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This Editorial describes PLOS Biology's experiment with contextualised publication, whereby potentially controversial studies are published with an Editor’s Note and an accompanying Primer in which the Academic Editor guides the reader, dissects the study, and lays out the thinking behind the decision to publish. [ABSTRACT FROM AUTHOR]

Determining the distribution of the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, YFP) in the Yangtze River has to date relied on traditional visual and counting methods, but such field surveys are time-consuming and expensive. Analyses using environmental DNA (eDNA) to investigate the presence and range of endangered aquatic species have proven to be more economical and effective detection methods, and are a non-invasive approach to sampling. A challenge of relying on eDNA for YFP monitoring is that the Yangtze River is characterized by high turbidity and a strong current. Here, we used an eDNA-based approach to estimate the presence of YFP at 18 sites in the Yangtze River in August 2017 and at an additional 11 sites in January 2018. At each sampling site, we filtered six 1 L water samples with 5 µm pore size filter paper and quantified the amount of YFP eDNA in each water sample using quantitative real-time polymerase chain reaction (qPCR). In addition, YFP eDNA was successfully detected in locations where we visually observed YFP, as well as in locations where YFP were not observed directly. We found that our eDNA-based method had higher detection rates than traditional field survey methods. Although YFP was visually observed in the Yangtze River in winter, water samples collected during the summer contained significantly higher YFP eDNA than winter water samples. Our results demonstrate the potential effectiveness of eDNA detection methods in determining the distribution of YFP in the Yangtze River. [ABSTRACT FROM AUTHOR]

The mitochondrion is the power plant of the eukaryotic cell, and tRNAs are the fundamental components of its translational machinery. In the present paper, the evolution of mitochondrial tRNAs was investigated in the Cetacea, a clade of Cetartiodactyla that retuned to water and thus had to adapt its metabolism to a different medium than that of its mainland ancestors. Our analysis focussed on identifying the factors that influenced the evolution of Cetacea tRNA double-helix elements, which play a pivotal role in the formation of the secondary and tertiary structures of each tRNA and consequently manipulate the whole translation machinery of the mitochondrion. Our analyses showed that the substitution pathways in the stems of different tRNAs were influenced by various factors, determining a molecular evolution that was unique to each of the 22 tRNAs. Our data suggested that the composition, AT-skew, and GC-skew of the tRNA stems were the main factors influencing the substitution process. In particular, the range of variation and the fluctuation of these parameters affected the fate of single tRNAs. Strong heterogeneity was observed among the different species of Cetacea. Finally, it appears that the evolution of mitochondrial tRNAs was also shaped by the environments in which the Cetacean taxa differentiated. This latter effect was particularly evident in toothed whales that either live in freshwater or are deep divers. [ABSTRACT FROM AUTHOR]

The pepper weevil, Anthonomus eugenii, is a major pest on Capsicum species. Apart from natural spread, there is a risk of spread via international pepper trade. In the Netherlands, a pepper weevil outbreak occurred in 2012 and affected six greenhouses producing different sweet pepper varieties. The following year, a pepper weevil outbreak occurred in Italy. To trace the origin of the Dutch outbreak and to establish if the Dutch and Italian outbreaks were linked, we determined the mitogenomes of A. eugenii specimens collected at outbreak locations, and compared these with specimens from the native area, and other areas where the pest was introduced either by natural dispersal or via trade. The circular 17,257 bp A. eugenii mitogenome comprises thirteen mitochondrial genes typically found in insect species. Intra-species variation of these mitochondrial genes revealed four main mitochondrial lineages encompassing 41 haplotypes. The highest diversity was observed for specimens from its presumed native area (i.e. Mexico). The Dutch outbreak specimens represented three highly similar haplotypes, suggesting a single introduction of the pest. The major Dutch haplotype was also found in two specimens from New Jersey. As the Netherlands does not have pepper trade with New Jersey, it is likely that the specimens sampled in New Jersey and those sampled in the Netherlands originate from a shared source that was not included in this study. In addition, our analysis shows that the Italian and Dutch outbreaks were not linked. The mitochondrial genome is a useful tool to trace outbreak populations and the methodology presented in this paper could prove valuable for other invasive pest species, such as the African fruit moth Thaumatotibia leucotreta and emerald ash borer Agrilus planipennis. [ABSTRACT FROM AUTHOR]

Butterfly monitoring and Red List programs in Switzerland rely on a combination of observations and collection records to document changes in species distributions through time. While most butterflies can be identified using morphology, some taxa remain challenging, making it difficult to accurately map their distributions and develop appropriate conservation measures. In this paper, we explore the use of the DNA barcode (a fragment of the mitochondrial gene COI) as a tool for the identification of Swiss butterflies and forester moths (Rhopalocera and Zygaenidae). We present a national DNA barcode reference library including 868 sequences representing 217 out of 224 resident species, or 96.9% of Swiss fauna. DNA barcodes were diagnostic for nearly 90% of Swiss species. The remaining 10% represent cases of para- and polyphyly likely involving introgression or incomplete lineage sorting among closely related taxa. We demonstrate that integrative taxonomic methods incorporating a combination of morphological and genetic techniques result in a rate of species identification of over 96% in females and over 98% in males, higher than either morphology or DNA barcodes alone. We explore the use of the DNA barcode for exploring boundaries among taxa, understanding the geographical distribution of cryptic diversity and evaluating the status of purportedly endemic taxa. Finally, we discuss how DNA barcodes may be used to improve field practices and ultimately enhance conservation strategies. [ABSTRACT FROM AUTHOR]

With an estimate of around 9,000 species, the Neotropical region hosts the greatest diversity of freshwater fishes of the world. Genetic surveys have the potential to unravel isolated and unique lineages and may result in the identification of undescribed species, accelerating the cataloguing of extant biodiversity. In this paper, molecular diversity within the valuable and widespread Neotropical genus Hoplias was assessed by means of DNA Barcoding. The geographic coverage spanned 40 degrees of latitude from French Guiana to Argentina. Our analyses revealed 22 mitochondrial lineages fully supported by means of Barcode Index Number, Automatic Barcode Gap Discovery and phylogenetic analyses. This mtDNA survey revealed the existence of 15 fully supported mitochondrial lineages within the once considered to be the continentally distributed H. malabaricus. Only four of them are currently described as valid species however, leaving 11 mitochondrial lineages currently “masked” within this species complex. Mean genetic divergence was 13.1%. Barcoding gap analysis discriminated 20 out of the 22 lineages tested. Phylogenetic analyses showed that all taxonomically recognized species form monophyletic groups. Hoplias malabaricus sensu stricto clustered within a large clade, excluding the representatives of the La Plata River Basin. In the H. lacerdae group, all species but H. curupira showed a cohesive match between taxonomic and molecular identification. Two different genetic lineages were recovered for H. aimara. Given the unexpected hidden mitochondrial diversity within H. malabaricus, the COI sequence composition of specimens from Suriname (the type locality), identified as H. malabaricus sensu stricto, is of major importance. [ABSTRACT FROM AUTHOR]

