In silico design of context-responsive mammalian promoters with user-defined functionality

Comprehensive de novo-design of complex mammalian promoters is restricted by unpredictable combinatorial interactions between constituent transcription factor regulatory elements (TFREs). In this study, we show that modular binding sites that do not function cooperatively can be identified by analyzing host cell transcription factor expression profiles, and subsequently testing cognate TFRE activities in varying homotypic and heterotypic promoter architectures. TFREs that displayed position-insensitive, additive function within a specific expression context could be rationally combined together in silico to create promoters with highly predictable activities. As TFRE order and spacing did not affect the performance of these TFRE-combinations, compositions could be specifically arranged to preclude the formation of undesirable sequence features. This facilitated simple in silico-design of promoters with context-required, user-defined functionalities. To demonstrate this, we de novo-created promoters for biopharmaceutical production in CHO cells that exhibited precisely designed activity dynamics and long-term expression-stability, without causing observable retroactive effects on cellular performance. The design process described can be utilized for applications requiring context-responsive, customizable promoter function, particularly where co-expression of synthetic TFs is not suitable. Although the synthetic promoter structure utilized does not closely resemble native mammalian architectures, our findings also provide additional support for a flexible billboard model of promoter regulation

Investigation of macrocyclisation routes to 1,4,7-triazacyclononanes : efficient syntheses from 1,2-ditosylamides

Two routes to the synthesis of a cyclohexyl-fused 1,4,7-triazacyclononane involving macrocyclisations of tosamides have been investigated. In the first approach, using a classic Richman-Atkins-type cyclisation of a cyclohexyl-substituted 1,4,7-tritosamide with ethylene glycol ditosylate, afforded the cyclohexyl-fused 1,4,7-triazacyclononane in 5.86% overall yield in four steps. The second, more concise, approach involving the macrocyclisation of trans-cyclohexane-1,2-ditosamide with the tritosyl derivative of diethanolamine initially gave poor yields (< 25%). The well-documented problems with efficiencies in macrocyclisations using 1,2-ditosamides led to the use of a wider range of 1,2-ditosamides including ethane-1,2-ditosamide and propane-1,2-ditosamide. These extended studies led to the development of an efficient macrocyclisation protocol using lithium hydride. This new method afforded 1,4,7-tritosyl-1,4,7-triazacyclononanes in good yield (57-90%) from 1,2-ditosamides in a single step. These efficient methods were then applied to the preparation of a chiral cyclohexyl-fused 1,4,7-tritosyl-1,4,7-triazacyclononane (65-70%). This key chiral intermediate was then converted into a copper(II) complex following detosylation and N-methylation. The resulting chiral copper(II) complex catalysed the aziridination of styrene but it did so in a racemic fashion

Constructing 3D crystal templates for photonic band gap materials using holographic optical tweezers

A simple and robust method is presented for the construction of 3-dimensional crystals from silica and polystyrene microspheres. The crystals are suitable for use as templates in the production of three-dimensional photonic band gap (PBG) materials. Manipulation of the microspheres was achieved using a dynamic holographic assembler (DHA) consisting of computer controlled holographic optical tweezers. Attachment of the microspheres was achieved by adjusting their colloidal interactions during assembly. The method is demonstrated by constructing a variety of 3-dimensional crystals using spheres ranging in size from 3 µm down to 800 nm. A major advantage of the technique is that it may be used to build structures that cannot be made using self-assembly. This is illustrated through the construction of crystals in which line defects have been deliberately included, and by building simple cubic structures

Determination of optimal ultrasound planes for the initialisation of image registration during endoscopic ultrasound-guided procedures

Purpose Navigation of endoscopic ultrasound (EUS)-guided procedures of the upper gastrointestinal (GI) system can be technically challenging due to the small fields-of-view of ultrasound and optical devices, as well as the anatomical variability and limited number of orienting landmarks during navigation. Co-registration of an EUS device and a pre-procedure 3D image can enhance the ability to navigate. However, the fidelity of this contextual information depends on the accuracy of registration. The purpose of this study was to develop and test the feasibility of a simulation-based planning method for pre-selecting patient-specific EUS-visible anatomical landmark locations to maximise the accuracy and robustness of a feature-based multimodality registration method. Methods A registration approach was adopted in which landmarks are registered to anatomical structures segmented from the pre-procedure volume. The predicted target registration errors (TREs) of EUS-CT registration were estimated using simulated visible anatomical landmarks and a Monte Carlo simulation of landmark localisation error. The optimal planes were selected based on the 90th percentile of TREs, which provide a robust and more accurate EUS-CT registration initialisation. The method was evaluated by comparing the accuracy and robustness of registrations initialised using optimised planes versus non-optimised planes using manually segmented CT images and simulated (n=9) or retrospective clinical (n=1) EUS landmarks. Results The results show a lower 90th percentile TRE when registration is initialised using the optimised planes compared with a non-optimised initialisation approach (p value <0.01). Conclusions The proposed simulation-based method to find optimised EUS planes and landmarks for EUS-guided procedures may have the potential to improve registration accuracy. Further work will investigate applying the technique in a clinical setting

