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1.  The Respiratory Chain Supercomplex Organization Is Independent of COX7a2l Isoforms 
Cell Metabolism  2014;20(6):1069-1075.
The organization of individual respiratory chain complexes into supercomplexes or respirasomes has attracted great interest because of the implications for cellular energy conversion. Recently, it was reported that commonly used mouse strains harbor a short COX7a2l (SCAFI) gene isoform that supposedly precludes the formation of complex IV-containing supercomplexes. This claim potentially has serious implications for numerous mouse studies addressing important topics in metabolism, including adaptation to space flights. Using several complementary experimental approaches, we show that mice with the short COX7a2l isoform have normal biogenesis and steady-state levels of complex IV-containing supercomplexes and consequently have normal respiratory chain function. Furthermore, we use a mouse knockout of Lrpprc and show that loss of complex IV compromises respirasome formation. We conclude that the presence of the short COX7a2l isoform in the commonly used C57BL/6 mouse strains does not prevent their use in metabolism research.
Graphical Abstract
•C57BL/6J and C57BL/6N mouse strains contain a short COX7a2l gene isoform•The short COX7a2l isoform does not impair respiratory chain function or oxidative capacity•Complex IV-containing supercomplexes exist in mice with the short COX7a2l isoform•The biogenesis of respirasomes is normal in mice with the short COX7a2l isoform
In contrast to a recent report, Mourier et al. show that the presence of the short COX7a2l isoform in the commonly used C57BL/6 mouse strains does not affect the supramolecular organization and function of the mitochondrial respiratory chain.
PMCID: PMC4261080  PMID: 25470551
2.  Drosophila melanogaster LRPPRC2 is involved in coordination of mitochondrial translation 
Nucleic Acids Research  2014;42(22):13920-13938.
Members of the pentatricopeptide repeat domain (PPR) protein family bind RNA and are important for post-transcriptional control of organelle gene expression in unicellular eukaryotes, metazoans and plants. They also have a role in human pathology, as mutations in the leucine-rich PPR-containing (LRPPRC) gene cause severe neurodegeneration. We have previously shown that the mammalian LRPPRC protein and its Drosophila melanogaster homolog DmLRPPRC1 (also known as bicoid stability factor) are necessary for mitochondrial translation by controlling stability and polyadenylation of mRNAs. We here report characterization of DmLRPPRC2, a second fruit fly homolog of LRPPRC, and show that it has a predominant mitochondrial localization and interacts with a stem-loop interacting RNA binding protein (DmSLIRP2). Ubiquitous downregulation of DmLrpprc2 expression causes respiratory chain dysfunction, developmental delay and shortened lifespan. Unexpectedly, decreased DmLRPPRC2 expression does not globally affect steady-state levels or polyadenylation of mitochondrial transcripts. However, some mitochondrial transcripts abnormally associate with the mitochondrial ribosomes and some products are dramatically overproduced and other ones decreased, which, in turn, results in severe deficiency of respiratory chain complexes. The function of DmLRPPRC2 thus seems to be to ensure that mitochondrial transcripts are presented to the mitochondrial ribosomes in an orderly fashion to avoid poorly coordinated translation.
PMCID: PMC4267620  PMID: 25428350
3.  Loss of UCP2 Attenuates Mitochondrial Dysfunction without Altering ROS Production and Uncoupling Activity 
PLoS Genetics  2014;10(6):e1004385.
Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.
