Chromosome Evolution and Genome Reconstruction in Falcon Species

Falcons and falconry have become an essential part of life in the Middle East since ancient times. In the United Arab Emirates (UAE) itself, the number of trained falcons ranges from 8,000 to 10,000. Over the last five years, falcon racing, a traditional sport, has gained momentum in the UAE where captive falcons are competing for huge prizemoney. A proportion of the UAE economy goes into their care and conservation e.g. through establishing falcon hospitals with modern facilities for disease treatment and breeding as well as centers for diagnosis and research. Being the national bird of the UAE, any research on falcons is of significant interest for the country. Most of the world's falcon species are in decline. Moreover, Saker falcons are classified as 'endangered' according to the IUCN Red List of Threatened Species. From the chromosomal perspective falcons are very interesting, as they represent birds that have undergone significant genome rearrangement compared to the "norm" of 2n=~80. 'Molecular cytogenomics' in birds includes karyotyping, cross species comparisons, nuclear organization, BAC mapping, physical mapping and telomeric DNA profiling. This thesis makes use of the above approaches to define chromosome evolution and genome organization in falcon species with the following results: Firstly, successful conventional characterization of the Saker, Peregrine and Gyr falcon karyotypes (2n=50-52) was achieved producing improved karyotypes and ideograms than those previously published. Comparative genomic analyses among these three species using molecular cytogenetic approaches revealed differences between Peregrine and the other two species, but none between Saker falcon and Gyrfalcon. Also, this study has supported upgrading the fragmented Saker genome assembly to chromosome level using a novel approach hitherto only published for the Peregrine falcon (and pigeon). Secondly, a comparison of genome-wide BAC-based studies and bioinformatic analysis Multiple Genomes Rearrangement Algorithm 2 (MGRA2) revealed the chromosomal changes (inter- and intra-) that led to the falcon lineage. Also, the present study established that common mechanisms of chromosomal fusion do not recur in two different groups of species with rearranged karyotypes (falcons and parrots). This thesis also provided an overview of the telomeric DNA profile in the three species of interest. It established that the highly rearranged karyotypes studied (plus those of the budgerigar and crocodile) do not appear to possess interstitial telomeres at evolutionary fusion points. Also, this study demonstrated the existence of megatelomeres in falcon species, their nature differing between the Peregrine and the other two species studied. Finally, this thesis produced the first detailed description of nuclear organization in a bird species (Peregrine falcon) other than the Galloanserae. Non-fused macro and microchromosomes behave the same way in chickens and falcons. This implies that the same general nuclear organization mechanisms are present in falcons as well as in chickens, ducks and turkeys whose last common ancestor existed around 89 million years ago. Most notably, fused microchromosomes in the Peregrine falcon retain the same nuclear organization pattern despite being fused to a larger chromosome. The findings from this study give insight into the basic nature of chromosome territory patterns in bird species with highly rearranged karyotypes. Overall, results presented in this thesis provide significant insight into genome organization and evolution in the Falco genus, revealing previously undetected levels of chromosomal synteny between three species important to the UAE. Results generated here have also made a significant contribution to the chromosome-level genome assembly of the Saker falcon, providing tools for further study of avian species both within and beyond the falcon group

Views and experiences of opioid access amongst palliative care providers and public representatives in a low-resource setting: A qualitative interview study

AbstractOpioids (e.g. morphine) are affordable, effective interventions for cancer-related pain. However, equity of access to this key medication remains a global challenge, particularly in low- and middle-income countries. We aimed to explore views of palliative care providers and public-representatives about opioid analgesia access in two States in India. We conducted a qualitative study using semi-structured interviews. Transcribed audio-recordings were subjected to thematic analysis using a Framework Approach. Palliative care providers and public-representatives were purposively sampled from services reporting consistent opioid availability and prescribing (≥4kg per annum) from Karnataka and Kerala. Twenty participants (doctors (10), nurses (4), pharmacists (2), service managers (2) and public-representatives (2) were interviewed. Three themes were identified: 1) Attitudes and awareness: opioid treatments are perceived as end-of-life (last days/weeks) interventions; fears of addiction and misunderstanding of pain management goals limit access. 2) Expected and unexpected inequities: patients/carers from lower socioeconomic strata accept doctor recommendations if opioids are affordable, more educated patients/families have reservations about opioids, delay access and perceive expensive medicines as better. Non-palliative care specialist doctors have negative entrenched views and require specialist training. 3) Experiential learning–positive experiences can positively alter attitudes (e.g., participants in Kerala report improved attitudes, awareness and understanding influenced by exposure and community awareness, but experience can also reinforce perceptions as end-of-life care. Entrenched negative views are reinforced by poor experiences while positive experiences improve attitudes. To promote access, opioid prescribing must be needs-based rather than prognosis-based. Addressing the lack of training for non-palliative care workforce would help overcome a major barrier

Patterns of microchromosome organization remain highly conserved throughout avian evolution

