Mitochondrial DNA mutations attenuate Bleomycin-induced dermal fibrosis by inhibiting differentiation into myofibroblasts.

Post-mitotic, non-proliferative dermal fibroblasts have crucial functions in maintenance and restoration of tissue homeostasis. They are involved in essential processes such as wound healing, pigmentation and hair growth, but also tumor development and aging-associated diseases. These processes are energetically highly demanding and error prone when mitochondrial damage occurs. However, mitochondrial function in fibroblasts and the influence of mitochondrial dysfunction on fibroblast-specific demands are still unclear. To address these questions, we created a mouse model in which accelerated cell-specific mitochondrial DNA (mtDNA) damage accumulates. We crossed mice carrying a dominant-negative mutant of the mitochondrial replicative helicase Twinkle (RosaSTOP system) with mice that express fibroblast-specific Cre Recombinase (Collagen1A2 CreERT) which can be activated by Tamoxifen (TwinkleFIBRO). Thus, we are able to induce mtDNA deletions and duplications in specific cells, a process which resembles the physiological aging process in humans, where this damage accumulates in all tissues. Upon proliferation in vitro, Tamoxifen induced Twinkle fibroblasts deplete most of their mitochondrial DNA which, although not disturbing the stoichiometry of the respiratory chain complexes, leads to reduced ROS production and mitochondrial membrane potential as well as an anti-inflammatory and anti-fibrotic profile of the cells. In Sodium Azide treated wildtype fibroblasts, without a functioning respiratory chain, we observe the opposite, a rather pro-inflammatory and pro-fibrotic signature. Upon accumulation of mitochondrial DNA mutations in vivo the TwinkleFIBRO mice are protected from fibrosis development induced by intradermal Bleomycin injections. This is due to dampened differentiation of the dermal fibroblasts into α-smooth-muscle-actin positive myofibroblasts in TwinkleFIBRO mice. We thus provide evidence for striking differences of the impact that mtDNA mutations have in contrast to blunted mitochondrial function in dermal fibroblasts and skin homeostasis. These data contribute to improved understanding of mitochondrial function and dysfunction in skin and provide mechanistic insight into potential targets to treat skin fibrosis in the future.

Re-assessment of monoclonal antibodies against diclofenac for their application in the analysis of environmental waters.

The non-steroidal anti-inflammatory drug (NSAID) diclofenac (DCF) is an important environmental contaminant occurring in surface waters all over the world, because, after excretion, it is not adequately removed from wastewater in sewage treatment plants. To be able to monitor this pollutant, highly efficient analytical methods are needed, including immunoassays. In a medical research project, monoclonal antibodies against diclofenac and its metabolites had been produced. Based on this monoclonal anti-DCF antibody, a new indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed and applied for environmental samples. The introduction of a spacer between diclofenac and the carrier protein in the coating conjugate led to higher sensitivity. With a test midpoint of 3 µg L-1 and a measurement range of 1-30 µg L-1, the system is not sensitive enough for direct analysis of surface water. However, this assay is quite robust against matrix influences and can be used for wastewater. Without adjustment of the calibration, organic solvents up to 5%, natural organic matter (NOM) up to 10 mg L-1, humic acids up to 2.5 mg L-1, and salt concentrations up to 6 g L-1 NaCl and 75 mg L-1 CaCl2 are tolerated. The antibody is also stable in a pH range from 3 to 12. Cross-reactivity (CR) of 1% or less was determined for the metabolites 4'-hydroxydiclofenac (4'-OH-DCF), 5-hydroxydiclofenac (5-OH-DCF), DCF lactam, and other NSAIDs. Relevant cross-reactivity occurred only with an amide derivative of DCF, 6-aminohexanoic acid (DCF-Ahx), aceclofenac (ACF) and DCF methyl ester (DCF-Me) with 150%, 61% and 44%, respectively. These substances, however, have not been found in samples. Only DCF-acyl glucuronide with a cross-reactivity of 57% is of some relevance. For the first time, photodegradation products were tested for cross-reactivity. With the ELISA based on this antibody, water samples were analysed. In sewage treatment plant effluents, concentrations in the range of 1.9-5.2 µg L-1 were determined directly, with recoveries compared to HPLC-MS/MS averaging 136%. Concentrations in lakes ranged from 3 to 4.4 ng L-1 and were, after pre-concentration, determined with an average recovery of 100%.

