Single cell RNA-sequencing and RNA-tomography of the avian embryo extending body axis.

Introduction: Vertebrate body axis formation initiates during gastrulation and continues within the tail bud at the posterior end of the embryo. Major structures in the trunk are paired somites, which generate the musculoskeletal system, the spinal cord-forming part of the central nervous system, and the notochord, with important patterning functions. The specification of these different cell lineages by key signalling pathways and transcription factors is essential, however, a global map of cell types and expressed genes in the avian trunk is missing. Methods: Here we use high-throughput sequencing approaches to generate a molecular map of the emerging trunk and tailbud in the chick embryo. Results and Discussion: Single cell RNA-sequencing (scRNA-seq) identifies discrete cell lineages including somites, neural tube, neural crest, lateral plate mesoderm, ectoderm, endothelial and blood progenitors. In addition, RNA-seq of sequential tissue sections (RNA-tomography) provides a spatially resolved, genome-wide expression dataset for the avian tailbud and emerging body, comparable to other model systems. Combining the single cell and RNA-tomography datasets, we identify spatially restricted genes, focusing on somites and early myoblasts. Thus, this high-resolution transcriptome map incorporating cell types in the embryonic trunk can expose molecular pathways involved in body axis development.

Ancient and modern DNA track temporal and spatial population dynamics in the European fallow deer since the Eemian interglacial.

Anthropogenic factors have impacted the diversity and evolutionary trajectory of various species. This can be through factors such as pressure on population size or range, habitat fragmentation, or extensive manipulation and translocation. Here we use time-calibrated data to better understand the pattern and processes of evolution in the heavily manipulated European fallow deer (Dama dama). During the Pleistocene, fallow deer had a broad distribution across Europe and were found as far north as Britain during the Eemian interglacial. The last glacial period saw fallow deer retreat to southern refugia and they did not disperse north afterwards. Their recolonisation was mediated by people and, from northern Europe and the British Isles, fallow deer were transported around the world. We use ancient and modern mitochondrial DNA (mtDNA) and mitogenomic data from Eemian Britain to assess the pattern of change in distribution and lineage structure across Europe over time. We find founder effects and mixed lineages in the northern populations, and stability over time for populations in southern Europe. The Eemian sample was most similar to a lineage currently in Italy, suggesting an early establishment of the relevant refuge. We consider the implications for the integration of anthropogenic and natural processes towards a better understanding of the evolution of fallow deer in Europe.

Baseline demographics, clinical features and predictors of conversion among 200 individuals in a longitudinal prospective psychosis-risk cohort.

BACKGROUND: DSM-5 proposes an Attenuated Psychosis Syndrome (APS) for further investigation, based upon the Attenuated Positive Symptom Syndrome (APSS) in the Structured Interview for Psychosis-Risk Syndromes (SIPS). SIPS Unusual Thought Content, Disorganized Communication and Total Disorganization scores predicted progression to psychosis in a 2015 NAPLS-2 Consortium report. We sought to independently replicate this in a large single-site high-risk cohort, and identify baseline demographic and clinical predictors beyond current APS/APSS criteria. METHOD: We prospectively studied 200 participants meeting criteria for both the SIPS APSS and DSM-5 APS. SIPS scores, demographics, family history of psychosis, DSM Axis-I diagnoses, schizotypy, and social and role functioning were assessed at baseline, with follow-up every 3 months for 2 years. RESULTS: The conversion rate was 30% (n = 60), or 37.7% excluding participants who were followed under 2 years. This rate was stable across time. Conversion time averaged 7.97 months for 60% who developed schizophrenia and 15.68 for other psychoses. Mean conversion age was 20.3 for males and 23.5 for females. Attenuated odd ideas and thought disorder appear to be the positive symptoms which best predict psychosis in a logistic regression. Total negative symptom score, Asian/Pacific Islander and Black/African-American race were also predictive. As no Axis-I diagnosis or schizotypy predicted conversion, the APS is supported as a distinct syndrome. In addition, cannabis use disorder did not increase risk of conversion to psychosis. CONCLUSIONS: NAPLS SIPS findings were replicated while controlling for clinical and demographic factors, strongly supporting the validity of the SIPS APSS and DSM-5 APS diagnosis.