The structure and organization of a species genome at a karyotypic level, and in interphase nuclei, have broad functional significance. Although regular sized chromosomes are studied extensively in this regard, microchromosomes, which are present in many terrestrial vertebrates, remain poorly explored. Birds have more cytologically indistinguishable microchromosomes (~ 30 pairs) than other vertebrates; however, the degree to which genome organization patterns at a karyotypic and interphase level differ between species is unknown. In species where microchromosomes have fused to other chromosomes, they retain genomic features such as gene density and GC content; however, the extent to which they retain a central nuclear position has not been investigated. In studying 22 avian species from 10 orders, we established that, other than in species where microchromosomal fusion is obvious (Falconiformes and Psittaciformes), there was no evidence of microchromosomal rearrangement, suggesting an evolutionarily stable avian genome (karyotypic) organization. Moreover, in species where microchromosomal fusion has occurred, they retain a central nuclear location, suggesting that the nuclear position of microchromosomes is a function of their genomic features rather than their physical size

Chromosome-level assembly reveals extensive rearrangement in sakar falcon and budgerigar, but not ostrich, genomes

Background: The number of de novo genome sequence assemblies is increasing exponentially; however, relatively few contain one scaffold/contig per chromosome. Such assemblies are essential for studies of genotype-to-phenotype association, gross genomic evolution, and speciation. Inter-species differences can arise from chromosomal changes fixed during evolution, and we previously hypothesized that a higher fraction of elements under negative selection contributed to avian-specific phenotypes and avian genome organization stability. The objective of this study is to generate chromosome-level assemblies of three avian species (saker falcon, budgerigar, and ostrich) previously reported as karyotypically rearranged compared to most birds. We also test the hypothesis that the density of conserved non-coding elements is associated with the positions of evolutionary breakpoint regions. Results: We used reference-assisted chromosome assembly, PCR, and lab-based molecular approaches, to generate chromosome-level assemblies of the three species. We mapped inter- and intrachromosomal changes from the avian ancestor, finding no interchromosomal rearrangements in the ostrich genome, despite it being previously described as chromosomally rearranged. We found that the average density of conserved non-coding elements in evolutionary breakpoint regions is significantly reduced. Fission evolutionary breakpoint regions have the lowest conserved non-coding element density, and intrachromomosomal evolutionary breakpoint regions have the highest. Conclusions: The tools used here can generate inexpensive, efficient chromosome-level assemblies, with > 80% assigned to chromosomes, which is comparable to genomes assembled using high-density physical or genetic mapping. Moreover, conserved non-coding elements are important factors in defining where rearrangements, especially interchromosomal, are fixed during evolution without deleterious effects

Upgrading short read animal genome assemblies to chromosome level using comparative genomics and a universal probe set

Most recent initiatives to sequence and assemble new species’ genomes de novo fail to achieve the ultimate endpoint to produce contigs, each representing one whole chromosome. Even the best-assembled genomes (using contemporary technologies) consist of subchromosomal-sized scaffolds. To circumvent this problem, we developed a novel approach that combines computational algorithms to merge scaffolds into chromosomal fragments, PCR-based scaffold verification, and physical mapping to chromosomes. Multigenome-alignment-guided probe selection led to the development of a set of universal avian BAC clones that permit rapid anchoring of multiple scaffolds to chromosomes on all avian genomes. As proof of principle, we assembled genomes of the pigeon (Columbia livia) and peregrine falcon (Falco peregrinus) to chromosome levels comparable, in continuity, to avian reference genomes. Both species are of interest for breeding, cultural, food, and/or environmental reasons. Pigeon has a typical avian karyotype (2n = 80), while falcon (2n = 50) is highly rearranged compared to the avian ancestor. By using chromosome breakpoint data, we established that avian interchromosomal breakpoints appear in the regions of low density of conserved noncoding elements (CNEs) and that the chromosomal fission sites are further limited to long CNE “deserts.” This corresponds with fission being the rarest type of rearrangement in avian genome evolution. High-throughput multiple hybridization and rapid capture strategies using the current BAC set provide the basis for assembling numerous avian (and possibly other reptilian) species, while the overall strategy for scaffold assembly and mapping provides the basis for an approach that (provided metaphases can be generated) could be applied to any animal genome

Chromosome Level Genome Assembly and Comparative Genomics between Three Falcon Species Reveals an Unusual Pattern of Genome Organisation