Preserved striatal innervation maintains motor function despite severe loss of nigral dopaminergic neurons.

Degeneration of dopaminergic neurons in the substantia nigra and their striatal axon terminals causes cardinal motor symptoms of Parkinson's disease. In idiopathic cases, high levels of mitochondrial DNA alterations leading to mitochondrial dysfunction are a central feature of these vulnerable neurons. Here we present a mouse model expressing the K320E-variant of the mitochondrial helicase Twinkle in dopaminergic neurons, leading to accelerated mitochondrial DNA mutations. These K320E-TwinkleDaN mice showed normal motor function at 20 months of age, although ∼70% of nigral dopaminergic neurons had perished. Remaining neurons still preserved ∼75% of axon terminals in the dorsal striatum and enabled normal dopamine release. Transcriptome analysis and viral tracing confirmed compensatory axonal sprouting of the surviving neurons. We conclude that a small population of substantia nigra dopaminergic neurons is able to adapt to the accumulation of mitochondrial DNA mutations and maintain motor control.

Immortalised murine R349P desmin knock-in myotubes exhibit a reduced proton leak and decreased ADP/ATP translocase levels in purified mitochondria.

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy.

Divergent impacts of the neonicotinoid insecticide, clothianidin, on flight performance metrics in two species of migratory butterflies.

Long-distance flight is crucial for the survival of migratory insects, and disruptions to their flight capacity can have significant consequences for conservation. In this study, we examined how a widely used insecticide, clothianidin (class: neonicotinoid), impacted the flight performance of two species of migratory butterflies, monarchs (Danaus plexippus) and painted ladies (Vanessa cardui). To do this, we quantified the free-flight energetics and tethered-flight velocity and distance of the two species using flow-through respirometry and flight mill assays. Our findings show differential effects of the pesticide on the two species. For painted ladies, we found that clothianidin exposure reduced average free-flight metabolic rates, but did not affect either average velocity or total distance during tethered flight. Other studies have linked low flight metabolic rates with reduced dispersal capacity, indicating that clothianidin exposure may hinder painted lady flight performance in the wild. Conversely, for monarchs, we saw no significant effect of clothianidin exposure on average free-flight metabolic rates but did observe increases in the average velocity, and for large individuals, total distance achieved by clothianidin-exposed monarchs in tethered flight. This suggests a potential stimulatory response of monarchs to low-dose exposures to clothianidin. These findings indicate that clothianidin exposure has the potential to influence the flight performance of butterflies, but that not all species are impacted in the same way. This highlights the need to be thoughtful when selecting performance assays, as different assays can evaluate fundamentally distinct aspects of physiology, and as such may yield divergent results.

Body mass, temperature, and pathogen intensity differentially affect critical thermal maxima and their population-level variation in a solitary bee.

Climate change presents a major threat to species distribution and persistence. Understanding what abiotic or biotic factors influence the thermal tolerances of natural populations is critical to assessing their vulnerability under rapidly changing thermal regimes. This study evaluates how body mass, local climate, and pathogen intensity influence heat tolerance and its population-level variation (SD) among individuals of the solitary bee Xenoglossa pruinosa. We assess the sex-specific relationships between these factors and heat tolerance given the differences in size between sexes and the ground-nesting behavior of the females. We collected X. pruinosa individuals from 14 sites across Pennsylvania, USA, that varied in mean temperature, precipitation, and soil texture. We measured the critical thermal maxima (CTmax) of X. pruinosa individuals as our proxy for heat tolerance and used quantitative PCR to determine relative intensities of three parasite groups-trypanosomes, Spiroplasma apis (mollicute bacteria), and Vairimorpha apis (microsporidian). While there was no difference in CTmax between the sexes, we found that CTmax increased significantly with body mass and that this relationship was stronger for males than for females. Air temperature, precipitation, and soil texture did not predict mean CTmax for either sex. However, population-level variation in CTmax was strongly and negatively correlated with air temperature, which suggests that temperature is acting as an environmental filter. Of the parasites screened, only trypanosome intensity correlated with heat tolerance. Specifically, trypanosome intensity negatively correlated with the CTmax of female X. pruinosa but not males. Our results highlight the importance of considering size, sex, and infection status when evaluating thermal tolerance traits. Importantly, this study reveals the need to evaluate trends in the variation of heat tolerance within and between populations and consider implications of reduced variation in heat tolerance for the persistence of ectotherms in future climate conditions.

