Mitochondrial DNA variants and microbiota: An experimental strategy to identify novel therapeutic potential in chronic inflammatory diseases.

We previously demonstrated that mice carrying natural mtDNA variants of the FVB/NJ strain (m.7778 G>T in the mt-Atp8 gene in mitochondrial complex V), namely C57BL/6 J-mtFVB/NJ (B6-mtFVB), exhibited (i) partial protection from experimental skin inflammatory diseases in an anti-murine type VII collagen antibody-induced skin inflammation model and psoriasiform dermatitis model; (ii) significantly altered metabolites, including short-chain fatty acids, according to targeted metabolomics of liver, skin and lymph node samples; and (iii) a differential composition of the gut microbiota according to bacterial 16 S rRNA gene sequencing of stool samples compared to wild-type C57BL/6 J (B6) mice. To further dissect these disease-contributing factors, we induced an experimental antibody-induced skin inflammatory disease in gnotobiotic mice. We performed shotgun metagenomic sequencing of caecum contents and untargeted metabolomics of liver, CD4+ T cell, and caecum content samples from conventional B6-mtFVB and B6 mice. We identified D-glucosamine as a candidate mediator that ameliorated disease severity in experimental antibody-induced skin inflammation by modulating immune cell function in T cells, neutrophils and macrophages. Because mice carrying mtDNA variants of the FVB/NJ strain show differential disease susceptibility to a wide range of experimental diseases, including diet-induced atherosclerosis in low-density lipoprotein receptor knockout mice and collagen antibody-induced arthritis in DBA/1 J mice, this experimental approach is valuable for identifying novel therapeutic options for skin inflammatory conditions and other chronic inflammatory diseases to which mice carrying specific mtDNA variants show differential susceptibility.

Changes of Gut Microbiota by Natural mtDNA Variant Differences Augment Susceptibility to Metabolic Disease and Ageing.

We recently reported on two mouse strains carrying different single nucleotide variations in the mitochondrial complex I gene, i.e., B6-mtBPL mice carrying m.11902T>C and B6-mtALR carrying m.4738C>A. B6-mtBPL mice exhibited a longer lifespan and a lower metabolic disease susceptibility despite mild mitochondrial functional differences in steady-state. As natural polymorphisms in the mitochondrial DNA (mtDNA) are known to be associated with distinct patterns of gut microbial composition, we further investigated the gut microbiota composition in these mice strains. In line with mouse phenotypes, we found a significantly lower abundance of Proteobacteria, which is positively associated with pathological conditions, in B6-mtBPL compared to B6-mtALR mice. A prediction of functional profile of significantly differential bacterial genera between these strains revealed an involvement of glucose metabolism pathways. Whole transcriptome analysis of liver samples from B6-mtBPL and B6-mtALR mice confirmed these findings. Thus, both host gene expression and gut microbial changes caused by the mtDNA variant differences may contribute to the ageing and metabolic phenotypes observed in these mice strains. Since gut microbiota are easier to modulate, compared with mtDNA variants, identification of such mtDNA variants, specific gut bacterial species and bacterial metabolites may be a potential intervention to modulate common diseases, which are differentially susceptible to individuals with different mtDNA variants.

Telmisartan induces a specific gut microbiota signature which may mediate its antiobesity effect.

