CATANA: an online modelling environment for proteins and nucleic acid nanostructures.

In the last decade, significant advances have been made towards the rational design of proteins, DNA, and other organic nanostructures. The emerging possibility to precisely engineer molecular structures resulted in a wide range of new applications in fields such as biotechnology or medicine. The complexity and size of the artificial molecular systems as well as the number of interactions are greatly increasing and are manifesting the need for computational design support. In addition, a new generation of AI-based structure prediction tools provides researchers with completely new possibilities to generate recombinant proteins and functionalized DNA nanostructures. In this study, we present Catana, a web-based modelling environment suited for proteins and DNA nanostructures. User-friendly features were developed to create and modify recombinant fusion proteins, predict protein structures based on the amino acid sequence, and manipulate DNA origami structures. Moreover, Catana was jointly developed with the novel Unified Nanotechnology Format (UNF). Therefore, it employs a state-of-the-art coarse-grained data model, that is compatible with other established and upcoming applications. A particular focus was put on an effortless data export to allow even inexperienced users to perform in silico evaluations of their designs by means of molecular dynamics simulations. Catana is freely available at http://catana.ait.ac.at/.

Circulating serum microRNAs including senescent miR-31-5p are associated with incident fragility fractures in older postmenopausal women with type 2 diabetes mellitus.

Fragility fractures are an important hallmark of aging and an increasingly recognized complication of Type 2 diabetes (T2D). T2D individuals have been found to exhibit an increased fracture risk despite elevated bone mineral density (BMD) by dual x-ray absorptiometry (DXA). However, BMD and FRAX-scores tend to underestimate fracture risk in T2D. New, reliable biomarkers are therefore needed. MicroRNAs (miRNAs) are secreted into the circulation from cells of various tissues proportional to local disease severity. Serum miRNA-classifiers were recently found to discriminate T2D women with and without prevalent fragility fractures with high specificity and sensitivity (AUC > 0.90). However, the association of circulating miRNAs with incident fractures in T2D has not been examined yet. In 168 T2D postmenopausal women in the AGES-Reykjavik cohort, miRNAs were extracted from baseline serum and a panel of 10 circulating miRNAs known to be involved in diabetic bone disease and aging was quantified by qPCR and Ct-values extracted. Unadjusted and adjusted Cox proportional hazard models assessed the associations between serum miRNAs and incident fragility fracture. Additionally, Receiver operating curve (ROC) analyses were performed. Of the included 168 T2D postmenopausal women who were on average 77.2 ±â€¯5.6 years old, 70 experienced at least one incident fragility fracture during the mean follow-up of 5.8 ±â€¯2.7 years. We found that 3 serum miRNAs were significantly associated with incident diabetic fragility fracture: while low expression of miR-19b-1-5p was associated with significantly lower risk of incident fragility fracture (HR 0.84 (95% CI: 0.71-0.99, p = 0.0323)), low expression of miR-203a and miR-31-5p was each significantly associated with a higher risk of incident fragility fracture per unit increase in Ct-value (miR-203a: HR 1.29 (95% CI: 1.12-1.49), p = 0.0004, miR-31-5p HR 1.27 (95% CI: 1.06-1.52), p = 0.009). Hazard ratios of the latter two miRNAs remained significant after adjustments for age, body mass index (BMI), areal bone mineral density (aBMD), clinical FRAX or FRAXaBMD. Women with miR-203a and miR-31-5p serum levels in the lowest expression quartiles exhibited a 2.4-3.4-fold larger fracture risk than women with miR-31-5p and miR-203a serum expressions in the highest expression quartile (0.002 ≤ p ≤ 0.039). Women with both miR-203a and miR-31-5p serum levels below the median had a significantly increased fracture risk (Unadjusted HR 3.26 (95% CI: 1.57-6.78, p = 0.001) compared to those with both expression levels above the median, stable to adjustments. We next built a diabetic fragility signature consisting of the 3 miRNAs that showed the largest associations with incident fracture (miR-203a, miR-31-5p, miR-19b-1-5p). This 3-miRNA signature showed with an AUC of 0.722 comparable diagnostic accuracy in identifying incident fractures to any of the clinical parameters such as aBMD, Clinical FRAX or FRAXaBMD alone. When the 3 miRNAs were combined with aBMD, this combined 4-feature signature performed with an AUC of 0.756 (95% CI: 0.680, 0.823) significantly better than aBMD alone (AUC 0.666, 95% CI: 0.585, 0.741) (p = 0.009). Our data indicate that specific serum microRNAs including senescent miR-31-5p are associated with incident fragility fracture in older diabetic women and can significantly improve fracture risk prediction in diabetics when combined with aBMD measurements of the femoral neck.