Mitochondrial disorders associated with genetic defects of the ATP synthase are among the most deleterious diseases of the neuromuscular system that primarily manifest in newborns. Nevertheless, the number of established animal models for the elucidation of the molecular mechanisms behind such pathologies is limited. In this paper, we target the Drosophila melanogaster gene encoding for the ATP synthase subunit c, ATPsynC, in order to create a fruit fly model for investigating defects in mitochondrial bioenergetics and to better understand the comprehensive pathological spectrum associated with mitochondrial ATP synthase dysfunctions. Using P-element and EMS mutagenesis, we isolated a set of mutations showing a wide range of effects, from larval lethality to complex pleiotropic phenotypes encompassing developmental delay, early adult lethality, hypoactivity, sterility, hypofertility, aberrant male courtship behavior, locomotor defects and aberrant gonadogenesis. ATPsynC mutations impair ATP synthesis and mitochondrial morphology, and represent a powerful toolkit for the screening of genetic modifiers that can lead to potential therapeutic solutions. Furthermore, the molecular characterization of ATPsynC mutations allowed us to better understand the genetics of the ATPsynC locus and to define three broad pathological consequences of mutations affecting the mitochondrial ATP synthase functionality in Drosophila: i) pre-adult lethality; ii) multi-trait pathology accompanied by early adult lethality; iii) multi-trait adult pathology. We finally predict plausible parallelisms with genetic defects of mitochondrial ATP synthase in humans. [ABSTRACT FROM AUTHOR]

It is well known that the rRNA structure information is important to assist phylogenetic analysis through identifying homologous positions to improve alignment accuracy. In addition, the secondary structure of some conserved motifs is highly stable among distantly related taxa, which can provide potentially informative characters for estimating phylogeny. In this paper, we applied the high-throughput pooled sequencing approach to the determination of neuropteran mitogenomes. Four complete mitogenome sequences were obtained: Micromus angulatus (Hemerobiidae), Chrysoperla nipponensis (Chrysopidae), Rapisma sp. (Ithonidae), and Thaumatosmylus sp. (Osmylidae). This allowed us to sample more complete mitochondrial RNA gene sequences. Secondary structure diagrams for the complete mitochondrial small and large ribosomal subunit RNA genes of eleven neuropterid species were predicted. Comparative analysis of the secondary structures indicated a closer relationship of Megaloptera and Neuroptera. This result was congruent with the resulting phylogeny inferred from sequence alignments of all 37 mitochondrial genes, namely the hypothesis of (Raphidioptera + (Megaloptera + Neuroptera)). [ABSTRACT FROM AUTHOR]

In this paper, we compare ndh genes in the plastid genome of many Cymbidium species and three closely related taxa in Orchidaceae looking for evidence of ndh gene degradation. Among the 11 ndh genes, there were frequently large deletions in directly repeated or AT-rich regions. Variation in these degraded ndh genes occurs between individual plants, apparently at population levels in these Cymbidium species. It is likely that ndh gene transfers from the plastome to mitochondrial genome (chondriome) occurred independently in Orchidaceae and that ndh genes in the chondriome were also relatively recently transferred between distantly related species in Orchidaceae. Four variants of the ycf1-rpl32 region, which normally includes the ndhF genes in the plastome, were identified, and some Cymbidium species contained at least two copies of that region in their organellar genomes. The four ycf1-rpl32 variants seem to have a clear pattern of close relationships. Patterns of ndh degradation between closely related taxa and translocation of ndh genes to the chondriome in Cymbidium suggest that there have been multiple bidirectional intracellular gene transfers between two organellar genomes, which have produced different levels of ndh gene degradation among even closely related species. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer is one of the most common cancers worldwide, and is influenced by the interplay of various factors, including a very strong genetic component. For instance, incorrect mitochondrial biogenesis is correlated with increased risk of developing colorectal cancer. Thus, it is important to understand the consequences of changes in both the expression and the correct function of the transcription factors that regulate mitochondrial biogenesis, namely NRF2. Objectives: The main objective of this paper is to characterise the relationship between NRF2 and colorectal cancer by compiling data from an exhaustive literature search. Methods: Information was obtained by defining specific search terms and searching in several databases. After a strict selection procedure, data were tabulated and the relationships between articles were assessed by measuring heterogeneity and by constructing conceptual maps. Results and discussion: We found a general consensus in the literature that the presence of oxidizing agents as well as the inhibition of the NRF2 repressor Keap1 maintain NRF2 expression at basal levels. This predominantly exerts a cytoprotective effect on cells and decreases risk of colorectal cancer. However, if NRF2 is inhibited, protection against external agents disappears and risk of colorectal cancer increases. Interestingly, colorectal cancer risk is also increased when NRF2 becomes overexpressed. In this case, the increased risk arises from NRF2-induced inflammation and resistance to chemotherapy. Conclusion: The proper basal function of NRF2 and Keap1 are essential for preventing oncogenic processes in the colon. Consequently, any disruption to the expression of these genes can promote the genesis and progression of colon cancer. [ABSTRACT FROM AUTHOR]

The midgut in the freshwater shrimp Neocaridina davidi (previously named N. heteropoda) (Crustacea, Malacostraca) is composed of a tube-shaped intestine and a large hepatopancreas that is formed by numerous blind-ended tubules. The precise structure and ultrastructure of these regions were presented in our previous papers, while here we focused on the ultrastructural changes that occurred in the midgut epithelial cells (D-cells in the intestine, B- and F- cells in the hepatopancreas) after long-term starvation and re-feeding. We used transmission electron microscopy, light and confocal microscopes and flow cytometry to describe all of the changes that occurred due to the stressor with special emphasis on mitochondrial alterations. A quantitative assessment of cells with depolarized mitochondria helped us to establish whether there is a relationship between starvation, re-feeding and the inactivation/activation of mitochondria. The results of our studies showed that in the freshwater shrimp N. davidi that were analyzed, long-term starvation activates the degeneration of epithelial cells at the ultrastructural level and causes an increase of cells with depolarized (non-active) mitochondria. The process of re-feeding leads to the gradual regeneration of the cytoplasm of the midgut epithelial cells; however, these changes were observed at the ultrastructural level. Additionally, re-feeding causes the regeneration of mitochondrial ultrastructure. Therefore, we can state that the increase in the number of cells with polarized mitochondria occurs slowly and does not depend on ultrastructural alterations. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment. [ABSTRACT FROM AUTHOR]

Background: The lack of new anthelmintic agents is of growing concern because it affects human health and our food supply, as both livestock and plants are affected. Two principal factors contribute to this problem. First, nematode resistance to anthelmintic drugs is increasing worldwide and second, many effective nematicides pose environmental hazards. In this paper we address this problem by deploying a high throughput screening platform for anthelmintic drug discovery using the nematode Caenorhabditis elegans as a surrogate for infectious nematodes. This method offers the possibility of identifying new anthelmintics in a cost-effective and timely manner. Methods/Principal findings: Using our high throughput screening platform we have identified 14 new potential anthelmintics by screening more than 26,000 compounds from the Chembridge and Maybridge chemical libraries. Using phylogenetic profiling we identified a subset of the 14 compounds as potential anthelmintics based on the relative sensitivity of C. elegans when compared to yeast and mammalian cells in culture. We showed that a subset of these compounds might employ mechanisms distinct from currently used anthelmintics by testing diverse drug resistant strains of C. elegans. One of these newly identified compounds targets mitochondrial complex II, and we used structural analysis of the target to suggest how differential binding of this compound may account for its different effects in nematodes versus mammalian cells. Conclusions/Significance: The challenge of anthelmintic drug discovery is exacerbated by several factors; including, 1) the biochemical similarity between host and parasite genomes, 2) the geographic location of parasitic nematodes and 3) the rapid development of resistance. Accordingly, an approach that can screen large compound collections rapidly is required. C. elegans as a surrogate parasite offers the ability to screen compounds rapidly and, equally importantly, with specificity, thus reducing the potential toxicity of these compounds to the host and the environment. We believe this approach will help to replenish the pipeline of potential nematicides. [ABSTRACT FROM AUTHOR]

