkylated), and sediment 2 contained 401 µg/g total PAHs + APAHs (95 % alkylated). Fathead minnow larvae exposed to oil sands tailings pond sediments had decreased survival, decreased weight, and increased deformities. Fathead minnow survival was unaffected at the embryo stage and at hatch. Most deaths occurred at the larval stages 1-8 days after hatching, showing the importance of exposing the fish for at least a week after hatch. Toxicity was seen at 0.2 g/L of sediment, which was equivalent to the addition of 35 and 80 µg total PAHs + APAHs to 1 L of overlying water for sediment 1 and 2, respectively. When compared to embryo-larval northern pike and walleye results from previous studies, all three species of fish responded more strongly to sediment 2 compared to sediment 1. For effects on lethality, fathead minnow were equally sensitive to pike, but walleye were 5-28 times more sensitive to the lethal effects of the sediments compared to both fathead minnow and pike. The study (and comparisons to our previous studies) shows the difference in sensitivity between a model laboratory species (fathead minnow) and some species of wild fish that are highly relevant to the oil sands area of Alberta.

Oil sands tailings pond sediment toxicity to early life stages of northern pike (Esox lucius).

The Athabasca River in Alberta flows through natural sources of eroding oil sands bitumen and oil sands mining operations that may result in low level contamination of surface waters. Northern pike (Esox lucius) are apex predators and important food and game fish species native to the Athabasca River system. This species has the potential to be exposed to both natural and anthropogenic sources of contamination from oil sands related materials throughout its life cycle. Pike are difficult to rear in the laboratory and little information exists on the toxicity of oil sands related materials to this key indigenous fish species. In this study, the potential effects of two sediment samples collected from different areas of one tailings pond in the Athabasca oil sands area are assessed in a daily renewal bioassay on early life stages of northern pike. Gametes were collected from spawning wild pike captured from a reference site outside of the oil sands area. Fertilized eggs were exposed to control water or increasing concentrations of tailings pond sediments for 21days, coinciding with initiation of exogenous feeding and completion of yolk absorption. Developing fish were examined for survival and changes in body weight, length, and development. Embryos exhibited increased developmental abnormalities and decreased growth and survival with increasing sediment concentration. Both sediment samples had similar levels of naphthenic acids and similar types of PAHs, with alkylated PAHs dominating. However, concentrations of total and alkylated PAHs differed between sediment samples and were related to increasing developmental abnormalities and decreased growth and survival. This is consistent with developmental changes observed with exposure to PAHs in other fish species. These results provide information on the effects of tailings pond sediments comprising mixtures of PAHs and alkylated PAHs on the development and survival of a key species in the northern aquatic ecosystem.

Drug-induced progressive multifocal leukoencephalopathy in multiple sclerosis: European regulators' perspective.

Progressive multifocal leukoencephalopathy (PML) has been associated with the use of a number of multiple sclerosis (MS) immunomodulatory therapies and has assumed a critical place in the evaluation of their benefit/risk. In this review, we discuss the European Union regulatory approach to drug-induced PML in MS, highlight a number of key issues related to the current knowledge on PML, and outline possible paths to help progress the risk management of patients with MS at risk of PML.

The effect of oil sands tailings pond sediments on embryo-larval walleye (Sander vitreus).

Walleye (Sander vitreus) are a commercially important North American fish species that inhabit the Athabasca River. This river flows through the Athabasca oil sands where natural sources of bitumen erode from the McMurray formation. Little information is available on responses of walleye embryos to oil sands tailings pond sediments in a laboratory setting. The current study describes the design and implementation of a daily-renewal bioassay to assess the potential effects of tailings pond sediments from the Athabasca oil sands area on walleye development. Developing walleye embryos were exposed to increasing concentrations of two tailings pond sediments (collected in the Athabasca oil sands area) until the completion of yolk absorption in control fish. Sediments from the tailings pond represent a mixture of polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs. During the 31 day exposure, the walleye were examined for mortalities, weight, length and developmental abnormalities to provide an initial evaluation of the effects of the oil sands tailings pond sediments. Walleye embryo survival differed between the tailings pond sediments, and survival decreased with increasing sediment concentration. Alkylated PAH content differed between the two tailings pond sediments and lower embryo survival corresponded to higher total and alkylated PAH content. Tailings pond sediment-exposed walleye exhibited a delay in development, as well as increased percentages of larvae with heart and yolk sac edema, and cranial and spinal malformations. These abnormalities in development are often associated with PAH and alkylated PAH exposure. This study provides an exposure design that can be used to assess sediment toxicity to early developmental stages of a fish species not commonly tested in the lab, and lays the groundwork for future studies with this and other difficult-to-culture species. These results offer information on the potential effects of tailings pond sediments containing PAH/alkylated PAH mixtures on walleye development and survival.

