Interactions between cytoplasmic and nuclear genomes confer sex-specific effects on lifespan in Drosophila melanogaster.

Genetic variation outside of the cell nucleus can affect the phenotype. The cytoplasm is home to the mitochondria, and in arthropods often hosts intracellular bacteria such as Wolbachia. Although numerous studies have implicated epistatic interactions between cytoplasmic and nuclear genetic variation as mediators of phenotypic expression, two questions remain. Firstly, it remains unclear whether outcomes of cyto-nuclear interactions will manifest differently across the sexes, as might be predicted given that cytoplasmic genomes are screened by natural selection only through females as a consequence of their maternal inheritance. Secondly, the relative contribution of mitochondrial genetic variation to other cytoplasmic sources of variation, such as Wolbachia infection, in shaping phenotypic outcomes of cyto-nuclear interactions remains unknown. Here, we address these questions, creating a fully crossed set of replicated cyto-nuclear populations derived from three geographically distinct populations of Drosophila melanogaster, measuring the lifespan of males and females from each population. We observed that cyto-nuclear interactions shape lifespan and that the outcomes of these interactions differ across the sexes. Yet, we found no evidence that placing the cytoplasms from one population alongside the nuclear background of others (generating putative cyto-nuclear mismatches) leads to decreased lifespan in either sex. Although it was difficult to partition mitochondrial from Wolbachia effects, our results suggest at least some of the cytoplasmic genotypic contribution to lifespan was directly mediated by an effect of sequence variation in the mtDNA. Future work should explore the degree to which cyto-nuclear interactions result in sex differences in the expression of other components of organismal life history.

Sex-specific effects of mitochondrial haplotype on metabolic rate in Drosophila melanogaster support predictions of the Mother's Curse hypothesis.

Evolutionary theory proposes that maternal inheritance of mitochondria will facilitate the accumulation of mitochondrial DNA (mtDNA) mutations that are harmful to males but benign or beneficial to females. Furthermore, mtDNA haplotypes sampled from across a given species distribution are expected to differ in the number and identity of these 'male-harming' mutations they accumulate. Consequently, it is predicted that the genetic variation which delineates distinct mtDNA haplotypes of a given species should confer larger phenotypic effects on males than females (reflecting mtDNA mutations that are male-harming, but female-benign), or sexually antagonistic effects (reflecting mutations that are male-harming, but female-benefitting). These predictions have received support from recent work examining mitochondrial haplotypic effects on adult life-history traits in Drosophila melanogaster. Here, we explore whether similar signatures of male-bias or sexual antagonism extend to a key physiological trait-metabolic rate. We measured the effects of mitochondrial haplotypes on the amount of carbon dioxide produced by individual flies, controlling for mass and activity, across 13 strains of D. melanogaster that differed only in their mtDNA haplotype. The effects of mtDNA haplotype on metabolic rate were larger in males than females. Furthermore, we observed a negative intersexual correlation across the haplotypes for metabolic rate. Finally, we uncovered a male-specific negative correlation, across haplotypes, between metabolic rate and longevity. These results are consistent with the hypothesis that maternal mitochondrial inheritance has led to the accumulation of a sex-specific genetic load within the mitochondrial genome, which affects metabolic rate and that may have consequences for the evolution of sex differences in life history. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Paradoxical septal motion: A diagnostic approach and clinical relevance.

Abnormal septal motion (commonly referred to as septal bounce) is a common echocardiographic finding that occurs with several conditions, including the following: mitral stenosis, left bundle branch block, pericardial syndromes and severe pulmonary hypertension. We explore the subtle changes that occur on M-mode imaging of the septum, other associated echocardiographic features, the impact of inspiratory effort on septal motion and relevant clinical findings. Finally, we discuss the impact of abnormal septal motion on cardiac form and function, proposing there is a clinically significant impact on biventricular filling and ejection.

Application of updated guidelines on diastolic dysfunction in patients with severe sepsis and septic shock.