Pentatricopeptide repeat containing proteins (PPRs) bind to RNA transcripts originating from mitochondria and plastids. There are two classes of PPR proteins. The class contains tandem -type motif sequences, and the class contains alternating , and type sequences. In this paper, we describe a novel tool that predicts PPR-RNA interaction; specifically, our method, which we call aPPRove, determines where and how a -class PPR protein will bind to RNA when given a PPR and one or more RNA transcripts by using a combinatorial binding code for site specificity proposed by Barkan et al. Our results demonstrate that aPPRove successfully locates how and where a PPR protein belonging to the class can bind to RNA. For each binding event it outputs the binding site, the amino-acid-nucleotide interaction, and its statistical significance. Furthermore, we show that our method can be used to predict binding events for -class proteins using a known edit site and the statistical significance of aligning the PPR protein to that site. In particular, we use our method to make a conjecture regarding an interaction between CLB19 and the second intronic region of ycf3. The aPPRove web server can be found at . [ABSTRACT FROM AUTHOR]

We present a cost-effective approach to sequence whole mitochondrial genomes for hundreds of individuals. Our approach uses small reaction volumes and unmodified (non-phosphorylated) barcoded adaptors to minimize reagent costs. We demonstrate our approach by sequencing 383 Fundulus sp. mitochondrial genomes (192 F. heteroclitus and 191 F. majalis). Prior to sequencing, we amplified the mitochondrial genomes using 4–5 custom-made, overlapping primer pairs, and sequencing was performed on an Illumina HiSeq 2500 platform. After removing low quality and short sequences, 2.9 million and 2.8 million reads were generated for F. heteroclitus and F. majalis respectively. Individual genomes were assembled for each species by mapping barcoded reads to a reference genome. For F. majalis, the reference genome was built de novo. On average, individual consensus sequences had high coverage: 61-fold for F. heteroclitus and 57-fold for F. majalis. The approach discussed in this paper is optimized for sequencing mitochondrial genomes on an Illumina platform. However, with the proper modifications, this approach could be easily applied to other small genomes and sequencing platforms. [ABSTRACT FROM AUTHOR]

Multiple in vitro tests are widely applied to assess the anticancer activity of new compounds, including their combinations and interactions with other drugs. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is one of the most commonly used assays to assess the efficacy and interactions of anticancer agents. However, it can be significantly influenced by compounds that modify cell metabolism and reaction conditions. Therefore, several assays are sometimes used to screen for potential anticancer drugs. However, the majority of drug interactions are evaluated only with this single method. The aim of our studies was to verify whether the choice of an assay has an impact on determining the type of interaction and to identify the source of discrepancies. We compared the accuracy of MTT and CVS (crystal violet staining) assays in the interaction of two compounds characterized by similar anticancer activity: isothiocyanates (ITCs) and Selol. Confocal microscopy studies were carried out to assess the influence of these compounds on the reactive oxygen species (ROS) level, mitochondrial membrane potential, dead-to-live cell ratio and MTT-tetrazolium salt reduction rate. The MTT assay was less reliable than CVS. The MTT test of Selol and 2-oxoheptyl ITC, which affected the ROS level and MTT reduction rate, gave false negative (2-oxoheptyl ITC) or false positive (Selol) results. As a consequence, the MTT assay identified an antagonistic interaction between Selol and ITC, while the metabolism-independent CVS test identified an additive or synergistic interaction. In this paper, we show for the first time that the test assay may change the interpretation of the compound interaction. Therefore, the test method should be chosen with caution, considering the mechanism of action of the compound. [ABSTRACT FROM AUTHOR]

Many cells are cultured in media that contains an antibiotic to prevent bacterial contamination. Mycoplasma and other bacterial contamination is a serious problem for those involved in cell culture. Antibiotics in the media helps prevent this contamination and make life easier for the investigators; as performing cell culture experiments in antibiotic free media is difficult and requires vigorous sterile technique. There are many reports of antibiotics causing mitochondrial damage. In this study, we tested the effect of gentamicin in culture media on human mammary epithelial MCF-12A and breast cancer MCF-7 and MDA-MB-231 cell lines by real time PCR, immunofluorescent microscopy, lactate assay, DNA damage assay. We found that the addition of gentamicin in media upregulated the gene expression of hypoxia inducer factor 1 alpha (HIF1a), glycolytic enzymes and glucose transporters, compared to the cells cultured in gentamicin free media. Gentamicin also increased the lactate production and inhibited mitochondrial membrane potential of the cell lines. Furthermore, the antibiotics in media induced mitochondrial reactive oxygen species causing DNA damage. We found an increase of 8-hydroxy-2’-deoxyguanosine a product of DNA oxidative damage in the media of MCF-12A, MCF-7 and MDA-MB-231 cell lines. These results showed that normal epithelial and breast cancer cells cultured in the media with gentamicin had increased HIF1a, aerobic glycolysis and DNA oxidative damage. If we use these unhealthy cells in the experiment, all data will be different, compared to cells grown in gentamicin free media. We have studied the detrimental effects of three antibiotics on mitochondrial function in the untransformed MCF-12A human mammary cell line and two human mammary cancer cell lines, MCF-7 and MB-MDA-231. The metabolic changes in all cell lines were dramatically different between those in antibiotic free media versus antibiotic containing media. There was a marked difference in gene expression of glycolytic enzymes, reactive oxygen species production and effects on membrane potential. Ironically, our first studies were done in media containing gentamicin, and repeated studies were done in gentamicin free media. The results were very different. The purpose of this report is to emphasize that metabolic cell culture data may be inaccurate because experiments were performed in cell culture media containing antibiotics. We will present evidence to support this theory. [ABSTRACT FROM AUTHOR]

Biologists determine experimental effects by perturbing biological entities or units. When done appropriately, independent replication of the entity–intervention pair contributes to the sample size (N) and forms the basis of statistical inference. If the wrong entity–intervention pair is chosen, an experiment cannot address the question of interest. We surveyed a random sample of published animal experiments from 2011 to 2016 where interventions were applied to parents and effects examined in the offspring, as regulatory authorities provide clear guidelines on replication with such designs. We found that only 22% of studies (95% CI = 17%–29%) replicated the correct entity–intervention pair and thus made valid statistical inferences. Nearly half of the studies (46%, 95% CI = 38%–53%) had pseudoreplication while 32% (95% CI = 26%–39%) provided insufficient information to make a judgement. Pseudoreplication artificially inflates the sample size, and thus the evidence for a scientific claim, resulting in false positives. We argue that distinguishing between biological units, experimental units, and observational units clarifies where replication should occur, describe the criteria for genuine replication, and provide concrete examples of in vitro, ex vivo, and in vivo experimental designs. [ABSTRACT FROM AUTHOR]

Mitochondria have been increasingly recognized as a central regulatory nexus for multiple metabolic pathways, in addition to ATP production via oxidative phosphorylation (OXPHOS). Here we show that inducing mitochondrial DNA (mtDNA) stress in Drosophila using a mitochondrially-targeted Type I restriction endonuclease (mtEcoBI) results in unexpected metabolic reprogramming in adult flies, distinct from effects on OXPHOS. Carbohydrate utilization was repressed, with catabolism shifted towards lipid oxidation, accompanied by elevated serine synthesis. Cleavage and translocation, the two modes of mtEcoBI action, repressed carbohydrate rmetabolism via two different mechanisms. DNA cleavage activity induced a type II diabetes-like phenotype involving deactivation of Akt kinase and inhibition of pyruvate dehydrogenase, whilst translocation decreased post-translational protein acetylation by cytonuclear depletion of acetyl-CoA (AcCoA). The associated decease in the concentrations of ketogenic amino acids also produced downstream effects on physiology and behavior, attributable to decreased neurotransmitter levels. We thus provide evidence for novel signaling pathways connecting mtDNA to metabolism, distinct from its role in supporting OXPHOS. [ABSTRACT FROM AUTHOR]

