Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.     

Are Rod Outer Segment ATP-ase and ATP-Synthase Activity Expression of the Same Protein?

Vertebrate retinal rod outer segments (OS) consist of a stack of disks surrounded by the plasma membrane, where phototransduction takes place. Energetic metabolism in rod OS remains obscure. Literature described a so-called Mg²⁺-dependent ATPase activity, while our previous results demonstrated the presence of oxidative phosphorylation (OXPHOS) in OS, sustained by an ATP synthetic activity. Here we propose that the OS ATPase and ATP synthase are the expression of the same protein, i.e., of F₁F_o-ATP synthase. Imaging on bovine retinal sections showed that some OXPHOS proteins are expressed in the OS. Biochemical data on bovine purified rod OS, characterized for purity, show an ATP synthase activity, inhibited by classical F₁F_o-ATP synthase inhibitors. Moreover, OS possess a pH-dependent ATP hydrolysis, inhibited by pH values below 7, suggestive of the functioning of the inhibitor of F1 (IF1) protein. WB confirmed the presence of IF1 in OS, substantiating the expression of F₁F_o ATP synthase in OS. Data suggest that the OS F₁Fo ATP synthase is able to hydrolyze or synthesize ATP, depending on in vitro or in vivo conditions and that the role of IF1 would be pivotal in the prevention of the reversal of ATP synthase in OS, for example during hypoxia, granting photoreceptor survival.     

Fine scale movements and activity areas of white sharks (Carcharodon carcharias) in Mossel Bay, South Africa

Previous work on white sharks indicate the species show seasonally limited movement patters, at certain aggregation sites small areas may play vital roles in the life history of a large amount of the population. Acoustic telemetry was used to estimate habitat use of white sharks, Carcharodon carcharias, while aggregating at Mossel Bay, South Africa. Total range of all shark tracks combined accumulated 782 h and covered an area of 93.5 km² however, within this range, sharks were found to highly utilise a core habitat (50 % Kernel, K50) of just 1.05 km² over a reef system adjacent to a river mouth. Individual tracks revealed additional core habitats, some of which were previously undocumented and one adjacent to a commercial harbor. Much was found to be dependent on the size of the shark, with larger sharks (>400 cm) occupying smaller activity areas than subadult (300–399 cm) and juvenile (<300 cm) conspecifics, while Index of Reuse (IOR) and Index of Shared Space (IOSS) were both found to increase with shark size. Such results provide evidence that larger white sharks are more selective in habitat use, which indicates they have greater experience within aggregation sites. Furthermore, the focused nature of foraging means spatially restricted management strategies would offer a powerful tool to aid enforcement of current protective legislation for the white shark in similar environments of limited resources and capacity.     

Habitat selection in translocated gregarious ungulate species: An interplay between sociality and ecological requirements

The adaptation of translocated organisms to a new environment in the first years after their release is crucial in translocation programs because it may affect survival and reproductive success. Therefore, identifying the factors determining resource selection by the relocated animals is essential to improve the planning and the outcome of such programs. Using data collected in 2006–2009 in the framework of a restocking program, we studied the temporal variation of habitat selection in 14 translocated Alpine ibex (Capra ibex) during the year of their release and the following 3 years. We hypothesized a progressive adaptation of the translocated individuals, highlighted by a gradual decrease in the dissimilarities between translocated and resident individuals in ecological characteristics and social behavior. We tested the differences in habitat selection and home range size between the translocated and resident individuals and compared the spatial overlap between the groups. As expected, the dissimilarities decreased annually. The translocated and resident ibex almost immediately selected the same habitat resources, but the translocated individuals required 3 years to become fully socially assimilated. Our results indicated that habitat selection by gregarious species in a new environment is primarily driven by specific ecological requirements and that sociality plays a significant role. The translocated individuals tended to colonize areas already occupied by residents, either to fulfill social requirements and/or because the location of resident individuals may indicate high-quality habitat. This pattern of behavior must be considered in the planning of translocation programs because habitat selection can affect the outcomes of the programs. © 2013 The Wildlife Society.     

Despite Long-Term Compost Amendment Seasonal Changes are Main Drivers of Soil Fungal and Bacterial Population Dynamics in a Tuscan Vineyard

The effect of long-term (8 years) compost treatments (compost or compost plus mineral fertilizer) on genetic structure of bacterial and fungal populations in both bulk soil and rhizosphere of grapevine (Vitis vinifera) was analyzed in respect to a control constituted by the soil treated with mineral fertilization. Soils were sampled in early summer (July), mid-summer (August), and before harvest (October). Bacterial and fungal populations were characterized by genetic fingerprints generated by the application of 16S rDNA and ITS rDNA Multiplex Terminal Fragment Length Polymorphism (M-TRFLP) technique. Compost induced no significant differences at any time on microbial communities from bulk soil samples, whereas seasonal variations significantly affected both bacterial and fungal populations as indicated by the Multi Dimensional Scaling (MDS) ordination method of the M-TRFLPs results. MDS analysis of grapevine rhizosphere M-TRFLPs showed that temporal separation was significant for the bacterial population only. Results suggested that soil microbial populations in vineyard productive ecosystems may be sensitive to environmental changes induced by seasonal variations and show a certain degree of resilience to different agricultural practices.     

Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity

Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and β-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of β-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity.     

The Ebola Virus Matrix Protein Penetrates into the Plasma Membrane: A KEY STEP IN VIRAL PROTEIN 40 (VP40) OLIGOMERIZATION AND VIRAL EGRESS*

Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu²¹³, Ile²⁹³, Leu²⁹⁵, and Val²⁹⁸ demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.     

The Outer Membrane TolC-like Channel HgdD Is Part of Tripartite Resistance-Nodulation-Cell Division (RND) Efflux Systems Conferring Multiple-drug Resistance in the Cyanobacterium Anabaena sp. PCC7120

The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component “AB-D” systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox⁻ phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.     

Mdm10 is an ancient eukaryotic porin co-occurring with the ERMES complex

Mitochondrial β-barrel proteins fulfill central functions in the outer membrane like metabolite exchange catalyzed by the voltage-dependent anion channel (VDAC) and protein biogenesis by the central components of the preprotein translocase of the outer membrane (Tom40) or of the sorting and assembly machinery (Sam50). The mitochondrial division and morphology protein Mdm10 is another essential outer membrane protein with proposed β-barrel fold, which has so far only been found in Fungi. Mdm10 is part of the endoplasmic reticulum mitochondria encounter structure (ERMES), which tethers the ER to mitochondria and associates with the SAM complex. In here, we provide evidence that Mdm10 phylogenetically belongs to the VDAC/Tom40 superfamily. Contrary to Tom40 and VDAC, Mdm10 exposes long loops towards both sides of the membrane. Analyses of single loop deletion mutants of Mdm10 in the yeast Saccharomyces cerevisiae reveal that the loops are dispensable for Mdm10 function. Sequences similar to fungal Mdm10 can be found in species from Excavates to Fungi, but neither in Metazoa nor in plants. Strikingly, the presence of Mdm10 coincides with the appearance of the other ERMES components. Mdm10's presence in both unikonts and bikonts indicates an introduction at an early time point in eukaryotic evolution.