Longitudinal Metabolic Biomarker Profile of Hyperketonemic Cows from Dry-Off to Peak Lactation and Identification of Prognostic Classifiers.

Currently about 30% to 50% of all dairy cows are affected by a metabolic or infectious disease during the transition period. A key factor for preventive actions is the ability to precisely predict metabolic diseases at an early stage. We report the longitudinal metabolic profile of non-esterified fatty acids, beta-hydroxybutyrate (BHB), total bilirubin, and aspartate aminotransferase in hyperketonemic dairy cows. Aiming for a novel measurement regime to improve metabolic health in dairy cows, we evaluated prognostic classifiers for hyperketonemia. In the observational longitudinal study, 99 healthy adult primiparous and multiparous Simmental dairy cows were included. Every cow was monitored weekly for 14 consecutive weeks, beginning two weeks prior to the expected day of parturition until peak lactation. Cows with serum concentrations of BHB > 0.8 mmol/L were considered hyperketonemic. Biomarker profiles were fitted by the maximum likelihood method using a mixed effects natural cubic spline model. In the hyperketonemic group, the BHB profile remained significantly higher than that of the control group until the end of the study period. As a prognostic classifier, the cut-off level of 0.54 mmol/L BHB measured on the 10th day post partum had the highest area under the curve. These results provide new longitudinal insights into the metabolic biomarker progression of dairy cows and enable an early onset diagnosis of hyperketonemia.

Influence of Different Lactation Stages on Circadian Rhythmicity of Metabolic Biomarkers in Dairy Cows: A Pilot Study.

Currently, subclinical metabolic imbalances at the individual cow and herd level are detected by measuring biomarkers in single blood samples. However, diurnal variations have not been fully described yet but need to be considered when sampling for a robust ad consistent analysis. The study describes the influence of lactation phases on circadian rhythms and diurnal variations for non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHB), total bilirubin (tBIL) and aspartate aminotransferase (AST) in dairy cows. In an observational pilot study, we used 16 clinically healthy Simmental dairy cows subdivided in four different lactation stages (dry-off, fresh, high and late lactating). Every cow was monitored for 24 h, with blood sampling and assessment of clinical parameters every 2 h. Time and lactation stage influence the concentration of the biomarkers NEFA, BHB and tBIL in serum. Further, circadian rhythmicity was found in high lactating cows for NEFA peaking at 5:39 am and BHB peaking at 4:20 pm. We suggest blood sampling for single-point measurements within three hours after the first feeding until two hours after the last feeding of the day. The results provide a new insight into the physiology of circadian rhythms in dairy cows and enable improved metabolic monitoring.

Full pathogen characterisation: species identification including the detection of virulence factors and antibiotic resistance genes via multiplex DNA-assays.

Antibiotic resistances progressively cause treatment failures, and their spreading dynamics reached an alarming level. Some strains have already been classified as highly critical, e.g. the ones summarised by the acronym ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.). To restrain this trend and enable effective medication, as much information as possible must be obtained in the least possible time. Here, we present a DNA microarray-based assay that screens for the most important sepsis-relevant 44 pathogenic species, 360 virulence factors (mediate pathogenicity in otherwise non-pathogenic strains), and 409 antibiotic resistance genes in parallel. The assay was evaluated with 14 multidrug resistant strains, including all ESKAPE pathogens, mainly obtained from clinical isolates. We used a cost-efficient ligation-based detection platform designed to emulate the highly specific multiplex detection of padlock probes. Results could be obtained within one day, requiring approximately 4 h for amplification, application to the microarray, and detection.

The Brownian and Flow-Driven Rotational Dynamics of a Multicomponent DNA Origami-Based Rotor.