Calreticulin (CRT) is a high-capacity Ca2+ protein whose expression is up-regulated during cellular transformation and is associated with disease progression in multiple types of malignancies. At the same time, CRT has been characterized as an important stress-response protein capable of inducing immunogenic cell death (ICD) when translocated to the cell surface. It remains unclear why CRT expression is preserved by malignant cells during the course of transformation despite its immunogenic properties. In this study, we identify a novel, critical function of CRT as a cell survival factor in multiple types of human solid-tissue malignancies. CRT knockdown activates p53, which mediates cell-death response independent of executioner caspase activity and accompanied full-length poly ADP ribose polymerase (PARP) cleavage. Mechanistically, we show that down-regulation of CRT results in mitochondrial Ca2+ overload and induction of mitochondria permeability transition pore (mPTP)-dependent cell death, which can be significantly rescued by the mPTP inhibitor, Cyclosporin A (CsA). The clinical importance of CRT expression was revealed in the analysis of the large cohort of cancer patients (N = 2,058) to demonstrate that high levels of CRT inversely correlates with patient survival. Our study identifies intracellular CRT as an important therapeutic target for tumors whose survival relies on its expression. [ABSTRACT FROM AUTHOR]

Fragmentation of mitochondrial network has been implicated in many neurodegenerative, renal, and metabolic diseases. However, a quantitative measure of the microscopic parameters resulting in the impaired balance between fission and fusion of mitochondria and consequently the fragmented networks in a wide range of pathological conditions does not exist. Here we present a comprehensive analysis of mitochondrial networks in cells with Alzheimer’s disease (AD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), optic neuropathy (OPA), diabetes/cancer, acute kidney injury, Ca2+ overload, and Down Syndrome (DS) pathologies that indicates significant network fragmentation in all these conditions. Furthermore, we found key differences in the way the microscopic rates of fission and fusion are affected in different conditions. The observed fragmentation in cells with AD, HD, DS, kidney injury, Ca2+ overload, and diabetes/cancer pathologies results from the imbalance between the fission and fusion through lateral interactions, whereas that in OPA, PD, and ALS results from impaired balance between fission and fusion arising from longitudinal interactions of mitochondria. Such microscopic difference leads to major disparities in the fine structure and topology of the network that could have significant implications for the way fragmentation affects various cell functions in different diseases. [ABSTRACT FROM AUTHOR]

Iron homeostasis is important for growth, reproduction and other metabolic processes in all eukaryotes. However, the functions of ATP-binding cassette (ABC) transporters in iron homeostasis are largely unknown. Here, we found that one ABC transporter (named FgAtm1) is involved in regulating iron homeostasis, by screening sensitivity to iron stress for 60 ABC transporter mutants of Fusarium graminearum, a devastating fungal pathogen of small grain cereal crops worldwide. The lack of FgAtm1 reduces the activity of cytosolic Fe-S proteins nitrite reductase and xanthine dehydrogenase, which causes high expression of FgHapX via activating transcription factor FgAreA. FgHapX represses transcription of genes for iron-consuming proteins directly but activates genes for iron acquisition proteins by suppressing another iron regulator FgSreA. In addition, the transcriptional activity of FgHapX is regulated by the monothiol glutaredoxin FgGrx4. Furthermore, the phosphorylation of FgHapX, mediated by the Ser/Thr kinase FgYak1, is required for its functions in iron homeostasis. Taken together, this study uncovers a novel regulatory mechanism of iron homeostasis mediated by an ABC transporter in an important pathogenic fungus. [ABSTRACT FROM AUTHOR]

A physiologically relevant in vitro human-based model could be the ‘gold standard’ to clarify the pathological steps involved in glaucoma onset. In this regard, human 3D cultures may represent an excellent starting point to achieve this goal. Indeed, the 3D matrix allows to re-create the in vivo-like tissue architecture, maintaining its functionality and cellular behaviour, compared to the 2D model. Thus, we propose a comparison between the 2D and 3D in vitro models of human trabecular meshwork cells in terms of cellular responses after chronic stress exposure. Our results showed that 3D-cells are more sensitive to intracellular reactive oxidative specie production induced by hydrogen peroxide treatment, compared to 2D cultures. Additionally, in 3D cultures a more accurate regulation of the apoptosis trigger and cell adaptation mechanisms was detected than in 2D models. In line with these findings, the 3D-HTMC model shows the ability to better mimic the in vivo cell behaviour in adaptive responses to chronic oxidative stress than 2D. [ABSTRACT FROM AUTHOR]

Plant mitochondrial genomes are usually assembled and displayed as circular maps based on the widely-held view across the broad community of life scientists that circular genome-sized molecules are the primary form of plant mitochondrial DNA, despite the understanding by plant mitochondrial researchers that this is an inaccurate and outdated concept. Many plant mitochondrial genomes have one or more pairs of large repeats that can act as sites for inter- or intramolecular recombination, leading to multiple alternative arrangements (isoforms). Most mitochondrial genomes have been assembled using methods unable to capture the complete spectrum of isoforms within a species, leading to an incomplete inference of their structure and recombinational activity. To document and investigate underlying reasons for structural diversity in plant mitochondrial DNA, we used long-read (PacBio) and short-read (Illumina) sequencing data to assemble and compare mitochondrial genomes of domesticated (Lactuca sativa) and wild (L. saligna and L. serriola) lettuce species. We characterized a comprehensive, complex set of isoforms within each species and compared genome structures between species. Physical analysis of L. sativa mtDNA molecules by fluorescence microscopy revealed a variety of linear, branched, and circular structures. The mitochondrial genomes for L. sativa and L. serriola were identical in sequence and arrangement and differed substantially from L. saligna, indicating that the mitochondrial genome structure did not change during domestication. From the isoforms in our data, we infer that recombination occurs at repeats of all sizes at variable frequencies. The differences in genome structure between L. saligna and the two other Lactuca species can be largely explained by rare recombination events that rearranged the structure. Our data demonstrate that representations of plant mitochondrial genomes as simple, circular molecules are not accurate descriptions of their true nature and that in reality plant mitochondrial DNA is a complex, dynamic mixture of forms. [ABSTRACT FROM AUTHOR]

Multiple cellular organelles tightly orchestrate intracellular calcium (Ca2+) dynamics to regulate cellular activities and maintain homeostasis. The interplay between the endoplasmic reticulum (ER), a major store of intracellular Ca2+, and mitochondria, an important source of adenosine triphosphate (ATP), has been the subject of much research, as their dysfunction has been linked with metabolic diseases. Interestingly, throughout the cell’s cytosolic domain, these two organelles share common microdomains called mitochondria-associated ER membranes (MAMs), where their membranes are in close apposition. The role of MAMs is critical for intracellular Ca2+ dynamics as they provide hubs for direct Ca2+ exchange between the organelles. A recent experimental study reported correlation between obesity and MAM formation in mouse liver cells, and obesity-related cellular changes that are closely associated with the regulation of Ca2+ dynamics. We constructed a mathematical model to study the effects of MAM Ca2+ dynamics on global Ca2+ activities. Through a series of model simulations, we investigated cellular mechanisms underlying the altered Ca2+ dynamics in the cells under obesity. We predict that, as the dosage of stimulus gradually increases, liver cells from obese mice will reach the state of saturated cytosolic Ca2+ concentration at a lower stimulus concentration, compared to cells from healthy mice. [ABSTRACT FROM AUTHOR]

