Candidate gene polymorphisms are linked to dispersive and migratory behaviour: Searching for a mechanism behind the "paradox of the great speciators".

The "paradox of the great speciators" has puzzled evolutionary biologists for over half a century. A great speciator requires excellent dispersal propensity to explain its occurrence on multiple islands, but reduced dispersal ability to explain its high number of subspecies. A rapid reduction in dispersal ability is often invoked to solve this apparent paradox, but a proximate mechanism has not been identified yet. Here, we explored the role of six genes linked to migration and animal personality differences (CREB1, CLOCK, ADCYAP1, NPAS2, DRD4, and SERT) in 20 South Pacific populations of silvereye (Zosterops lateralis) that range from highly sedentary to partially migratory, to determine if genetic variation is associated with dispersal propensity and migration. We detected genetic associations in three of the six genes: (i) in a partial migrant population, migrant individuals had longer microsatellite alleles at the CLOCK gene compared to resident individuals from the same population; (ii) CREB1 displayed longer average microsatellite allele lengths in recently colonized island populations (<200 years), compared to evolutionarily older populations. Bayesian broken stick regression models supported a reduction in CREB1 length with time since colonization; and (iii) like CREB1, DRD4 showed differences in polymorphisms between recent and old colonizations but a larger sample is needed to confirm. ADCYAP1, SERT, and NPAS2 were variable but that variation was not associated with dispersal propensity. The association of genetic variants at three genes with migration and dispersal ability in silvereyes provides the impetus for further exploration of genetic mechanisms underlying dispersal shifts, and the prospect of resolving a long-running evolutionary paradox through a genetic lens.

Feasibility and impact of haplogroup matching for mitochondrial replacement treatment.

Mitochondrial replacement technology (MRT) aims to reduce the risk of serious disease in children born to women who carry pathogenic mitochondrial DNA (mtDNA) variants. By transplanting nuclear genomes from eggs of an affected woman to enucleated eggs from an unaffected donor, MRT creates new combinations of nuclear and mtDNA. Based on sets of shared sequence variants, mtDNA is classified into ~30 haplogroups. Haplogroup matching between egg donors and women undergoing MRT has been proposed as a means of reducing mtDNA sequence divergence between them. Here we investigate the potential effect of mtDNA haplogroup matching on clinical delivery of MRT and on mtDNA sequence divergence between donor/recipient pairs. Our findings indicate that haplogroup matching would limit the availability of egg donors such that women belonging to rare haplogroups may have to wait > 4 years for treatment. Moreover, we find that intra-haplogroup sequence variation is frequently within the range observed between randomly matched mtDNA pairs. We conclude that haplogroup matching would restrict the availability of MRT, without necessarily reducing mtDNA sequence divergence between donor/recipient pairs.

Species-wide genomics of kakapo provides tools to accelerate recovery.

The kakapo is a critically endangered, intensively managed, long-lived nocturnal parrot endemic to Aotearoa New Zealand. We generated and analysed whole-genome sequence data for nearly all individuals living in early 2018 (169 individuals) to generate a high-quality species-wide genetic variant callset. We leverage extensive long-term metadata to quantify genome-wide diversity of the species over time and present new approaches using probabilistic programming, combined with a phenotype dataset spanning five decades, to disentangle phenotypic variance into environmental and genetic effects while quantifying uncertainty in small populations. We find associations for growth, disease susceptibility, clutch size and egg fertility within genic regions previously shown to influence these traits in other species. Finally, we generate breeding values to predict phenotype and illustrate that active management over the past 45 years has maintained both genome-wide diversity and diversity in breeding values and, hence, evolutionary potential. We provide new pathways for informing future conservation management decisions for kakapo, including prioritizing individuals for translocation and monitoring individuals with poor growth or high disease risk. Overall, by explicitly addressing the challenge of the small sample size, we provide a template for the inclusion of genomic data that will be transformational for species recovery efforts around the globe.

Decolonisation initiatives at the Institute of Tropical Medicine, Antwerp, Belgium: ready for change?

