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Background: Rising surgery rates have raised questions about the indications for spinal surgery. The study investigated patient-level and regional factors associated with spinal surgery for patients with spinal diseases., Methods: We undertook a cohort study based on routine healthcare data from Germany of 18.4 million patients within 60.9 million episodes of two patient-years before a possible spinal surgery in the time period 2008 to 2016. Using a Poisson model, the effects of a broad range of patient-related (sociodemographic, morbidity, social status), disease- and healthcare-related (physicians' specialty, conservative treatments) and regional variables were analyzed., Results: There was substantial regional heterogeneity in the occurrence of spinal surgery which decreased by only one quarter when controlling for the various determinants assessed. Previous musculoskeletal and mental health disorders as well as physical therapy were associated with a lower probability of surgery in the fully-adjusted model. Prescriptions for pain medication and consultations of specialists were associated with a higher probability of surgery. However, the specific severity of the vertebral diseases could not be taken into account in the analysis. Furthermore, a substantial proportion of patients with surgery did not receive a consultation with an outpatient specialist (29.5%), preoperative diagnostics (37.0%) or physical therapy (48.3%) before hospital admission., Conclusion: This large study on spinal diseases in Germany highlights important patterns in medical care of spinal diseases and their association with the probability of spinal surgery. However, only a relatively small proportion of the regional heterogeneity in spinal surgery could be explained by the extensive consideration of confounders, which suggests the relevance of other unmeasured factors like physicians' preferences., (© 2022. The Author(s).)

Background: Specifically young women are at risk for a poor outcome after ST-elevation myocardial infarction (STEMI). We aimed to investigate sex- and age-specific differences in outcome and associate these results with adherence to a guideline-directed optimal medical therapy (OMT). Methods: Administrative insurance data (≈26 million insured) were screened for patients aged 18–60 years with STEMI. Patient demographics, details on in-hospital treatment, adherence to OMT and its effect on mortality were assessed. Adherence to OMT was analyzed using multistate models and an association of those with death was fitted using multivariable Cox regression models with time-dependent co-variables. Results: Overall, 59,401 patients (19.3% women), median age 52 (interquartile range 48, 56) presented with STEMI. Female sex was associated with a poor outcome early after STEMI (90-day mortality: odds ratio 1.22, 95% confidence interval (CI) 1.12–1.32, p < 0.001). Overall survival was reduced in women compared to same-aged men. The ten-year survival rate was 19.7% (18.1–21.2%) versus 19.6% (18.9–20.4%) in men (p < 0.001). Although long-term drug adherence was low, its intake was associated with a better outcome. Specifically younger women showed a markedly lower mortality when on OMT (hazard ratio (HR) 0.22 (95% CI 0.19–0.26) versus HR 0.31 (95% CI 0.28–0.33) in men, pint < 0.001). Conclusions: Specifically young women were at risk for a poor outcome in the early phase after STEMI. Although long-term adherence to OMT was low, it was generally associated with a lower mortality, specifically in women. Our findings emphasize on early and long-term preventive measures in all patients after STEMI.

Background: The global rise of obesity and its association with cardiovascular risk factors (CVRF) have highlighted its connection to chronic heart failure (CHF). Paradoxically, obese CHF patients often experience better outcomes, a phenomenon known as the ‘obesity paradox’. This study evaluated the ‘obesity paradox’ within a large cohort in Germany and explored how varying degrees of obesity affect HF outcome. Methods: Anonymized health claims data from the largest German insurer (AOK) for the years 2014–2015 were utilized to analyze 88,247 patients hospitalized for myocardial infarction. This analysis encompassed baseline characteristics, comorbidities, interventions, complications, and long-term outcomes, including overall survival, freedom from CHF, and CHF-related rehospitalization. Patients were categorized based on body mass index. Results: Obese patients encompassed 21.3% of our cohort (median age 68.69 years); they exhibited a higher prevalence of CVRF (p < 0.001) and comorbidities than non-obese patients (median age 70.69 years). Short-term outcomes revealed lower complication rates and mortality (p < 0.001) in obese compared to non-obese patients. Kaplan–Meier estimations for long-term analysis illustrated increased incidences of CHF and rehospitalization rates among the obese, yet with lower overall mortality. Multivariable Cox regression analysis indicated that obese individuals faced a higher risk of developing CHF and being rehospitalized due to CHF but demonstrated better overall survival for those classified as having low-level obesity (p < 0.001). Conclusions: This study underscores favorable short-term outcomes among obese individuals. The ‘obesity paradox’ was confirmed, with more frequent CHF cases and rehospitalizations in the long term, alongside better overall survival for certain degrees of obesity.

