Hyperbaric oxygen treatment reveals spatiotemporal OXPHOS plasticity in the porcine heart.

Cardiomyocytes meet their high ATP demand almost exclusively by oxidative phosphorylation (OXPHOS). Adequate oxygen supply is an essential prerequisite to keep OXPHOS operational. At least two spatially distinct mitochondrial subpopulations facilitate OXPHOS in cardiomyocytes, i.e. subsarcolemmal (SSM) and interfibrillar mitochondria (IFM). Their intracellular localization below the sarcolemma or buried deep between the sarcomeres suggests different oxygen availability. Here, we studied SSM and IFM isolated from piglet hearts and found significantly lower activities of electron transport chain enzymes and F1FO-ATP synthase in IFM, indicative for compromised energy metabolism. To test the contribution of oxygen availability to this outcome, we ventilated piglets under hyperbaric hyperoxic (HBO) conditions for 240 min. HBO treatment raised OXPHOS enzyme activities in IFM to the level of SSM. Complexome profiling analysis revealed that a high proportion of the F1FO-ATP synthase in the IFM was in a disassembled state prior to the HBO treatment. Upon increased oxygen availability, the enzyme was found to be largely assembled, which may account for the observed increase in OXPHOS complex activities. Although HBO also induced transcription of genes involved in mitochondrial biogenesis, a full proteome analysis revealed only minimal alterations, meaning that HBO-mediated tissue remodeling is an unlikely cause for the observed differences in OXPHOS. We conclude that a previously unrecognized oxygen-regulated mechanism endows cardiac OXPHOS with spatiotemporal plasticity that may underlie the enormous metabolic and contractile adaptability of the heart.

Species-specific variation in mitochondrial genome tandem repeat polymorphisms in hares (Lepus spp., Lagomorpha, Leporidae) provides insight into their evolution.

The non-coding regions of the mitochondrial DNAs (mtDNAs) of hares, rabbits, and pikas (Lagomorpha) contain short (∼20 bp) and long (130-160 bp) tandem repeats, absent in related mammalian orders. In the presented study, we provide in-depth analysis for mountain hare (Lepus timidus) and brown hare (L. europaeus) mtDNA non-coding regions, together with a species- and population-level analysis of tandem repeat variation. Mountain hare short tandem repeats (SRs) as well as other analyzed hare species consist of two conserved 10 bp motifs, with only brown hares exhibiting a single, more variable motif. Long tandem repeats (LRs) also differ in sequence and copy number between species. Mountain hares have four to seven LRs, median value five, while brown hares exhibit five to nine LRs, median value six. Interestingly, introgressed mountain hare mtDNA in brown hares obtained an intermediate LR length distribution, with median copy number being the same as with conspecific brown hare mtDNA. In contrast, transfer of brown hare mtDNA into cultured mtDNA-less mountain hare cells maintained the original LR number, whereas the reciprocal transfer caused copy number instability, suggesting that cellular environment rather than the nuclear genomic background plays a role in the LR maintenance. Due to their dynamic nature and separation from other known conserved sequence elements on the non-coding region of hare mitochondrial genomes, the tandem repeat elements likely to represent signatures of ancient genetic rearrangements. clarifying the nature and dynamics of these rearrangements may shed light on the possible role of NCR repeated elements in mitochondria and in species evolution.

Linear DNA-driven recombination in mammalian mitochondria.

Mitochondrial DNA (mtDNA) recombination in animals has remained enigmatic due to its uniparental inheritance and subsequent homoplasmic state, which excludes the biological need for genetic recombination, as well as limits tools to study it. However, molecular recombination is an important genome maintenance mechanism for all organisms, most notably being required for double-strand break repair. To demonstrate the existence of mtDNA recombination, we took advantage of a cell model with two different types of mitochondrial genomes and impaired its ability to degrade broken mtDNA. The resulting excess of linear DNA fragments caused increased formation of cruciform mtDNA, appearance of heterodimeric mtDNA complexes and recombinant mtDNA genomes, detectable by Southern blot and by long range PacBio® HiFi sequencing approach. Besides utilizing different electrophoretic methods, we also directly observed molecular complexes between different mtDNA haplotypes and recombination intermediates using transmission electron microscopy. We propose that the known copy-choice recombination by mitochondrial replisome could be sufficient for the needs of the small genome, thus removing the requirement for a specialized mitochondrial recombinase. The error-proneness of this system is likely to contribute to the formation of pathological mtDNA rearrangements.

Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells.

Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

Analysis of Mitochondrial DNA Replication by Two-Dimensional Agarose Gel Electrophoresis.

