Conveniently Pre-Tagged and Pre-Packaged: Extended Molecular Identification and Metagenomics Using Complete Metazoan Mitochondrial Genomes

Background

Researchers sorely need markers and approaches for biodiversity exploration (both specimen linked and metagenomics) using the full potential of next generation sequencing technologies (NGST). Currently, most studies rely on expensive multiple tagging, PCR primer universality and/or the use of few markers, sometimes with insufficient variability.

Methodology/Principal Findings

We propose a novel approach for the isolation and sequencing of a universal, useful and popular marker across distant, non-model metazoans: the complete mitochondrial genome. It relies on the properties of metazoan mitogenomes for enrichment, on careful choice of the organisms to multiplex, as well as on the wide collection of accumulated mitochondrial reference datasets for post-sequencing sorting and identification instead of individual tagging. Multiple divergent organisms can be sequenced simultaneously, and their complete mitogenome obtained at a very low cost. We provide in silico testing of dataset assembly for a selected set of example datasets.

Conclusions/Significance

This approach generates large mitogenome datasets. These sequences are useful for phylogenetics, molecular identification and molecular ecology studies, and are compatible with all existing projects or available datasets based on mitochondrial sequences, such as the Barcode of Life project. Our method can yield sequences both from identified samples and metagenomic samples. The use of the same datasets for both kinds of studies makes for a powerful approach, especially since the datasets have a high variability even at species level, and would be a useful complement to the less variable 18S rDNA currently prevailing in metagenomic studies.     

Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy

Adult skeletal muscles adapt their fiber size to workload. We show that serum response factor (Srf) is required for satellite cell-mediated hypertrophic muscle growth. Deletion of Srf from myofibers and not satellite cells blunts overload-induced hypertrophy, and impairs satellite cell proliferation and recruitment to pre-existing fibers. We reveal a gene network in which Srf within myofibers modulates interleukin-6 and cyclooxygenase-2/interleukin-4 expressions and therefore exerts a paracrine control of satellite cell functions. In Srf-deleted muscles, in vivo overexpression of interleukin-6 is sufficient to restore satellite cell proliferation but not satellite cell fusion and overall growth. In contrast cyclooxygenase-2/interleukin-4 overexpression rescue satellite cell recruitment and muscle growth without affecting satellite cell proliferation, identifying altered fusion as the limiting cellular event. These findings unravel a role for Srf in the translation of mechanical cues applied to myofibers into paracrine signals, which in turn will modulate satellite cell functions and support muscle growth.     

Extensive natural variation for cellular hydrogen peroxide release is genetically controlled

Natural variation in DNA sequence contributes to individual differences in quantitative traits. While multiple studies have shown genetic control over gene expression variation, few additional cellular traits have been investigated. Here, we investigated the natural variation of NADPH oxidase-dependent hydrogen peroxide (H₂O₂ release), which is the joint effect of reactive oxygen species (ROS) production, superoxide metabolism and degradation, and is related to a number of human disorders. We assessed the normal variation of H₂O₂ release in lymphoblastoid cell lines (LCL) in a family-based 3-generation cohort (CEPH-HapMap), and in 3 population-based cohorts (KORA, GenCord, HapMap). Substantial individual variation was observed, 45% of which were associated with heritability in the CEPH-HapMap cohort. We identified 2 genome-wide significant loci of Hsa12 and Hsa15 in genome-wide linkage analysis. Next, we performed genome-wide association study (GWAS) for the combined KORA-GenCord cohorts (n = 279) using enhanced marker resolution by imputation (>1.4 million SNPs). We found 5 significant associations (p<5.00×10−8) and 54 suggestive associations (p<1.00×10−5), one of which confirmed the linked region on Hsa15. To replicate our findings, we performed GWAS using 58 HapMap individuals and ∼2.1 million SNPs. We identified 40 genome-wide significant and 302 suggestive SNPs, and confirmed genome signals on Hsa1, Hsa12, and Hsa15. Genetic loci within 900 kb from the known candidate gene p67phox on Hsa1 were identified in GWAS in both cohorts. We did not find replication of SNPs across all cohorts, but replication within the same genomic region. Finally, a highly significant decrease in H₂O₂ release was observed in Down Syndrome (DS) individuals (p<2.88×10−12). Taken together, our results show strong evidence of genetic control of H₂O₂ in LCL of healthy and DS cohorts and suggest that cellular phenotypes, which themselves are also complex, may be used as proxies for dissection of complex disorders.     