Author Summary
Mitochondria produce the majority of the energy needed for numerous cell functions through oxidative phosphorylation. However, this comes with the cost in the form of potentially harmful reactive oxygen species (ROS) that could damage all kinds of biological macromolecules. Changes in mitochondrial membrane potential through mild uncoupling could alter ROS production in the cell (“uncoupling to survive”). Mitochondrial uncoupling proteins (UCPs) are believed to play a central role in this process. We detected increased amounts of UCP2 in mtDNA mutator mice, a model for premature aging. Depletion of UCP2 in mtDNA mutator mice led to further shortening of the lifespan with earlier signs of mitochondrial cardiomyopathy accompanied with high systemic lactic acidosis, often used as a marker of mitochondrial diseases. Remarkably, our results demonstrate that the presence of UCP2 wields beneficial effect on respiratory deficient mitochondria without affecting ROS production or uncoupling. Instead, UCP2 protein seems to mediate a valuable upregulation of fatty acid metabolism detected in mtDNA mutator hearts. Our results provide a novel mechanism of adaptation of mitochondria to respiratory deficiency mediated by UCP2 that clearly argues against the “uncoupling to survive” theory.
PMCID: PMC4063685  PMID: 24945157
4.  Characterization of a Novel Conus bandanus Conopeptide Belonging to the M-Superfamily Containing Bromotryptophan 
Marine Drugs  2014;12(6):3449-3465.
A novel conotoxin (conopeptide) was biochemically characterized from the crude venom of the molluscivorous marine snail, Conus bandanus (Hwass in Bruguière, 1792), collected in the south-central coast of Vietnam. The peptide was identified by screening bromotryptophan from chromatographic fractions of the crude venom. Tandem mass spectrometry techniques were used to detect and localize different post-translational modifications (PTMs) present in the BnIIID conopeptide. The sequence was confirmed by Edman’s degradation and mass spectrometry revealing that the purified BnIIID conopeptide had 15 amino acid residues, with six cysteines at positions 1, 2, 7, 11, 13, and 14, and three PTMs: bromotryptophan, γ-carboxy glutamate, and amidated aspartic acid, at positions “4”, “5”, and “15”, respectively. The BnIIID peptide was synthesized for comparison with the native peptide. Homology comparison with conopeptides having the III-cysteine framework (–CCx1x2x3x4Cx1x2x3Cx1CC–) revealed that BnIIID belongs to the M-1 family of conotoxins. This is the first report of a member of the M-superfamily containing bromotryptophan as PTM.
PMCID: PMC4071585  PMID: 24905483
Conus bandanus; cone snail venom; mass spectrometry; BnIIID; bromotryptophan; γ-carboxy glutamate; post-translational modifications
5.  Transposable elements in cancer as a by-product of stress-induced evolvability 
Frontiers in Genetics  2014;5:156.
Transposable elements (TEs) are ubiquitous in eukaryotic genomes. Barbara McClintock’s famous notion of TEs acting as controlling elements modifying the genetic response of an organism upon exposure to stressful environments has since been solidly supported in a series of model organisms. This requires the TE activity response to possess an element of specificity and be targeted toward certain parts of the genome. We propose that a similar TE response is present in human cells, and that this stress response may drive the onset of human cancers. As such, TE-driven cancers may be viewed as an evolutionary by-product of organisms’ abilities to genetically adapt to environmental stress.
PMCID: PMC4038923  PMID: 24910642
transposable elements; stress response; evolution; evolvability; cancer
7.  Loss of LRPPRC causes ATP synthase deficiency 
Human Molecular Genetics  2014;23(10):2580-2592.
Defects of the oxidative phosphorylation system, in particular of cytochrome-c oxidase (COX, respiratory chain complex IV), are common causes of Leigh syndrome (LS), which is a rare neurodegenerative disorder with severe progressive neurological symptoms that usually present during infancy or early childhood. The COX-deficient form of LS is commonly caused by mutations in genes encoding COX assembly factors, e.g. SURF1, SCO1, SCO2 or COX10. However, other mutations affecting genes that encode proteins not directly involved in COX assembly can also cause LS. The leucine-rich pentatricopeptide repeat containing protein (LRPPRC) regulates mRNA stability, polyadenylation and coordinates mitochondrial translation. In humans, mutations in Lrpprc cause the French Canadian type of LS. Despite the finding that LRPPRC deficiency affects the stability of most mitochondrial mRNAs, its pathophysiological effect has mainly been attributed to COX deficiency. Surprisingly, we show here that the impaired mitochondrial respiration and reduced ATP production observed in Lrpprc conditional knockout mouse hearts is caused by an ATP synthase deficiency. Furthermore, the appearance of inactive subassembled ATP synthase complexes causes hyperpolarization and increases mitochondrial reactive oxygen species production. Our findings shed important new light on the bioenergetic consequences of the loss of LRPPRC in cardiac mitochondria.
