Evolutionary Analysis of Mitogenomes from Parasitic and Free-Living Flatworms

Mitochondrial genomes (mitogenomes) are useful and relatively accessible sources of molecular data to explore and understand the evolutionary history and relationships of eukaryotic organisms across diverse taxonomic levels. The availability of complete mitogenomes from Platyhelminthes is limited; of the 40 or so published most are from parasitic flatworms (Neodermata). Here, we present the mitogenomes of two free-living flatworms (Tricladida): the complete genome of the freshwater species Crenobia alpina (Planariidae) and a nearly complete genome of the land planarian Obama sp. (Geoplanidae). Moreover, we have reanotated the published mitogenome of the species Dugesia japonica (Dugesiidae). This contribution almost doubles the total number of mtDNAs published for Tricladida, a species-rich group including model organisms and economically important invasive species. We took the opportunity to conduct comparative mitogenomic analyses between available free-living and selected parasitic flatworms in order to gain insights into the putative effect of life cycle on nucleotide composition through mutation and natural selection. Unexpectedly, we did not find any molecular hallmark of a selective relaxation in mitogenomes of parasitic flatworms; on the contrary, three out of the four studied free-living triclad mitogenomes exhibit higher A+T content and selective relaxation levels. Additionally, we provide new and valuable molecular data to develop markers for future phylogenetic studies on planariids and geoplanids.     

In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis

Activity of the oxidative phosphorylation system (OXPHOS) is decreased in humans and mice with nonalcoholic steatohepatitis. Nitro-oxidative stress seems to be involved in its pathogenesis. The aim of this study was to determine whether fatty acids are implicated in the pathogenesis of this mitochondrial defect. In HepG2 cells, we analyzed the effect of saturated (palmitic and stearic acids) and monounsaturated (oleic acid) fatty acids on: OXPHOS activity; levels of protein expression of OXPHOS complexes and their subunits; gene expression and half-life of OXPHOS complexes; nitro-oxidative stress; and NADPH oxidase gene expression and activity. We also studied the effects of inhibiting or silencing NADPH oxidase on the palmitic-acid-induced nitro-oxidative stress and subsequent OXPHOS inhibition. Exposure of cultured HepG2 cells to saturated fatty acids resulted in a significant decrease in the OXPHOS activity. This effect was prevented in the presence of a mimic of manganese superoxide dismutase. Palmitic acid reduced the amount of both fully-assembled OXPHOS complexes and of complex subunits. This reduction was due mainly to an accelerated degradation of these subunits, which was associated with a 3-tyrosine nitration of mitochondrial proteins. Pretreatment of cells with uric acid, an antiperoxynitrite agent, prevented protein degradation induced by palmitic acid. A reduced gene expression also contributed to decrease mitochondrial DNA (mtDNA)-encoded subunits. Saturated fatty acids induced oxidative stress and caused mtDNA oxidative damage. This effect was prevented by inhibiting NADPH oxidase. These acids activated NADPH oxidase gene expression and increased NADPH oxidase activity. Silencing this oxidase abrogated totally the inhibitory effect of palmitic acid on OXPHOS complex activity. We conclude that saturated fatty acids caused nitro-oxidative stress, reduced OXPHOS complex half-life and activity, and decreased gene expression of mtDNA-encoded subunits. These effects were mediated by activation of NADPH oxidase. That is, these acids reproduced mitochondrial dysfunction found in humans and animals with nonalcoholic steatohepatitis.KEY WORDS: Mitochondrial respiratory chain, Nonalcoholic steatohepatitis, NADPH oxidase, Oxidative phosphorylation, Proteomic, Nitro-oxidative stress, OXPHOS     

A γ-Secretase Inhibitor,but Not a γ-Secretase Modulator,Induced Defects in BDNF Axonal Trafficking and Signaling: Evidence for a Role for APP