Evolution and dispersal of mammoths across the Northern Hemisphere.

Mammoths provide a detailed example of species origins and dispersal, but understanding has been impeded by taxonomic confusion, especially in North America. The Columbian mammoth Mammuthus columbi was thought to have evolved in North America from a more primitive Eurasian immigrant. The earliest American mammoths (1.5 million years ago), however, resemble the advanced Eurasian M. trogontherii that crossed the Bering land bridge around that time, giving rise directly to M. columbi. Woolly mammoth M. primigenius later evolved in Beringia and spread into Europe and North America, leading to a diversity of morphologies as it encountered endemic M. trogontherii and M. columbi, respectively. In North America, this included intermediates ("M. jeffersonii"), suggesting introgression of M. primigenius with M. columbi. The lineage illustrates the dynamic interplay of local adaptation, dispersal, and gene flow in the evolution of a widely distributed species complex.

Episodic sucrose intake during food restriction increases synaptic abundance of AMPA receptors in nucleus accumbens and augments intake of sucrose following restoration of ad libitum feeding.

Weight-loss dieting often leads to loss of control, rebound weight gain, and is a risk factor for binge pathology. Based on findings that food restriction (FR) upregulates sucrose-induced trafficking of glutamatergic AMPA receptors to the nucleus accumbens (NAc) postsynaptic density (PSD), this study was an initial test of the hypothesis that episodic "breakthrough" intake of forbidden food during dieting interacts with upregulated mechanisms of synaptic plasticity to increase reward-driven feeding. Ad libitum (AL) fed and FR subjects consumed a limited amount of 10% sucrose, or had access to water, every other day for 10 occasions. Beginning three weeks after return of FR rats to AL feeding, when 24-h chow intake and rate of body weight gain had normalized, subjects with a history of sucrose intake during FR consumed more sucrose during a four week intermittent access protocol than the two AL groups and the group that had access to water during FR. In an experiment that substituted noncontingent administration of d-amphetamine for sucrose, FR subjects displayed an enhanced locomotor response during active FR but a blunted response, relative to AL subjects, during recovery from FR. This result suggests that the enduring increase in sucrose consumption is unlikely to be explained by residual enhancing effects of FR on dopamine signaling. In a biochemical experiment which paralleled the sucrose behavioral experiment, rats with a history of sucrose intake during FR displayed increased abundance of pSer845-GluA1, GluA2, and GluA3 in the NAc PSD relative to rats with a history of FR without sucrose access and rats that had been AL throughout, whether they had a history of episodic sucrose intake or not. A history of FR, with or without a history of sucrose intake, was associated with increased abundance of GluA1. A terminal 15-min bout of sucrose intake produced a further increase in pSer845-GluA1 and GluA2 in subjects with a history of sucrose intake during FR. Generally, neither a history of sucrose intake nor a terminal bout of sucrose intake affected AMPA receptor abundance in the NAc PSD of AL subjects. Together, these results are consistent with the hypothesis, but the functional contribution of increased synaptic incorporation of AMPA receptors remains to be established.

A study to investigate dose escalation of doxorubicin in ABVD chemotherapy for Hodgkin lymphoma incorporating biomarkers of response and toxicity.