Abstract Despite antagonizing attempts from the tobacco industry, passive inhalation of tobacco smoke is known to be cancerogenic and toxic to human health for decades. Nonetheless, millions of non-smoking adults and children are still victims of second-hand smoke. Accumulation of particulate matter (PM) in confined spaces such as the car are particularly harmful due to high concentrations. We here aimed to analyze the specific effects of ventilation conditions in the setting of a car. By the use of the measuring platform TAPaC (tobacco-associated particulate matter emissions inside a car cabin), 3R4F reference cigarettes, Marlboro red, and Marlboro gold were smoked in a car interior with a volume of 3.709 m3. Seven different ventilation conditions (C1–C7) were analyzed. Under C1, all windows were closed. Under C2–C7, the car ventilation was turned on power level 2/4 with the air directed towards the windshield. Only the passenger side window was opened, where an outer placed fan could create an airstream speed of 15.9–17.4 km/h at one meter distance to simulate a driving car. C2: Window 10 cm opened. C3: Window 10 cm opened with the fan turned on. C4: Window half-opened. C5: Window half-opened with the fan turned on. C6: Window fully opened. C7: Window fully opened with the fan turned on. Cigarettes were remotely smoked by an automatic environmental tobacco smoke emitter and a cigarette smoking device. Depending on the ventilation condition the cigarettes emitted different mean PM concentrations after 10 min under condition C1 (PM10: 1272–1697 µg/m3, PM2.5: 1253–1659 µg/m3, PM1: 964–1263 µg/m3) under C2, C4, and C6 (PM10: 68.7–196.2 µg/m3, PM2.5: 68.2–194.7 µg/m3, PM1: 66.1–183.8 µg/m3) C3, C5, and C7 (PM10: 73.7–139 µg/m3, PM2.5: 72–137.9 µg/m3, PM1:68.9–131.9 µg/m3). Vehicle ventilation is insufficient to protect passengers from toxic second-hand smoke completely. Brand-specific variations of tobacco ingredients and mixtures markedly influence PM emissions under ventilation conditions. The most efficient ventilation mode to reduce PM exposure was achieved by opening the passenger´s window 10 cm and turning the onboard ventilation on power level 2/4. In-vehicle smoking should be banned to preserve innocent risk groups (e.g., children) from harm.

Background: Temporary mechanical circulatory support devices (tMCS) are increasingly being used in patients with infarct-associated cardiogenic shock (AMICS). Evidence on patient selection, complications and long-term outcomes is lacking. We aim to investigate differences in clinical characteristics, complications and outcomes between patients receiving no tMCS or either intra-aortic balloon pump (IABP), veno-arterial extracorporeal membrane oxygenation (V-A ECMO) or Impella® for AMICS, with a particular focus on long-term outcomes. Methods: Using health claim data from AOK—Die Gesundheitskasse (local health care funds), we retrospectively analysed complications and outcomes of all insured patients with AMICS between 1 January 2010 and 31 December 2017. Results: A total of 39,864 patients were included (IABP 5451; Impella 776; V-A ECMO 833; no tMCS 32,804). In-hospital complications, including renal failure requiring dialysis (50.3% V-A ECMO vs. 30.5% Impella vs. 29.2 IABP vs. 12.1% no tMCS), major bleeding (38.1% vs. 20.9% vs. 18.0% vs. 9.3%) and sepsis (22.5% vs. 15.9% vs. 13.9% vs. 9.3%) were more common in V-A ECMO patients. In a multivariate analysis, the use of both V-A ECMO (HR 1.57, p < 0.001) and Impella (HR 1.25, p < 0.001) were independently associated with long-term mortality, whereas use of IABP was not (HR 0.89, p < 0.001). Kaplan–Meier estimates showed better survival for patients on IABP compared with Impella, V-A ECMO and no-tMCS. Short- and long-term mortality was high across all groups. Conclusions: Our data show noticeably more in-hospital complications in patients on tMCS and higher mortality with V-A ECMO and Impella. The use of both devices is an independent risk factor for mortality, whereas the use of IABP is associated with a survival benefit.