Whole genome assemblies are crucial for understanding a wide range of aspects of falcon biology, including morphology, ecology, and physiology, and are thus essential for their care and conservation. A key aspect of the genome of any species is its karyotype, which can then be linked to the whole genome sequence to generate a so-called chromosome-level assembly. Chromosome-level assemblies are essential for marker assisted selection and genotype-phenotype correlations in breeding regimes, as well as determining patterns of gross genomic evolution. To date, only two falcon species have been sequenced and neither initially were assembled to the chromosome level. Falcons have atypical avian karyotypes with fewer chromosomes than other birds, presumably brought about by wholesale fusion. To date, however, published chromosome preparations are of poor quality, few chromosomes have been distinguished and standard ideograms have not been made. The purposes of this study were to generate analyzable karyotypes and ideograms of peregrine, saker, and gyr falcons, report on our recent generation of chromosome level sequence assemblies of peregrine and saker falcons, and for the first time, sequence the gyr falcon genome. Finally, we aimed to generate comparative genomic data between all three species and the reference chicken genome. Results revealed a diploid number of 2n = 50 for peregrine falcon and 2n = 52 for saker and gyr through high quality banded chromosomes. Standard ideograms that are generated here helped to map predicted chromosomal fragments (PCFs) from the genome sequences directly to chromosomes and thus generate chromosome level sequence assemblies for peregrine and saker falcons. Whole genome sequencing was successful in gyr falcon, but read depth and coverage was not sufficient to generate a chromosome level assembly. Nonetheless, comparative genomics revealed no differences in genome organization between gyr and saker falcons. When compared to peregrine falcon, saker/gyr differed by one interchromosomal and seven intrachromosomal rearrangements (a fusion plus seven inversions), whereas peregrine and saker/gyr differ from the reference chicken genome by 14/13 fusions (11 microchromosomal) and six fissions. The chromosomal differences between the species could potentially provide the basis of a screening test for hybrid animals

Effect of a Quality Improvement Intervention on Clinical Outcomes in Patients in India With Acute Myocardial Infarction: The ACS QUIK Randomized Clinical Trial.

Importance: Wide heterogeneity exists in acute myocardial infarction treatment and outcomes in India. Objective: To evaluate the effect of a locally adapted quality improvement tool kit on clinical outcomes and process measures in Kerala, a southern Indian state. Design, Setting, and Participants: Cluster randomized, stepped-wedge clinical trial conducted between November 10, 2014, and November 9, 2016, in 63 hospitals in Kerala, India, with a last date of follow-up of December 31, 2016. During 5 predefined steps over the study period, hospitals were randomly selected to move in a 1-way crossover from the control group to the intervention group. Consecutively presenting patients with acute myocardial infarction were offered participation. Interventions: Hospitals provided either usual care (control group; n = 10 066 participants [step 0: n = 2915; step 1: n = 2649; step 2: n = 2251; step 3: n = 1422; step 4; n = 829; step 5: n = 0]) or care using a quality improvement tool kit (intervention group; n = 11 308 participants [step 0: n = 0; step 1: n = 662; step 2: n = 1265; step 3: n = 2432; step 4: n = 3214; step 5: n = 3735]) that consisted of audit and feedback, checklists, patient education materials, and linkage to emergency cardiovascular care and quality improvement training. Main Outcomes and Measures: The primary outcome was the composite of all-cause death, reinfarction, stroke, or major bleeding using standardized definitions at 30 days. Secondary outcomes included the primary outcome's individual components, 30-day cardiovascular death, medication use, and tobacco cessation counseling. Mixed-effects logistic regression models were used to account for clustering and temporal trends. Results: Among 21 374 eligible randomized participants (mean age, 60.6 [SD, 12.0] years; n = 16 183 men [76%] ; n = 13 689 [64%] with ST-segment elevation myocardial infarction), 21 079 (99%) completed the trial. The primary composite outcome was observed in 5.3% of the intervention participants and 6.4% of the control participants. The observed difference in 30-day major adverse cardiovascular event rates between the groups was not statistically significant after adjustment (adjusted risk difference, -0.09% [95% CI, -1.32% to 1.14%]; adjusted odds ratio, 0.98 [95% CI, 0.80-1.21]). The intervention group had a higher rate of medication use including reperfusion but no effect on tobacco cessation counseling. There were no unexpected adverse events reported. Conclusions and Relevance: Among patients with acute myocardial infarction in Kerala, India, use of a quality improvement intervention compared with usual care did not decrease a composite of 30-day major adverse cardiovascular events. Further research is needed to understand the lack of efficacy. Trial Registration: clinicaltrials.gov Identifier: NCT02256657

Development of a surveillance scheme for equine influenza in the UK and characterisation of viruses isolated in Europe, Dubai and the USA from 2010-2012

Equine influenza viruses are a major cause of respiratory disease in horses worldwide and undergo antigenic drift. Several outbreaks of equine influenza occurred worldwide during 2010-2012, including in vaccinated animals, highlighting the importance of surveillance and virus characterisation. Virus isolates were characterised from more than 20 outbreaks over a 3-year period, including strains from the UK, Dubai, Germany and the USA. The haemagglutinin-1 (HA1) sequence of all isolates was determined and compared with OIE-recommended vaccine strains. Viruses from Florida clades 1 and 2 showed continued divergence from each other compared with 2009 isolates. The antigenic inter-relationships among viruses were determined using a haemagglutination-inhibition (HI) assay with ferret antisera and visualised using antigenic cartography. All European isolates belonged to Florida clade 2, all those from the USA belonged to Florida clade 1. Two subpopulations of clade 2 viruses were isolated, with either substitution A144V or I179V. Isolates from Dubai, obtained from horses shipped from Uruguay, belonged to Florida clade 1 and were similar to viruses isolated in the USA the previous year. The neuraminidase (NA) sequence of representative strains from 2007 and 2009 to 2012 was also determined and compared with that of earlier isolates dating back to 1963. Multiple changes were observed at the amino acid level and clear distinctions could be made between viruses belonging to Florida clade 1 and clade 2