BACKGROUND: Myelotoxicity during initial cycles of chemotherapy for Hodgkin lymphoma is associated with better outcome, supporting the concept of individualised dosing based on pharmacodynamic end points to optimise results. This study was performed to identify the maximum tolerated dose (MTD) of doxorubicin within cycles 1-3 ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine). Circulating biomarkers of response (nucleosomal DNA, nDNA) and epithelial toxicity (Cytokeratin 18, CK18) were also measured. METHODS: Dose escalation of doxorubicin in cycles 1-3 ABVD supported by pegfilgrastim was performed on a six-patient cohort basis (35, 45 and 55 mg m(-2)) with doxorubicin reduced to 25 mg m(-2) or omitted in cycles 4-6 to maintain cumulative exposure of 103-130% standard ABVD. BVD was given at standard doses throughout. Six additional subjects were recruited at the MTD. RESULTS: Twenty-four subjects were recruited. Dose-limiting toxicities (DLTs) of grade 3 neuropathy, pneumonitis, palmar-plantar erythema and neutropenic infection were observed at 55 mg m(-2), so 45 mg m(-2) was declared the MTD. In patients who subsequently experienced DLT at any time, large increases in CK18 were seen on day 3 of cycle 1 ABVD. CONCLUSION: Escalated ABVD incorporating doxorubicin at 45 mg m(-2) in cycles 1-3 can be delivered safely with pegfilgrastim support. Circulating cell death biomarkers may assist in the development of future individualised dosing strategies.

Immunohistochemical prognostic markers in diffuse large B-cell lymphoma: validation of tissue microarray as a prerequisite for broad clinical applications (a study from the Lunenburg Lymphoma Biomarker Consortium).

BACKGROUND AND AIMS: The results of class prediction and the determination of prognostic markers in diffuse large B-cell lymphoma (DLBCL) have been variably reported. Apart from biological variations, this may be caused by differences in laboratory techniques, scoring definitions and inter- and intra-observer variation. In this study, an international collaboration of clinical lymphoma research groups has concentrated on validation and standardisation of immunohistochemistry of the currently potentially interesting prognostic markers in DLBCL. METHODS: Sections of a tissue microarray with 36 cases of DLBCL were stained in eight laboratories with antibodies to CD20, CD5, bcl-2, bcl-6, CD10, HLA-DR, MUM-1 and Ki-67 according to local methods. The study was performed in two rounds, firstly focused on the evaluation of laboratory staining variation, and secondly on the scoring variation. RESULTS: Different techniques resulted in highly variable results and poor reproducibility for almost all markers. Reproducibility of the nuclear markers was highly sensitive to technical variations, including immunological enhancement techniques (agreements 34%). With elimination of variation due to staining and uniformly agreed on scoring criteria, significant improvement was seen; however less so for bcl-6 and Ki-67 (agreement 53-58%). Absence of internal controls that preclude scoring, significantly influenced the results for CD10 and bcl-6. CONCLUSION: Semi-quantitative immunohistochemistry for subclassification of DLBCL is feasible, but with varying rates of concordance for different markers and only using optimised techniques and strict scoring criteria. These findings may explain the wide variation in prognostic impact reported in the literature. Harmonisation of techniques and centralised consensus review appears mandatory when using immunohistochemical biomarkers for treatment stratification.

An investigation into the role of prostaglandins in zebrafish oocyte maturation and ovulation.

This study explored the potential for ovarian-derived prostaglandins (PGs) to be involved in the regulation of oocyte maturation and ovulation in zebrafish. It was demonstrated that cultured vitellogenic follicles have the capacity to produce prostaglandin E(2) (PGE(2)) and PGF(2alpha) in response to arachidonic acid (AA) in a concentration-dependent manner, and that AA stimulates the in vitro production of 17beta-estradiol (E(2)). The production of AA-stimulated PGF(2alpha) was significantly reduced by treatment with the non-selective cyclooxygenase (COX) inhibitor, indomethacin (INDO). Treatment of full-grown follicles with AA did not induce oocyte maturation as assessed by germinal vesicle breakdown, but INDO significantly decreased the rate of spontaneous maturation. Using Real-Time PCR, it was shown that follicles of different developmental size classes (primary growth and pre-vitellogenic, early-vitellogenic, and mid- to full-grown vitellogenic) express enzymes that release (cytosolic phospholipase A(2) (cPLA(2)); phospholipase Cgamma1) or metabolize (COX-1, COX-2, and prostaglandin synthase-2) AA to PG metabolites. The expression of cPLA(2) was found to be significantly greater in full-grown follicles compared to follicles of the pre- and early-vitellogenic stages. In vivo studies demonstrated that breeding groups of zebrafish exposed to 100 microg/L INDO exhibited reduced spawning rates and clutch sizes compared with control and 1 microg/L INDO exposed fish. In other studies, it was shown that naturally spawning groups of females exhibit increased ovarian levels of PGF(2alpha), E(2), and 17alpha,20beta-dihydroxy-4-pregnen-3-one (a maturation-inducing hormone in zebrafish) near the time of ovulation compared with non-breeding females. Collectively, these experiments indicate that the AA pathway in zebrafish ovaries is involved in the regulation of oocyte maturation and ovulation and a non-selective inhibitor of COX disrupts these processes.