Etazolate, a neuroprotective drug linking GABA(A) receptor pharmacology to amyloid precursor protein processing

Pharmacological modulation of the GABA_A receptor has gained increasing attention as a potential treatment for central processes affected in Alzheimer disease (AD), including neuronal survival and cognition. The proteolytic cleavage of the amyloid precursor protein (APP) through the α-secretase pathway decreases in AD, concurrent with cognitive impairment. This APP cleavage occurs within the β-amyloid peptide (Aβ) sequence, precluding formation of amyloidogenic peptides and leading to the release of the soluble N-terminal APP fragment (sAPPα) which is neurotrophic and procognitive. In this study, we show that at nanomolar-low micromolar concentrations, etazolate, a selective GABA_A receptor modulator, stimulates sAPPα production in rat cortical neurons and in guinea pig brains. Etazolate (20 nM–2 μM) dose-dependently protected rat cortical neurons against Aβ-induced toxicity. The neuroprotective effects of etazolate were fully blocked by GABA_A receptor antagonists indicating that this neuroprotection was due to GABA_A receptor signalling. Baclofen, a GABA_B receptor agonist failed to inhibit the Aβ-induced neuronal death. Furthermore, both pharmacological α-secretase pathway inhibition and sAPPα immunoneutralization approaches prevented etazolate neuroprotection against Aβ, indicating that etazolate exerts its neuroprotective effect via sAPPα induction. Our findings therefore indicate a relationship between GABA_A receptor signalling, the α-secretase pathway and neuroprotection, documenting a new therapeutic approach for AD treatment.     

High Virulence of Wolbachia after Host Switching: When Autophagy Hurts

Wolbachia are widespread endosymbionts found in a large variety of arthropods. While these bacteria are generally transmitted vertically and exhibit weak virulence in their native hosts, a growing number of studies suggests that horizontal transfers of Wolbachia to new host species also occur frequently in nature. In transfer situations, virulence variations can be predicted since hosts and symbionts are not adapted to each other. Here, we describe a situation where a Wolbachia strain (wVulC) becomes a pathogen when transfected from its native terrestrial isopod host species (Armadillidium vulgare) to another species (Porcellio d. dilatatus). Such transfer of wVulC kills all recipient animals within 75 days. Before death, animals suffer symptoms such as growth slowdown and nervous system disorders. Neither those symptoms nor mortalities were observed after injection of wVulC into its native host A. vulgare. Analyses of wVulC''s densities in main organs including Central Nervous System (CNS) of both naturally infected A. vulgare and transfected P. d. dilatatus and A. vulgare individuals revealed a similar pattern of host colonization suggesting an overall similar resistance of both host species towards this bacterium. However, for only P. d. dilatatus, we observed drastic accumulations of autophagic vesicles and vacuoles in the nerve cells and adipocytes of the CNS from individuals infected by wVulC. The symptoms and mortalities could therefore be explained by this huge autophagic response against wVulC in P. d. dilatatus cells that is not triggered in A. vulgare. Our results show that Wolbachia (wVulC) can lead to a pathogenic interaction when transferred horizontally into species that are phylogenetically close to their native hosts. This change in virulence likely results from the autophagic response of the host, strongly altering its tolerance to the symbiont and turning it into a deadly pathogen.     