BACKGROUND: Left ventricular diastolic dysfunction is suggested to be associated with higher mortality in severe sepsis and septic shock, yet the methods of diagnosis described in the literature are often inconsistent. The recently published 2016 American Society of Echocardiography and European Association of Cardiovascular Imaging (ASE/EACVI) guidelines offer the opportunity to apply a simple pragmatic diagnostic algorithm for the detection of diastolic dysfunction; however, it has not been tested in this cohort. AIMS: We sought to assess the applicability in septic patients of recently published 2016 ASE/EACVI guidelines on diastolic dysfunction compared with the 2009 ASE guidelines. Our hypothesis was that there would be poor agreement in classifying patients. METHODS: Prospective observational study includes patients identified as having severe sepsis and septic shock. Patients underwent transthoracic echocardiography on day 1 and day 3 of their ICU admission. Patients with normal and abnormal (ejection fraction < 52%) systolic function had their diastolic function stratified according to both the 2009 ASE and 2016 ASE/EACVI guidelines. RESULTS: On day 1 echocardiography, of the 62 patients analysed, 37 (60%) had diastolic dysfunction according to the 2016 ASE/EACVI guideline with a further 23% having indeterminate diastolic function, compared to the 2009 ASE guidelines where only 13 (21%) had confirmed diastolic dysfunction with 46 (74%) having indeterminate diastolic dysfunction. On day 3, of the 55 patients studied, 22 patients (40%) were defined as having diastolic dysfunction, with 6 (11%) having indeterminate diastolic dysfunction according to the 2016 ASE/EACVI guidelines, compared to the 2009 guidelines where 11 (20%) were confirmed to have diastolic dysfunction and 41 (75%) had indeterminate diastolic function. Systolic dysfunction was identified in 18 of 62 patients (29%) on day 1 and 18 of 55 (33%) on day 3. These patients were classified as having abnormal diastolic function in 94 and 89% with the 2016 guidelines on day 1 and day 3, respectively, compared with 50 and 28% using the 2009 guidelines. The 2016 guidelines had less patients with indeterminate diastolic function on days 1 and 3 (11 and 6%) compared to the 2009 guidelines (50 and 72%). Normal systolic function was identified in 44 patients on day 1 and 37 on day 3. In this group, abnormal diastolic function was present in 45 and 54% on days 1 and 3 according to the 2016 ASE/EACVI guidelines, compared with 9 and 16% using the 2009 guidelines, respectively. In those with normal systolic function, the 2016 guidelines had less indeterminate patients with 30 and 16% on days 1 and 3, respectively, compared to 84 and 76% in the 2009 guidelines. CONCLUSION: The 2016 ASE/EACVI diastolic function guidelines identify a significantly higher incidence of dysfunction in patients with severe sepsis and septic shock compared to the previous 2009 guidelines. Although the new guidelines seem to be an improvement, issues remain with the application of guidelines using traditional measures of diastolic dysfunction in this cohort.

Tension pneumomediastinum: A literal form of chest tightness.

Tension pneumomediastinum is a rare and life-threatening complication of mediastinal emphysema which can occur with mechanical ventilation. We present a case of tension mediastinum associated with mechanical ventilation in a patient with Acute Respiratory Distress Syndrome due to Pneumocystis jirovecii pneumonia. We discuss the mechanism and pathophysiology of tension pneumomediastinum, the potential association with Pneumocystis jirovecii pneumonia and recruitment manouvres, and its definitive emergency treatment.

Detecting impaired myocardial relaxation in sepsis with a novel tissue Doppler parameter (septal e'/s').

BACKGROUND: Left ventricular diastolic dysfunction is associated with mortality outcomes in severe sepsis and septic shock. There are ongoing issues with diagnosing diastolic dysfunction in this cohort, partly owing to the poor applicability of traditional parameters in the hyperdynamic circulation. In this feasibility study, we sought to assess the utility of a novel parameter (septal e'/s') to identify diastolic dysfunction in patients with severe sepsis and septic shock who had normal systolic function against the 2016 American Society Echocardiography and European Association of Cardiovascular Imaging (ASE/EACI) guidelines on diastolic dysfunction. METHODS: In this prospective observational pilot study, patients identified as having severe sepsis and septic shock underwent transthoracic echocardiography on day 1 and day 3 of their intensive care unit admission. In patients with normal systolic function, septal e'/s' was calculated using the peak modal velocity of the s' compared with the e' from the septal annulus tissue Doppler imaging and compared with their diastolic grade according to the 2016 ASE/EACI guidelines on diastolic dysfunction. RESULTS: On day 1 of admission, 44 of 62 patients with severe sepsis and septic shock had normal systolic function. There was a strong association of those with diastolic dysfunction having a reduced septal e'/s' compared with patients with normal diastolic function (AUC 0.91). A similar relationship was seen with patients who had indeterminate diastolic dysfunction. On day 3, 37 patients had normal systolic function. Again, there was a strong association of those with diastolic dysfunction and a reduced septal e'/s' (AUC 0.95). CONCLUSIONS: A reduction in septal e'/s' may indicate diastolic dysfunction in patients with severe sepsis and septic shock who have normal systolic function. As opposed to limited traditional measures of diastolic dysfunction, it is applicable in those with hyperdynamic systolic function.