Acetabular version and long posterior wall cup in total hip replacement.

This study discusses the definition of operative, anatomic and radiographic acetabular anteversion and their effect on a long posterior wall cup. In this cadaveric study, anteversion was modelled radiographically with a Charnley double wire marker Long Posterior Wall (LPW) cup and the position of the long posterior wall was studied with relation to different anteversion angles, and also correlated with computer generated line diagrams of the acetabular cup. The position of a long posterior wall varies when different types of anteversion are applied. By increasing the operative anteversion the long posterior wall comes to lie inferiorly and the advantage of the long posterior wall is lost. In light of prior confusion with definitions and in an attempt to make the terms more relevant to the surgical technique we propose the simplified alternate terms of 'flexion-anteversion' previously called 'operative anteversion', 'internal rotation-anteversion' previously called 'anatomic anteversion' and 'combined anteversion' previously called 'radiographic anteversion'. If the surgeon elects to use LPW it is important to understand the effect of each type of anteversion on eventual position of the raised wall.

Reproductive and stress hormone levels in goldfish (Carassius auratus) exposed to oil sands process-affected water.

Athabasca oil sands mining in northern Alberta produces process-affected waters that are characterized by the presence of naphthenic acids, polycyclic aromatic hydrocarbons, and high salinity. The purpose of this study was to examine the impact of these process-affected waters on reproductive and stress related endpoints in mature goldfish, Carassius auratus. In two separate studies, testosterone and 17beta-estradiol levels in the plasma were significantly reduced in both male and female goldfish caged for 19 days in process-affected waters relative to controls. This effect was most pronounced in goldfish caged at a site containing mature fine tailing and tailings pond water (P5). Ovarian and testicular tissues from fish in the caging studies were incubated in vitro to evaluate potential differences in basal steroid production levels and responsiveness to human chorionic gonadotropin (hCG). Basal levels of testosterone were reduced significantly in males and females from P5 compared with the control pond (P1) demonstrating that the gonads from exposed fish had a diminished steroidogenic capacity. Gonadal tissues of fish from all ponds responded similarly to hCG suggesting that the steroid biosynthetic pathway remained functionally intact. Plasma cortisol levels were significantly higher in male goldfish caged in a pond containing mature fine tailings and capped with uncontaminated water (P3) and in P5 compared with P1. Collectively, these studies suggest that waste products of oil sands mining have the potential to disrupt the normal endocrine functioning in exposed fish through alterations to both reproductive and glucocorticoid hormone biosynthesis. In additional laboratory studies, exposure of goldfish to a naphthenic acid extract for 7 days failed to replicate the effects of processes-affected waters on plasma steroid levels and the causative agent(s) responsible for the effects on steroid biosynthesis remains to be identified.

Gill and liver histopathological changes in yellow perch (Perca flavescens) and goldfish (Carassius auratus) exposed to oil sands process-affected water.