In a global context, the pernicious effects of colonialism and coloniality are increasingly being recognised in many sectors. As a result, calls to reverse colonial aphasia and amnesia, and decolonise, are getting stronger. This raises a number of questions, particularly for entities that acted as agents of (previous) colonising countries and worked to further the progress of the colonial project: What does decolonisation mean for such historically colonial entities? How can they confront their (forgotten) arsonist past while addressing their current role in maintaining coloniality, at home and abroad? Given the embeddedness of many such entities in current global (power) structures of coloniality, do these entities really want change, and if so, how can such entities redefine their future to ensure that they are and remain 'decolonised'? We attempt to answer these questions, by reflecting on our efforts to think through and start the process of decolonisation at the Institute of Tropical Medicine (ITM) in Antwerp, Belgium. The overarching aim is to contribute to closing the gap in the literature when it comes to documenting practical efforts at decolonisation, particularly in contexts similar to ITM and to share our experience and engage with others who are undertaking or planning to undertake similar initiatives.

Associations with age and glomerular filtration rate in a referred population with chronic kidney disease: methods and baseline data from a UK multicentre cohort study (NURTuRE-CKD).

BACKGROUND: Chronic kidney disease (CKD) is common but heterogenous and is associated with multiple adverse outcomes. The National Unified Renal Translational Research Enterprise (NURTuRE)-CKD cohort was established to investigate risk factors for clinically important outcomes in persons with CKD referred to secondary care. METHODS: Eligible participants with CKD stages G3-4 or stages G1-2 plus albuminuria >30 mg/mmol were enrolled from 16 nephrology centres in England, Scotland and Wales from 2017 to 2019. Baseline assessment included demographic data, routine laboratory data and research samples. Clinical outcomes are being collected over 15 years by the UK Renal Registry using established data linkage. Baseline data are presented with subgroup analysis by age, sex and estimated glomerular filtration rate (eGFR). RESULTS: A total of 2996 participants was enrolled. Median (interquartile range) age was 66 (54-74) years, eGFR 33.8 (24.0-46.6) mL/min/1.73 m2 and urine albumin to creatinine ratio 209 (33-926) mg/g; 58.5% were male. Of these participants, 1883 (69.1%) were in high-risk CKD categories. Primary renal diagnosis was CKD of unknown cause in 32.3%, glomerular disease in 23.4% and diabetic kidney disease in 11.5%. Older participants and those with lower eGFR had higher systolic blood pressure and were less likely to be treated with renin-angiotensin system inhibitors (RASi) but were more likely to receive a statin. Female participants were less likely to receive a RASi or statin. CONCLUSIONS: NURTuRE-CKD is a prospective cohort of persons who are at relatively high risk of adverse outcomes. Long-term follow-up and a large biorepository create opportunities for research to improve risk prediction and to investigate underlying mechanisms to inform new treatment development.

Evaluating Ultrasonicator Performance for Cyanobacteria Management at Freshwater Sources.

Algal blooms consisting of potentially toxic cyanobacteria are a growing source water management challenge faced by water utilities globally. Commercially available sonication devices are designed to mitigate this challenge by targeting cyanobacteria-specific cellular features and aim to inhibit cyanobacterial growth within water bodies. There is limited available literature evaluating this technology; therefore, a sonication trial was conducted in a drinking water reservoir within regional Victoria, Australia across an 18-month period using one device. The trial reservoir, referred to as Reservoir C, is the final reservoir in a local network of reservoirs managed by a regional water utility. Sonicator efficacy was evaluated through qualitative and quantitative analysis of algal and cyanobacterial trends within Reservoir C and surrounding reservoirs using field data collected across three years preceding the trial and during the 18-month duration of the trial. Qualitative assessment revealed a slight increase in eukaryotic algal growth within Reservoir C following device installation, which is likely due to local environmental factors such as rainfall-driven nutrient influx. Post-sonication quantities of cyanobacteria remained relatively consistent, which may indicate that the device was able to counteract favorable phytoplankton growth conditions. Qualitative assessments also revealed minimal prevalence variations of the dominant cyanobacterial species within the reservoir following trial initiation. Since the dominant species were potential toxin producers, there is no strong evidence that sonication altered Reservoir C's water risk profiles during this trial. Statistical analysis of samples collected within the reservoir and from the intake pipe to the associated treatment plant supported qualitative observations and revealed a significant elevation in eukaryotic algal cell counts during bloom and non-bloom periods post-installation. Corresponding cyanobacteria biovolumes and cell counts revealed that no significant changes occurred, excluding a significant decrease in bloom season cell counts measured within the treatment plant intake pipe and a significant increase in non-bloom season biovolumes and cell counts as measured within the reservoir. One technical disruption occurred during the trial; however, this had no notable impacts on cyanobacterial prevalence. Acknowledging the limitations of the experimental conditions, data and observations from this trial indicate there is no strong evidence that sonication significantly reduced cyanobacteria occurrence within Reservoir C.