Complete mitochondrial genome sequences of thirteen globally sourced strains of fruit fly (Drosophila melanogaster) form a powerful model for mitochondrial research.

The complete mitogenomes of 13 strains of the fruit fly Drosophila melanogaster were sequenced. Haplotypes varied between 19 532 and 19 537 bp in length, and followed standard dipteran mitogenome content and organization. We detected a total of 354 variable sites between all thirteen haplotypes, while single pairs of haplotypes were separated by an average of 123 variable sites. The sequenced fly strains form a powerful model for mitochondrial research, when it comes to elucidating the links between the mitochondrial genotype and the phenotype.

Mitochondria, maternal inheritance, and male aging.

The maternal transmission of mitochondrial genomes invokes a sex-specific selective sieve, whereby mutations in mitochondrial DNA can only respond to selection acting directly on females. In theory, this enables male-harming mutations to accumulate in mitochondrial genomes when these same mutations are neutral, beneficial, or only slightly deleterious in their effects on females. Ultimately, this evolutionary process could result in the evolution of male-specific mitochondrial mutation loads; an idea previously termed Mother's Curse. Here, we present evidence that the effects of this process are broader than hitherto realized, and that it has resulted in mutation loads affecting patterns of aging in male, but not female Drosophila melanogaster. Furthermore, our results indicate that the mitochondrial mutation loads affecting male aging generally comprise numerous mutations over multiple sites. Our findings thus suggest that males are subject to dramatic consequences that result from the maternal transmission of mitochondrial genomes. They implicate the diminutive mitochondrial genome as a hotspot for mutations that affect sex-specific patterns of aging, thus promoting the idea that a sex-specific selective sieve in mitochondrial genome evolution is a contributing factor to sexual dimorphism in aging, commonly observed across species.

DmWRNexo is a 3'-5' exonuclease: phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease.

The premature human ageing Werner's syndrome is caused by loss or mutation of the WRN helicase/exonuclease. We have recently identified the orthologue of the WRN exonuclease in flies, DmWRNexo, encoded by the CG7670 locus, and showed very high levels of mitotic recombination in a hypomorphic PiggyBac insertional mutant. Here, we report a novel allele of CG7670, with a point mutation resulting in the change of the conserved aspartate (229) to valine. Flies bearing this mutation show levels of mitotic recombination 20-fold higher than wild type. Molecular modelling suggests that D229 lies towards the outside of the molecule distant from the nuclease active site. We have produced recombinant protein of the D229V mutant, assayed its nuclease activity in vitro, and compared activity with that of wild type DmWRNexo and a D162A E164A double active site mutant we have created. We show for the first time that DmWRNexo has 3'-5' exonuclease activity and that mutation within the presumptive active site disrupts exonuclease activity. Furthermore, we show that the D229V mutant has very limited exonuclease activity in vitro. Using Drosophila, we can therefore analyse WRN exonuclease from enzyme activity in vitro through to fly phenotype, and show that loss of exonuclease activity contributes to genome instability.

Variation in mitochondrial genotype has substantial lifespan effects which may be modulated by nuclear background.

Mitochondria are thought to play a central role in aging. In humans, specific naturally occurring mitochondrial genetic variants are overrepresented among centenarians, but only in certain populations; therefore, we cannot tell whether this effect is due solely to mitochondrial genetics or to nuclear-mitochondrial gene complexes, nor do we know the magnitude of the effect in terms we can relate to, such as mean lifespan differences. To examine the effects of natural mitochondrial DNA (mtDNA) variation on lifespan, we need to vary the mitochondrial genotype while controlling the nuclear genotype. Here, nuclear genome replacement is achieved using strains of Drosophila melanogaster bearing multiply inverted 'balancer' chromosomes that suppress recombination, and an isogenic donor strain, thus forcing replacement of entire chromosomes in a single cross while suppressing recombination. Lifespans of wild-type mtDNA variants on the chromosome replacement background vary substantially, and sequencing of the entire protein coding mitochondrial genomes indicates that these lifespan differences are sometimes associated with single amino acid differences. On other nuclear genetic backgrounds, the magnitude and direction of these lifespan effects can change dramatically, and this can be due to changes in baseline mortality risk, rate of aging and/or time of onset of aging. The limited mtDNA variation in D. melanogaster makes it an ideal organism for biochemical studies to link genotype and aging phenotype.

Identification and characterization of a Drosophila ortholog of WRN exonuclease that is required to maintain genome integrity.