Telmisartan prevents diet-induced obesity (DIO) in rodents. Given that the precise underlying mechanism is not known, we examined whether a gut-related mechanism might be involved. Sprague-Dawley rats received cafeteria diet (CD) for 3 months to develop DIO and were administered either telmisartan (8 mg/kgbw) or vehicle. In addition, pair-fed (PF) rats received CD adjusted to TEL and control rats (CON) only received chow. Stool samples were analysed by 16 S rRNA gene amplicon sequencing. CD-fed rats became obese while TEL, PF and CON rats remained lean. Alpha diversity analyses indicated that bacterial diversity was similar before the study but changed over time. Beta diversity revealed a time-, CD- and telmisartan-dependent effect. The Firmicutes/Bacteroidetes ratio and the abundance of Blautia, Allobaculum and Parasutterella were higher in DIO and PF than in CON, but not in TEL. Enterotype (ET)-like clustering analyses, Kleinberg's hub network scoring and random forest analyses also indicated that telmisartan induced a specific signature of gut microbiota. In response to stool transfer from telmisartan-pre-treated donor to high-fat fed acceptor mice, body weight gain was slightly attenuated. We attribute the anti-obesity action of telmisartan treatment to diet-independent alterations in gut microbiota as the microbiota from telmisartan-treated, CD-fed rats clearly differed from those of DIO and PF rats. ET-like clustering network, random forest classification and the higher stability in bacterial co-occurrence network analyses indicate that there is more than one indicator species for telmisartan's specific signature, which is further strengthened by the fact that we cannot identify a single indicator species.

A Mitochondrial Polymorphism Alters Immune Cell Metabolism and Protects Mice from Skin Inflammation.

Several genetic variants in the mitochondrial genome (mtDNA), including ancient polymorphisms, are associated with chronic inflammatory conditions, but investigating the functional consequences of such mtDNA polymorphisms in humans is challenging due to the influence of many other polymorphisms in both mtDNA and the nuclear genome (nDNA). Here, using the conplastic mouse strain B6-mtFVB, we show that in mice, a maternally inherited natural mutation (m.7778G > T) in the mitochondrially encoded gene ATP synthase 8 (mt-Atp8) of complex V impacts on the cellular metabolic profile and effector functions of CD4+ T cells and induces mild changes in oxidative phosphorylation (OXPHOS) complex activities. These changes culminated in significantly lower disease susceptibility in two models of inflammatory skin disease. Our findings provide experimental evidence that a natural variation in mtDNA influences chronic inflammatory conditions through alterations in cellular metabolism and the systemic metabolic profile without causing major dysfunction in the OXPHOS system.

A Natural mtDNA Polymorphism in Complex III Is a Modifier of Healthspan in Mice.

In this study, we provide experimental evidence that a maternally inherited polymorphism in the mitochondrial cytochrome b gene (mt-Cytb; m.15124A>G, Ile-Val) in mitochondrial complex III resulted in middle-aged obesity and higher susceptibility to diet-induced obesity, as well as age-related inflammatory disease, e.g., ulcerative dermatitis, in mice. As a consequence of the gene variation, we observed alterations in body composition, metabolism and mitochondrial functions, i.e., increased mitochondrial oxygen consumption rate and higher levels of reactive oxygen species, as well as in the commensal bacterial composition in the gut, with higher abundance of Proteobacteria in mice carrying the variant. These observations are in line with the previously described links of the mitochondrial complex III gene with obesity and metabolic diseases in humans. Given that these functional changes by the G variant at m.15124 in the mt-Cytb are already present in young mice that were kept under normal condition, it is plausible that the m.15124A>G variant is a disease susceptibility modifier to the diseases induced by additional stressors, i.e., dietary and/or aging stress, and that the variant results in the higher incidence of clinical diseases presentation in C57BL/6J-mt129S1/SvlmJ than C57BL/6J mice. Thus, mtDNA variants could be potential biomarkers to evaluate the healthspan.

Reduced skin blistering in experimental epidermolysis bullosa acquisita after anti-TNF treatment.