Background: Depression and anxiety (DA) are common mental disorders in patients with chronic diseases, but the research regarding their prevalence in heart transplantation (HTx) is still limited. Methods: We performed an analysis of the prevalence and prognostic relevance of DA in patients who underwent HTx between 2010 and 2018 in Germany. Data were obtained from Allgemeine Ortskrankenkasse (AOK), which is the largest public health insurance provider. Results: Overall, 694 patients were identified. More than a third of them were diagnosed with DA before undergoing HTx (n = 260, 37.5%). Patients with DA more often had an ischaemic cardiomyopathy (p < 0.001) and a history of previous myocardial infarction (p = 0.001) or stroke (p = 0.002). The prevalence of hypertension (p < 0.001), diabetes (p = 0.004), dyslipidaemia (p < 0.001) and chronic kidney disease (p = 0.003) was higher amongst transplant recipients with DA. Patients with DA were more likely to suffer an ischaemic stroke (p < 0.001) or haemorrhagic stroke (p = 0.032), or develop septicaemia (p = 0.050) during hospitalisation for HTx. Our analysis found no significant differences between the groups with respect to in-hospital mortality. The female sex and mechanical circulatory support were associated with an inferior prognosis. Pretransplant non-ischaemic cardiomyopathy was related to a favourable outcome. Conclusions: DA affect up to a third of the population undergoing HTx, with a greater prevalence in patients with comorbidities. DA are associated with a higher incidence of stroke and septicaemia after HTx.

Seamless DNA vectors derived from bacterial plasmids are devoid of bacterial genetic elements and represent attractive alternatives for biomedical applications including DNA vaccines. Larger scale production of seamless vectors employs engineered Escherichia coli strains in order to enable tightly regulated expression of site-specific DNA recombinases which precisely delete unwanted sequences from bacterial plasmids. As a novel component of a developing lambda integrase genome editing platform, we describe here strain MG1655-ISC as a means to easily produce different scales of seamless vectors, ranging in size from a few hundred base pairs to more than ten kilo base pairs. Since we employed an engineered lambda integrase that is able to efficiently recombine pairs of DNA crossover sites that differ in sequence, the resulting seamless vectors will be useful for subsequent genome editing in higher eukaryotes to accommodate variations in target site sequences. Future inclusion of single cognate sites for other genome targeting systems could enable modularity. These features, together with the demonstrated simplicity of in vivo seamless vector production, add to their utility in the biomedical space.

Abstract HMGA2 belongs to the family of the high mobility group (HMG) proteins. It binds DNA via three AT-hook domains to the minor groove of adenine-thymine (AT) rich DNA. Recently, a new function of HMGA2 as a replication fork chaperone that protects stem and cancer cells from replication fork collapse induced by chemotherapeutic agents was uncovered, suggesting a previously uncharacterized binding at replication forks. In this study, we examined HMGA2 binding to four DNA structures relevant to replication forks, namely ds DNA, ss DNA, forked DNA and supercoiled DNA plectonemes. We detected HMGA2 binding to supercoiled DNA at the lowest concentration and this binding mode transiently stabilizes the supercoiled plectonemes against relaxation by type I topoisomerase. Together, these findings suggest a plausible mechanism how fork regression and collapse are attenuated by HMGA2 during replication stress, i.e. through transient stabilization of positively supercoiled plectonemes in the parental duplex.