Microcarrier-based fluorescent yeast estrogen screen assay for fast determination of endocrine disrupting compounds.

The presence of endocrine-disrupting compounds (EDCs) in water poses a significant threat to human and animal health, as recognized by regulatory agencies throughout the world. The Yeast Estrogen Screen (YES) assay is an excellent method to evaluate the presence of these compounds in water due to its simplicity and capacity to assess the bioaccessible forms/fractions of these compounds. In the presence of a compound with estrogenic activity, Saccharomyces cerevisiae cells, containing a lacZ reporter gene encoding the enzyme ß-galactosidase, are induced, the enzyme is synthesised, and released to the extracellular medium. In this work, a YES-based approach encompassing the use of a lacZ reporter gene modified strain of S. cerevisiae, microcarriers as solid support, and a fluorescent substrate, fluorescein di-ß-d-galactopyranoside, is proposed, allowing for the assessment of EDCs' presence after only 2 h of incubation. The proposed method provided an EC50 of 0.17 ± 0.03 nM and an LLOQ of 0.03 nM, expressed as 17ß-estradiol. The assessment of different EDCs provided EC50 values between 0.16 and 1.2 × 103 nM. After application to wastewaters, similar results were obtained for EDCs screening, much faster, compared to the conventional 45 h spectrophotometric procedure using a commercial kit, showing potential for onsite high-throughput screening of environmental contamination.

BPA Endocrine Disruptor Detection at the Cutting Edge: FPIA and ELISA Immunoassays.

BPA is a chemical commonly used in the production of polymer-based materials that can have detrimental effects on the thyroid gland and impact human reproductive health. Various expensive methods, such as liquid and gas chromatography, have been suggested for detecting BPA. The fluorescence polarization immunoassay (FPIA) is an inexpensive and efficient homogeneous mix-and-read method that allows for high-throughput screening. FPIA offers high specificity and sensitivity and can be carried out in a single phase within a timeframe of 20-30 min. In this study, new tracer molecules were designed that linked the fluorescein fluorophore with and without a spacer to the bisphenol A moiety. To assess the influence of the C6 spacer on the sensitivity of an assay based on the respective antibody, hapten-protein conjugates were synthesized and assessed for performance in an ELISA setup, and this resulted in a highly sensitive assay with a detection limit of 0.05 g/L. The lowest limit of detection was reached by employing the spacer derivate in the FPIA and was 1.0 µg/L, working range from 2 to 155 µg/L. The validation of the methods was conducted using actual samples compared to LC-MS/MS, which served as the reference method. The FPIA and ELISA both demonstrated satisfactory concordance.

Deployment of machine learning algorithms to predict sepsis: systematic review and application of the SALIENT clinical AI implementation framework.