Epidermolysis bullosa acquisita (EBA) is a difficult-to-treat subepidermal autoimmune blistering skin disease (AIBD) with circulating and tissue-bound anti-type VII collagen antibodies. Different reports have indicated an increased concentration of tumor necrosis factor alpha (TNF) in the serum and blister fluid of patients with subepidermal AIBDs. Furthermore, successful anti-TNF treatment has been reported for individual patients with AIBDs. Here, we show that in mice, induction of experimental EBA by repeated injections of rabbit-anti mouse type VII collagen antibodies led to increased expression of TNF in skin, as determined by real-time PCR and immunohistochemistry. To investigate if the increased TNF expression is of functional relevance in experimental EBA, we inhibited TNF function using the soluble TNF receptor fusion protein etanercept (Enbrel®) or a monoclonal antibody to murine TNF. Interestingly, mice receiving either of these two treatments showed significantly milder disease progression than controls. In addition, immunohistochemical staining demonstrated reduced numbers of macrophages in lesional skin in mice treated with TNF inhibitors compared to controls. Furthermore, etanercept treatment significantly reduced the disease progression in immunization-induced EBA. In conclusion, the increased expression of TNF in experimental EBA is of functional relevance, as both the prophylactic blockade of TNF and the therapeutic use of etanercept impaired the induction and progression of experimental EBA. Thus, TNF is likely to serve as a new therapeutic target for EBA and AIBDs with a similar pathogenesis.

mtDNA polymorphism and metabolic inhibition affect sperm performance in conplastic mice.

Whereas a broad link exists between nucleotide substitutions in the mitochondrial genome (mtDNA) and a range of metabolic pathologies, exploration of the effect of specific mtDNA genotypes is on-going. Mitochondrial DNA mutations are of particular relevance for reproductive traits, since they are expected to have profound effects on male specific processes as a result of the strict maternal inheritance of mtDNA. Sperm motility is crucially dependent on ATP in most systems studied. However, the importance of mitochondrial function in the production of the ATP necessary for sperm function remains uncertain. In this study, we test the effect of mtDNA polymorphisms upon mouse sperm performance and bioenergetics by using five conplastic inbred strains that share the same nuclear background while differing in their mitochondrial genomes. We found that, while genetic polymorphisms across distinct mtDNA haplotypes are associated with modification in sperm progressive velocity, this effect is not related to ATP production. Furthermore, there is no association between the number of mtDNA polymorphisms and either (a) the magnitude of sperm performance decrease, or (b) performance response to specific inhibition of the main sperm metabolic pathways. The observed variability between strains may be explained in terms of additive effects of single nucleotide substitutions on mtDNA coding sequences, which have been stabilized through genetic drift in the different laboratory strains. Alternatively, the decreased sperm performance might have arisen from the disruption of the nuclear DNA/mtDNA interactions that have coevolved during the radiation of Mus musculus subspecies.

Dissecting genetics of cutaneous miRNA in a mouse model of an autoimmune blistering disease.

BACKGROUND: MicroRNAs (miRNAs) are small endogenous non-coding RNAs that control genes at post-transcriptional level. They are essential for development and tissue differentiation, and such altered miRNA expression patterns are linked to the pathogenesis of inflammation and cancer. There is evidence that miRNA expression is genetically controlled similar to the transcription of protein-coding genes and previous studies identified quantitative trait loci (QTL) for miRNA expression in the liver. So far, little attention has been paid to miRNA expression in the skin. Moreover, epistatic control of miRNA expression remains unknown. In this study, we characterize genetic regulation of cutaneous miRNA and their correlation with skin inflammation using a previously established murine autoimmune-prone advanced intercross line. RESULTS: We identified in silico 42 eQTL controlling the expression of 38 cutaneous miRNAs and furthermore found two chromosomal hot-spots on chromosomes 2 and 8 that control the expression of multiple miRNAs. Moreover, for 8 miRNAs an interacting effect from pairs of SNPs was observed. Combining the constraints on genes from the statistical interaction of their loci and further using curated protein interaction networks, the number of candidate genes for association of miRNAs was reduced to a set of several genes. A cluster analysis identified miR-379 and miR-223 to be associated with EBA severity/onset, where miR-379 was observed to be associated to loci on chromosome 6. CONCLUSION: The murine advanced intercross line allowed us to identify the genetic loci regulating multiple miRNA in skin. The recurrence of trans-eQTL and epistasis suggest that cutaneous miRNAs are regulated by yet an unexplored complex gene networks. Further, using co-expression analysis of miRNA expression levels we showed that multiple miRNA contribute to multiple pathways that might be involved in pathogenesis of autoimmune skin blistering disease. Specifically, we provide evidence that miRNA such as miR-223 and miR-379 may play critical role in disease progression and severity.