Genomic trajectories of a near-extinction event in the Chatham Island black robin.

BACKGROUND: Understanding the micro--evolutionary response of populations to demographic declines is a major goal in evolutionary and conservation biology. In small populations, genetic drift can lead to an accumulation of deleterious mutations, which will increase the risk of extinction. However, demographic recovery can still occur after extreme declines, suggesting that natural selection may purge deleterious mutations, even in extremely small populations. The Chatham Island black robin (Petroica traversi) is arguably the most inbred bird species in the world. It avoided imminent extinction in the early 1980s and after a remarkable recovery from a single pair, a second population was established and the two extant populations have evolved in complete isolation since then. Here, we analysed 52 modern and historical genomes to examine the genomic consequences of this extreme bottleneck and the subsequent translocation. RESULTS: We found evidence for two-fold decline in heterozygosity and three- to four-fold increase in inbreeding in modern genomes. Moreover, there was partial support for temporal reduction in total load for detrimental variation. In contrast, compared to historical genomes, modern genomes showed a significantly higher realised load, reflecting the temporal increase in inbreeding. Furthermore, the translocation induced only small changes in the frequency of deleterious alleles, with the majority of detrimental variation being shared between the two populations. CONCLUSION: Our results highlight the dynamics of mutational load in a species that recovered from the brink of extinction, and show rather limited temporal changes in mutational load. We hypothesise that ancestral purging may have been facilitated by population fragmentation and isolation on several islands for thousands of generations and may have already reduced much of the highly deleterious load well before human arrival and introduction of pests to the archipelago. The majority of fixed deleterious variation was shared between the modern populations, but translocation of individuals with low mutational load could possibly mitigate further fixation of high-frequency deleterious variation.

Genomic signatures of inbreeding in a critically endangered parrot, the kakapo.

Events of inbreeding are inevitable in critically endangered species. Reduced population sizes and unique life-history traits can increase the severity of inbreeding, leading to declines in fitness and increased risk of extinction. Here, we investigate levels of inbreeding in a critically endangered flightless parrot, the kakapo (Strigops habroptilus), wherein a highly inbred island population and one individual from the mainland of New Zealand founded the entire extant population. Genotyping-by-sequencing (GBS), and a genotype calling approach using a chromosome-level genome assembly, identified a filtered set of 12,241 single-nucleotide polymorphisms (SNPs) among 161 kakapo, which together encompass the total genetic potential of the extant population. Multiple molecular-based estimates of inbreeding were compared, including genome-wide estimates of heterozygosity (FH), the diagonal elements of a genomic-relatedness matrix (FGRM), and runs of homozygosity (RoH, FRoH). In addition, we compared levels of inbreeding in chicks from a recent breeding season to examine if inbreeding is associated with offspring survival. The density of SNPs generated with GBS was sufficient to identify chromosomes that were largely homozygous with RoH distributed in similar patterns to other inbred species. Measures of inbreeding were largely correlated and differed significantly between descendants of the two founding populations. However, neither inbreeding nor ancestry was found to be associated with reduced survivorship in chicks, owing to unexpected mortality in chicks exhibiting low levels of inbreeding. Our study highlights important considerations for estimating inbreeding in critically endangered species, such as the impacts of small population sizes and admixture between diverse lineages.