The transient build-up of DNA supercoiling during the translocation of replication forks threatens genome stability and is controlled by DNA topoisomerases (TOPs). This crucial process has been exploited with TOP poisons for cancer chemotherapy. However, pinpointing cellular determinants of the best clinical response to TOP poisons still remains enigmatic. Here, we present an integrated approach and demonstrate that endogenous and exogenous expression of the oncofetal high-mobility group AT-hook 2 (HMGA2) protein exhibited broad protection against the formation of hydroxyurea-induced DNA breaks in various cancer cells, thus corroborating our previously proposed model in which HMGA2 functions as a replication fork chaperone that forms a protective DNA scaffold at or close to stalled replication forks. We now further demonstrate that high levels of HMGA2 also protected cancer cells against DNA breaks triggered by the clinically important TOP1 poison irinotecan. This protection is most likely due to the recently identified DNA supercoil constraining function of HMGA2 in combination with exclusion of TOP1 from binding to supercoiled substrate DNA. In contrast, low to moderate HMGA2 protein levels surprisingly potentiated the formation of irinotecan-induced genotoxic covalent TOP1-DNA cleavage complexes. Our data from cell-based and several in vitro assays indicate that, mechanistically, this potentiating role involves enhanced drug-target interactions mediated by HMGA2 in ternary complexes with supercoiled DNA. Subtelomeric regions were found to be extraordinarily vulnerable to these genotoxic challenges induced by TOP1 poisoning, pointing at strong DNA topological barriers located at human telomeres. These findings were corroborated by an increased irinotecan sensitivity of patient-derived xenografts of colorectal cancers exhibiting low to moderate HMGA2 levels. Collectively, we uncovered a therapeutically important control mechanism of transient changes in chromosomal DNA topology that ultimately leads to enhanced human subtelomere stability.

Ungulate populations face declines across the globe, and populations are commonly conserved by using protected areas. However, assessing the effectiveness of protected areas in conserving ungulate populations has remained difficult. Using herd size data from four years of line transect surveys and distance sampling models, we modeled population densities of four important herbivore species across a gradient of protection on the edge of Zambia's South Luangwa National Park (SLNP) while accounting for the role of various ecological and anthropogenic variables. Our goal was to test whether protection was responsible for density dynamics in this protection gradient, and whether a hunting moratorium impacted herbivore densities during the studies. For all four species, we estimated lower densities in partially protected buffer areas adjacent to SLNP (ranging from 4.5-fold to 13.2-fold lower) compared to protected parklands. Density trends through the study period were species-specific, with some species increasing, decreasing, or remaining stable in all or some regions of the protection gradient. Surprisingly, when controlling for other covariates, we found that these observed differences were not always detectably related to the level of protection or year. Our findings highlight the importance of accounting for variables beyond strata of interest in evaluating the effectiveness of a protected area. This study highlights the importance of comprehensively modeling ungulate population density across protection gradients, identifies lands within an important protection gradient for targeted conservation and monitoring, documents prey depletion and expands our understanding on the drivers in a critical buffer area in Zambia.

Telomerase RNA (TR) provides the template for DNA repeat synthesis at telomeres and is essential for genome stability in continuously dividing cells. We mapped the RNA interactome of human TR (hTR) and identified a set of non-coding and coding hTR-interacting RNAs, including the histone 1C mRNA (HIST1H1C). Disruption of the hTR-HIST1H1C RNA association resulted in markedly increased telomere elongation without affecting telomerase enzymatic activity. Conversely, over-expression of HIST1H1C led to telomere attrition. By using a combination of mutations to disentangle the effects of histone 1 RNA synthesis, protein expression, and hTR interaction, we show that HIST1H1C RNA negatively regulates telomere length independently of its protein coding potential. Taken together, our data provide important insights into a surprisingly complex hTR-RNA interaction network and define an unexpected non-coding RNA role for HIST1H1C in regulating telomere length homeostasis, thus offering a glimpse into the mostly uncharted, vast space of non-canonical messenger RNA functions.