Two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE) has been employed for nearly two decades in the analysis of replication and maintenance processes of animal mitochondrial DNA, but the method's potential has not been fully exploited. Here, we describe the various steps involved in this technique, from DNA isolation, to two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE), Southern hybridization and interpretation. We also provide examples of the applicability of 2D-AGE to investigate the different features of mtDNA maintenance and regulation.

Molecular phenotyping uncovers differences in basic housekeeping functions among closely related species of hares (Lepus spp., Lagomorpha: Leporidae).

Speciation is a fundamental evolutionary process, which results in genetic differentiation of populations and manifests as discrete morphological, physiological and behavioural differences. Each species has travelled its own evolutionary trajectory, influenced by random drift and driven by various types of natural selection, making the association of genetic differences between the species with the phenotypic differences extremely complex to dissect. In the present study, we have used an in vitro model to analyse in depth the genetic and gene regulation differences between fibroblasts of two closely related mammals, the arctic/subarctic mountain hare (Lepus timidus Linnaeus) and the temperate steppe-climate adapted brown hare (Lepus europaeus Pallas). We discovered the existence of a species-specific expression pattern of 1623 genes, manifesting in differences in cell growth, cell cycle control, respiration, and metabolism. Interspecific differences in the housekeeping functions of fibroblast cells suggest that speciation acts on fundamental cellular processes, even in these two interfertile species. Our results help to understand the molecular constituents of a species difference on a cellular level, which could contribute to the maintenance of the species boundary.

Mountain- and brown hare genetic polymorphisms to survey local adaptations and conservation status of the heath hare (Lepus timidus sylvaticus, Nilsson 1831).

We provide the first whole genome sequences from three specimens of the mountain hare subspecies the heath hare (Lepus timidus sylvaticus), along with samples from two mountain hares (Lepus timidus timidus) and two brown hares (Lepus europaeus) from Sweden. The heath hare has a unique grey winter pelage as compared to other mountain hares (white) and brown hares (mostly brown), and face regional extinction, likely due to competitive exclusion from the non-native brown hare. Whole genome resequencing from the seven hare specimens were mapped to the Lepus timidus pseudoreference genome and used for detection of 11,363,883 polymorphic nucleotide positions. The data presented here could be useful for addressing local adaptations and conservation status of mountain hares and brown hares in Sweden, including unique subspecies.

Top3α is the replicative topoisomerase in mitochondrial DNA replication.

Mitochondrial DNA has been investigated for nearly fifty years, but many aspects of the maintenance of this essential small genome remain unknown. Like any genome, mammalian mitochondrial DNA requires the function of topoisomerases to counter and regulate the topological tension arising during replication, transcription, segregation, and repair. However, the functions of the different mitochondrial topoisomerases are poorly understood. Here, we investigate the role of Topoisomerase 3α (Top3α) in mtDNA replication and transcription, providing evidence that this enzyme, previously reported to act in mtDNA segregation, also participates in mtDNA replication fork progression. Top3α knockdown caused replication fork stalling, increased mtDNA catenation and decreased mtDNA levels. Overexpression in contrast induced abundant double-strand breaks around the replication origin OH and abortion of early replication, while at the same time improving the resolution of mtDNA replication termination intermediates. Both Top3α knockdown and overexpression affected mitochondrial RNA transcription, leading to a decrease in steady-state levels of mitochondrial transcripts. Together, our results indicate that the mitochondrial isoform of Top3α is not only involved in mtDNA segregation, as reported previously, but also supports the progression of the replication fork. Mitochondrial Top3α is also influencing the progression of transcription, with its absence affecting downstream transcript levels.

Heterozygous p.Y955C mutation in DNA polymerase γ leads to alterations in bioenergetics, complex I subunit expression, and mtDNA replication.

In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared with the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2'-3'-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction.

Adaptive and Pathological Outcomes of Radiation Stress-Induced Redox Signaling.

Significance: Ionizing radiation can damage cells either directly or through oxidative damage caused by ionization. Although radiation exposure from natural sources is very limited, ionizing radiation in nuclear disaster zones and long spaceflights causes inconspicuous, yet measurable physiological effects in men and animals, whose significance remains poorly known. Understanding the physiological impacts of ionizing radiation has a wide importance due to the increased use of medical imaging and radiotherapy. Recent Advances: Radiation exposure has been traditionally investigated from the perspective of DNA damage and its consequences. However, recent studies from Chernobyl as well as spaceflights have provided interesting insights into oxidative stress-induced metabolic alterations and disturbances in the circadian regulation. Critical Issues: In this review, we discuss the physiological consequences of radiation exposure in the light of oxidative stress signaling. Radiation exposure likely triggers many converging or interconnecting signaling pathways, some of which mimic mitochondrial dysfunction and might explain the observed metabolic changes. Future Directions: Better understanding of the different radiation-induced signaling pathways might help to devise strategies for mitigation of the long-term effects of radiation exposure. The utility of fibroblast growth factor 21 (FGF21) as a radiation exposure biomarker and the use of radiation hormesis as a method to protect astronauts on a prolonged spaceflight, such as a mission to Mars, should be investigated. Antioxid. Redox Signal. 37, 336-348.