Analysis of factors contributing to variation in the C57BL/6J fecal microbiota across German animal facilities.

The intestinal microbiota is involved in many physiological processes and it is increasingly recognized that differences in community composition can influence the outcome of a variety of murine models used in biomedical research. In an effort to describe and account for the variation in intestinal microbiota composition across the animal facilities of participating members of the DFG Priority Program 1656 "Intestinal Microbiota", we performed a survey of C57BL/6J mice from 21 different mouse rooms/facilities located at 13 different institutions across Germany. Fresh feces was sampled from five mice per room/facility using standardized procedures, followed by extraction and 16S rRNA gene profiling (V1-V2 region, Illumina MiSeq) at both the DNA and RNA (reverse transcribed to cDNA) level. In order to determine the variables contributing to bacterial community differences, we collected detailed questionnaires of animal husbandry practices and incorporated this information into our analyses. We identified considerable variation in a number of descriptive aspects including the proportions of major phyla, alpha- and beta diversity, all of which displayed significant associations to specific aspects of husbandry. Salient findings include a reduction in alpha diversity with the use of irradiated chow, an increase in inter-individual variability (beta diversity) with respect to barrier access and open cages and an increase in bacterial community divergence with time since importing from a vendor. We further observe a high degree of facility-level individuality, which is likely due to each facility harboring its own unique combination of multiple varying attributes of animal husbandry. While it is important to account and control for such differences between facilities, the documentation of such diversity may also serve as a valuable future resource for investigating the origins of microbial-driven host phenotypes.

Mitochondrial gene polymorphisms that protect mice from colitis.

BACKGROUND & AIMS: Dysregulated energy homeostasis in the intestinal mucosa frequently is observed in patients with ulcerative colitis (UC). Intestinal tissues from these patients have reduced activity of the mitochondrial oxidative phosphorylation (OXPHOS) complex, so mitochondrial dysfunction could contribute to the pathogenesis of UC. However, little is known about the mechanisms by which OXPHOS activity could be altered. We used conplastic mice, which have identical nuclear but different mitochondrial genomes, to investigate activities of the OXPHOS complex. METHODS: Colitis was induced in C57BL/6J wild-type (B6.B6) and 3 strains of conplastic mice (B6.NZB, B6.NOD, and B6.AKR) by administration of dextran sodium sulfate or rectal application of trinitrobenzene sulfonate. Colon tissues were collected and analyzed by histopathology, immunohistochemical analysis, and immunoblot analysis; we also measured mucosal levels of adenosine triphosphate (ATP) and reactive oxygen species, OXPHOS complex activity, and epithelial cell proliferation and apoptosis. RESULTS: We identified mice with increased mucosal OXPHOS complex activities and levels of ATP. These mice developed less-severe colitis after administration of dextran sodium sulfate or trinitrobenzene sulfonate than mice with lower mucosal levels of ATP. Colon tissues from these mice also had increased enterocyte proliferation and transcription factor nuclear factor-κB activity, which have been shown to protect the mucosal barrier-defects in these processes have been associated with inflammatory bowel disease. CONCLUSIONS: Variants in mitochondrial DNA that increase mucosal levels of ATP protect mice from colitis. Increasing mitochondrial ATP synthesis in intestinal epithelial cells could be a therapeutic approach for UC.

Chronic Inflammation Disrupts Circadian Rhythms in Splenic CD4+ and CD8+ T Cells in Mice.