Factors that limit African lion populations are manifold and well-recognized, but their relative demographic effects remain poorly understood, particularly trophy hunting near protected areas. We identified and monitored 386 individual lions within and around South Luangwa National Park, Zambia, for five years (2008-2012) with trophy hunting and for three additional years (2013-2015) during a hunting moratorium. We used these data with mark-resight models to estimate the effects of hunting on lion survival, recruitment, and abundance. The best survival models, accounting for imperfect detection, revealed strong positive effects of the moratorium, with survival increasing by 17.1 and 14.0 percentage points in subadult and adult males, respectively. Smaller effects on adult female survival and positive effects on cub survival were also detected. The sex-ratio of cubs shifted from unbiased during trophy-hunting to female-biased during the moratorium. Closed mark-recapture models revealed a large increase in lion abundance during the hunting moratorium, from 116 lions in 2012 immediately preceding the moratorium to 209 lions in the last year of the moratorium. More cubs were produced each year of the moratorium than in any year with trophy hunting. Lion demographics shifted from a male-depleted population consisting mostly of adult (≥4 years) females to a younger population with more (>29%) adult males. These data show that the three-year moratorium was effective at growing the Luangwa lion population and increasing the number of adult males. The results suggest that moratoria may be an effective tool for improving the sustainability of lion trophy hunting, particularly where systematic monitoring, conservative quotas, and age-based harvesting are difficult to enforce.

Background. Metagenomics is an approach for characterizing environmental microbial communities in situ, it allows their functional and taxonomic characterization and to recover sequences from uncultured taxa. This is often achieved by a combination of sequence assembly and binning, where sequences are grouped into ‘bins’ representing taxa of the underlying microbial community. Assignment to low-ranking taxonomic bins is an important challenge for binning methods as is scalability to Gb-sized datasets generated with deep sequencing techniques. One of the best available methods for species bins recovery from deep-branching phyla is the expert-trained PhyloPythiaS package, where a human expert decides on the taxa to incorporate in the model and identifies ‘training’ sequences based on marker genes directly from the sample. Due to the manual effort involved, this approach does not scale to multiple metagenome samples and requires substantial expertise, which researchers who are new to the area do not have. Results. We have developed PhyloPythiaS+, a successor to our PhyloPythia(S) software. The new (+) component performs the work previously done by the human expert. PhyloPythiaS+ also includes a new k-mer counting algorithm, which accelerated the simultaneous counting of 4–6-mers used for taxonomic binning 100-fold and reduced the overall execution time of the software by a factor of three. Our software allows to analyze Gb-sized metagenomes with inexpensive hardware, and to recover species or genera-level bins with low error rates in a fully automated fashion. PhyloPythiaS+ was compared to MEGAN, taxator-tk, Kraken and the generic PhyloPythiaS model. The results showed that PhyloPythiaS+ performs especially well for samples originating from novel environments in comparison to the other methods. Availability. PhyloPythiaS+ in a virtual machine is available for installation under Windows, Unix systems or OS X on: https://github.com/algbioi/ppsp/wiki.

13-Lipoxygenase-derived oxylipins, such as jasmonates act as potent signaling molecules in plants. Although experimental evidence supports the impact of oxylipins generated by the 9-Lipoxygenase (9-LOX) pathway in root development and pathogen defense, their signaling function in plants remains largely elusive. Based on the root growth inhibiting properties of the 9-LOX-oxylipin 9-HOT (9-hydroxy-10,12,15-octadecatrienoic acid), we established a screening approach aiming at identifying transcription factors (TFs) involved in signaling and/or metabolism of this oxylipin. Making use of the AtTORF-Ex (Arabidopsis thaliana Transcription Factor Open Reading Frame Expression) collection of plant lines overexpressing TF genes, we screened for those TFs which restore root growth on 9-HOT. Out of 6,000 lines, eight TFs were recovered at least three times and were therefore selected for detailed analysis. Overexpression of the basic leucine Zipper (bZIP) TF TGA5 and its target, the monoxygenase CYP81D11 reduced the effect of added 9-HOT, presumably due to activation of a detoxification pathway. The highly related ETHYLENE RESPONSE FACTORs ERF106 and ERF107 induce a broad detoxification response towards 9-LOX-oxylipins and xenobiotic compounds. From a set of 18 related group S-bZIP factors isolated in the screen, bZIP11 is known to participate in auxin-mediated root growth and may connect oxylipins to root meristem function. The TF candidates isolated in this screen provide starting points for further attempts to dissect putative signaling pathways involving 9-LOX-derived oxylipins.