The first Linguatula serrata case in an imported dog in Finland.

Linguatula serrata is a pentastomid parasite infecting carnivores as definitive hosts and herbivores as intermediate hosts. In carnivores, including dogs, it usually parasitises the nasal cavity and sinuses, causing upper respiratory signs. This case report presents the first canine Linguatula case in Finland in an imported dog originating from Spain. In addition to the unremarkable clinical history of the dog, the treatment, parasite's morphology and molecular analysis are described, and the zoonotic potential is discussed.

TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts.

The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.

Scenopinusjerei, a new species of window fly (Diptera, Scenopinidae) from Finland.

A new species of window fly (Diptera: Scenopinidae), Scenopinusjerei sp. nov., with characteristic bicoloured legs and completely black halteres, is described from Finland. To exclude potential previously named species, a survey of the relevant type specimens as well as original descriptions of the Palearctic and Nearctic Scenopinus species has been conducted, including old Scenopinusfenestralis (Linnaeus) synonyms. Scenopinusjerei sp. nov. is likely to be an overlooked, boreal forest specialist living in the nests of cavity-nesting birds. An identification key to the European species is provided.

Hybridization with mountain hares increases the functional allelic repertoire in brown hares.

Brown hares (Lepus europaeus Pallas) are able to hybridize with mountain hares (L. timidus Linnaeus) and produce fertile offspring, which results in cross-species gene flow. However, not much is known about the functional significance of this genetic introgression. Using targeted sequencing of candidate loci combined with mtDNA genotyping, we found the ancestral genetic diversity in the Finnish brown hare to be small, likely due to founder effect and range expansion, while gene flow from mountain hares constitutes an important source of functional genetic variability. Some of this variability, such as the alleles of the mountain hare thermogenin (uncoupling protein 1, UCP1), might have adaptive advantage for brown hares, whereas immunity-related MHC alleles are reciprocally exchanged and maintained via balancing selection. Our study offers a rare example where an expanding species can increase its allelic variability through hybridization with a congeneric native species, offering a route to shortcut evolutionary adaptation to the local environmental conditions.

Review of the Nordic Gymnocheta Robineau-Desvoidy (Diptera, Tachinidae) with report of two species new to Europe.

The genus Gymnocheta Robineau-Desvoidy, 1830 (Diptera, Tachinidae) has until now been represented by two species in Europe, G.viridis (Fallén, 1810) and G.magna Zimin, 1958. Two species are newly recorded from Finland and Sweden, Gymnochetalucida Zimin, 1958 and G.zhelochovtsevi Zimin, 1958, both previously known only from the Russian Far East and Japan. These four European species are redescribed and illustrated, including the first description of the female of G.zhelochovtsevi. A key is provided to seven of the eight described species of Palaearctic Gymnocheta. The holotype of G.viridis was examined and found to differ from the present concept of the species, instead matching the concept of the more recently described G.magna. In the interests of nomenclatural stability, the two names are maintained in their current usage pending a request to the International Commission on Zoological Nomenclature to replace the current holotype of G.viridis with a neotype that corresponds to the long-established concept of that species.

Promiscuous specialists: Host specificity patterns among generalist louse flies.

Ectoparasites such as louse flies (Diptera: Hippoboscidae) have tendency for host specialization, which is driven by adaptation to host biology as well as competition avoidance between parasites of the same host. However, some louse fly species, especially in genera attacking birds, show wide range of suitable hosts. In the presented study, we have surveyed the current status of bird specific louse flies in Finland to provide comprehensive host association data to analyse the ecological requirements of the generalist species. A thorough sampling of 9342 birds, representing 134 species, recovered 576 specimens of louse flies, belonging to six species: Crataerina hirundinis, C. pallida, Ornithomya avicularia, O. chloropus, O. fringillina and Ornithophila metallica. Despite some overlapping hosts, the three Ornithomya species showed a notable pattern in their host preference, which was influenced not only by the host size but also by the habitat and host breeding strategy. We also provide DNA barcodes for ten Finnish species of Hippoboscidae, which can be used as a resource for species identification as well as metabarcoding studies in the future.

