ALBERT

Description: Plastid genomes of photosynthetic flowering plants are usually highly conserved in both structure and gene content. However, the plastomes of parasitic and mycoheterotrophic plants may be released from selective constraint due to the reduction or loss of photosynthetic ability. Here we present the greatly reduced and highly divergent, yet functional, plastome of the nonphotosynthetic holoparasite Hydnora visseri (Hydnoraceae, Piperales). The plastome is 27 kb in length, with 24 genes encoding ribosomal proteins, ribosomal RNAs, tRNAs, and a few nonbioenergetic genes, but no genes related to photosynthesis. The inverted repeat and the small single copy region are only approximately 1.5 kb, and intergenic regions have been drastically reduced. Despite extreme reduction, gene order and orientation are highly similar to the plastome of Piper cenocladum , a related photosynthetic plant in Piperales. Gene sequences in Hydnora are highly divergent and several complementary approaches using the highest possible sensitivity were required for identification and annotation of this plastome. Active transcription is detected for all of the protein-coding genes in the plastid genome, and one of two introns is appropriately spliced out of rps 12 transcripts. The whole-genome shotgun read depth is 1,400 x coverage for the plastome, whereas the mitochondrial genome is covered at 40 x and the nuclear genome at 2 x . Despite the extreme reduction of the genome and high sequence divergence, the presence of syntenic, long transcriptionally active open-reading frames with distant similarity to other plastid genomes and a high plastome stoichiometry relative to the mitochondrial and nuclear genomes suggests that the plastome remains functional in H. visseri . A four-stage model of gene reduction, including the potential for complete plastome loss, is proposed to account for the range of plastid genomes in nonphotosynthetic plants.

Description: The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer’s disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i 6 A) into 2-methylthio-N6-isopentenyladenosine (ms 2 i 6 A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.

Description: Early successional stands composed of naturally regenerated hardwood and conifer species are abundant in the forests of northeastern North America. Substantial improvements in the composition and growth of these stands may be possible with early management intervention. Unfortunately, stand responses to early management inputs are poorly understood since many of these stands are rarely manipulated. We examined the response of early successional stands to combinations of two management intensities (with and without enrichment planting and different levels of vegetation control) and three compositional objectives (hardwood, mixedwood and conifer). The treatments were designed to represent management options available in the region, including hardwood thinning, conifer release and a combination of treatments to promote hardwood–conifer mixedwood stands. Seven years after treatment, yields of the two hardwood thinning treatments ranged from 43.4 to 56.6 Mg ha –1 , which were similar to the 52.9 Mg ha –1 yield of the untreated control but with 17 and 46% lower densities, respectively. In the conifer release treatments, the removal of hardwoods promoted conifer dominance and resulted in yields between 19.9 and 30.4 Mg ha –1 7 years after treatment. The conifer release treatments will likely be dominated by conifers in the future with varying hardwood densities due to hardwoods establishing in gaps without conifers. After 7 years, yields of the mixedwood treatments were between 19 and 47% greater than the conifer release treatments due to the retention of thinned hardwood stems and represent stands that dominate much of the forestland in the region. Results from this study demonstrated that early successional stands can be effectively managed during early stand development to improve growth and the longer term composition.

Description: This study aimed to compare the frequencies of nuclear anomalies in buccal cells between diabetic and non-diabetic individuals and to assess the impact of a ‘healthy diet’—a cornerstone in the treatment of diabetes. Seventy-six diabetic and 21 non-diabetic individuals participated in this parallel, randomised, intervention trial. All participants received information about the importance of a healthy diet, while participants randomly assigned to the intervention group received additionally 300g of vegetables and 25ml of plant oil rich in polyunsaturated fatty acids (PUFA) per day for 8 weeks. Cytogenetic damage in buccal cells was assessed at baseline and after 8 weeks using the buccal micronucleus cytome assay. Micronucleus (MN) frequency at baseline was significantly higher in participants with diabetes (0.58±0.30) compared with non-diabetic individuals (0.28±0.29). Further analysis of baseline data revealed significantly higher MN levels in participants of the highest tertile of waist circumference (+40%), fasting plasma glucose (+55%), glycated haemoglobin (+41%) and cardiovascular disease risk (+39%) relative to participants of the lowest tertile. The dietary intervention had no effect on MN frequencies. Glycated haemoglobin and biomarkers reflecting cytokinetic defect or acute cell death were reduced in both the intervention and ‘information only’ groups. The results of this study suggest a strong impact of abdominal obesity and glucose metabolism on genomic stability. Similar effects on nuclear anomalies were observed in the ‘information only’ group and the intervention group receiving vegetables and PUFA-rich plant oil.

Description: Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans.