Nanomechanical devices are becoming increasingly popular due to the very diverse field of potential applications, including nanocomputing, robotics, and drug delivery. DNA is one of the most promising building materials to realize complex 3D structures at the nanoscale level. Several mechanical DNA origami structures have already been designed capable of simple operations such as a DNA box with a controllable lid, bipedal walkers, and cargo sorting robots. However, the nanomechanical properties of mechanically interlinked DNA nanostructures that are in general highly deformable have yet to be extensively experimentally evaluated. In this work, a multicomponent DNA origami-based rotor is created and fully characterized by electron microscopy under negative stain and cryo preparations. The nanodevice is further immobilized on a microfluidic chamber and its Brownian and flow-driven rotational behaviors are analyzed in real time by single-molecule fluorescence microscopy. The rotation in previous DNA rotors based either on strand displacement, electric field or Brownian motion. This study is the first to attempt to manipulate the dynamics of an artificial nanodevice with fluidic flow as a natural force.

Preventive sublingual immunotherapy with House Dust Mite extract modulates epitope diversity in pre-school children.

BACKGROUND: The preventive effect of allergen immunotherapy (AIT) on allergy and asthma development is currently assessed using primary and secondary AIT approaches. Knowledge of the immunological effects of these interventions is limited and the impact on epitope diversity remains to be defined. METHODS: We used high-density peptide arrays that included all known Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) allergens and the whole proteome of Der f to study changes in House Dust Mite (HDM) linear peptide recognition during a 2-year preventive double-blind placebo-controlled sublingual HDM AIT pilot study in 2-5-year-old children with sensitization to HDM but without symptoms. RESULTS: Preventive AIT-treated patients showed significantly higher IgG epitope diversity to HDM allergens compared to placebo-treated individuals at 24 months of treatment (P < 0.05), while no increase in IgE diversity was seen. At 24 months of treatment, IgG4 diversity for HDM allergens was significantly higher in the pAIT-treated patients compared to placebo group (P < 0.05). Potentially beneficial changes in epitope recognition throughout the treatment are also seen in peptides derived from Der f proteome. CONCLUSION: These data suggest a beneficial immunomodulation of preventive sublingual immunotherapy at a molecular level by favoring a broader blocking repertoire and inhibiting epitope spreading.

Serum miRNA Signatures Are Indicative of Skeletal Fractures in Postmenopausal Women With and Without Type 2 Diabetes and Influence Osteogenic and Adipogenic Differentiation of Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro.

Standard DXA measurements, including Fracture Risk Assessment Tool (FRAX) scores, have shown limitations in assessing fracture risk in Type 2 Diabetes (T2D), underscoring the need for novel biomarkers and suggesting that other pathomechanisms may drive diabetic bone fragility. MicroRNAs (miRNAs) are secreted into the circulation from cells of various tissues proportional to local disease severity and were recently found to be crucial to bone homeostasis and T2D. Here, we studied, if and which circulating miRNAs or combinations of miRNAs can discriminate best fracture status in a well-characterized study of diabetic bone disease and postmenopausal osteoporosis (n = 80 postmenopausal women). We then tested the most discriminative and most frequent miRNAs in vitro. Using miRNA-qPCR-arrays, we showed that 48 miRNAs can differentiate fracture status in T2D women and that several combinations of four miRNAs can discriminate diabetes-related fractures with high specificity and sensitivity (area under the receiver-operating characteristic curve values [AUCs], 0.92 to 0.96; 95% CI, 0.88 to 0.98). For the osteoporotic study arm, 23 miRNAs were fracture-indicative and potential combinations of four miRNAs showed AUCs from 0.97 to 1.00 (95% CI, 0.93 to 1.00). Because a role in bone homeostasis for those miRNAs that were most discriminative and most present among all miRNA combinations had not been described, we performed in vitro functional studies in human adipose tissue-derived mesenchymal stem cells to investigate the effect of miR-550a-5p, miR-188-3p, and miR-382-3p on osteogenesis, adipogenesis, and cell proliferation. We found that miR-382-3p significantly enhanced osteogenic differentiation (p < 0.001), whereas miR-550a-5p inhibited this process (p < 0.001). Both miRNAs, miR-382-3p and miR-550a-5p, impaired adipogenic differentiation, whereas miR-188-3p did not exert an effect on adipogenesis. None of the miRNAs affected significantly cell proliferation. Our data suggest for the first time that miRNAs are linked to fragility fractures in T2D postmenopausal women and should be further investigated for their diagnostic potential and their detailed function in diabetic bone. © 2016 American Society for Bone and Mineral Research.