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Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites.

Activating epidermal growth factor receptor (EGFR) mutations are recognized biomarkers for patients with metastatic non-small cell lung cancer (NSCLC) treated with EGFR tyrosine kinase inhibitors (TKIs). EGFR TKIs can also have activity against NSCLC without EGFR mutations, requiring the identification of additional relevant biomarkers. Previous studies on tumor EGFR protein levels and EGFR gene copy number revealed inconsistent results. The aim of the study was to identify novel biomarkers of the response to TKIs in NSCLC by investigating whole genome expression at the exon-level. We used exon arrays and clinical samples from a previous trial (SAKK19/05) to investigate the expression variations at the exon-level of 3 genes potentially playing a key role in modulating treatment response: EGFR, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and vascular endothelial growth factor (VEGFA). We identified the expression of EGFR exon 18 as a new predictive marker for patients with untreated metastatic NSCLC treated with bevacizumab and erlotinib in the first line setting. The overexpression of EGFR exon 18 in tumor was significantly associated with tumor shrinkage, independently of EGFR mutation status. A similar significant association could be found in blood samples. In conclusion, exonic EGFR expression particularly in exon 18 was found to be a relevant predictive biomarker for response to bevacizumab and erlotinib. Based on these results, we propose a new model of EGFR testing in tumor and blood.

The integration host factor (IHF) is an abundant nucleoid-associated protein and an essential co-factor for phage λ site-specific recombination and gene regulation in E. coli. Introduction of a sharp DNA kink at specific cognate sites is critical for these functions. Interestingly, the intracellular concentration of IHF is much higher than the concentration needed for site-specific interactions, suggesting that non-specific binding of IHF to DNA plays a role in the physical organization of bacterial chromatin. However, it is unclear how non-specific DNA association contributes to DNA organization. By using a combination of single DNA manipulation and atomic force microscopy imaging methods, we show here that distinct modes of non-specific DNA binding of IHF result in complex global DNA conformations. Changes in KCl and IHF concentrations, as well as tension applied to DNA, dramatically influence the degree of DNA-bending. In addition, IHF can crosslink DNA into a highly compact DNA meshwork that is observed in the presence of magnesium at low concentration of monovalent ions and high IHF-DNA stoichiometries. Our findings provide important insights into how IHF contributes to bacterial chromatin organization, gene regulation, and biofilm formation.

Neuroglobin (Ngb) is a hexacoordinated globin expressed mainly in the central and peripheral nervous system of vertebrates. Although several hypotheses have been put forward regarding the role of neuroglobin, its definite function remains uncertain. Ngb appears to have a neuro-protective role enhancing cell viability under hypoxia and other types of oxidative stress. Ngb is phylogenetically ancient and has a substitution rate nearly four times lower than that of other vertebrate globins, e.g. hemoglobin. Despite its high sequence conservation among vertebrates Ngb seems to be elusive in invertebrates.We determined candidate orthologs in invertebrates and identified a globin of the placozoan Trichoplax adhaerens that is most likely orthologous to vertebrate Ngb and confirmed the orthologous relationship of the polymeric globin of the sea urchin Strongylocentrotus purpuratus to Ngb. The putative orthologous globin genes are located next to genes orthologous to vertebrate POMT2 similarly to localization of vertebrate Ngb. The shared syntenic position of the globins from Trichoplax, the sea urchin and of vertebrate Ngb strongly suggests that they are orthologous. A search for conserved transcription factor binding sites (TFBSs) in the promoter regions of the Ngb genes of different vertebrates via phylogenetic footprinting revealed several TFBSs, which may contribute to the specific expression of Ngb, whereas a comparative analysis with myoglobin revealed several common TFBSs, suggestive of regulatory mechanisms common to globin genes.Identification of the placozoan and echinoderm genes orthologous to vertebrate neuroglobin strongly supports the hypothesis of the early evolutionary origin of this globin, as it shows that neuroglobin was already present in the placozoan-bilaterian last common ancestor. Computational determination of the transcription factor binding sites repertoire provides on the one hand a set of transcriptional factors that are responsible for the specific expression of the Ngb genes and on the other hand a set of factors potentially controlling expression of a couple of different globin genes.