PMCID: PMC3990160  PMID: 24399447
8.  Adipose-Specific Deletion of TFAM Increases Mitochondrial Oxidation and Protects Mice against Obesity and Insulin Resistance 
Cell metabolism  2012;16(6):765-776.
Obesity and type 2 diabetes are associated with mitochondrial dysfunction in adipose tissue, but the role for adipose tissues mitochondria in the development of these disorders is currently unknown. To understand the impact of adipose tissue mitochondria on whole body metabolism, we have generated a mouse model with disruption of the mitochondrial transcription factor A (TFAM) specifically in fat. F-TFKO adipose tissue exhibit decreased mtDNA copy number, altered levels of proteins of the electron transport chain, and perturbed mitochondrial function with decreased Complex I activity and greater oxygen consumption and uncoupling. As a result, F-TFKO mice exhibit higher energy expenditure and are protected from age- and diet-induced obesity, insulin resistance and hepatosteatosis, despite a greater food intake. Thus, TFAM deletion in the adipose tissue increases mitochondrial oxidation that has positive metabolic effects suggesting that regulation of adipose tissue mitochondria may be a potential therapeutic target for the treatment of obesity.
PMCID: PMC3529641  PMID: 23168219
Obesity; Brown adipose tissue; Mitochondrial function; mitochondrial bioenergetics; White adipose tissue; Insulin resistance; Diabetes
9.  Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing 
Scientific Reports  2013;3:2468.
Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent “primer walking” combined with Sanger sequencing validated the RS platform's utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only completely sequenced HPIV4a subtype. However, it also exhibits various structural features present in one of the HPIV4b reference strains, opening questions regarding their lifecycle and evolutionary relationships among these viruses. Clinical data from patients infected with the strain, as well as viral prevalence estimates using real-time PCR, is also described.
PMCID: PMC3760282  PMID: 24002378
10.  Genetic Network and Breeding Patterns of a Sicklefin Lemon Shark (Negaprion acutidens) Population in the Society Islands, French Polynesia 
PLoS ONE  2013;8(8):e73899.
Human pressures have put many top predator populations at risk of extinction. Recent years have seen alarming declines in sharks worldwide, while their resilience remains poorly understood. Studying the ecology of small populations of marine predators is a priority to better understand their ability to withstand anthropogenic and environmental stressors. In the present study, we monitored a naturally small island population of 40 adult sicklefin lemon sharks in Moorea, French Polynesia over 5 years. We reconstructed the genetic relationships among individuals and determined the population’s mating system. The genetic network illustrates that all individuals, except one, are interconnected at least through one first order genetic relationship. While this species developed a clear inbreeding avoidance strategy involving dispersal and migration, the small population size, low number of breeders, and the fragmented environment characterizing these tropical islands, limits its complete effectiveness.
PMCID: PMC3742621  PMID: 23967354
11.  Sex and Breed-Dependent Organ Development and Metabolic Responses in Foetuses from Lean and Obese/Leptin Resistant Swine 
PLoS ONE  2013;8(7):e66728.
