Quantitative characterization of translational riboregulators using an in vitro transcription–translation system

Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo. However, riboregulators are generally designed in silico and tested in vivo, which provides little quantitative information about their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX–TL) system provides valuable information about the performances of in silico designed riboregulators. We first propose a simple model that provides a quantitative definition of the dynamic range of a riboregulator. We further characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off, in agreement with our model, and that relative dynamic ranges of riboregulators determined in vitro are in agreement with published in vivo data. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX–TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico

Structure and relaxations in liquid and amorphous Selenium

We report a molecular dynamics simulation of selenium, described by a three-body interaction. The temperatures T_g and T_c and the structural properties are in agreement with experiment. The mean nearest neighbor coordination number is 2.1. A small pre-peak at about 1 AA^-1 can be explained in terms of void correlations. In the intermediate self-scattering function, i.e. the density fluctuation correlation, classical behavior, alpha- and beta-regimes, is found. We also observe the plateau in the beta-regime below T_g. In a second step, we investigated the heterogeneous and/or homogeneous behavior of the relaxations. At both short and long times the relaxations are homogeneous (or weakly heterogeneous). In the intermediate time scale, lowering the temperature increases the heterogeneity. We connect these different domains to the vibrational (ballistic), beta- and alpha-regimes. We have also shown that the increase in heterogeneity can be understood in terms of relaxations

A framework for co-construction of knowledge within eLearning

Instructional designers, course developers and academics require ways to create eLearning courses rapidly and support learners to be engaged with authentic learning tasks. Social media tools within eLearning courses are in the discovery phase and rely heavily on social constructivist design that includes meta-cognition, collaborative learning, active knowledge-making and recursive feedback. Emergent models and frameworks that allow for optimisation of social networking and the co-construction of knowledge are required for sound eLearning design. This research study investigates the effectiveness of learning design elements and identifies components informed by a constructivist instructional design (C-ID) approach. The study explores how learning design elements that facilitates the co-construction of knowledge can be implemented within a framework applicable to the Australian eLearning higher education context. Pragmatism as a research paradigm views knowledge as constructed and based on the reality of the world one experiences and lives in, and aligns with a social constructivist approach. Following on from a pragmatic viewpoint, this study selected exploratory sequential design within a mixed methods approach as it enables both narrative data collection and numerical analysis. A group of subject-matter experts from Australian higher education institutions were purposefully invited to participate in an eDelphi expert panel. A total of 53 (n = 53) instructional designers and academics that adhered to the selection criteria were contacted by means of email. Seventeen (17) respondents agreed to the study, resulting in a response rate of 32%. Qualitative data sets of semi-structured interviews with respondents were analysed to determine emergent themes and topics. An electronic questionnaire was administered to a larger sample. The survey was sent out to 434 prospective respondents, of which 143 started the survey and n = 113 respondents completed all Likert scale questions in the survey. The Wilcoxon rank-sum test and Kruskal–Wallis process were applied to investigate if the perception of respondents was different depending on variables identified for the research. Complex, multi-layered eLearning modalities are a new field within instructional design, and there are minimal empirical studies for instructional designers and course developers to find shared meaning of critical learning design elements. The results from the study indicated that learning design elements associated with constructivist concepts such as social learning presence, learning interaction, meta-cognitive load, knowledge-sharing space and knowledge construction are conducive to social networking and the co-construction of knowledge. The categories emerging from coding of the data sets were learning activities and interactivities, social communication, collaboration, diversity, fears and the student-lecturer relationship. These categories need to be considered when designing for social learning interaction as they are aimed at addressing human interaction with technology. Relationships surrounding activities therefore need to be taken into account when designing the learning activities, and not merely the learning content and outcomes. The instructional guidelines considered most important when designing for online social interaction were authentic, meaningful and relevant instruction; conscious modelling of behaviour; rules for engagement; user-centred design; and spontaneous design. Establishing a strong social learning presence fosters the building of trust and opportunities to create social learning experiences critical for social networking. The eLearning framework presented by this study underpins a shared meaning, categories and recommended learning activities that can be utilised by instructional designers, academics and course developers when creating multi-layered complex online learning spaces

The diversity of cyanobacterial metabolism: genome analysis of multiple phototrophic microorganisms