Clues to Alzheimer disease (AD) pathogenesis come from a variety of different sources including studies of clinical and neuropathological features, biomarkers, genomics and animal and cellular models. An important role for amyloid precursor protein (APP) and its processing has emerged and considerable interest has been directed at the hypothesis that Aβ peptides induce changes central to pathogenesis. Accordingly, molecules that reduce the levels of Aβ peptides have been discovered such as γ-secretase inhibitors (GSIs) and modulators (GSMs). GSIs and GSMs reduce Aβ levels through very different mechanisms. However, GSIs, but not GSMs, markedly increase the levels of APP CTFs that are increasingly viewed as disrupting neuronal function. Here, we evaluated the effects of GSIs and GSMs on a number of neuronal phenotypes possibly relevant to their use in treatment of AD. We report that GSI disrupted retrograde axonal trafficking of brain-derived neurotrophic factor (BDNF), suppressed BDNF-induced downstream signaling pathways and induced changes in the distribution within neuronal processes of mitochondria and synaptic vesicles. In contrast, treatment with a novel class of GSMs had no significant effect on these measures. Since knockdown of APP by specific siRNA prevented GSI-induced changes in BDNF axonal trafficking and signaling, we concluded that GSI effects on APP processing were responsible, at least in part, for BDNF trafficking and signaling deficits. Our findings argue that with respect to anti-amyloid treatments, even an APP-specific GSI may have deleterious effects and GSMs may serve as a better alternative.     

Construction of High Density Sweet Cherry (Prunus avium L.) Linkage Maps Using Microsatellite Markers and SNPs Detected by Genotyping-by-Sequencing (GBS)

Linkage maps are valuable tools in genetic and genomic studies. For sweet cherry, linkage maps have been constructed using mainly microsatellite markers (SSRs) and, recently, using single nucleotide polymorphism markers (SNPs) from a cherry 6K SNP array. Genotyping-by-sequencing (GBS), a new methodology based on high-throughput sequencing, holds great promise for identification of high number of SNPs and construction of high density linkage maps. In this study, GBS was used to identify SNPs from an intra-specific sweet cherry cross. A total of 8,476 high quality SNPs were selected for mapping. The physical position for each SNP was determined using the peach genome, Peach v1.0, as reference, and a homogeneous distribution of markers along the eight peach scaffolds was obtained. On average, 65.6% of the SNPs were present in genic regions and 49.8% were located in exonic regions. In addition to the SNPs, a group of SSRs was also used for construction of linkage maps. Parental and consensus high density maps were constructed by genotyping 166 siblings from a ‘Rainier’ x ‘Rivedel’ (Ra x Ri) cross. Using Ra x Ri population, 462, 489 and 985 markers were mapped into eight linkage groups in ‘Rainier’, ‘Rivedel’ and the Ra x Ri map, respectively, with 80% of mapped SNPs located in genic regions. Obtained maps spanned 549.5, 582.6 and 731.3 cM for ‘Rainier’, ‘Rivedel’ and consensus maps, respectively, with an average distance of 1.2 cM between adjacent markers for both ‘Rainier’ and ‘Rivedel’ maps and of 0.7 cM for Ra x Ri map. High synteny and co-linearity was observed between obtained maps and with Peach v1.0. These new high density linkage maps provide valuable information on the sweet cherry genome, and serve as the basis for identification of QTLs and genes relevant for the breeding of the species.     

Some Like It Fat: Comparative Ultrastructure of the Embryo in Two Demosponges of the Genus Mycale (Order Poecilosclerida) from Antarctica and the Caribbean

During embryogenesis, organisms with lecithotrophic indirect development usually accumulate large quantities of energetic reserves in the form of yolk that are necessary for larval survival. Since all sponges have lecithotrophic development, yolk formation is an ineludible step of their embryogenesis. Sponge yolk platelets have a wide range of morphological forms, from entirely lipid or protein platelets to a combined platelet showing both lipids and proteins and even glycogen. So far, there are no comparative studies on the nature and content of yolk in congeneric species of sponges inhabiting contrasting environments, which could have putative effects on the larval adaptation to environmental conditions. Here, we have taken advantage of the worldwide distribution of the sponge genus Mycale, in order to compare the embryogenesis and yolk formation in two species inhabiting contrasting latitudinal areas: M. acerata from Antarctic waters and M. laevis from the Caribbean. We have compared their brooded embryos and larvae using scanning and transmission electron microscopy, and calculated their energetic signatures based on the nature of their yolk. While the general morphological feature of embryos and larvae of both species were very similar, the main difference resided in the yolk nature. The Antarctic species, M. acerata, showed exclusively lipid yolk, whereas the Caribbean species, M. laevis, showed combined platelets of lipids and proteins and less frequently protein yolk platelets. The larvae of M. acerata were estimated to possess a two-fold energetic signature compared to that of M. laevis, which may have important ecological implications for their survival and for maintaining large population densities in the cold waters of the Southern Ocean.     