The present study aimed to determine the effects of breed and sex on growth patterns and metabolic features of advanced-pregnancy foetuses exposed to the same environmental conditions. Thus, at Day 62 of pregnancy, swine foetuses from an obese breed with leptin resistance (Iberian breed) were compared to lean crossbred foetuses (25% Large White ×25% Landrace ×50% Pietrain). There were differential developmental patterns in foetuses with leptin resistance, mainly a higher relative weight of the brain resembling “brain-sparing effect”. Prioritization of brain growth may be protective for the adequate growth and postnatal survival of the Iberian individuals, an ancient breed reared in extensive semi-feral conditions for centuries. There were also clear sex-related differences in foetal development and metabolism in the Iberian breed. Female Iberian foetuses were similar in size and weight to male littermates but had a significantly higher relative liver to body weight ratio resembling “liver-sparing effect” and a trend for a higher relative intestine to body ratio. Moreover, the availability of triglycerides, cholesterol and IL-6 in female Iberian foetuses was similar to that of lean crossbred foetuses. Overall, these features may favour a better postnatal survival and development of females, the sex more critical for the species survival. These findings set the basis for future translational studies aimed at increasing the knowledge on the interaction between genetic and environmental factors in the early programming of the adult phenotype.
PMCID: PMC3720837  PMID: 23935823
12.  Variation in germ line mtDNA heteroplasmy is determined prenatally but modified during subsequent transmission 
Nature genetics  2012;44(11):1282-1285.
A genetic bottleneck explains the marked changes in mitochondrial DNA (mtDNA) heteroplasmy observed during the transmission of pathogenic mutations, but the precise timing remains controversial, and it is not clear whether selection plays a role. These issues are critically important for the genetic counseling of prospective mothers, and developing treatments aimed at disease prevention. By studying mice transmitting a heteroplasmic single base-pair deletion in the mitochondrial tRNAMet gene, we show that mammalian mtDNA heteroplasmy levels are principally determined prenatally within the developing female germ line. Although we saw no evidence of mtDNA selection prenatally, skewed heteroplasmy levels were observed in the offspring of the next generation, consistent with purifying selection. High percentage levels of the tRNAMet mutation were linked to a compensatory increase in overall mitochondrial RNAs, ameliorating the biochemical phenotype, and explaining why fecundity is not compromised.
PMCID: PMC3492742  PMID: 23042113
13.  Microsatellite Analyses of Blacktip Reef Sharks (Carcharhinus melanopterus) in a Fragmented Environment Show Structured Clusters 
PLoS ONE  2013;8(4):e61067.
The population dynamics of shark species are generally poorly described because highly mobile marine life is challenging to investigate. Here we investigate the genetic population structure of the blacktip reef shark (Carcharhinus melanopterus) in French Polynesia. Five demes were sampled from five islands with different inter-island distances (50–1500 km). Whether dispersal occurs between islands frequently enough to prevent moderate genetic structure is unknown. We used 11 microsatellites loci from 165 individuals and a strong genetic structure was found among demes with both F-statistics and Bayesian approaches. This differentiation is correlated with the geographic distance between islands. It is likely that the genetic structure seen is the result of all or some combination of the following: low gene flow, time since divergence, small effective population sizes, and the standard issues with the extent to which mutation models actually fit reality. We suggest low levels of gene flow as at least a partial explanation of the level of genetic structure seen among the sampled blacktip demes. This explanation is consistent with the ecological traits of blacktip reef sharks, and that the suitable habitat for blacktips in French Polynesia is highly fragmented. Evidence for spatial genetic structure of the blacktip demes we studied highlights that similar species may have populations with as yet undetected or underestimated structure. Shark biology and the market for their fins make them highly vulnerable and many species are in rapid decline. Our results add weight to the case that total bans on shark fishing are a better conservation approach for sharks than marine protected area networks.
PMCID: PMC3622011  PMID: 23585872
14.  Succinate Dehydrogenase Upregulation Destabilize Complex I and Limits the Lifespan of gas-1 Mutant 
PLoS ONE  2013;8(3):e59493.