Recent molecular systematic studies of arvicoline voles of the genera Neodon, Lasiopodomys, Phaiomys, and Microtus from Central Asia suggest the inclusion of Phaiomys leucurus, Microtus clarkei, and Lasiopodomys fuscus into Neodon and moving Neodon juldaschi into Microtus (Blanfordimys). In addition, three new species of Neodon (N. linzhiensis, N. medogensis, and N. nyalamensis) have recently been described from Tibet. Analyses of concatenated mitochondrial (Cytb, COI) and nuclear (Ghr, Rbp3) genes recovered Neodon as a well-supported monophyletic clade including all the recently described and relocated species. Kimura-2-parameter distance between Neodon from western Nepal compared to N. sikimensis (K2P = 13.1) and N. irene (K2P = 13.4) was equivalent to genetic distances observed between recognized species of this genus. The specimens sampled from western Nepal were recovered sister to N. sikimensis in the concatenated analysis. However, analyses conducted exclusively with mitochondrial loci did not support this relationship. The occlusal patterns of the first lower (m1) and third upper (M3) molars were simpler in specimens from western Nepal in comparison to N. sikimensis from eastern Nepal and India. Twelve craniodental characters and four external field measurements were examined from specimens of N. sikimensis from eastern Nepal and India, N. irene, and Neodon from western Nepal. Neodon from western Nepal were significantly different from N. sikimensis from eastern Nepal and India in ten out of 16 characters measured and from N. irene for all characters except ear height. Specimens from western Nepal were smaller in size than N. sikimensis from Eastern Nepal and India and larger than N. irene. Together the results of the molecular and morphological analyses indicate that Neodon from western Nepal are distinct under the phylogenetic, genetic and morpho species concepts. [ABSTRACT FROM AUTHOR]

Larix populations at the tundra-taiga ecotone in northern Siberia are highly under-represented in population genetic studies, possibly due to the remoteness of these regions that can only be accessed at extraordinary expense. The genetic signatures of populations in these boundary regions are therefore largely unknown. We aim to generate organelle reference genomes for the detection of single nucleotide polymorphisms (SNPs) that can be used for paleogenetic studies. We present 19 complete chloroplast genomes and mitochondrial genomic sequences of larches from the southern lowlands of the Taymyr Peninsula (northernmost range of Larix gmelinii (Rupr.) Kuzen.), the lower Omoloy River, and the lower Kolyma River (both in the range of Larix cajanderi Mayr). The genomic data reveal 84 chloroplast SNPs and 213 putatively mitochondrial SNPs. Parsimony-based chloroplast haplotype networks show no spatial structure of individuals from different geographic origins, while the mitochondrial haplotype network shows at least a slight spatial structure with haplotypes from the Omoloy and Kolyma populations being more closely related to each other than to most of the haplotypes from the Taymyr populations. Whole genome alignments with publicly available complete chloroplast genomes of different Larix species show that among official plant barcodes only the rcbL gene contains sufficient polymorphisms, but has to be sequenced completely to distinguish the different provenances. We provide 8 novel mitochondrial SNPs that are putatively diagnostic for the separation of L. gmelinii and L. cajanderi, while 4 chloroplast SNPs have the potential to distinguish the L. gmelinii/L. cajanderi group from other Larix species. Our organelle references can be used for a targeted primer and probe design allowing the generation of short amplicons. This is particularly important with regard to future investigations of, for example, the biogeographic history of Larix by screening ancient sedimentary DNA of Larix. [ABSTRACT FROM AUTHOR]

Mung bean (Vigna radiata), an important legume crop, has the property of desiccation tolerance (DT), which is lost in the final stage of germination (preimbibition, 18 h-24 h). We compared parameters related to the programmed cell death (PCD) of mung bean seeds before and after dehydration at different imbibition stages through various detection methods. The results of Evans blue and TTC staining methods showed that the dehydration process could lead to cell death. The results of optical and subcellular morphology showed that PCD occurred after dehydration. The destruction of DNA integrity and the activity changes in caspase and total nuclease in mung bean seeds after dehydration treatment indicated that the loss of desiccation tolerance was related to PCD. Dehydration resulted in the destruction of the mitochondrial structure, reversal of the membrane potential, and the entrance of cytochrome C into the cytoplasm. These processes all indicate that the mitochondrial apoptosis pathway was the main form of dehydration-induced PCD. The results of cytoplasmic Ca2+ concentration showed that Ca2+ signaling also played a role in inducing PCD, with the upstream signal being dehydration-induced changes in water potential and the downstream signal being the ROS and mitochondrial PT channel, according to the order in which these signals happened. The mitochondrial apoptosis pathway can be considered the main mechanism of dehydration-induced PCD based on our analysis of the sequence of major events in PCD. The main processes include dehydration induction, changes in Ca2+ and mitochondrial respiratory electron transport, the reversal of mitochondrial membrane potential induced by ROS and Ca2+, and the transmission and execution of PCD downstream signals induced by cytochrome C release. [ABSTRACT FROM AUTHOR]

The dynamics of the cellular proportion of mutant mtDNA molecules is crucial for mitochondrial diseases. Cellular populations of mitochondria are under homeostatic control, but the details of the control mechanisms involved remain elusive. Here, we use stochastic modelling to derive general results for the impact of cellular control on mtDNA populations, the cost to the cell of different mtDNA states, and the optimisation of therapeutic control of mtDNA populations. This formalism yields a wealth of biological results, including that an increasing mtDNA variance can increase the energetic cost of maintaining a tissue, that intermediate levels of heteroplasmy can be more detrimental than homoplasmy even for a dysfunctional mutant, that heteroplasmy distribution (not mean alone) is crucial for the success of gene therapies, and that long-term rather than short intense gene therapies are more likely to beneficially impact mtDNA populations. [ABSTRACT FROM AUTHOR]

In this study, we report on the synthesis of silver nanoparticles (AgNPs) from the leaf extracts of Cynara scolymus (Artichoke) using microwave irradiation and the evaluation of its anti-cancer potential with photodynamic therapy (PDT). Silver nanoparticles formation was characterized by scanning electron microscopy with energy dispersive x-ray spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Silver nanoparticles formation was also investigated the surface charge, particle size and distribution using zetasizer analysis. The cytotoxic effect of AgNPs and/or PDT was studied by MTT assay and migration by the scratch assay. The apoptotic inducing ability of the AgNPs and/or PDT was investigated by intracellular ROS analysis, antioxidant enzyme levels (SOD, CAT, GPx and GSH), Hoechst staining and Bax/Bcl-2 analysis using western blotting. The mean particle size of produced AgNPs was found 98.47±2.04 nm with low polydispersity (0.301±0.033). Zeta potential values of AgNPs show -32.3± 0.8 mV. These results clearly indicate the successful formation of AgNPs for cellular uptake. Mitochondrial damage and intracellular ROS production were observed upon treatment with AgNPs (10μg/mL) and PDT (0.5 mJ/cm2) showed significant reducing cell migration, expression of Bax and suppression of Bcl-2. Significantly, biosynthesized AgNPs showed a broad-spectrum anti-cancer activity with PDT therapy and therefore represent promoting ROS generation by modulating mitochondrial apoptosis induction in MCF7 breast cancer cells. [ABSTRACT FROM AUTHOR]