Internal circadian clocks coordinate 24 h rhythms in behavior and physiology. Many immune functions show daily oscillations, and cellular circadian clocks can impact immune functions and disease outcome. Inflammation may disrupt circadian clocks in peripheral tissues and innate immune cells. However, it remains elusive if chronic inflammation impacts adaptive immune cell clock, e.g., in CD4+ and CD8+ T lymphocytes. We studied this in the experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis, as an established experimental paradigm for chronic inflammation. We analyzed splenic T cell circadian clock and immune gene expression rhythms in mice with late-stage EAE, CFA/PTx-treated, and untreated mice. In both treatment groups, clock gene expression rhythms were altered with differential effects for baseline expression and peak phase compared with control mice. Most immune cell marker genes tested in this study did not show circadian oscillations in either of the three groups, but time-of-day- independent alterations were observed in EAE and CFA/PTx compared to control mice. Notably, T cell effects were likely independent of central clock function as circadian behavioral rhythms in EAE mice remained intact. Together, chronic inflammation induced by CFA/PTx treatment and EAE immunization has lasting effects on circadian rhythms in peripheral immune cells.

Mitochondrial haplotype and mito-nuclear matching drive somatic mutation and selection throughout ageing.

Mitochondrial genomes co-evolve with the nuclear genome over evolutionary timescales and are shaped by selection in the female germline. Here we investigate how mismatching between nuclear and mitochondrial ancestry impacts the somatic evolution of the mitochondrial genome in different tissues throughout ageing. We used ultrasensitive duplex sequencing to profile ~2.5 million mitochondrial genomes across five mitochondrial haplotypes and three tissues in young and aged mice, cataloguing ~1.2 million mitochondrial somatic and ultralow-frequency inherited mutations, of which 81,097 are unique. We identify haplotype-specific mutational patterns and several mutational hotspots, including at the light strand origin of replication, which consistently exhibits the highest mutation frequency. We show that rodents exhibit a distinct mitochondrial somatic mutational spectrum compared with primates with a surfeit of reactive oxygen species-associated G > T/C > A mutations, and that somatic mutations in protein-coding genes exhibit signatures of negative selection. Lastly, we identify an extensive enrichment in somatic reversion mutations that 're-align' mito-nuclear ancestry within an organism's lifespan. Together, our findings demonstrate that mitochondrial genomes are a dynamically evolving subcellular population shaped by somatic mutation and selection throughout organismal lifetimes.

Azathioprine promotes intestinal epithelial cell differentiation into Paneth cells and alleviates ileal Crohn's disease severity.

Paneth cells (PCs), a subset of intestinal epithelial cells (IECs) found at the base of small intestinal crypts, play an essential role in maintaining intestinal homeostasis. Altered PCs function is associated with diverse intestinal pathologies, including ileal Crohn's disease (CD). CD patients with ileal involvement have been previously demonstrated to display impairment in PCs and decreased levels of anti-microbial peptides. Although the immunosuppressive drug Azathioprine (AZA) is widely used in CD therapy, the impact of AZA on IEC differentiation remains largely elusive. In the present study, we hypothesized that the orally administered drug AZA also exerts its effect through modulation of the intestinal epithelium and specifically via modulation of PC function. AZA-treated CD patients exhibited an ileal upregulation of AMPs on both mRNA and protein levels compared to non-AZA treated patients. Upon in vitro AZA stimulation, intestinal epithelial cell line MODE-K exhibited heightened expression levels of PC marker in concert with diminished cell proliferation but boosted mitochondrial OXPHOS activity. Moreover, differentiation of IECs, including PCs differentiation, was boosted in AZA-treated murine small intestinal organoids and was associated with decreased D-glucose consumption and decreased growth rates. Of note, AZA treatment strongly decreased Lgr5 mRNA expression as well as Ki67 positive cells. Further, AZA restored dysregulated PCs associated with mitochondrial dysfunction. AZA-dependent inhibition of IEC proliferation is accompanied by boosted mitochondria function and IEC differentiation into PC.