Many Caenorhabditis elegans mutants with dysfunctional mitochondrial electron transport chain are surprisingly long lived. Both short-lived (gas-1(fc21)) and long-lived (nuo-6(qm200)) mutants of mitochondrial complex I have been identified. However, it is not clear what are the pathways determining the difference in longevity. We show that even in a short-lived gas-1(fc21) mutant, many longevity assurance pathways, shown to be important for lifespan prolongation in long-lived mutants, are active. Beside similar dependence on alternative metabolic pathways, short-lived gas-1(fc21) mutants and long-lived nuo-6(qm200) mutants also activate hypoxia-inducible factor –1α (HIF-1α) stress pathway and mitochondrial unfolded protein response (UPRmt). The major difference that we detected between mutants of different longevity, is in the massive loss of complex I accompanied by upregulation of complex II levels, only in short-lived, gas-1(fc21) mutant. We show that high levels of complex II negatively regulate longevity in gas-1(fc21) mutant by decreasing the stability of complex I. Furthermore, our results demonstrate that increase in complex I stability, improves mitochondrial function and decreases mitochondrial stress, putting it inside a “window” of mitochondrial dysfunction that allows lifespan prolongation.
PMCID: PMC3610896  PMID: 23555681
15.  MTERF3 Regulates Mitochondrial Ribosome Biogenesis in Invertebrates and Mammals 
PLoS Genetics  2013;9(1):e1003178.
Regulation of mitochondrial DNA (mtDNA) expression is critical for the control of oxidative phosphorylation in response to physiological demand, and this regulation is often impaired in disease and aging. We have previously shown that mitochondrial transcription termination factor 3 (MTERF3) is a key regulator that represses mtDNA transcription in the mouse, but its molecular mode of action has remained elusive. Based on the hypothesis that key regulatory mechanisms for mtDNA expression are conserved in metazoans, we analyzed Mterf3 knockout and knockdown flies. We demonstrate here that decreased expression of MTERF3 not only leads to activation of mtDNA transcription, but also impairs assembly of the large mitochondrial ribosomal subunit. This novel function of MTERF3 in mitochondrial ribosomal biogenesis is conserved in the mouse, thus we identify a novel and unexpected role for MTERF3 in coordinating the crosstalk between transcription and translation for the regulation of mammalian mtDNA gene expression.
Author Summary
One of the main functions of the mitochondrial network is to provide the energy currency ATP to drive a large array of cellular metabolic processes. The formation of the mitochondrial respiratory chain, which allows this energy supply, is under the control of two separate genetic systems, the nuclear and the mitochondrial genomes, whose expressions have to be tightly coordinated to ensure efficient mitochondrial function. The regulation of mitochondrial genome expression is still poorly understood despite the profound importance of this process in human physiology, disease, and aging. Here, we make one step forward by unraveling a new role for the mitochondrial transcription termination factor 3 (MTERF3), which was initially characterized as a factor able to decrease mitochondrial transcription. Using gene invalidation approaches, we show in two distinct model organisms, the fruit fly and the mouse, that MTERF3 is not only involved in mitochondrial transcription but also favors the assembly of the mitochondrial ribosome and thereby reinforces the coordination between transcription and translation events, two key steps in mitochondrial genome expression.
PMCID: PMC3536695  PMID: 23300484
16.  Probable Transmission of Coxsackie B3 Virus from Human to Chimpanzee, Denmark 
Emerging Infectious Diseases  2012;18(7):1163-1165.
In 2010, a chimpanzee died at Copenhagen Zoo following an outbreak of respiratory disease among chimpanzees in the zoo. Identification of coxsackie B3 virus, a common human pathogen, as the causative agent, and its severe manifestation, raise questions about pathogenicity and transmissibility among humans and other primates.