Biogas production from renewable resources is attracting increased attention as an alternative energy source due to the limited availability of traditional fossil fuels. Many countries are promoting the use of alternative energy sources for sustainable energy production. In this study, a metagenome from a production-scale biogas fermenter was analysed employing Roche's GS FLX Titanium technology and compared to a previous dataset obtained from the same community DNA sample that was sequenced on the GS FLX platform. Taxonomic profiling based on 16S rRNA-specific sequences and an Environmental Gene Tag (EGT) analysis employing CARMA demonstrated that both approaches benefit from the longer read lengths obtained on the Titanium platform. Results confirmed Clostridia as the most prevalent taxonomic class, whereas species of the order Methanomicrobiales are dominant among methanogenic Archaea. However, the analyses also identified additional taxa that were missed by the previous study, including members of the genera Streptococcus, Acetivibrio, Garciella, Tissierella, and Gelria, which might also play a role in the fermentation process leading to the formation of methane. Taking advantage of the CARMA feature to correlate taxonomic information of sequences with their assigned functions, it appeared that Firmicutes, followed by Bacteroidetes and Proteobacteria, dominate within the functional context of polysaccharide degradation whereas Methanomicrobiales represent the most abundant taxonomic group responsible for methane production. Clostridia is the most important class involved in the reductive CoA pathway (Wood-Ljungdahl pathway) that is characteristic for acetogenesis. Based on binning of 16S rRNA-specific sequences allocated to the dominant genus Methanoculleus, it could be shown that this genus is represented by several different species. Phylogenetic analysis of these sequences placed them in close proximity to the hydrogenotrophic methanogen Methanoculleus bourgensis. While rarefaction analyses still indicate incomplete coverage, examination of the GS FLX Titanium dataset resulted in the identification of additional genera and functional elements, providing a far more complete coverage of the community involved in anaerobic fermentative pathways leading to methane formation.

The glutamine/asparagine (Q/N)-rich yeast prion protein Sup35 has a low intrinsic propensity to spontaneously self-assemble into ordered, beta-sheet-rich amyloid fibrils. In yeast cells, de novo formation of Sup35 aggregates is greatly facilitated by high protein concentrations and the presence of preformed Q/N-rich protein aggregates that template Sup35 polymerization. Here, we have investigated whether aggregation-promoting polyglutamine (polyQ) tracts can stimulate the de novo formation of ordered Sup35 protein aggregates in the absence of Q/N-rich yeast prions. Fusion proteins with polyQ tracts of different lengths were produced and their ability to spontaneously self-assemble into amlyloid structures was analyzed using in vitro and in vivo model systems. We found that Sup35 fusions with pathogenic (>or=54 glutamines), as opposed to non-pathogenic (19 glutamines) polyQ tracts efficiently form seeding-competent protein aggregates. Strikingly, polyQ-mediated de novo assembly of Sup35 protein aggregates in yeast cells was independent of pre-existing Q/N-rich protein aggregates. This indicates that increasing the content of aggregation-promoting sequences enhances the tendency of Sup35 to spontaneously self-assemble into insoluble protein aggregates. A similar result was obtained when pathogenic polyQ tracts were linked to the yeast prion protein Rnq1, demonstrating that polyQ sequences are generic inducers of amyloidogenesis. In conclusion, long polyQ sequences are powerful molecular tools that allow the efficient production of seeding-competent amyloid structures.