The Isolation and Deep Sequencing of Mitochondrial DNA.

In recent years, next-generation sequencing (NGS) has become a powerful tool for studying both inherited and somatic heteroplasmic mitochondrial DNA (mtDNA) variation. NGS has proved particularly powerful when combined with single-cell isolation techniques, allowing the investigation of low-level heteroplasmic variants both between cells and within tissues. Nevertheless, there remain significant challenges, especially around the selective enrichment of mtDNA from total cellular DNA and the avoidance of nuclear pseudogenes. This chapter summarizes the techniques needed to enrich, amplify, sequence, and analyse mtDNA using NGS .

Ninjaflex vs Superflab: A comparison of dosimetric properties, conformity to the skin surface, Planning Target Volume coverage and positional reproducibility for external beam radiotherapy.

BACKGROUND AND PURPOSE: When planning and delivering radiotherapy, ideally bolus should be in direct contact with the skin surface. Varying air gaps between the skin surface and bolus material can result in discrepancies between the intended and delivered dose. This study assessed a three-dimensional (3D) printed flexible bolus to determine whether it could improve conformity to the skin surface, reduce air gaps, and improve planning target volume coverage, compared to a commercial bolus material, Superflab. MATERIALS AND METHODS: An anthropomorphic head phantom was CT scanned to generate photon and electron treatment plans using virtual bolus. Two 3D printing companies used the material Ninjaflex to print bolus for the head phantom, which we designated Ninjaflex1 and Ninjaflex2. The phantom was scanned a further 15 more times with the different bolus materials in situ allowing plan comparison of the virtual to physical bolus in terms of planning target volume coverage, dose at the prescription point, skin dose, and air gap volumes. RESULTS: Superflab produced a larger volume and a greater number of air gaps compared to both Ninjaflex1 and Ninjaflex2, with the largest air gap volume of 12.02 cm3 . Our study revealed that Ninjaflex1 produced the least variation from the virtual bolus clinical goal values for all modalities, while Superflab displayed the largest variances in conformity, positional accuracy, and clinical goal values. For PTV coverage Superflab produced significant percentage differences for the VMAT and Electron3 plans when compared to the virtual bolus plans. Superflab also generated a significant difference in prescription point dose for the 3D conformal plan. CONCLUSION: Compared to Superflab, both Ninjaflex materials improved conformity and reduced the variance between the virtual and physical bolus clinical goal values. Results illustrate that custom-made Ninjaflex bolus could be useful clinically and may improve the accuracy of the delivered dose.

Population genomics of the critically endangered kakapo.

The kakapo is a flightless parrot endemic to New Zealand. Once common in the archipelago, only 201 individuals remain today, most of them descending from an isolated island population. We report the first genome-wide analyses of the species, including a high-quality genome assembly for kakapo, one of the first chromosome-level reference genomes sequenced by the Vertebrate Genomes Project (VGP). We also sequenced and analyzed 35 modern genomes from the sole surviving island population and 14 genomes from the extinct mainland population. While theory suggests that such a small population is likely to have accumulated deleterious mutations through genetic drift, our analyses on the impact of the long-term small population size in kakapo indicate that present-day island kakapo have a reduced number of harmful mutations compared to mainland individuals. We hypothesize that this reduced mutational load is due to the island population having been subjected to a combination of genetic drift and purging of deleterious mutations, through increased inbreeding and purifying selection, since its isolation from the mainland ∼10,000 years ago. Our results provide evidence that small populations can survive even when isolated for hundreds of generations. This work provides key insights into kakapo breeding and recovery and more generally into the application of genetic tools in conservation efforts for endangered species.

Mitochondrial DNA Damage and Brain Aging in Human Immunodeficiency Virus.