PMCID: PMC3376799  PMID: 22709557
viruses; coxsackie B3 virus; coxsackievirus; nonhuman primate; human; chimpanzee; myocarditis; Copenhagen Zoo; Denmark; transmission; zoonoses
17.  Engineering of Three-Finger Fold Toxins Creates Ligands with Original Pharmacological Profiles for Muscarinic and Adrenergic Receptors 
PLoS ONE  2012;7(6):e39166.
Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test “loop grafting,” a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α1A-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α1A-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles.
PMCID: PMC3375269  PMID: 22720062
18.  Comparative Genomics of the Apicomplexan Parasites Toxoplasma gondii and Neospora caninum: Coccidia Differing in Host Range and Transmission Strategy 
PLoS Pathogens  2012;8(3):e1002567.
Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species.
Author Summary
Coccidian parasites have a major impact on human and animal health world-wide and are among the most successful and widespread parasitic protozoa. They include Neospora caninum which is a leading cause of abortion in cattle and one of its nearest relatives, Toxoplasma gondii. Despite its close phylogenetic relationship to Toxoplasma, Neospora has a far more restricted host range, does not infect humans and its epidemiology depends predominantly on efficient vertical transmission. The divergent biology of these two closely related species provides a unique opportunity to study the mechanisms of host specificity, pathogenesis and zoonotic potential not only in these, but other Coccidia. We have sequenced the genome of Neospora and the transcriptomes of both species to show that despite diverging some 28 million years ago, both genome and gene expression remain remarkably conserved. Evolution has focused almost exclusively on molecules which control the interaction of the parasite with the host cell. We show that some secreted invasion-related proteins and surface genes which are known to control virulence and host cell interactions in Toxoplasma are dramatically altered in their expression and functionality in Neospora and propose that evolution of these genes may underpin the ecological niches inhabited by coccidian parasites.
PMCID: PMC3310773  PMID: 22457617
19.  A Tissue-Specific Approach to the Analysis of Metabolic Changes in Caenorhabditis elegans 
PLoS ONE  2011;6(12):e28417.
The majority of metabolic principles are evolutionarily conserved from nematodes to humans. Caenorhabditis elegans has widely accelerated the discovery of new genes important to maintain organismic metabolic homeostasis. Various methods exist to assess the metabolic state in worms, yet they often require large animal numbers and tend to be performed as bulk analyses of whole worm homogenates, thereby largely precluding a detailed studies of metabolic changes in specific worm tissues. Here, we have adapted well-established histochemical methods for the use on C. elegans fresh frozen sections and demonstrate their validity for analyses of morphological and metabolic changes on tissue level in wild type and various mutant strains. We show how the worm presents on hematoxylin and eosin (H&E) stained sections and demonstrate their usefulness in monitoring and the identification of morphological abnormalities. In addition, we demonstrate how Oil-Red-O staining on frozen worm cross-sections permits quantification of lipid storage, avoiding the artifact-prone fixation and permeabilization procedures of traditional whole-mount protocols. We also adjusted standard enzymatic stains for respiratory chain subunits (NADH, SDH, and COX) to monitor metabolic states of various C. elegans tissues. In summary, the protocols presented here provide technical guidance to obtain robust, reproducible and quantifiable tissue-specific data on worm morphology as well as carbohydrate, lipid and mitochondrial energy metabolism that cannot be obtained through traditional biochemical bulk analyses of worm homogenates. Furthermore, analysis of worm cross-sections overcomes the common problem with quantification in three-dimensional whole-mount specimens.
PMCID: PMC3230600  PMID: 22162770
20.  The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial mRNAs in Drosophila melanogaster 
PLoS Genetics  2011;7(10):e1002324.
The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation.