Depth-Related Effects on a Meiofaunal Community Dwelling in the Periphyton of a Mesotrophic Lake.

Periphyton is a complex assemblage of micro- and meiofauna embedded in the organic matrix that coats most submerged substrate in the littoral of lakes. The aim of this study was to better understand the consequences of depth-level fluctuation on a periphytic community. The effects of light and wave disturbance on the development of littoral periphyton were evaluated in Lake Erken (Sweden) using an experimental design that combined in situ shading with periphyton depth transfers. Free-living nematodes were a major contributor to the meiofaunal community. Their species composition was therefore used as a proxy to distinguish the contributions of light- and wave-related effects. The periphyton layer was much thicker at a depth of 30 cm than at 200 cm, as indicated by differences in the amounts of organic and phototrophic biomass and meiofaunal and nematode densities. A reduction of the depth-level of periphyton via a transfer from a deep to a shallow location induced rapid positive responses by its algal, meiofaunal, and nematode communities. The slower and weaker negative responses to the reverse transfer were attributed to the potentially higher resilience of periphytic communities to increases in the water level. In the shallow littoral of the lake, shading magnified the effects of phototrophic biomass erosion by waves, as the increased exposure to wave shear stress was not compensated for by an increase in photosynthesis. This finding suggests that benthic primary production will be strongly impeded in the shallow littoral zones of lakes artificially shaded by construction or embankments. However, regardless of the light constraints, an increased exposure to wave action had a generally positive short-term effect on meiofaunal density, by favoring the predominance of species able to anchor themselves to the substrate, especially the Chromadorid nematode Punctodora ratzeburgensis.

Life cycle and population growth rate of Caenorhabditis elegans studied by a new method.

BACKGROUND: The free-living nematode Caenorhabditis elegans is the predominant model organism in biological research, being used by a huge number of laboratories worldwide. Many researchers have evaluated life-history traits of C. elegans in investigations covering quite different aspects such as ecotoxicology, inbreeding depression and heterosis, dietary restriction/supplement, mutations, and ageing. Such traits include juvenile growth rates, age at sexual maturity, adult body size, age-specific fecundity/mortality, total reproduction, mean and maximum lifespan, and intrinsic population growth rates. However, we found that in life-cycle experiments care is needed regarding protocol design. Here, we test a recently developed method that overcomes some problems associated with traditional cultivation techniques. In this fast and yet precise approach, single individuals are maintained within hanging drops of semi-fluid culture medium, allowing the simultaneous investigation of various life-history traits at any desired degree of accuracy. Here, the life cycles of wild-type C. elegans strains N2 (Bristol, UK) and MY6 (Münster, Germany) were compared at 20 degrees C with 5 x 10(9) Escherichia coli ml-1 as food source. RESULTS: High-resolution life tables and fecundity schedules of the two strains are presented. Though isolated 700 km and 60 years apart from each other, the two strains barely differed in life-cycle parameters. For strain N2 (n = 69), the intrinsic rate of natural increase (r m d(-1)), calculated according to the Lotka equation, was 1.375, the net reproductive rate (R 0) 291, the mean generation time (T) 90 h, and the minimum generation time (T min) 73.0 h. The corresponding values for strain MY6 (n = 72) were r m = 1.460, R0 = 289, T = 84 h, and T min = 67.3 h. Peak egg-laying rates in both strains exceeded 140 eggs d(-1). Juvenile and early adulthood mortality was negligible. Strain N2 lived, on average, for 16.7 d, while strain MY6 died 2 days earlier; however, differences in survivorship curves were statistically non-significant. CONCLUSION: We found no evidence that adaptation to the laboratory altered the life history traits of C. elegans strain N2. Our results, discussed in the light of earlier studies on C. elegans, demonstrate certain advantages of the hanging drop method in investigations of nematode life cycles. Assuming that its reproducibility is validated in further studies, the method will reduce the inter-laboratory variability of life-history estimates and may ultimately prove to be more convenient than the current standard methods used by C. elegans researchers.