Hyperthermia (HT) has shown feasibility and potency as an anticancer therapy. Administration of HT in the chemotherapy has previously enhanced the cytotoxicity of drugs against pancreatic cancer. However, the drugs used when conducting these studies are substantially conventional chemotherapeutic agents that may cause unwanted side effects. Additionally, the thermal dosage in the treatment of cancer cells could also probably harm the healthy cells. The purpose of this work was to investigate the potential of the two natural polyphenolic compounds, epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), as heat synergizers in the thermal treatment of the PANC-1 cells. Furthermore, we have introduced a unique strategy entitled the thermal cycling-hyperthermia (TC-HT) that is capable of providing a maximum synergy and minimal side effect with the anticancer compounds. Our results demonstrate that the combination of the TC-HT and the CGA or EGCG markedly exerts the anticancer effect against the PANC-1 cells, while none of the single treatment induced such changes. The synergistic activity was attributed to the cell cycle arrest at the G2/M phase and the induction of the ROS-dependent mitochondria-mediated apoptosis. These findings not only represent the first in vitro thermal synergistic study of natural compounds in the treatment of pancreatic cancer, but also highlight the potential of the TC-HT as an alternative strategy in thermal treatment. [ABSTRACT FROM AUTHOR]

Mutations in or dys-regulation of the TDP-43 gene have been associated with TDP-43 proteinopathy, a spectrum of neurodegenerative diseases including Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS). The underlying molecular and cellular defects, however, remain unclear. Here, we report a systematic study combining analyses of patient brain samples with cellular and animal models for TDP-43 proteinopathy. Electron microscopy (EM) analyses of patient samples revealed prominent mitochondrial impairment, including abnormal cristae and a loss of cristae; these ultrastructural changes were consistently observed in both cellular and animal models of TDP-43 proteinopathy. In these models, increased TDP-43 expression induced mitochondrial dysfunction, including decreased mitochondrial membrane potential and elevated production of reactive oxygen species (ROS). TDP-43 expression suppressed mitochondrial complex I activity and reduced mitochondrial ATP synthesis. Importantly, TDP-43 activated the mitochondrial unfolded protein response (UPRmt) in both cellular and animal models. Down-regulating mitochondrial protease LonP1 increased mitochondrial TDP-43 levels and exacerbated TDP-43-induced mitochondrial damage as well as neurodegeneration. Together, our results demonstrate that TDP-43 induced mitochondrial impairment is a critical aspect in TDP-43 proteinopathy. Our work has not only uncovered a previously unknown role of LonP1 in regulating mitochondrial TDP-43 levels, but also advanced our understanding of the pathogenic mechanisms for TDP-43 proteinopathy. Our study suggests that blocking or reversing mitochondrial damage may provide a potential therapeutic approach to these devastating diseases. [ABSTRACT FROM AUTHOR]

In this study, the prebiotic potential of soluble dietary fibre extracted from plantain inflorescence (PIF) was investigated. PIF demonstrated prebiotic potential by enhancing the growth of the probiotics under study and thereby hindered colon cancer development. The soluble dietary fibre from Musa paradisiaca inflorescence (PIF) was fermented using Lactobacillus casei and Bifidobacterium bifidum. The fermentation supernatants (LS and BS) were enriched with short chain fatty acids (SCFA) and were able to initiate apoptotic signalling in HT29 colon cancer cells leading to cell death. Both BS and LS exhibited cytotoxic effect; induced DNA damage and enhanced generation of reactive oxygen species in HT29 cells leading to apoptosis. The induction of apoptosis was facilitated by the reduction of membrane potential of mitochondria and ATP synthesis; enhanced delivery of cytochrome c and interference with the expression of pro/antiapoptotic proteins. BS, which exhibited better activity, was further analysed for the identification of differentially regulated proteins by performing two dimensional electrophoresis and MALDI-TOF mass spectrometry. Results emphasized on the fact that, the exposure to BSalteredthe HT29 proteins expression, particularly the upregulation of apoptosis- inducing factor-AIFM1 leading to apoptosis of HT29 cells. [ABSTRACT FROM AUTHOR]

The introduction and establishment of fall armyworm (Spodoptera frugiperda) in Africa presents a major threat to agriculture in that continent and potentially to the entire Eastern Hemisphere. The species is subdivided into two subpopulations called the R-strain and C-strain that differ in their distribution on different plant hosts. This means that the scope of the economic risk posed by invasive fall armyworm is influenced by whether one or both strains are present. Multiple studies have found mitochondrial markers diagnostic of the two strains throughout Africa but there is substantial disagreement with a nuclear strain marker that makes conclusions about strain composition uncertain. In this study the issue of whether both strains are present in Africa was tested by an assay that can detect strain-biased mating behaviors. Western Hemisphere fall armyworm consistently showed evidence of strain-specific assortative mating in the field that was not found in surveys from multiple locations in Africa. The absence of strain mating biases and the disagreements between the strain diagnostic genetic markers indicates that the R-strain is rare (<1% of the population) or absent in Africa. Instead, it appears that the African fall armyworm populations are dominated by two groups, the C-strain and the descendants of interstrain hybrids. These results suggest that plant hosts associated with the R-strain may not be at high risk of fall armyworm infestation in Africa. [ABSTRACT FROM AUTHOR]

Background: Some case-control studies reported that mitochondrial haplogroups could be associated with the onset of cardiovascular diseases (CVD), but the literature regarding this topic is limited. We aimed to investigate whether any mitochondrial haplogroup carried a higher or lower risk of CVD in a large cohort of North American people affected by knee osteoarthritis or at high risk for this condition. Materials and methods: A longitudinal cohort study including individuals from the Osteoarthritis Initiative was done. Haplogroups were assigned through a combination of sequencing and PCR-RFLP techniques. All the mitochondrial haplogroups have been named following this nomenclature: HV, JT, UK, IWX, and superHV/others. The strength of the association between mitochondrial haplogroups and incident CVD was evaluated through a Cox’s regression analysis, adjusted for potential confounders, and reported as hazard ratios (HRs) with their 95% confidence intervals (CIs). Results: Overall, 3,288 Caucasian participants (56.8% women) with a mean age of 61.3±9.2 years without CVD at baseline were included. During a median follow-up of 8 years, 322 individuals (= 9.8% of baseline population) developed a CVD. After adjusting for 11 potential confounders at baseline and taking those with the HV haplotype as reference (the most frequent), those with JT carried a significant lower risk of CVD (HR = 0.75; 95%CI: 0.54–0.96; p = 0.03). Participants with the J haplogroup had the lowest risk of CVD (HR = 0.71; 95%CI: 0.46–0.95; p = 0.02). Conclusions: The presence of JT haplogroups (particularly J) may be associated with a reduced risk of CVD. However, this result was not based on a high level of statistical significance. Thus, future research with larger sample size is needed to assess whether our results can be corroborated. [ABSTRACT FROM AUTHOR]

Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated “mutation negative” probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA–and by implication rI production—correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA. [ABSTRACT FROM AUTHOR]