Preferential Targeting of Nav1.6 Voltage-Gated Na+ Channels to the Axon Initial Segment during Development

During axonal maturation, voltage-gated sodium (Na_v) channels accumulate at the axon initial segment (AIS) at high concentrations. This localization is necessary for the efficient initiation of action potentials. The mechanisms underlying channel trafficking to the AIS during axonal development have remained elusive due to a lack of Na_v reagents suitable for high resolution imaging of channels located specifically on the cell surface. Using an optical pulse-chase approach in combination with a novel Na_v1.6 construct containing an extracellular biotinylation domain we demonstrate that Na_v1.6 channels are preferentially inserted into the AIS membrane during neuronal development via direct vesicular trafficking. Single-molecule tracking illustrates that axonal channels are immediately immobilized following delivery, while channels delivered to the soma are often mobile. Neither a Na_v1.6 channel lacking the ankyrin-binding motif nor a chimeric K_v2.1 channel containing the Na_v ankyrinG-binding domain show preferential AIS insertion. Together these data support a model where ankyrinG-binding is required for preferential Na_v1.6 insertion into the AIS plasma membrane. In contrast, ankyrinG-binding alone does not confer the preferential delivery of proteins to the AIS.     

Trisomy 8, a Cytogenetic Abnormality in Myelodysplastic Syndromes,Is Constitutional or Not?

Isolated trisomy 8 is not considered presumptive evidence of myelodysplastic syndrome (MDS) in cases without minimal morphological criteria. One reason given is that trisomy 8 (+8) can be found as a constitutional mosaicism (cT8M). We tried to clarify the incidence of cT8M in myeloid neoplasms, specifically in MDS, and the diagnostic value of isolated +8 in MDS. Twenty-two MDS and 10 other myeloid neoplasms carrying +8 were studied. Trisomy 8 was determined in peripheral blood by conventional cytogenetics (CC) and on granulocytes, CD3+ lymphocytes and oral mucosa cells by fluorescence in situ hybridization (FISH). In peripheral blood CC, +8 was seen in 4/32 patients. By FISH, only one patient with chronic myelomonocytic leukemia showed +8 in all cell samples and was interpreted as a cT8M. In our series +8 was acquired in all MDS. Probably, once discarded cT8M by FISH from CD3+ lymphocytes and non-hematological cells, +8 should be considered with enough evidence to MDS.     

Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production

Sucrose is one of the most promising carbon sources for industrial fermentation. To achieve sucrose catabolism, the sucrose utilization operons have been introduced into microorganisms that are not able to utilize sucrose. However, the rates of growth and sucrose uptake of these engineered strains were relatively low to be successfully employed for industrial applications. Here, we report a practical example of developing sucrose-utilizing microorganisms using Escherichia coli K-12 as a model system. The sucrose utilizing ability was acquired by introducing only β-fructofuranosidase from three different sucrose-utilizing organisms (Mannheimia succiniciproducens, E. coli W, and Bacillus subtilis). Among them, the M. succiniciproducens β-fructofuranosidase was found to be the most effective for sucrose utilization. Analyses of the underlying mechanism revealed that sucrose was hydrolyzed into glucose and fructose in the extracellular space and both liberated hexoses could be transported by their respective uptake systems in E. coli K-12. To prove that this system can also be applied for the production of useful metabolites, the M. succiniciproducens β-fructofuranosidase was introduced into the engineered l-threonine production strain of E. coli K-12. This recombinant strain was able to produce 51.1 g/L l-threonine by fed-batch culture, resulting in an overall yield of 0.284 g l-threonine per g sucrose. This simple approach to make E. coli K-12 to acquire sucrose-utilizing ability and its successful biotechnological application can be employed to develop sustainable bioprocesses using renewable biomass.