The extraction of bitumen from the Athabasca oil sands (Alberta, Canada) produces significant volumes of process-affected water containing elevated levels of naphthenic acids (NAs), ions, and polycyclic aromatic hydrocarbons (PAHs). The sublethal response of aquatic organisms exposed to oil sands constituents in experimental aquatic environments that represent possible reclamation options has been studied. In this study, the effects of process-affected waters on gill and liver tissues in yellow perch (Perca flavescens) and caged goldfish (Carassius auratus) held in several reclamation ponds at Syncrude's Mildred Lake site have been assessed. Following a 3-week exposure, significant gill (epithelial cell necrosis, mucous cell proliferation) and liver (hepatocellular degeneration, inflammatory cell infiltration) histopathological changes were noted in fish held in waters containing high levels of oil sands process-affected water. In addition, measurements of gill dimensions (gill morphometrical indices) proved sensitive and provided evidence of a physiological disturbance (gas exchange) with exposure to oil sands materials. Due to the complexity of oil sands process-affected water, the cause of the alterations could not be attributed to specific oil sands constituents. However, the histopathological parameters were strong indicators of exposure to oil sands process-affected water and morphometrical data were sensitive indicators of pathological response, which can be used to identify the interactive effects of ionic content, NAs, and PAHs in future laboratory studies.

Sex steroid regulation of brain glutamic acid decarboxylase (GAD) mRNA is season-dependent and sexually dimorphic in the goldfish Carassius auratus.

GABA, the major inhibitory neurotransmitter of the vertebrate brain, has been shown to play an important role in vertebrate reproduction by regulating LH release and sexual behavior. We have studied the expression of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), in goldfish throughout the reproductive cycle in May (mature), November (early gonadal recrudescence) and February (late gonadal recrudescence) and in response to implanted sex steroids. Levels of GAD67 and GAD65 mRNA levels in the hypothalamus of both males and females were highest in the early stages of gonadal recrudescence. In the telencephalon, a different seasonal pattern of GAD expression was evident. The telencephalic expression GAD67, GAD65 and a novel isoform, GAD3, were highest in sexually mature fish in May. Five-day intraperitoneal implantation of gonad-intact fish with testosterone (T), estradiol (E2) or progesterone (P4) did not affect GAD expression in November and February. This is in contrast to results in May when sex differences in steroid responsiveness were evident. Progesterone decreased hypothalamic GAD67 and GAD65 in females and was without effect in males. All other treatments did not alter GAD67, GAD65 or GAD3 expression in the hypothalamus. Both T and P4 decreased GAD67 and GAD65 levels in the telencephalon of male goldfish but had no effect in females. Serum sex steroid levels in control and implanted mature males and females in May were similar so it is unlikely that sex differences in the GAD responses were a result of differences in serum sex steroid levels. These contrasting effects of sex steroids on males and females suggest important sex differences in the regulation of the GADs in sexually mature goldfish.

The phylogenetic position of the 'giant deer' Megaloceros giganteus.

The giant deer, or 'Irish elk', has featured extensively in debates on adaptation, sexual selection, and extinction. Its huge antlers--the largest of any deer species, living or extinct--formed a focus of much past work. Yet the phylogenetic position of the giant deer has remained an enigma. On the basis of its flattened antlers, the species was previously regarded as closely related to the living fallow deer. Recent morphological studies, however, have challenged that view and placed the giant deer closer to the living red deer or wapiti. Here we present a new phylogenetic analysis encompassing morphological and DNA sequence evidence, and find that both sets of data independently support a sister-group relationship of giant and fallow deer. Our results include the successful extraction and sequencing of DNA from this extinct species, and highlight the value of a joint molecular and morphological approach.

Pleistocene to Holocene extinction dynamics in giant deer and woolly mammoth.

The extinction of the many well-known large mammals (megafauna) of the Late Pleistocene epoch has usually been attributed to 'overkill' by human hunters, climatic/vegetational changes or to a combination of both. An accurate knowledge of the geography and chronology of these extinctions is crucial for testing these hypotheses. Previous assumptions that the megafauna of northern Eurasia had disappeared by the Pleistocene/Holocene transition were first challenged a decade ago by the discovery that the latest woolly mammoths on Wrangel Island, northeastern Siberia, were contemporaneous with ancient Egyptian civilization. Here we show that another spectacular megafaunal species, the giant deer or 'Irish elk', survived to around 6,900 radiocarbon yr bp (about 7,700 yr ago) in western Siberia-more than three millennia later than its previously accepted terminal date-and therefore, that the reasons for its ultimate demise are to be sought in Holocene not Pleistocene events. Before their extinction, both giant deer and woolly mammoth underwent dramatic shifts in distribution, driven largely by climatic/vegetational changes. Their differing responses reflect major differences in ecology.

