Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity

Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by rapamycin could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.     

BlackOPs: increasing confidence in variant detection through mappability filtering

Identifying variants using high-throughput sequencing data is currently a challenge because true biological variants can be indistinguishable from technical artifacts. One source of technical artifact results from incorrectly aligning experimentally observed sequences to their true genomic origin (‘mismapping’) and inferring differences in mismapped sequences to be true variants. We developed BlackOPs, an open-source tool that simulates experimental RNA-seq and DNA whole exome sequences derived from the reference genome, aligns these sequences by custom parameters, detects variants and outputs a blacklist of positions and alleles caused by mismapping. Blacklists contain thousands of artifact variants that are indistinguishable from true variants and, for a given sample, are expected to be almost completely false positives. We show that these blacklist positions are specific to the alignment algorithm and read length used, and BlackOPs allows users to generate a blacklist specific to their experimental setup. We queried the dbSNP and COSMIC variant databases and found numerous variants indistinguishable from mapping errors. We demonstrate how filtering against blacklist positions reduces the number of potential false variants using an RNA-seq glioblastoma cell line data set. In summary, accounting for mapping-caused variants tuned to experimental setups reduces false positives and, therefore, improves genome characterization by high-throughput sequencing.     

The response of peatlands to climate warming: A review

Peatlands hold a large portion of the Earth’s terrestrial organic carbon and serve as important pools in the global carbon cycle. Due to their strong feedbacks, peatlands are one of the most important ecosystems with respect to climate warming. This paper reviews the effects of climate warming on peatland ecosystems. Climate warming will shift the point in time when vascular peatland plants flower and reach maximum biomass to an earlier date. Flower production for some plants will increase, but how the phenology of peatland bryophytes will react is still unknown. Climate warming may increase productivity of peatlands, especially ombrotrophic Sphagnum bogs, but in the long run the negative effects from decreased water availability may prevail. Climate warming will change the basic characteristics of peatlands: their wetness and the related cold environment and nutrient shortage. By increased mineralization and nitrogen and phosphorus availability, climate warming will facilitate the growth of vascular plants. This will suppress endangered plant species (which usually grow in low-productive, phosphorus-limited habitats) and lead to a change in vegetation composition and a decrease in peatland biodiversity. Climate warming will change the competitive balance between bryophytes and between Sphagnum and vascular plants. Climate warming in the Late Pleistocene facilitated the initiation of peatland formation, but most current experiments show an obvious tendency for climate warming to drive many peatlands to regressive succession with a shift in dominance from Sphagnum to vascular plants. This change in vegetation will increase the flux of CH₄ and possibly also CO₂. The effect of accelerated peat decay as a result of climate warming will vary between types of peatlands. Since climate warming will generally enhance peat respiration more than net primary production, more and more peatlands will become carbon sources rather than carbon sinks, which will aggravate climate warming by positive feedback. Finally, this paper addresses some problems with current manipulative experimental studies on peatland response to climate warming and makes suggestions for further studies.     

Invertebrates in field margins: taxonomic group diversity and functional group abundance in relation to age

Sown, temporary field margins are a common agri-environment scheme (AES) in the Netherlands. Despite their wide application, though, there has been scarcely any long-term monitoring of the succession of invertebrates. In the field margins of 40 farms, invertebrate diversity and the abundance of three functional groups were assessed in relation to age. The diversity in terms of number of species groups was found to increase with the age of the margins. The abundance of herbivores and detritivores also showed a positive correlation with the age of the margins. However, the abundance of predators decreased with increasing age. Older margins showed a higher total vegetation cover and fewer plant species, also resulting in lower plant species evenness. We suggest several changes to the current AES regulations. For the conservation of invertebrate diversity, longer-lasting field margins are desirable. In addition, old margins are favoured by detritivores, a group that has particular difficulty finding suitable habitats in agricultural landscapes. However, such margins are less favourable from an agricultural perspective, as they appear unsuitable for high abundances of potentially useful predators and the high vegetation cover attracts many potentially harmful herbivores. To circumvent this, the AES might be extended by incorporating hay-making, which would reduce standing biomass and might lead to more predators and fewer herbivores.     