<p>Abstract</p> <p>Background</p> <p>Cyanobacteria are among the most abundant organisms on Earth and represent one of the oldest and most widespread clades known in modern phylogenetics. As the only known prokaryotes capable of oxygenic photosynthesis, cyanobacteria are considered to be a promising resource for renewable fuels and natural products. Our efforts to harness the sun's energy using cyanobacteria would greatly benefit from an increased understanding of the genomic diversity across multiple cyanobacterial strains. In this respect, the advent of novel sequencing techniques and the availability of several cyanobacterial genomes offers new opportunities for understanding microbial diversity and metabolic organization and evolution in diverse environments.</p> <p>Results</p> <p>Here, we report a whole genome comparison of multiple phototrophic cyanobacteria. We describe genetic diversity found within cyanobacterial genomes, specifically with respect to metabolic functionality. Our results are based on pair-wise comparison of protein sequences and concomitant construction of clusters of likely ortholog genes. We differentiate between core, shared and unique genes and show that the majority of genes are associated with a single genome. In contrast, genes with metabolic function are strongly overrepresented within the core genome that is common to all considered strains. The analysis of metabolic diversity within core carbon metabolism reveals parts of the metabolic networks that are highly conserved, as well as highly fragmented pathways.</p> <p>Conclusions</p> <p>Our results have direct implications for resource allocation and further sequencing projects. It can be extrapolated that the number of newly identified genes still significantly increases with increasing number of new sequenced genomes. Furthermore, genome analysis of multiple phototrophic strains allows us to obtain a detailed picture of metabolic diversity that can serve as a starting point for biotechnological applications and automated metabolic reconstructions.</p

A motif-based search in bacterial genomes identifies the ortholog of the small RNA Yfr1 in all lineages of cyanobacteria

<p>Abstract</p> <p>Background</p> <p>Non-coding RNAs (ncRNA) are regulators of gene expression in all domains of life. They control growth and differentiation, virulence, motility and various stress responses. The identification of ncRNAs can be a tedious process due to the heterogeneous nature of this molecule class and the missing sequence similarity of orthologs, even among closely related species. The small ncRNA Yfr1 has previously been found in the <it>Prochlorococcus/Synechococcus </it>group of marine cyanobacteria.</p> <p>Results</p> <p>Here we show that screening available genome sequences based on an RNA motif and followed by experimental analysis works successfully in detecting this RNA in all lineages of cyanobacteria. Yfr1 is an abundant ncRNA between 54 and 69 nt in size that is ubiquitous for cyanobacteria except for two low light-adapted strains of <it>Prochlorococcus</it>, MIT 9211 and SS120, in which it must have been lost secondarily. Yfr1 consists of two predicted stem-loop elements separated by an unpaired sequence of 16–20 nucleotides containing the ultraconserved undecanucleotide 5'-ACUCCUCACAC-3'.</p> <p>Conclusion</p> <p>Starting with an ncRNA previously found in a narrow group of cyanobacteria only, we show here the highly specific and sensitive identification of its homologs within all lineages of cyanobacteria, whereas it was not detected within the genome sequences of <it>E. coli </it>and of 7 other eubacteria belonging to the alpha-proteobacteria, chlorobiaceae and spirochaete. The integration of RNA motif prediction into computational pipelines for the detection of ncRNAs in bacteria appears as a promising step to improve the quality of such predictions.</p

Identification of cyanobacterial non-coding RNAs by comparative genome analysis

BACKGROUND: Whole genome sequencing of marine cyanobacteria has revealed an unprecedented degree of genomic variation and streamlining. With a size of 1.66 megabase-pairs, Prochlorococcus sp. MED4 has the most compact of these genomes and it is enigmatic how the few identified regulatory proteins efficiently sustain the lifestyle of an ecologically successful marine microorganism. Small non-coding RNAs (ncRNAs) control a plethora of processes in eukaryotes as well as in bacteria; however, systematic searches for ncRNAs are still lacking for most eubacterial phyla outside the enterobacteria. RESULTS: Based on a computational prediction we show the presence of several ncRNAs (cyanobacterial functional RNA or Yfr) in several different cyanobacteria of the Prochlorococcus-Synechococcus lineage. Some ncRNA genes are present only in two or three of the four strains investigated, whereas the RNAs Yfr2 through Yfr5 are structurally highly related and are encoded by a rapidly evolving gene family as their genes exist in different copy numbers and at different sites in the four investigated genomes. One ncRNA, Yfr7, is present in at least seven other cyanobacteria. In addition, control elements for several ribosomal operons were predicted as well as riboswitches for thiamine pyrophosphate and cobalamin. CONCLUSION: This is the first genome-wide and systematic screen for ncRNAs in cyanobacteria. Several ncRNAs were both computationally predicted and their presence was biochemically verified. These RNAs may have regulatory functions and each shows a distinct phylogenetic distribution. Our approach can be applied to any group of microorganisms for which more than one total genome sequence is available for comparative analysis

Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species

Bax is a key regulator of apoptosis that mediates the release of cytochrome c to the cytosol via oligomerization in the outer mitochondrial membrane before pore formation. However, the molecular mechanism of Bax assembly and regulation by other Bcl-2 members remains obscure. Here, by analysing the stoichiometry of Bax oligomers at the single-molecule level, we find that Bax binds to the membrane in a monomeric state and then self-assembles in &lt;1min. Strikingly, active Bax does not exist in a unique oligomeric state, but as several different species based on dimer units. Moreover, we show that cBid activates Bax without affecting its assembly, while Bcl-xL induces the dissociation of Bax oligomers. On the basis of our experimental data and theoretical modelling, we propose a new mechanism for the molecular pathway of Bax assembly to form the apoptotic pore

Unmet needs in the treatment of idiopathic pulmonary fibrosis―insights from patient chart review in five European countries

Background: Two antifibrotic drugs, pirfenidone and nintedanib, are approved by the European Medicines Agency and the US Food and Drug Administration for the treatment of idiopathic pulmonary fibrosis (IPF). In this analysis, treatment patterns of European patients with IPF were investigated to understand antifibrotic prescribing and identify unmet needs in IPF treatment practice. Methods: Between February and March 2016, respiratory physicians from France, Germany, Italy, Spain, and the UK participated in an online questionnaire designed to collect information on IPF treatment patterns in patients under their care. Patients were categorized as treated (received approved antifibrotics) or untreated (did not receive approved antifibrotics, but may have received other unapproved therapies). Classification of IPF diagnosis (confirmed/suspected) and severity ('mild'/'moderate'/'severe') for each patient was based on the individual physician's report. Patients' perspectives were not recorded in this study. Results: In total, 290 physicians responded to the questionnaire. Overall, 54% of patients with IPF did not receive treatment with an approved antifibrotic. More patients had a confirmed IPF diagnosis in the treated (84%) versus the untreated (51%) population. Of patients with a confirmed diagnosis, 40% did not receive treatment. The treated population was younger than the untreated population (67 vs 70 years, respectively; p = 0.01), with more frequent multidisciplinary team evaluation (83% vs 57%, respectively; p = 0.01). A higher proportion of untreated patients had forced vital capacity > 80% at diagnosis versus treated patients. Of patients with 'mild' IPF, 71% did not receive an approved antifibrotic versus 41% and 60% of patients with 'moderate' and 'severe' IPF, respectively. Conclusions: Despite the availability of antifibrotic therapies, many European patients with confirmed IPF do not receive approved antifibrotic treatment. Importantly, there appears to be a reluctance to treat patients with 'mild' or 'stable' disease, and instead adopt a 'watch and wait' approach. More education is required to address diagnostic uncertainty, poor understanding of IPF and its treatments, and issues of treatment access. There is a need to increase physician awareness of the benefits associated with antifibrotic treatment across the spectrum of IPF severity

Food Loss and Waste country profile for Colombia - Estimates of Food Loss and Waste, associated GHG emissions and nutritional losses

The UN and the Champions 12.3 Coalition launched the ‘Target-Measure-Act approach’ calling on all governments and companies to set FLW reduction targets, measure FLW, identify hotspots, and to take action to reduce FLW accordingly (Lipinski, 2020). Lack of data makes it particularly difficult for low- and middle-income countries (LMIC) to specify the hotspot food products and chain stages, to define smart targets and to identify adequate interventions. In order to contribute to this essential information we developed and used a mass flow model based on secondary data (see next section for details). This approach allows to present an indicative country profile showing per food product category and chain stage not only the amount of FLW but also the GHGEs related to producing the FLW and induced nutrient losses. This integrated approach towards FLW reduction can support policy makers and other food system actors taking informed decisions contributing to several sustainability objectives in parallel