Author Summary
The majority of the cellular energy currency ATP is formed in a tubular network, termed mitochondria, present within virtually all eukaryotic cells. The mitochondria are unique among cellular organelles in that they contain their own genome, which encodes critical proteins necessary for cellular energy production. However, the vast majority of mitochondrial proteins are encoded in the nucleus and imported into mitochondria. Gene expression thus needs to be coordinated between the two genomes to ensure efficient mitochondrial function and sufficient adaptation to different physiological demands. The regulation of the mitochondrial genome is poorly understood, with many of the basic regulators not yet being characterized. We used RNAi in the fruit fly to study the in vivo function of the bicoid stability factor (BSF), previously thought to be a cytoplasmic and nuclear protein important for fly development. We show here that BSF is mainly localized to mitochondria, where it is essential for mtDNA gene expression, regulating the polyadenylation and maturation of specific mRNAs. Furthermore, BSF coordinates the translation and assembly of mitochondrial peptides in the inner mitochondrial membrane.
PMCID: PMC3192837  PMID: 22022283
21.  Retrotransposon-centered analysis of piRNA targeting shows a shift from active to passive retrotransposon transcription in developing mouse testes 
BMC Genomics  2011;12:440.
Piwi-associated RNAs (piRNAs) bind transcripts from retrotransposable elements (RTE) in mouse germline cells and seemingly act as guides for genomic methylation, thereby repressing the activity of RTEs. It is currently unknown if and how Piwi proteins distinguish RTE transcripts from other cellular RNAs. During germline development, the main target of piRNAs switch between different types of RTEs. Using the piRNA targeting of RTEs as an indicator of RTE activity, and considering the entire population of genomic RTE loci along with their age and location, this study aims at further elucidating the dynamics of RTE activity during mouse germline development.
Due to the inherent sequence redundancy between RTE loci, assigning piRNA targeting to specific loci is problematic. This limits the analysis, although certain features of piRNA targeting of RTE loci are apparent. As expected, young RTEs display a much higher level of piRNA targeting than old RTEs. Further, irrespective of age, RTE loci near protein-coding coding genes are targeted to a greater extent than RTE loci far from genes. During development, a shift in piRNA targeting is observed, with a clear increase in the relative piRNA targeting of RTEs residing within boundaries of protein-coding gene transcripts.
Reanalyzing published piRNA sequences and taking into account the features of individual RTE loci provide novel insight into the activity of RTEs during development. The obtained results are consistent with some degree of proportionality between what transcripts become substrates for Piwi protein complexes and the level by which the transcripts are present in the cell. A transition from active transcription of RTEs to passive co-transcription of RTE sequences residing within protein-coding transcripts appears to take place in postnatal development. Hence, the previously reported increase in piRNA targeting of SINEs in postnatal testis development does not necessitate widespread active transcription of SINEs, but may simply be explained by the prevalence of SINEs residing in introns.
PMCID: PMC3175481  PMID: 21884594
22.  Tracing the Trail of Protons through Complex I of the Mitochondrial Respiratory Chain 
PLoS Biology  2011;9(8):e1001129.
Mitochondria are the structures that produce the bulk part of the cellular energy currency ATP, which drives numerous energy requiring processes in the cell. This process involves a series of large enzyme complexes—the respiratory chain—that couples the transfer of electrons to the creation of a concentration gradient of protons across the inner mitochondrial membrane, which drives ATP synthesis. Complex I (or NADH-quinone oxidoreductase) is the largest and by far the most complicated of the respiratory chain enzyme complexes. The molecular mechanism whereby it couples electron transfer to proton extrusion has remained mysterious until very recently. Low-resolution X-ray structures of complex I have, surprisingly, suggested that electron transfer in the hydrophilic arm, protruding into the mitochondrial matrix, causes movement of a coupling rod that influences three putative proton pumps within the hydrophobic arm embedded in the inner mitochondrial membrane. In this Primer, we will briefly introduce the recent progress made in this area and highlight the road ahead that likely will unravel the detailed molecular mechanisms of complex I function.
PMCID: PMC3160332  PMID: 21886481
23.  CD25 Appears Non Essential for Human Peripheral Treg Maintenance In Vivo 
PLoS ONE  2010;5(7):e11784.