Hepatopathy-thrombocytopenia syndrome--a complication of dactinomycin therapy for Wilms' tumor: a report from the United Kingdom Childrens Cancer Study Group.

We have observed hepatopathy, associated with thrombocytopenia, in children receiving chemotherapy for Wilms' tumor. We have studied this hepatopathy-thrombocytopenia syndrome (HTS) in patients enrolled in the United Kingdom Childrens' Cancer Study Group (UKCCSG) Wilms' tumor trials (UKW1 and UKW2). At the time of this study, 501 patients had completed therapy. Treatment flow sheets were examined for evidence of hepatopathy (hepatomegaly with abnormal liver function tests) and severe thrombocytopenia (platelet count less than 25 x 10(9)/L). No child who developed the syndrome had received irradiation. HTS was seen in five of 355 (1.4%) of patients treated with combination chemotherapy but in none of the 146 patients treated with vincristine alone. In each instance, the onset was less than 10 weeks after diagnosis. In two children, hepatopathy was severe with jaundice, ascites, transaminases greater than 1,000 IU/L, and prolongation of prothrombin time. On average, HTS lasted 12 days, and resolved with supportive treatment. After recovery, the children tolerated chemotherapy, mostly at reduced dosage, without recurrence. There was no evident long-term morbidity. Dactinomycin is the probable cause of this syndrome. We conclude that the HTS is a rare but important complication of dactinomycin-containing combination chemotherapy for Wilms' tumor. Children developing "isolated" thrombocytopenia following dactinomycin are "at risk" of developing the full-blown syndrome and should have their treatment modified accordingly.

From adaptive licensing to adaptive pathways: delivering a flexible life-span approach to bring new drugs to patients.

The concept of adaptive licensing (AL) has met with considerable interest. Yet some remain skeptical about its feasibility. Others argue that the focus and name of AL should be broadened. Against this background of ongoing debate, we examine the environmental changes that will likely make adaptive pathways the preferred approach in the future. The key drivers include: growing patient demand for timely access to promising therapies, emerging science leading to fragmentation of treatment populations, rising payer influence on product accessibility, and pressure on pharma/investors to ensure sustainability of drug development. We also discuss a number of environmental changes that will enable an adaptive paradigm. A life-span approach to bringing innovation to patients is expected to help address the perceived access vs. evidence trade-off, help de-risk drug development, and lead to better outcomes for patients.

Continuous negative extrathoracic pressure in neonatal respiratory failure.

OBJECTIVE: In uncontrolled clinical trials, negative extrathoracic pressure has been shown to be an effective respiratory support. We aimed to assess its role in the context of current neonatal intensive care. DESIGN: A randomized controlled trial, with sequential analysis of matched pairs of infants. Matching was undertaken by stratified randomization from 15 groups divided according to gestational age, oxygen requirement, and whether patients were intubated at 4 hours of age. SETTING: Two neonatal intensive care units. PATIENTS: Two hundred forty-four patients (birth weight 1.53 +/- 0.69 kg (mean +/- SD); gestational age 30.4 +/- 3.5 weeks) with respiratory failure. INTERVENTIONS: Patients were randomized at 4 hours of age to receive either standard neonatal intensive care, or standard care plus continuous negative extrathoracic pressure (CNEP, -4 to -6 cmH2O) applied within a purpose-designed neonatal incubator. OUTCOME SCORES: Clinical scores were calculated for each infant at 56 days of age, or death if earlier. Scores included measures for mortality, respiratory outcome, the presence of cerebral ultrasound abnormalities, patent arterial duct, necrotizing enterocolitis, and retinopathy. The treatment given for the higher score for each pair was recorded and the cumulative net number of pairs favoring CNEP plotted in the sequential analysis to provide an ethical early termination strategy. Individual components of the outcome score and other secondary measurements were analyzed on completion of the trial. RESULTS: The sequential analysis reached a decision boundary after 122 out of a possible maximum of 124 pairs were completed. The overall outcome score showed an overall significant benefit for CNEP. Secondary analysis showed that the use of CNEP was associated with an increase in mortality, cranial ultrasound abnormalities, and pneumothoraces, which were not statistically significant. However, 5% fewer patients were intubated (95% confidence interval [CI], 0-10), and the total duration of oxygen therapy among surviving infants at 56 days was lower (20.5 days, compared with 38.9 in controls; difference 18.4 days, 95% CI 3.8 to 33.0). Among all infants, the mean total duration of oxygen therapy was 18.3 days among CNEP-treated infants compared with 33.6 days among the controls (difference -15.3 days, 95% CI -0.2 to -30.4). This reduction in mean levels is entirely attributable to substantially fewer patients requiring prolonged oxygen therapy, the median duration of treatment being very similar in the two groups. As a result, commensurately fewer surviving infants showed chronic lung disease of prematurity. CONCLUSIONS: The use of continuous negative pressure improves the respiratory outcome for neonates with respiratory failure.