BACKGROUND: Neurocognitive impairment (NCI) remains common in people living with human immunodeficiency virus (PLWH), despite suppressive antiretroviral therapy (ART), but the reasons remain incompletely understood. Mitochondrial dysfunction is a hallmark of aging and of neurodegenerative diseases. We hypothesized that human immunodeficiency virus (HIV) or ART may lead to mitochondrial abnormalities in the brain, thus contributing to NCI. METHODS: We studied postmortem frozen brain samples from 52 PLWH and 40 HIV-negative controls. Cellular mitochondrial DNA (mtDNA) content and levels of large-scale mtDNA deletions were measured by real-time polymerase chain reaction. Heteroplasmic mtDNA point mutations were quantified by deep sequencing (Illumina). Neurocognitive data were taken within 48 months antemortem. RESULTS: We observed a decrease in mtDNA content, an increase in the mtDNA "common deletion," and an increase in mtDNA point mutations with age (all P < .05). Each of these changes was exacerbated in HIV-positive cases compared with HIV-negative controls (all P < .05). ART exposures, including nucleoside analogue reverse transcriptase inhibitors, were not associated with changes in mtDNA. The number of mtDNA point mutations was associated with low CD4/CD8 ratio (P = .04) and with NCI (global T-score, P = .007). CONCLUSIONS: In people with predominantly advanced HIV infection, there is exacerbation of age-associated mtDNA damage. This change is driven by HIV per se rather than by ART toxicity and may contribute to NCI. These data suggest that mitochondrial dysfunction may be a mediator of adverse aging phenotypes in PLWH.

Age-associated mitochondrial DNA mutations cause metabolic remodelling that contributes to accelerated intestinal tumorigenesis.

Oxidative phosphorylation (OXPHOS) defects caused by somatic mitochondrial DNA (mtDNA) mutations increase with age in human colorectal epithelium and are prevalent in colorectal tumours, but whether they actively contribute to tumorigenesis remains unknown. Here we demonstrate that mtDNA mutations causing OXPHOS defects are enriched during the human adenoma/carcinoma sequence, suggesting they may confer a metabolic advantage. To test this we deleted the tumour suppressor Apc in OXPHOS deficient intestinal stem cells in mice. The resulting tumours were larger than in control mice due to accelerated cell proliferation and reduced apoptosis. We show that both normal crypts and tumours undergo metabolic remodelling in response to OXPHOS deficiency by upregulating the de novo serine synthesis pathway (SSP). Moreover, normal human colonic crypts upregulate the SSP in response to OXPHOS deficiency prior to tumorigenesis. Our data show that age-associated OXPHOS deficiency causes metabolic remodelling that can functionally contribute to accelerated intestinal cancer development.

Recent advances in understanding the molecular genetic basis of mitochondrial disease.

Mitochondrial disease is hugely diverse with respect to associated clinical presentations and underlying genetic causes, with pathogenic variants in over 300 disease genes currently described. Approximately half of these have been discovered in the last decade due to the increasingly widespread application of next generation sequencing technologies, in particular unbiased, whole exome-and latterly, whole genome sequencing. These technologies allow more genetic data to be collected from patients with mitochondrial disorders, continually improving the diagnostic success rate in a clinical setting. Despite these significant advances, some patients still remain without a definitive genetic diagnosis. Large datasets containing many variants of unknown significance have become a major challenge with next generation sequencing strategies and these require significant functional validation to confirm pathogenicity. This interface between diagnostics and research is critical in continuing to expand the list of known pathogenic variants and concomitantly enhance our knowledge of mitochondrial biology. The increasing use of whole exome sequencing, whole genome sequencing and other "omics" techniques such as transcriptomics and proteomics will generate even more data and allow further interrogation and validation of genetic causes, including those outside of coding regions. This will improve diagnostic yields still further and emphasizes the integral role that functional assessment of variant causality plays in this process-the overarching focus of this review.

Peritoneal Ultrafiltration for Heart Failure: Lessons from a Randomized Controlled Trial.