OBJECTIVE: To retrieve and appraise studies of deployed artificial intelligence (AI)-based sepsis prediction algorithms using systematic methods, identify implementation barriers, enablers, and key decisions and then map these to a novel end-to-end clinical AI implementation framework. MATERIALS AND METHODS: Systematically review studies of clinically applied AI-based sepsis prediction algorithms in regard to methodological quality, deployment and evaluation methods, and outcomes. Identify contextual factors that influence implementation and map these factors to the SALIENT implementation framework. RESULTS: The review identified 30 articles of algorithms applied in adult hospital settings, with 5 studies reporting significantly decreased mortality post-implementation. Eight groups of algorithms were identified, each sharing a common algorithm. We identified 14 barriers, 26 enablers, and 22 decision points which were able to be mapped to the 5 stages of the SALIENT implementation framework. DISCUSSION: Empirical studies of deployed sepsis prediction algorithms demonstrate their potential for improving care and reducing mortality but reveal persisting gaps in existing implementation guidance. In the examined publications, key decision points reflecting real-word implementation experience could be mapped to the SALIENT framework and, as these decision points appear to be AI-task agnostic, this framework may also be applicable to non-sepsis algorithms. The mapping clarified where and when barriers, enablers, and key decisions arise within the end-to-end AI implementation process. CONCLUSIONS: A systematic review of real-world implementation studies of sepsis prediction algorithms was used to validate an end-to-end staged implementation framework that has the ability to account for key factors that warrant attention in ensuring successful deployment, and which extends on previous AI implementation frameworks.

Implementation frameworks for end-to-end clinical AI: derivation of the SALIENT framework.

OBJECTIVE: To derive a comprehensive implementation framework for clinical AI models within hospitals informed by existing AI frameworks and integrated with reporting standards for clinical AI research. MATERIALS AND METHODS: (1) Derive a provisional implementation framework based on the taxonomy of Stead et al and integrated with current reporting standards for AI research: TRIPOD, DECIDE-AI, CONSORT-AI. (2) Undertake a scoping review of published clinical AI implementation frameworks and identify key themes and stages. (3) Perform a gap analysis and refine the framework by incorporating missing items. RESULTS: The provisional AI implementation framework, called SALIENT, was mapped to 5 stages common to both the taxonomy and the reporting standards. A scoping review retrieved 20 studies and 247 themes, stages, and subelements were identified. A gap analysis identified 5 new cross-stage themes and 16 new tasks. The final framework comprised 5 stages, 7 elements, and 4 components, including the AI system, data pipeline, human-computer interface, and clinical workflow. DISCUSSION: This pragmatic framework resolves gaps in existing stage- and theme-based clinical AI implementation guidance by comprehensively addressing the what (components), when (stages), and how (tasks) of AI implementation, as well as the who (organization) and why (policy domains). By integrating research reporting standards into SALIENT, the framework is grounded in rigorous evaluation methodologies. The framework requires validation as being applicable to real-world studies of deployed AI models. CONCLUSIONS: A novel end-to-end framework has been developed for implementing AI within hospital clinical practice that builds on previous AI implementation frameworks and research reporting standards.

Ergometrine sensing in rye flour by a magnetic bead-based immunoassay followed by flow injection analysis with amperometric detection.

A certain group of mycotoxins, the ergot alkaloids, has caused countless deaths throughout human history. They are found in rye and other cereals and ingesting contaminated foods can cause serious health problems. To identify contaminated food exceeding the legal limits for ergot alkaloids, a portable and cost-effective test system is of great interest to the food industry. Rapid analysis can be achieved by screening for a marker compound, for which we chose ergometrine. We developed a magnetic bead-based immunoassay for ergometrine with amperometric detection in a flow injection system using a handheld potentiostat and a smartphone. With this assay a limit of detection of 3 nM (1 µg L-1) was achieved. In spiked rye flour, ergometrine levels from 25 to 250 µg kg-1 could be quantified. All results could be verified by optical detection. The developed assay offers great promise to meet the demand for on-site ergometrine detection in the food industry.

Factors affecting the hydrolysis of the antibiotic amoxicillin in the aquatic environment.