The Type and Source of Reactive Oxygen Species Influences the Outcome of Oxidative Stress in Cultured Cells.

Oxidative stress can be modeled using various different experimental approaches, such as exposing the cells or organisms to oxidative chemicals. However, the actual effects of these chemicals, outside of the immediate measured effect, have attracted relatively little attention. We show here that three commonly used oxidants, menadione, potassium bromate, and hydrogen peroxide, while known to function differently, also elicit different types of responses in HEK293T cells. Menadione and bromate exposure mainly trigger an integrated stress response, whereas hydrogen peroxide affects cellular processes more diversely. Interestingly, acute oxidative stress does not universally cause notable induction of DNA repair or antioxidant defense mechanisms. We also provide evidence that cells with previous experience of oxidative stress show adaptive changes in their responses when the stress is renewed. Our results urge caution when comparing studies where different sources of oxidative stress have been used or when generalizing the findings of these studies to other oxidant types or tissues.

Variation in breeding practices and geographic isolation drive subpopulation differentiation, contributing to the loss of genetic diversity within dog breed lineages.

BACKGROUND: Discrete breed ideals are not restricted to delimiting dog breeds from another, but also are key drivers of subpopulation differentiation. As genetic differentiation due to population fragmentation results in increased rates of inbreeding and loss of genetic diversity, detecting and alleviating the reasons of population fragmentation can provide effective tools for the maintenance of healthy dog breeds. RESULTS: Using a genome-wide SNP array, we detected genetic differentiation to subpopulations in six breeds, Belgian Shepherd, English Greyhound, Finnish Lapphund, Italian Greyhound, Labrador Retriever and Shetland Sheepdog, either due to geographical isolation or as a result of differential breeding strategies. The subpopulation differentiation was strongest in show dog lineages. CONCLUSIONS: Besides geographical differentiation caused by founder effect and lack of gene flow, selection on champion looks or restricted pedigrees is a strong driver of population fragmentation. Artificial barriers for gene flow between the different subpopulations should be recognized, their necessity evaluated critically and perhaps abolished in order to maintain genetic diversity within a breed. Subpopulation differentiation might also result in false positive signals in genome-wide association studies of different traits. LAY SUMMARY: Purebred dogs are, by definition, reproductively isolated from other breeds. However, similar isolation can also occur within a breed due to conflicting breeder ideals and geographic distances between the dog populations. We show here that both of these examples can contribute to breed division, with subsequent loss of genetic variation in the resulting breed lineages. Breeders should avoid creating unnecessary boundaries between breed lineages and facilitate the exchange of dogs between countries.

Replication fork rescue in mammalian mitochondria.

Replication stalling has been associated with the formation of pathological mitochondrial DNA (mtDNA) rearrangements. Yet, almost nothing is known about the fate of stalled replication intermediates in mitochondria. We show here that replication stalling in mitochondria leads to replication fork regression and mtDNA double-strand breaks. The resulting mtDNA fragments are normally degraded by a mechanism involving the mitochondrial exonuclease MGME1, and the loss of this enzyme results in accumulation of linear and recombining mtDNA species. Additionally, replication stress promotes the initiation of alternative replication origins as an apparent means of rescue by fork convergence. Besides demonstrating an interplay between two major mechanisms rescuing stalled replication forks - mtDNA degradation and homology-dependent repair - our data provide evidence that mitochondria employ similar mechanisms to cope with replication stress as known from other genetic systems.

Twist and Turn-Topoisomerase Functions in Mitochondrial DNA Maintenance.

Like any genome, mitochondrial DNA (mtDNA) also requires the action of topoisomerases to resolve topological problems in its maintenance, but for a long time, little was known about mitochondrial topoisomerases. The last years have brought a closer insight into the function of these fascinating enzymes in mtDNA topology regulation, replication, transcription, and segregation. Here, we summarize the current knowledge about mitochondrial topoisomerases, paying special attention to mammalian mitochondrial genome maintenance. We also discuss the open gaps in the existing knowledge of mtDNA topology control and the potential involvement of mitochondrial topoisomerases in human pathologies. While Top1mt, the only exclusively mitochondrial topoisomerase in mammals, has been studied intensively for nearly a decade, only recent studies have shed some light onto the mitochondrial function of Top2ß and Top3α, enzymes that are shared between nucleus and mitochondria. Top3α mediates the segregation of freshly replicated mtDNA molecules, and its dysfunction leads to mtDNA aggregation and copy number depletion in patients. Top2ß, in contrast, regulates mitochondrial DNA replication and transcription through the alteration of mtDNA topology, a fact that should be acknowledged due to the frequent use of Topoisomerase 2 inhibitors in medical therapy.