Structure-based function inference using protein family-specific fingerprints

We describe a method to assign a protein structure to a functional family using family-specific fingerprints. Fingerprints represent amino acid packing patterns that occur in most members of a family but are rare in the background, a nonredundant subset of PDB; their information is additional to sequence alignments, sequence patterns, structural superposition, and active-site templates. Fingerprints were derived for 120 families in SCOP using Frequent Subgraph Mining. For a new structure, all occurrences of these family-specific fingerprints may be found by a fast algorithm for subgraph isomorphism; the structure can then be assigned to a family with a confidence value derived from the number of fingerprints found and their distribution in background proteins. In validation experiments, we infer the function of new members added to SCOP families and we discriminate between structurally similar, but functionally divergent TIM barrel families. We then apply our method to predict function for several structural genomics proteins, including orphan structures. Some predictions have been corroborated by other computational methods and some validated by subsequent functional characterization.     

Effects of vegetation management by mowing on ground-dwelling arthropods

Species-rich grasslands are rare in the Netherlands and need consistent vegetation management to retain their characteristic biodiversity. Roadside verges are important refuges for grassland plants since the mowing management no longer aims at traffic safety only but also strives for botanical diversity. Although arthropods are highly abundant in roadside verges, the effect of different mowing practices on this group is largely unknown. During 4 years, we studied ground beetles, weevils, ants and ground-dwelling spiders with pitfall traps in experimental plots in roadside verges with five different mowing treatments: (i) no management, (ii) and (iii) mowing once a year with and without hay removal, (iv) and (v) mowing twice a year with and without hay removal. This was done in a plant productivity gradient; the experiment was repeated in low-, medium- and high-productive verges. In the low-productive site, the effect of management on the arthropods only existed in a higher abundance in plots mown twice per year with hay removal. In the medium- and high-productive sites, mowing twice a year with hay removal resulted not only in highest abundances but also in highest arthropod species richness. Mowing twice without hay removal and mowing once with removal showed intermediate values, while mowing once per year without removal and particularly the absence of management resulted in low diversity and low abundance. To promote ground-dwelling arthropods in medium-to-high-productive grassland verges, we recommend a management of mowing twice a year with the removal of hay. It is reasoned that some form of rotational management, aiming at leaving some vegetation refuges intact after mowing events, may further promote arthropod survival. However, caution should be taken that these refuges are not too large, as overall suitability for ground-dwelling arthropod decreases rapidly in such patches. Out of several studied vegetation characteristics, the number of flowering plant species (medium-productive verge) and total flower abundance (high-productive verge) appeared to represent suitable, and easily monitored, proxies that significantly mirror arthropod diversity.     

Analysis of Subcellular Localization and Pathogenicity of <i>Plum Bark Necrosis Stem-Pitting Associated Virus</i> Protein P6

Infection of plum bark necrosis stem pitting associated virus (PBNSPaV) has been reported in many Prunus species in several countries, causing significant economic losses. The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance. However, numerous studies have indicated that they might play important roles in the pathogenesis of virus infection. The role of small hydrophobic protein P6, encoded by the open reading frame 2 of PBNSPaV, has not been well explored. In this study, we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain. Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes to the cytomembrane and nuclear membrane. To further clarify the pathogenicity of P6 proteins, we constructed a PVX-P6 expression vector by inserting the p6 fragment into a potato virus X (PVX)-based vector and transformed it into Agrobacterium tumefaciens GV3101. Infiltration of Nicotiana benthamiana (N. benthamiana) with the PVX vector-transformed A. tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation. Meanwhile, infiltration with the PVX-P6 vector-transformed A. tumefaciens resulted in no significant symptoms. These results demonstrated that heterologous expression of P6 in N. benthamiana could not enhance the pathogenicity of PVX. Our study indicates that P6 may not be a potential pathogenic factor associate with the causing of symptoms, and the mode of action of PBNSPaV-P6 protein remains to be further studied.