Accumulation of mutations in mitochondrial DNA (mtDNA) is thought to be responsible for mitochondrial, and other, diseases and biological phenomena, such as diabetes, cancer, neurodegenerative diseases, and aging. Mouse models may elucidate the relationship between mutations in mtDNA and these abnormalities. However, because of the difficulty of mtDNA manipulation, generation of mouse models has not sufficiently progressed to enable such studies. To overcome this difficulty and to establish a source of diverse mtDNA mutations, we here generated cultured mouse cells containing mtDNA derived from an mtDNA mutator mouse that accumulates random mtDNA mutations with age. Mutation analysis of the obtained transmitochondrial cytoplasmic hybrid cells (cybrids) revealed that the cells harbored diverse mtDNA mutations occurring at a higher frequency than in mouse tissues, and exhibited severe respiration defects that would be lethal in tissues or organs. Abnormal respiratory complex formation and high stress on the mitochondrial protein quality control system appeared to be involved in these severe respiration defects. The mutation rates of the majority of highly accumulated mutations converged to either approximately 5%, 10%, or 40%, suggesting that these mutations are linked on the respective mtDNA molecules, and mtDNA in cybrid cells likely consisted of mtDNA molecules clonally expanded from the small population of introduced mtDNAs. Thus, the linked mutations in these cybrid cells cannot be evaluated individually. In addition, mtDNA mutations homologous to confirmed pathogenic mutations in human were rarely observed in our generated cybrids. However, the transmitochondrial cybrids constitute a useful tool for concentrating pathogenic mtDNA mutations and as a source of diverse mtDNA mutations to elucidate the relationship between mtDNA mutations and diseases. [ABSTRACT FROM AUTHOR]

The only way to visually observe cellular viscosity, which can greatly influence biological reactions and has been linked to several human diseases, is through viscosity imaging. Imaging cellular viscosity has allowed the mapping of viscosity in cells, and the next frontier is targeted viscosity imaging of organelles and their microenvironments. Here we present a fluorescent molecular rotor/FLIM framework to image both organellar viscosity and membrane fluidity, using a combination of chemical targeting and organelle extraction. For demonstration, we image matrix viscosity and membrane fluidity of mitochondria, which have been linked to human diseases, including Alzheimer’s Disease and Leigh’s syndrome. We find that both are highly dynamic and responsive to small environmental and physiological changes, even under non-pathological conditions. This shows that neither viscosity nor fluidity can be assumed to be fixed and underlines the need for single-cell, and now even single-organelle, imaging. [ABSTRACT FROM AUTHOR]

Hybridization capture with in-solution oligonucleotide probes has quickly become the preferred method for enriching specific DNA loci from degraded or ancient samples prior to high-throughput sequencing (HTS). Several companies synthesize sets of probes for in-solution hybridization capture, but these commercial reagents are usually expensive. Methods for economical in-house probe synthesis have been described, but they do not directly address one of the major advantages of commercially synthesised probes: that probe sequences matching many species can be synthesised in parallel and pooled. The ability to make “phylogenetically diverse” probes increases the cost-effectiveness of commercial probe sets, as they can be used across multiple projects (or for projects involving multiple species). However, it is labour-intensive to replicate this with in-house methods, as template molecules must first be generated for each species of interest. While it has been observed that probes can be used to enrich for phylogenetically distant targets, the ability of this effect to compensate for the lack of phylogenetically diverse probes in in-house synthesised probe sets has not been tested. In this study, we present a refined protocol for in-house RNA probe synthesis and evaluated the ability of probes generated using this method from a single species to successfully enrich for the target locus in phylogenetically distant species. We demonstrated that probes synthesized using long-range PCR products from a placental mammal mitochondrion (Bison spp.) could be used to enrich for mitochondrial DNA in birds and marsupials (but not plants). Importantly, our results were obtained for approximately a third of the cost of similar commercially available reagents. [ABSTRACT FROM AUTHOR]

Numerous plant viruses that cause significant agricultural problems are persistently transmitted by insect vectors. We wanted to see if apoptosis was involved in viral infection process in the vector. We found that a plant reovirus (rice gall dwarf virus, RGDV) induced typical apoptotic response during viral replication in the leafhopper vector and cultured vector cells, as demonstrated by mitochondrial degeneration and membrane potential decrease. Fibrillar structures formed by nonstructural protein Pns11 of RGDV targeted the outer membrane of mitochondria, likely by interaction with an apoptosis-related mitochondrial protein in virus-infected leafhopper cells or nonvector insect cells. Such association of virus-induced fibrillar structures with mitochondria clearly led to mitochondrial degeneration and membrane potential decrease, suggesting that RGDV Pns11 was the inducer of apoptotic response in insect vectors. A caspase inhibitor treatment and knockdown of caspase gene expression using RNA interference each reduced apoptosis and viral accumulation, while the knockdown of gene expression for the inhibitor of apoptosis protein improved apoptosis and viral accumulation. Thus, RGDV exploited caspase-dependent apoptotic response to promote viral infection in insect vectors. For the first time, we directly confirmed that a nonstructural protein encoded by a persistent plant virus can induce the typical apoptotic response to benefit viral transmission by insect vectors. [ABSTRACT FROM AUTHOR]

Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages. [ABSTRACT FROM AUTHOR]

Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3′-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria. [ABSTRACT FROM AUTHOR]

Cognitive decline is a common occurrence of the natural aging process in animals and studying age-related changes in gene expression in the brain might shed light on disrupted molecular pathways that play a role in this decline. The fruit fly is a useful neurobiological model for studying aging due to its short generational time and relatively small brain size. We investigated age-dependent changes in the Drosophila melanogaster whole-brain transcriptome by comparing 5-, 20-, 30- and 40-day-old flies of both sexes. We used RNA-Sequencing of dissected brain samples followed by differential expression, temporal clustering, co-expression network and gene ontology enrichment analyses. We found an overall decline in expression of genes from the mitochondrial oxidative phosphorylation pathway that occurred as part of aging. We also detected, in females, a pattern of continuously declining expression for many neuronal function genes, which was unexpectedly reversed later in life. This group of genes was highly enriched in memory-impairing genes previously identified through an RNAi screen. We also identified deficits in short-term olfactory memory performance in older flies of both sexes, some of which matched the timing of certain changes in the brain transcriptome. Our study provides the first transcriptome profile of aging brains from fruit flies of both sexes, and it will serve as an important resource for those who study aging and cognitive decline in this model. [ABSTRACT FROM AUTHOR]

Genetic characterization of southern African cichlids has not received much attention. Here, we describe the mitogenome sequences and phylogenetic positioning of Oreochromis andersonii and O. macrochir among the African cichlids. The complete mitochondrial DNA sequences were determined for O. andersonii and O. macrochir, two important aquaculture and fisheries species endemic to southern Africa. The complete mitogenome sequence lengths were 16642 bp and 16644 bp for O. andersonii and O. macrochir respectively. The general structural organization follows that of other teleost species with 13 protein–coding genes, 2 rRNAs, 22 tRNAs and a non-coding control region. Phylogenetic placement of the two species among other African cichlids was performed using Maximum Likelihood (ML) and Bayesian Markov-Chain-Monte-Carlo (MCMC). The consensus trees confirmed the relative positions of the two cichlid species with O. andersonii being very closely related to O. mossambicus and O. macrochir showing a close relation to both species. Among the 13 mitochondrial DNA protein coding genes ND6 may have evolved more rapidly and COIII was the most conserved. There are signs that ND6 may have been subjected to positive selection in order for these cichlid lineages to diversify and adapt to new environments. More work is needed to characterize the southern Africa cichlids as they are important species for capture fisheries, aquaculture development and understanding biogeographic history of African cichlids. Bio-conservation of some endangered cichlids is also essential due to the threat by invasive species. [ABSTRACT FROM AUTHOR]