The premature human aging Werner syndrome (WS) is caused by mutation of the RecQ-family WRN helicase, which is unique in possessing also 3'-5' exonuclease activity. WS patients show significant genomic instability with elevated cancer incidence. WRN is implicated in restraining illegitimate recombination, especially during DNA replication. Here we identify a Drosophila ortholog of the WRN exonuclease encoded by the CG7670 locus. The predicted DmWRNexo protein shows conservation of structural motifs and key catalytic residues with human WRN exonuclease, but entirely lacks a helicase domain. Insertion of a piggyBac element into the 5' UTR of CG7670 severely reduces gene expression. DmWRNexo mutant flies homozygous for this insertional allele of CG7670 are thus severely hypomorphic; although adults show no gross morphological abnormalities, females are sterile. Like human WS cells, we show that the DmWRNexo mutant flies are hypersensitive to the topoisomerase I inhibitor camptothecin. Furthermore, these mutant flies show highly elevated rates of mitotic DNA recombination resulting from excessive reciprocal exchange. This study identifies a novel WRN ortholog in flies and demonstrates an important role for WRN exonuclease in maintaining genome stability.

Modeling Werner Syndrome in Drosophila melanogaster: hyper-recombination in flies lacking WRN-like exonuclease.

Human progeroid Werner syndrome provides the current best model for analysis of human aging, recapitulating many aspects of normal aging as a result of mutation of the WRN gene. This gene encodes a RecQ-type helicase with additional exonuclease activity. While biochemical studies in vitro have proven invaluable in determining substrate specificities of the WRN exonuclease and helicase, it has been difficult to dissociate the two key enzyme activities in vivo. We are developing Drosophila as a model system for analysis of WRN function; the suitability of Drosophila for extensive and sophisticated genetic manipulation permits us to investigate regulatory pathways and the impact of WRN loss at organismal, cellular, and molecular levels. BLASTP screening of the Drosophila genome with human WRN sequence allowed us to identify three RecQ helicases with strong homology to human WRN, a presumed helicase component of the spliceosome, and two DEAH-box putative RNA helicases with weaker WRN homology. None of these helicases contain a WRN-like exonuclease domain, but two potential WRN-like exonucleases in flies encoded by the loci CG7670 and CG6744 were also identified in the BLAST search. CG6744 and CG7670 are more closely related to human WRN than to each other. We have obtained a fly strain with a piggyBac insertional mutation within the CG6744 locus, which decreases expression of the encoded mRNA. Such flies show elevated levels of somatic recombination. We suggest that WRN-like exonuclease activity is critical in maintaining genomic integrity in flies.

Metabolic rate is not reduced by dietary-restriction or by lowered insulin/IGF-1 signalling and is not correlated with individual lifespan in Drosophila melanogaster.

The link between resting metabolic rate and aging, measured as adult lifespan, was investigated in Drosophila melanogaster by (i) comparing lifespan and metabolic rate of individual flies, (ii) examining the effect of dietary-restriction on the metabolic rate of adult flies, and (iii) comparing the metabolic rate of wild-type and insulin/IGF-1 signalling mutant chico1 flies. The resting oxygen consumption of 65 individually housed and fully fed Drosophila was measured weekly throughout their lifetime. There was no significant difference in the mass-specific rate of oxygen consumption between cohorts that differed in lifespan. Nor was there any statistical correlation between mass-specific oxygen consumption and lifespan of individual Drosophila. The average mass-specific rate of oxygen consumption at 25 degrees C was 3.52+/-0.07 microl O2 mg(-1) h(-1). Variation in mass-specific metabolic rate explained only 4% of variation in individual life span in these flies. Contrary to predictions from the 'rate of living' theory of aging lifetime oxygen consumption was not constant and the lifespan of individual flies accounted for 91% of their lifetime oxygen consumption. An average Drosophila consumes about 3 ml O2 during its adult life. Dietary-restriction had no effect on mass-specific resting metabolic rate both when measured as oxygen consumption by respirometry and when measured as heat production by microcalorimetry. The mass-specific resting heat production of fully fed adult flies at 25 degrees C averaged 17.3+/-0.3 microW mg(-1). Similarly there was no difference in mass-specific metabolic rate of wild-type flies and longliving chico1 insulin/IGF-1 signalling mutant flies, either when measured as oxygen consumption or heat production. Thus, individual variation in lifespan in wild-type flies, and life extension by dietary-restriction and reduced insulin/IGF-1 signalling is not attributable to differences in metabolic rate.