Peritoneal ultrafiltration (PuF) has been employed for severe heart failure (HF), but evidence for its benefit is lacking. The Peritoneal Dialysis for Heart Failure (PDHF) study was a multicenter prospective randomized controlled trial which aimed to investigate this issue. The trial stopped early due to inadequate recruitment. We describe methods, trial activity, and lessons learned.The trial aimed to recruit 130 participants with severe diuretic-resistant HF (New York Heart Association [NYHA] 3/4) and chronic kidney disease (CKD) stage 3/4 on optimal medical treatment for ≥ 4 weeks from 6 UK centers. Participants were randomized to either continuation of conventional HF treatment or to additionally receiving PuF (1 overnight exchange using Icodextrin dialysate). Primary outcome was change in 6-minute walk test (6MWT) between baseline and 28 weeks (end of trial). Secondary outcomes were changes in patient reported quality of life as assessed by the Kansas City Cardiomyopathy Questionnaire, short form 36 (SF 36) health survey results, hospitalization, and mortality.Over a 2-year period, 290 patients were screened from which only 20 met inclusion criteria and 10 were recruited. Reasons for ineligibility were fluctuating estimated glomerular filtration rate (eGFR), suboptimal HF treatment, frailty, and patients being too unwell for randomization. Barriers to recruitment included patient frailty, with some participants considered only when they were at end of life, unwillingness to engage in an invasive therapy, and suboptimal coordination between cardiology and renal services. This is a challenging patient group in which to perform research, and lessons learned from the peritoneal dialysis (PD)-HF trial will be helpful in the planning of future studies in this area.

Dynamical differential expression (DyDE) reveals the period control mechanisms of the Arabidopsis circadian oscillator.

The circadian oscillator, an internal time-keeping device found in most organisms, enables timely regulation of daily biological activities by maintaining synchrony with the external environment. The mechanistic basis underlying the adjustment of circadian rhythms to changing external conditions, however, has yet to be clearly elucidated. We explored the mechanism of action of nicotinamide in Arabidopsis thaliana, a metabolite that lengthens the period of circadian rhythms, to understand the regulation of circadian period. To identify the key mechanisms involved in the circadian response to nicotinamide, we developed a systematic and practical modeling framework based on the identification and comparison of gene regulatory dynamics. Our mathematical predictions, confirmed by experimentation, identified key transcriptional regulatory mechanisms of circadian period and uncovered the role of blue light in the response of the circadian oscillator to nicotinamide. We suggest that our methodology could be adapted to predict mechanisms of drug action in complex biological systems.

OXA1L mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect.

OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle. Whole exome sequencing identified biallelic OXA1L variants (c.500_507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) that segregated with disease. Patient muscle and fibroblasts showed decreased OXA1L and subunits of complexes IV and V. Crucially, expression of wild-type human OXA1L in patient fibroblasts rescued the complex IV and V defects. Targeted depletion of OXA1L in human cells or Drosophila melanogaster caused defects in the assembly of complexes I, IV and V, consistent with patient data. Immunoprecipitation of OXA1L revealed the enrichment of mtDNA-encoded subunits of complexes I, IV and V. Our data verify the pathogenicity of these OXA1L variants and demonstrate that OXA1L is required for the assembly of multiple respiratory chain complexes.

Circadian oscillations of cytosolic free calcium regulate the Arabidopsis circadian clock.

In the last decade, the view of circadian oscillators has expanded from transcriptional feedback to incorporate post-transcriptional, post-translational, metabolic processes and ionic signalling. In plants and animals, there are circadian oscillations in the concentration of cytosolic free Ca2+ ([Ca2+]cyt), though their purpose has not been fully characterized. We investigated whether circadian oscillations of [Ca2+]cyt regulate the circadian oscillator of Arabidopsis thaliana. We report that in Arabidopsis, [Ca2+]cyt circadian oscillations can regulate circadian clock function through the Ca2+-dependent action of CALMODULIN-LIKE24 (CML24). Genetic analyses demonstrate a linkage between CML24 and the circadian oscillator, through pathways involving the circadian oscillator gene TIMING OF CAB2 EXPRESSION1 (TOC1).