Inactin is a long lasting anesthetic agent commonly used in rat studies, but is also shown to exert physiological effects such as reducing renal blood flow, glomerular filtration rate and depressing tubular transport capacity. The effect of inactin on isolated kidney mitochondria is unknown and may be important when studying related topics in anaesthetized animals. The aim of this study was to determine whether inactin exerts effects on mitochondrial function and production of reactive oxygen species. Kidney mitochondrial function and production of reactive oxygen after acutely (5 min) or longer (1.5 hour) anesthetizing rats with inactin was evaluated using high-resolution respirometry. The results demonstrate that inactin significantly improves respiratory control ratio, inhibits complex I in the mitochondrial respiratory chain, reduce both unregulated proton leak and time dependently reduce the regulated proton leak via uncoupling protein-2 and adenine nucleotide translocase. Inactin also contributes to increased mitochondrial hydrogen peroxide production. In conclusion, inactin exerts persistent effects on mitochondrial function and these profound effects on mitochondrial function should to be considered when studying mitochondria isolated from animals anesthesized with inactin. [ABSTRACT FROM AUTHOR]

Background: Mitochondria are energy-producing structure of the cell and help to maintain redox environment. In cardiovascular disease, the number of mitochondrial DNA (mtDNA) will changes accordingly compare to normal condition. Some investigators ask whether it has a clear association between mtDNA and cardiovascular disease with its adverse events. Thus, we conduct the meta-analysis to assess the role of circulating mtDNA in evaluating cardiovascular disease. Methods: The meta-analysis was conducted in accordance with a predetermined protocol following the recommendations of Cochrane Handbook of Systematic Reviews. We searched the Pubmed, Embase, the Cochrane Central Register of Controlled Trials and World Health Organization clinical trials registry center to identify relevant studies up to the end of October 2017. Data were analyzed using STATA. Besides, publication bias and meta-regression analysis were also conducted. Results: We collected results from 5 articles for further analyses with 8,252 cases and 20,904 control. The normalized mtDNA copy number level is lower in cardiovascular disease (CVD) than the control groups with a pooled standard mean difference (SMD) of -0.36(95%CI,-0.65 to -0.08); The pooled odds ratio (OR) for CVD proportion associated with a 1-SD (standard deviation) decrease in mtDNA copy number level is 1.23 (95% CI,1.06–1.42); The OR for CVD patients with mtDNA copy number lower than median level is 1.88(95% CI,1.65–2.13); The OR for CVD patients with mtDNA copy number located in the lowest quartile part is 2.15(95% CI, 1.46–3.18); the OR between mtDNA copy number and the risk of sudden cardiac death (SCD) is 1.83(95% CI, 1.22–2.74). Conclusion: Although inter-study variability, the overall performance test of mtDNA for evaluating CVD and SCD revealed that the mtDNA copy number presented the potential to be a biomarker for CVD and SCD prediction. Given that, the fewer copies of mtDNA, the higher the risk of CVD. [ABSTRACT FROM AUTHOR]

Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. All these interactions have the potential to alter the frequencies of mtDNA haplotypes (mitotypes) in nature and may impact human health. In a model laboratory system, we fed four diets varying in Protein: Carbohydrate (P:C) ratio (1:2, 1:4, 1:8 and 1:16 P:C) to four homoplasmic Drosophila melanogaster mitotypes (nuclear genome standardised) and assayed their frequency in population cages. When fed a high protein 1:2 P:C diet, the frequency of flies harbouring Alstonville mtDNA increased. In contrast, when fed the high carbohydrate 1:16 P:C food the incidence of flies harbouring Dahomey mtDNA increased. This result, driven by differences in larval development, was generalisable to the replacement of the laboratory diet with fruits having high and low P:C ratios, perturbation of the nuclear genome and changes to the microbiome. Structural modelling and cellular assays suggested a V161L mutation in the ND4 subunit of complex I of Dahomey mtDNA was mildly deleterious, reduced mitochondrial functions, increased oxidative stress and resulted in an increase in larval development time on the 1:2 P:C diet. The 1:16 P:C diet triggered a cascade of changes in both mitotypes. In Dahomey larvae, increased feeding fuelled increased β-oxidation and the partial bypass of the complex I mutation. Conversely, Alstonville larvae upregulated genes involved with oxidative phosphorylation, increased glycogen metabolism and they were more physically active. We hypothesise that the increased physical activity diverted energy from growth and cell division and thereby slowed development. These data further question the use of mtDNA as an assumed neutral marker in evolutionary and population genetic studies. Moreover, if humans respond similarly, we posit that individuals with specific mtDNA variations may differentially metabolise carbohydrates, which has implications for a variety of diseases including cardiovascular disease, obesity, and perhaps Parkinson’s Disease. [ABSTRACT FROM AUTHOR]

Obesity and insulin resistance are primary risk factors for Non-Alcoholic Fatty Liver Disease (NAFLD). NAFLD is generally exhibited by non-progressive simple steatosis. However, a significant subset of patient’s progress to nonalcoholic steatohepatitis (NASH) that is defined by the presence of steatosis, inflammation and hepatocyte injury with fibrosis. Unfortunately, there are no approved therapies for NAFLD or NASH and therefore therapeutic approaches are urgently needed. Niclosamide is an U.S. Food and Drug Administration (FDA)-approved anthelmintic drug that mediates its effect by uncoupling oxidative phosphorylation. Niclosamide and its salt forms, Niclosamide Ethanolamine (NEN), and Niclosamide Piperazine (NPP) have shown efficacy in murine models of diet induced obesity characterized by attenuation of the prominent fatty liver disease phenotype and improved glucose metabolism. While the exact mechanism(s) underlying these changes remains unclear, the ability to uncouple oxidative phosphorylation leading to increased energy expenditure and lipid metabolism or attenuation of PKA mediated glucagon signaling in the liver have been proposed. Unfortunately, niclosamide has very poor water solubility, leading to low oral bioavailability. This, in addition to mitochondrial uncoupling activity and potential genotoxicity have reduced enthusiasm for its clinical use. More recently, salt forms of niclosamide, NEN and NPP, have demonstrated improved oral bioavailability while retaining activity. This suggests that development of safer more effective niclosamide derivatives for the treatment of NAFLD and Type 2 Diabetes may be possible. Herein we explored the ability of a series of N-substituted phenylbenzamide derivatives of the niclosamide salicylanilide chemotype to attenuate hepatic steatosis using a novel phenotypic in vitro model of fatty liver and the high fat diet-fed mouse model of diet induced obesity. These studies identified novel compounds with improved pre-clinical properties that attenuate hepatic steatosis in vitro and in vivo. These compounds with improved drug properties may be useful in alleviating symptoms and protection against disease progression in patients with metabolic syndrome and NAFLD. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA depletion syndromes (MDDS) are a genetically and clinically heterogeneous group of human diseases caused by mutations in nuclear genes and characterized by a severe reduction in mitochondrial DNA (mtDNA) copy number leading to impaired energy production in affected tissues and organs. Mutations in the MPV17 gene, whose role is still elusive, were described as cause of the hepatocerebral form of MDDS and Navajo neuro-hepathopathy. The high degree of conservation observed between MPV17 and its yeast homolog SYM1 made the latter a good model for the study of the pathology. Here, we used Saccharomyces cerevisiae to elucidate the molecular consequences of seven MPV17 missense mutations identified in patients and localized in different protein domains. The phenotypic analysis of the appropriate sym1 mutant strains created demonstrated deleterious effect for all mutations regarding OXPHOS metabolism and mtDNA stability. We deepened the pathogenic effect of the mutations by investigating whether they prevented the correct protein localization into the mitochondria or affected the stability of the proteins. All the Sym1 mutant proteins correctly localized into the mitochondria and only one mutation predominantly affects protein stability. All the other mutations compromised the formation of the high molecular weight complex of unknown composition, previously identified both in yeast, cell cultures and mouse tissues, as demonstrated by the consistent fraction of the Sym1 mutant proteins found free or in not fully assembled complex, strengthening its role as protein forming part of a high molecular weight complex. [ABSTRACT FROM AUTHOR]