Genetic identification and functional validation of FcγRIV as key molecule in autoantibody-induced tissue injury.

Autoantibody-mediated diseases are clinically heterogeneous and often fail conventional therapeutic strategies. Gene expression profiling has helped to identify new molecular pathways in these diseases, although their potential as treatment targets largely remains to be functionally validated. Based on weighted gene co-expression network analysis, we determined the transcriptional network in experimental epidermolysis bullosa acquisita (EBA), a paradigm of an antibody-mediated organ-specific autoimmune disease characterized by autoantibodies directed against type VII collagen. We identified 33 distinct and differentially expressed modules, including Fcγ receptor (FcγR) IV and components of the neutrophil-associated enzyme system in autoantibody transfer-induced EBA. Validation experiments, including functional analysis, demonstrated that FcγRIV expression on neutrophils crucially contributes to autoantibody-induced tissue injury in the transfer model of EBA. Mice lacking the common γ-chain of activating FcγRs, deficient in FcγRIV or treated with FcγRIV function blocking antibody, but not mice deficient in FcγRI, FcγRIIB, FcγRIII or both FcγRI and FcγRIII, were effectively protected from EBA. Skin disease was restored in γ-chain-deficient mice locally reconstituted with neutrophils from wild-type, but not from γ-chain-deficient, mice. Our findings both genetically and functionally identify a novel disease-related molecule, FcγRIV, in an autoantibody-mediated disorder, which may be of importance for the development of novel targeted therapies.

Repetitive immunization breaks tolerance to type XVII collagen and leads to bullous pemphigoid in mice.

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease of the elderly associated with considerable morbidity and mortality. As unspecific immunosuppressants are still the mainstay of BP therapy, several animal models, based on the passive transfer of autoantibodies or immune cells, have been developed to obtain a better understanding of the pathogenesis of BP and evaluate novel therapeutic interventions. We describe in this study an experimental model inducing BP by immunization of immunocompetent mice with a recombinant form of the immunodominant 15th noncollagenous domain of murine BP180 (type XVII collagen). The homologous noncollagenous 16A domain of human BP180 has previously been identified as an immunodominant region in human BP. Immunization of female SJL/J mice with the murine peptide led to clinical disease within 14 wk in 56% of mice. In contrast, none of the other strains developed blisters despite the presence of autoantibodies. The clinical disease manifested for at least 8 wk without further manipulation. This novel immunization-induced model reflects key immunopathological characteristics of human BP, including binding of complement-fixing autoantibodies along the dermal-epidermal junction, elevated total IgE serum levels, and infiltration of skin lesions with eosinophilic granulocytes. The use of immunocompetent mice and the induction of sustained clinical disease not requiring additional interventions make this immunization-induced mouse model most suitable to further explore the pathogenesis of BP and novel therapeutic interventions for this and other autoantibody-mediated diseases.

Interaction between mitochondria and microbiota modulating cellular metabolism in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a prototypic complex disease in the gastrointestinal tract that has been increasing in incidence and prevalence in recent decades. Although the precise pathophysiology of IBD remains to be elucidated, a large body of evidence suggests the critical roles of mitochondria and intestinal microbiota in the pathogenesis of IBD. In addition to their contributions to the disease, both mitochondria and gut microbes may interact with each other and modulate disease-causing cell activities. Therefore, we hypothesize that dissecting this unique interaction may help to identify novel pathways involved in IBD, which will further contribute to discovering new therapeutic approaches to the disease. As poorly treated IBD significantly affects the quality of life of patients and is associated with risks and complications, successful treatment is crucial. In this review, we stratify previously reported experimental and clinical observations of the role of mitochondria and intestinal microbiota in IBD. Additionally, we review the intercommunication between mitochondria, and the intestinal microbiome in patients with IBD is reviewed along with the potential mediators for these interactions. We specifically focus on their roles in cellular metabolism in intestinal epithelial cells and immune cells. To this end, we propose a potential therapeutic intervention strategy for IBD.