The environmental fate of the frequently used broad-spectrum ß-lactam antibiotic amoxicillin (AMX) is of high concern regarding the potential evolution of antimicrobial resistance (AMR). Moreover, it is known that AMX is prone to hydrolysis, yielding a variety of hydrolysis products (HPs) with yet unknown effects. Studies to identify those HPs and investigate their formation mechanisms have been reported but a long-term study on their stability in real water samples was missing. In this regard, we investigated the hydrolysis of AMX at two concentration levels in four distinct water types under three different storage conditions over two months. Concentrations of AMX and four relevant HPs were monitored by an LC-MS/MS method revealing pronounced differences in the hydrolysis rate of AMX in tap water and mineral water on the one hand (fast) and surface water on the other (slow). In this context, the occurrence, relative intensities, and stability of certain HPs are more dependent on the water type than on the storage condition. As clarified by ICP-MS, the main difference between the water types was the content of the metals copper and zinc which are supposed to catalyze AMX hydrolysis demonstrating an effective method to degrade AMX at ambient conditions.

Squash bees host high diversity and prevalence of parasites in the northeastern United States.

The squash bee Eucera (Peponapis) pruinosa is emerging as a model species to study how stressors impact solitary wild bees in North America. Here, we describe the prevalence of trypanosomes, microsporidians and mollicute bacteria in E. pruinosa and two other species, Bombus impatiens and Apis mellifera, that together comprise over 97% of the pollinator visitors of Cucurbita agroecosystems in Pennsylvania (United States). Our results indicate that all three parasite groups are commonly detected in these bee species, but E. pruinosa often exhibit higher prevalences. We further describe novel trypanosome parasites detected in E. pruinosa, however it is unknown how these parasites impact these bees. We suggest future work investigates parasite replication and infection outcomes.

Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA.

Understanding the mechanisms governing selective turnover of mutation-bearing mtDNA is fundamental to design therapeutic strategies against mtDNA diseases. Here, we show that specific mtDNA damage leads to an exacerbated mtDNA turnover, independent of canonical macroautophagy, but relying on lysosomal function and ATG5. Using proximity labeling and Twinkle as a nucleoid marker, we demonstrate that mtDNA damage induces membrane remodeling and endosomal recruitment in close proximity to mitochondrial nucleoid sub-compartments. Targeting of mitochondrial nucleoids is controlled by the ATAD3-SAMM50 axis, which is disrupted upon mtDNA damage. SAMM50 acts as a gatekeeper, influencing BAK clustering, controlling nucleoid release and facilitating transfer to endosomes. Here, VPS35 mediates maturation of early endosomes to late autophagy vesicles where degradation occurs. In addition, using a mouse model where mtDNA alterations cause impairment of muscle regeneration, we show that stimulation of lysosomal activity by rapamycin, selectively removes mtDNA deletions without affecting mtDNA copy number, ameliorating mitochondrial dysfunction. Taken together, our data demonstrates that upon mtDNA damage, mitochondrial nucleoids are eliminated outside the mitochondrial network through an endosomal-mitophagy pathway. With these results, we unveil the molecular players of a complex mechanism with multiple potential benefits to understand mtDNA related diseases, inherited, acquired or due to normal ageing.

Transcriptomics and protein biomarkers reveal the detoxifying mechanisms of UV radiation for nebivolol toward zebrafish (Danio rerio) embryos/larvae.

Nebivolol (NEB), a ß-blocker frequently used to treat cardiovascular diseases, has been widely detected in aquatic environments, and can be degraded under exposure to UV radiation, leading to the formation of certain transformation products (UV-TPs). Thus, the toxic effects of NEB and its UV-TPs on aquatic organisms are of great importance for aquatic ecosystems. In the present study, the degradation pathway of NEB under UV radiation was investigated. Subsequently, zebrafish embryos/larvae were used to assess the median lethal concentration (LC50) of NEB, and to clarify the sub-lethal effects of NEB and its UV-TPs for the first time. It was found that UV radiation could reduce the toxic effects of NEB on the early development of zebrafish. Transcriptomic analysis identified the top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in zebrafish larvae exposed to NEB, most of which were associated with the antioxidant, nervous, and immune systems. The number of differentially expressed genes (DEGs) in the pathways were reduced after UV radiation. Furthermore, the analysis of protein biomarkers, including CAT and GST (antioxidant response), AChE and ACh (neurotoxicity), CRP and LYS (immune response), revealed that NEB exposure reduced the activity of these biomarkers, whereas UV radiation could alleviate the effects. The present study provides initial insights into the mechanisms underlying toxic effects of NEB and the detoxification effects of UV radiation on the early development of zebrafish. It highlights the necessity of considering the toxicity of UV-TPs when evaluating the toxicity of emerging pollutants in aquatic systems.

Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling the flux of the TCA cycle.

To elucidate the function of oxidative phosphorylation (OxPhos) during B cell differentiation, we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA, and, consequently, respiratory chain super-complexes in activated B cells. Whereas B cell development in DNT mice is normal, B cell proliferation, germinal centers, class switch to IgG, plasma cell maturation, and T cell-dependent as well as T cell-independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B cell receptor-activated cells. Lipopolysaccharide-activated DNT-B cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed, whereas HIF1α is stabilized. Hence, mitochondrial DNA controls the metabolism of activated B cells via OxPhos to foster humoral immunity.

Combined fibre atrophy and decreased muscle regeneration capacity driven by mitochondrial DNA alterations underlie the development of sarcopenia.

BACKGROUND: Mitochondrial dysfunction caused by mitochondrial (mtDNA) deletions have been associated with skeletal muscle atrophy and myofibre loss. However, whether such defects occurring in myofibres cause sarcopenia is unclear. Also, the contribution of mtDNA alterations in muscle stem cells (MuSCs) to sarcopenia remains to be investigated. METHODS: We expressed a dominant-negative variant of the mitochondrial helicase, which induces mtDNA alterations, specifically in differentiated myofibres (K320Eskm mice) and MuSCs (K320Emsc mice), respectively, and investigated their impact on muscle structure and function by immunohistochemistry, analysis of mtDNA and respiratory chain content, muscle transcriptome and functional tests. RESULTS: K320Eskm mice at 24 months of age had higher levels of mtDNA deletions compared with controls in soleus (SOL, 0.07673% vs. 0.00015%, P = 0.0167), extensor digitorum longus (EDL, 0.0649 vs. 0.000925, P = 0.0015) and gastrocnemius (GAS, 0.09353 vs. 0.000425, P = 0.0004). K320Eskm mice revealed a progressive increase in the proportion of cytochrome c oxidase deficient (COX- ) fibres in skeletal muscle cross sections, reaching a maximum of 3.03%, 4.36%, 13.58%, and 17.08% in EDL, SOL, tibialis anterior (TA) and GAS, respectively. However, mice did not show accelerated loss of muscle mass, muscle strength or physical performance. Histological analyses revealed ragged red fibres but also stimulated regeneration, indicating activation of MuSCs. RNAseq demonstrated enhanced expression of genes associated with protein synthesis, but also degradation, as well as muscle fibre differentiation and cell proliferation. In contrast, 7 days after destruction by cardiotoxin, regenerating TA of K320Emsc mice showed 30% of COX- fibres. Notably, regenerated muscle showed dystrophic changes, increased fibrosis (2.5% vs. 1.6%, P = 0.0003), increased abundance of fat cells (2.76% vs. 0.23%, P = 0.0144) and reduced muscle mass (regenerated TA: 40.0 mg vs. 60.2 mg, P = 0.0171). In contrast to muscles from K320Eskm mice, freshly isolated MuSCs from aged K320Emsc mice were completely devoid of mtDNA alterations. However, after passaging, mtDNA copy number as well as respiratory chain subunits and p62 levels gradually decreased. CONCLUSIONS: Taken together, accumulation of large-scale mtDNA alterations in myofibres alone is not sufficient to cause sarcopenia. Expression of K320E-Twinkle is tolerated in quiescent MuSCs, but progressively leads to mtDNA and respiratory chain depletion upon activation, in vivo and in vitro, possibly caused by an increased mitochondrial removal. Altogether, our results suggest that the accumulation of mtDNA alterations in myofibres activates regeneration during aging, which leads to sarcopenia if such alterations have expanded in MuSCs as well.