IL-2 has been reported to be critical for peripheral Treg survival in mouse models. Here, we examined Treg maintenance in a series of paediatric liver transplant recipients who received basiliximab, a therapeutic anti-CD25 monoclonal antibody.
Methodology/Principal Findings
FoxP3+ CD4 T cells were analyzed by flow cytometry before liver grafting and more than 9 months later. We found that in vivo CD25 blockade did not lead to Treg depletion: the proportion of FoxP3+ cells among CD4 T cells and the level of FoxP3 expression were both unchanged. IL-2Rβ expression was enhanced in FoxP3+ cells both before and after basiliximab treatment, while the level of IL-2Rγ expression was similar in Tregs and non-Tregs. No significant change in the weak or absent expression of IL-7Rα and IL-15Rα expression on FoxP3+ cells was observed. Although the proportion of FoxP3+ cells among CD4 T cells did not vary, food allergies occurred more rapidly after liver grafting in patients who received basiliximab, raising questions as to Treg functionality in vivo in the absence of functional CD25.
CD25 appears non essential for human Treg peripheral maintenance in vivo. However, our results raise questions as to Treg functionality after therapeutic CD25 targeting.
PMCID: PMC2912775  PMID: 20689592
24.  Large-scale transcriptome data reveals transcriptional activity of fission yeast LTR retrotransposons 
BMC Genomics  2010;11:167.
Retrotransposons are transposable elements that proliferate within eukaryotic genomes through a process involving reverse transcription. The numbers of retrotransposons within genomes and differences between closely related species may yield insight into the evolutionary history of the elements. Less is known about the ongoing dynamics of retrotransposons, as analysis of genome sequences will only reveal insertions of retrotransposons that are fixed - or near fixation - in the population or strain from which genetic material has been extracted for sequencing. One pre-requisite for retrotransposition is transcription of the elements. Given their intrinsic sequence redundancy, transcriptome-level analyses of transposable elements are scarce. We have used recently published transcriptome data from the fission yeast Schizosaccharomyces pombe to assess the ability to detect and describe transcriptional activity from Long Terminal Repeat (LTR) retrotransposons. LTR retrotransposons are normally flanked by two LTR sequences. However, the majority of LTR sequences in S. pombe exist as solitary LTRs, i.e. as single terminal repeat sequences not flanking a retrotransposon. Transcriptional activity was analysed for both full-length LTR retrotransposons and solitary LTRs.
Two independent sets of transcriptome data reveal the presence of full-length, polyadenylated transcripts from LTR retrotransposons in S. pombe during growth phase in rich medium. The redundancy of retrotransposon sequences makes it difficult to assess which elements are transcriptionally active, but data strongly indicates that only a subset of the LTR retrotransposons contribute significantly to the detected transcription. A considerable level of reverse strand transcription is also detected. Equal levels of transcriptional activity are observed from both strands of solitary LTR sequences. Transcriptome data collected during meiosis suggests that transcription of solitary LTRs is correlated with the transcription of nearby protein-coding genes.
Presumably, the host organism negatively regulates proliferation of LTR retrotransposons. The finding of considerable transcriptional activity of retrotransposons suggests that part of this regulation is likely to take place at a post-transcriptional level. Alternatively, the transcriptional activity may signify a hitherto unrecognized activity level of retrotransposon proliferation. Our findings underline the usefulness of transcriptome data in elucidating dynamics in retrotransposon transcription.
PMCID: PMC2848245  PMID: 20226011
25.  The genome of the simian and human malaria parasite Plasmodium knowlesi 
Nature  2008;455(7214):799-803.
Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the ‘kra’ monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia1,2. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated3, and it has a close phylogenetic relationship to Plasmodium vivax​4, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or ‘hypnozoite’ in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone5) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome4 and other sequenced Plasmodium genomes6-8. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs9, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.
PMCID: PMC2656934  PMID: 18843368

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