Fire ant venom alkaloid, isosolenopsin A, a potent and selective inhibitor of neuronal nitric oxide synthase.

Massive, multiple fire ant, Solenopsis invicta, stings are often treated aggressively, particularly in the elderly, despite limited evidence of systemic toxicity due to the venom. Over 95% of the S. invicta venom is composed of piperidine alkaloid components, whose toxicity, if any, is unknown. To assess a possible pharmacological basis for systemic toxicity, an alkaloid-rich, protein-free methanol extract of the venom from whole ants was assayed for inhibitory activity on the following nitric oxide synthase (NOS) isoforms, rat cerebellar neuronal (nNOS), bovine recombinant endothelial (eNOS), and murine recombinant immunologic (iNOS). Cytosolic NOS activity was determined by measuring the conversion of [(3)H]arginine to [(3)H]citrulline in vitro. Rat nNOS activity was inhibited significantly and in a concentration-dependent manner by the alkaloid-rich venom extract. For nNOS, enzyme activity was inhibited by approximately 50% with 0.33 +/- 0.06 microg of this venom extract, and over 95% inhibition of the three isoforms, nNOS, eNOS, and iNOS, was found with doses of 60 microg in 60 microl reaction mixture. These results indicate that the alkaloid components of S. invicta venom can produce potent inhibition of all three major NOS isoforms. Isosolenopsin A (cis-2-methyl-6-undecylpiperidine), a naturally occurring fire ant piperidine alkaloid, was synthesized and tested for inhibitory activity against the three NOS isoforms. Enzyme activities for nNOS and eNOS were over 95% inhibited with 1000 microM of isosolenopsin A, whereas the activity of iNOS was inhibited by only about 20% at the same concentration. The IC(50) for each of three NOS isoforms was approximately 18 +/- 3.9 microM for nNOS, 156 +/- 10 microM for eNOS, and >1000 microM for iNOS, respectively. Kinetic studies showed isosolenopsin A inhibition to be noncompetitive with L-arginine (K(i) = 19 +/- 2 microM). The potency of isosolenopsin A as an inhibitor of nNOS compares favorably with the inhibitory potency of widely used nNOS inhibitors. Inhibition of NOS isoforms by isosolenopsin A and structurally similar compounds may have toxicological significance with respect to adverse reactions to fire ant stings.

Stevens-Johnson syndrome after treatment with rituximab.

Rituximab is a chimeric mouse/human anti-CD20 antibody licensed for the treatment of low-grade non-Hodgkin's lymphoma and has recently also been shown to have a role in the treatment of diffuse large B-cell lymphoma. We report a case of Stevens-Johnson syndrome after treatment with rituximab, which occurred in a 36-year-old man with relapsed follicular lymphoma. The patient developed mucositis and fevers after the first two injections, followed by a florid maculopapular rash with severe orogenital ulceration after the third infusion. Over several weeks his symptoms progressed with severe cutaneous, orogenital and conjunctival ulceration, leading to visual problems and malnutrition. No improvement occurred with steroids and immunosuppressant therapy. A review of the literature reveals this to be the first reported case of Stevens-Johnson syndrome associated with rituximab therapy.

The origin and evolution of the woolly mammoth.

The mammoth lineage provides an example of rapid adaptive evolution in response to the changing environments of the Pleistocene. Using well-dated samples from across the mammoth's Eurasian range, we document geographical and chronological variation in adaptive morphology. This work illustrates an incremental (if mosaic) evolutionary sequence but also reveals a complex interplay of local morphological innovation, migration, and extirpation in the origin and evolution of a mammalian species. In particular, northeastern Siberia is identified as an area of successive allopatric innovations that apparently spread to Europe, where they contributed to a complex pattern of stasis, replacement, and transformation.