Honeybee Colony Vibrational Measurements to Highlight the Brood Cycle

Insect pollination is of great importance to crop production worldwide and honey bees are amongst its chief facilitators. Because of the decline of managed colonies, the use of sensor technology is growing in popularity and it is of interest to develop new methods which can more accurately and less invasively assess honey bee colony status. Our approach is to use accelerometers to measure vibrations in order to provide information on colony activity and development. The accelerometers provide amplitude and frequency information which is recorded every three minutes and analysed for night time only. Vibrational data were validated by comparison to visual inspection data, particularly the brood development. We show a strong correlation between vibrational amplitude data and the brood cycle in the vicinity of the sensor. We have further explored the minimum data that is required, when frequency information is also included, to accurately predict the current point in the brood cycle. Such a technique should enable beekeepers to reduce the frequency with which visual inspections are required, reducing the stress this places on the colony and saving the beekeeper time.     

Cuticular differences of the exoskeleton relative to habitat preferences among three terrestrial isopods

Terrestrial isopods have adapted to land life by diverse morphological, physiological and behavioral changes. Isopod species exhibit a large variety of preferences ranging from moist to dry habitats. Isopod cuticle is an interesting model for studying adaptations to terrestrial life. The cuticle consists of a hierarchically organized material which provides protection and sites for muscle attachment. The present paper aimed to investigate the structure of the cuticle in three isopod species in order to relate some peculiar features to an adaptive process to environmental constraints. Results showed that general thickness of Ligia italica cuticle was quite similar to that observed in Porcellio lamellatus. Nevertheless, this cuticle was half as thick as in Porcellio laevis. Although the positive relationship found between total body lengths and the total thickness of the cuticle, the exocuticle did not show any differences between the three species. However, it is the development of a thicker endocuticle in P. laevis which make its total cuticle thickness more important.     

The cost of growing large: costs of post‐weaning growth on body mass senescence in a wild mammal

Individual body mass often positively correlates with survival and reproductive success, whereas fitness costs of growing large are rarely detected in vertebrates in the wild. Evidence that adult body mass progressively declines with increasing age is accumulating across mammalian populations. Growing fast to a large body can increase the cellular damage accumulated throughout life, leading body growth in early life to be negatively associated with the rate of body mass senescence. Moreover, the onset of mass senescence may strongly depend on both sex‐specific reproductive tactics and environmental conditions. Assessing the timing and the rate of body mass decline with increasing age thus offers an opportunity to look for costs of having grown fast, especially after a poor start during early life, in both sexes and in different environments. Using a unique dataset including 30 years of longitudinal data on age‐specific body mass collected in two roe deer Capreolus capreolus populations subjected to contrasted environmental conditions, we looked for potential costs of high post‐weaning growth rate in terms of steeper rate of body mass senescence. Our analyses of body mass senescence accounted for the potential variation in the onset of senescence and allowed explicit comparisons of this variable between sexes and populations. Higher growth rates late in the growing period (after weaning) were associated with a steeper rate of body mass senescence, regardless of early mass (gained before weaning), but at different extents depending on sex and environmental conditions. Body mass senescence occurred earlier in males than in females, especially in the population facing limiting resources. In the wild, although heavy individuals generally survive better than small ones, the costs of growing large late in the growing period only became apparent late in life through mass senescence.     

Design, synthesis and preliminary biological evaluation of acridine compounds as potential agents for a combined targeted chemo-radionuclide therapy approach to melanoma

Various iodo-acridone and acridine carboxamides have been prepared and evaluated as agents for targeted radionuclide and/or chemotherapy for melanoma, due to their structural similarity to benzamides which are known to possess specific affinity for melanin. Three of these carboxamides selected for their in vitro cytotoxic properties were radioiodinated with [(125)I]NaI at high specific activity. Biodistribution studies carried out in B16F0 murine melanoma tumour-bearing mice highlighted that acridone 8f and acridine 9d, presented high, long-lasting tumour concentrations together with an in vivo kinetic profile favourable to application in targeted radionuclide therapy.