Impacts of larval host plant species on dispersal traits and free-flight energetics of adult butterflies.

Animals derive resources from their diet and allocate them to organismal functions such as growth, maintenance, reproduction, and dispersal. How variation in diet quality can affect resource allocation to life-history traits, in particular those important to locomotion and dispersal, is poorly understood. We hypothesize that, particularly for specialist herbivore insects that are in co-evolutionary arms races with host plants, changes in host plant will impact performance. From their coevolutionary arms-race with plants, to a complex migratory life history, Monarch butterflies are among the most iconic insect species worldwide. Population declines initiated international conservation efforts involving the replanting of a variety of milkweed species. However, this practice was implemented with little regard for how diverse defensive chemistry of milkweeds experienced by monarch larvae may affect adult fitness traits. We report that adult flight muscle investment, flight energetics, and maintenance costs depend on the host plant species of larvae, and correlate with concentration of milkweed-derived cardenolides sequestered by adults. Our findings indicate host plant species can impact monarchs by affecting fuel requirements for flight.

Zinc Ammonio-dodecaborates: Synthesis, Lewis Acid Strength, and Reactivity.

Two series of zinc salts, [EtZn][A] and Zn[A]2, with weakly coordinating anions [A]- as counterions have been prepared, and their activities as catalysts for hydrosilylation reactions of 1-hexene, benzophenone, and acetophenone have been investigated. The counterions and per- and partially chlorinated 1-ammonio-closo-dodecaborate anions [Me3NB12Cl11]- [1]-, [Pr3NB12H5Cl6]- [2]-, [Bu3NB12H4Cl7]- [3]-, and [Hex3NB12H5Cl6]- [4]- were chosen as potential and more readily available alternatives to carborate anions such as [CHB11Cl11]- and [HexCB11Cl11]-. The basicity of anion [4]- was determined as being close to that of the triflimide anion [N(SO2CF3)2]-, and the fluoride ion affinities (FIAs) of compounds [EtZn][2] and Zn[2]2 are lower than those of the Lewis acids B(C6F5)3 and Zn[HexCB11Cl11]2. The higher anion basicity and the resulting lower Lewis acidity of the zinc centers result in low activity in 1-hexene hydrosilylation catalysis and only moderate activity in the hydrosilylation catalysis of benzophenone and acetophenone.

Novel Pathogenic Sequence Variation m.5789T>C Causes NARP Syndrome and Promotes Formation of Deletions of the Mitochondrial Genome.

BACKGROUND AND OBJECTIVES: We report the pathogenic sequence variant m.5789T>C in the anticodon stem of the mitochondrial tRNA for cysteine as a novel cause of neuropathy, ataxia, and retinitis pigmentosa (NARP), which is usually associated with pathogenic variants in the MT-ATP6 gene. METHODS: To address the correlation of oxidative phosphorylation deficiency with mutation loads, we performed genotyping on single laser-dissected skeletal muscle fibers. Stability of the mitochondrial tRNACys was investigated by Northern blotting. Accompanying deletions of the mitochondrial genome were detected by long-range PCR and their breakpoints were determined by sequencing of single-molecule amplicons. RESULTS: The sequence variant m.5789T>C, originating from the patient's mother, decreases the stability of the mitochondrial tRNA for cysteine by disrupting the anticodon stem, which subsequently leads to a combined oxidative phosphorylation deficiency. In parallel, we observed a prominent cluster of low-abundance somatic deletions with breakpoints in the immediate vicinity of the m.5789T>C variant. Strikingly, all deletion-carrying mitochondrial DNA (mtDNA) species, in which the corresponding nucleotide position was not removed, harbored the mutant allele, and none carried the wild-type allele. DISCUSSION: In addition to providing evidence for the novel association of a tRNA sequence alteration with NARP syndrome, our observations support the hypothesis that single nucleotide changes can lead to increased occurrence of site-specific mtDNA deletions through the formation of an imperfect repeat. This finding might be relevant for understanding mechanisms of deletion generation in the human mitochondrial genome.