North and East African mitochondrial genetic variation needs further characterization towards precision medicine.

INTRODUCTION: Mitochondria are maternally inherited cell organelles with their own genome, and perform various functions in eukaryotic cells such as energy production and cellular homeostasis. Due to their inheritance and manifold biological roles in health and disease, mitochondrial genetics serves a dual purpose of tracing the history as well as disease susceptibility of human populations across the globe. This work requires a comprehensive catalogue of commonly observed genetic variations in the mitochondrial DNAs for all regions throughout the world. So far, however, certain regions, such as North and East Africa have been understudied. OBJECTIVES: To address this shortcoming, we have created the most comprehensive quality-controlled North and East African mitochondrial data set to date and use it for characterizing mitochondrial genetic variation in this region. METHODS: We compiled 11 published cohorts with novel data for mitochondrial genomes from 159 Sudanese individuals. We combined these 641 mitochondrial sequences with sequences from the 1000 Genomes (n = 2504) and the Human Genome Diversity Project (n = 828) and used the tool haplocheck for extensive quality control and detection of in-sample contamination, as well as Nanopore long read sequencing for haplogroup validation of 18 samples. RESULTS: Using a subset of high-coverage mitochondrial sequences, we predict 15 potentially novel haplogroups in North and East African subjects and observe likely phylogenetic deviations from the established PhyloTree reference for haplogroups L0a1 and L2a1. CONCLUSION: Our findings demonstrate common hitherto unexplored variants in mitochondrial genomes of North and East Africa that lead to novel phylogenetic relationships between haplogroups present in these regions. These observations call for further in-depth population genetic studies in that region to enable the prospective use of mitochondrial genetic variation for precision medicine.

Mitochondrial haplotype and mito-nuclear matching drive somatic mutation and selection throughout aging.

Mitochondrial genomes co-evolve with the nuclear genome over evolutionary timescales and are shaped by selection in the female germline. Here, we investigate how mismatching between nuclear and mitochondrial ancestry impacts the somatic evolution of the mt-genome in different tissues throughout aging. We used ultra-sensitive Duplex Sequencing to profile ~2.5 million mt-genomes across five mitochondrial haplotypes and three tissues in young and aged mice, cataloging ~1.2 million mitochondrial somatic and ultra low frequency inherited mutations, of which 81,097 are unique. We identify haplotype-specific mutational patterns and several mutational hotspots, including at the Light Strand Origin of Replication, which consistently exhibits the highest mutation frequency. We show that rodents exhibit a distinct mitochondrial somatic mutational spectrum compared to primates with a surfeit of reactive oxygen species-associated G>T/C>A mutations, and that somatic mutations in protein coding genes exhibit signatures of negative selection. Lastly, we identify an extensive enrichment in somatic reversion mutations that "re-align" mito-nuclear ancestry within an organism's lifespan. Together, our findings demonstrate that mitochondrial genomes are a dynamically evolving subcellular population shaped by somatic mutation and selection throughout organismal lifetimes.

Proton pump inhibitor treatment aggravates bacterial translocation in patients with advanced cirrhosis and portal hypertension.

IMPORTANCE: Long-term prescription of proton pump inhibitors (PPIs) in patients with cirrhosis is common practice. However, in recent years, several observational studies have reported increased complications and negative prognostic effects of PPI treatment in these patients. Judging the significance of these associations is complicated by the fact that a plausible underlying pathomechanism has not been identified so far. In the present study, we address this important issue by investigating the impact of PPI treatment on subclinical bacterial translocation from the gut into the blood stream in patients with advanced cirrhosis and portal hypertension. Indeed, we report significantly aggravated bacterial translocation in cirrhosis patients receiving PPI treatment. This finding is highly relevant, as bacterial translocation is known to promote the development of complications and impair prognosis in patients with cirrhosis. Hence, the present study could establish a plausible link between PPI treatment and adverse effects in cirrhosis.