Description: Hereditarily finite sets (sets which are finite and have only hereditarily finite sets as members) are basic mathematical and computational objects, and also stand at the basis of some programming languages. We solve an open problem proposed by Kirby in 2008 concerning a recurrence relation for the cardinality $${a}_{n}$$ of the $$n$$ -th level of the adjunctive hierarchy of hereditarily finite sets; in this hierarchy, new sets are formed by the addition of a new single element drawn from the already existing sets to an already existing set. We also show that our results can be generalized to sets with atoms, or can be refined by rank, cardinality, or by the maximum level from where the new adjoined element is drawn. We also show that $${a}_{n}$$ satisfies the asymptotic formula $${a}_{n}={C}^{{2}^{n}}+O({C}^{{2}^{n-1}})$$ , for a constant $$C\approx 1.3399$$ , which is a too fast asymptotic growth for practical purposes. We thus propose a very natural variant of the adjunctive hierarchy, whose asymptotic behaviour we prove to be $$\Theta ({2}^{n})$$ .

Description: This work is a summary of the X-ray spectral studies of 29 TeV (10 12 eV, tera-electron-volt) -ray emitting blazars observed with Swift /XRT, especially focusing on sources for which the X-ray regime allows us to study the low- and the high-energy ends of the particle distribution function. Variability studies require simultaneous coverage, ideally sampling different flux states of each source. This is achieved using X-ray observations by disentangling the high-energy end of the synchrotron emission and the low-energy end of the Compton emission, which are produced by the same electron population. We focused on a sample of 29 TeV -ray emitting blazars with the best signal-to-noise X-ray observations collected with Swift /XRT in the energy range 0.3–10 keV during 10 yr of Swift /XRT operations. We investigate the X-ray spectral shapes and the effects of different corrections for neutral hydrogen absorption and decompose the synchrotron and inverse Compton components. For five sources (3C 66A, S5 0716+714, W Comae, 4C +21.35 and BL Lacertae) a superposition of both components is observed in the X-ray band, permitting simultaneous, time-resolved studies of both ends of the electron distribution. The analysis of multi-epoch observations revealed that the break energy of the X-ray spectrum varies only by a small factor with flux changes. Flux variability is more pronounced in the synchrotron domain (high-energy end of the electron distribution) than in the Compton domain (low-energy end of the electron distribution). The spectral shape of the Compton domain is stable, while the flux of the synchrotron domain is variable. These changes cannot be described by simple variations of the cut-off energy, suggesting that the high-energy end of the electron distribution is not generally well described by cooling only.

Description: Current therapy approaches in melanoma targeting have met with the development of resistance and tumour recurrence with a more aggressive phenotype. In a quest for alternative therapy targets, we had previously identified Signal Sequence Receptor 2 (SSR2) as a gene with high expression in a subgroup of human primary melanomas. Now we show that SSR2 exerts a prosurvival functionality in human melanoma cells and that high expression levels of SSR2 are associated with an unfavourable disease outcome in primary melanoma patients. Consistent with SSR’s role in translocation of proteins from the ribosome across the endoplasmic reticulum (ER) membrane, our data supports induction of SSR2 as a part of the ER stress response. This response included SSR2 upregulation upon development of therapy resistance to BRAF inhibitors, as well as the dependency of cell survival of BRAF inhibitor-resistant melanoma cells on SSR2. Complementary gain and loss of function data showed the Unfolded Protein Response (UPR) to ER stress as an inducer of SSR2 via transcriptional regulation through X-Box Binding Protein 1s (XBP1s) and support an ER stress-UPR-Transcription Factor XBP1s-SSR2 response axis in human melanocytic cells. Together with its dispensability for survival in normal human cells, these data propose SSR2 as a potential therapeutic target in (therapy-resistant) human melanoma.

Description: Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo . Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1 G107R but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

Description: Repetitive nucleic acid sequences are often prone to form secondary structures distinct from B-DNA. Prominent examples of such structures are DNA triplexes. We observed that certain intrastrand triplex motifs are highly conserved and abundant in prokaryotic genomes. A systematic search of 5246 different prokaryotic plasmids and genomes for intrastrand triplex motifs was conducted and the results summarized in the ITxF database available online at http://bioinformatics.uni-konstanz.de/utils/ITxF/ . Next we investigated biophysical and biochemical properties of a particular G/C-rich triplex motif (TM) that occurs in many copies in more than 260 bacterial genomes by CD and nuclear magnetic resonance spectroscopy as well as in vivo footprinting techniques. A characterization of putative properties and functions of these unusually frequent nucleic acid motifs demonstrated that the occurrence of the TM is associated with a high degree of genomic instability. TM-containing genomic loci are significantly more rearranged among closely related Escherichia coli strains compared to control sites. In addition, we found very high frequencies of TM motifs in certain Enterobacteria and Cyanobacteria that were previously described as genetically highly diverse. In conclusion we link intrastrand triplex motifs with the induction of genomic instability. We speculate that the observed instability might be an adaptive feature of these genomes that creates variation for natural selection to act upon.