Whole synthetic pathway engineering of recombinant protein production

The output from protein biomanufacturing systems is a function of total host cell biomass synthetic capacity and recombinant protein production per unit cell biomass. In this study, we describe how these two properties can be simultaneously optimized via design of a product-specific combination of synthetic DNA parts to maximize flux through the protein synthetic pathway and the use of a host cell chassis with an increased capability to synthesize both cell and product biomass. Using secreted alkaline phosphatase (SEAP) production in Chinese hamster ovary cells as our example, we demonstrate how an optimal composition of input components can be assembled from a minimal toolbox containing rationally designed promoters, untranslated regions, signal peptides, product coding sequences, cell chassis, and genetic effectors. Product titer was increased 10-fold, compared with a standard reference system by (a) identifying genetic components that acted in concert to maximize the rates of SEAP transcription, translation, and translocation, (b) selection of a cell chassis with increased biomass synthetic capacity, and (c) engineering the host cell factory's capacity for protein folding and secretion. This whole synthetic pathway engineering process to design optimal expression cassette-chassis combinations should be applicable to diverse recombinant protein and host cell-type contexts

The visual, the auditory and the haptic – A user study on combining modalities in virtual worlds

Fröhlich J, Wachsmuth I. The visual, the auditory and the haptic – A user study on combining modalities in virtual worlds. In: Shumaker R, ed. Virtual Augmented and Mixed Reality. Designing and Developing Augmented and Virtual Environments. Lecture Notes in Computer Science. Vol 8021. Berlin, Heidelberg: Springer Berlin Heidelberg; 2013: 159-168.In order to make a step further towards understanding the impact of multi-modal stimuli in Virtual Reality we conducted a user study with 80 participants performing tasks in a virtual pit environment. Participants were divided into four groups, each presented a different combination of multi-sensory stimuli. Those included real-time 3D graphics, audio stimuli (ambient, static and event sounds), and haptics consisting of wind and tactile feedback when touching objects. A presence questionnaire was used to evaluate subjectively reported presence on the one hand, and on the other physiological sensors were used to measure heart rate and skin conductance as an objective measure. Results strongly indicate that an increase of modalities does not automatically result in an increase of presence

Dietary iron intakes based on food composition data may underestimate the contribution of potentially exchangeable contaminant iron from soil

Iron intakes calculated from one-day weighed records were compared with those from same day analyzed duplicate diet composites collected from 120 Malawian women living in two rural districts with contrasting soil mineralogy and where threshing may contaminate cereals with soil iron. Soils and diet composites from the two districts were then subjected to a simulated gastrointestinal digestion and iron availability in the digests measured using a Caco-2 cell model. Median analyzed iron intakes (mg/d) were higher (p < 0.001) than calculated intakes in both Zombwe (16.6 vs. 10.1 mg/d) and Mikalango (29.6 vs. 19.1 mg/d), attributed to some soil contaminant iron based on high Al and Ti concentrations in diet composites. A small portion of iron in acidic soil from Zombwe, but not Mikalango calcareous soil, was bioavailable, as it induced ferritin expression in the cells, and may have contributed to higher plasma ferritin and total body iron for the Zombwe women reported earlier, despite lower iron intakes. In conclusion, iron intakes calculated from food composition data were underestimated, highlighting the importance of analyzing duplicate diet composites where extraneous contaminant iron from soil is likely. Acidic contaminant soil may make a small but useful contribution to iron nutrition

A systematic review of reviews identifying UK validated dietary assessment tools for inclusion on an interactive guided website for researchers: www.nutritools.org

Background: Health researchers may struggle to choose suitable validated dietary assessment tools (DATs) for their target population. The aim of this review was to identify and collate information on validated UK DATs and validation studies for inclusion on a website to support researchers to choose appropriate DATs. Design: A systematic review of reviews of DATs was undertaken. DATs validated in UK populations were extracted from the studies identified. A searchable website was designed to display these data. Additionally, mean differences and limits of agreement between test and comparison methods were summarized by a method, weighting by sample size. Results: Over 900 validation results covering 5 life stages, 18 nutrients, 6 dietary assessment methods, and 9 validation method types were extracted from 63 validated DATs which were identified from 68 reviews. These were incorporated into www.nutritools.org. Limits of agreement were determined for about half of validations. Thirty four DATs were FFQs. Only 17 DATs were validated against biomarkers, and only 19 DATs were validated in infant/children/adolescents. Conclusions: The interactive www.nutritools.org website holds extensive validation data identified from this review and can be used to guide researchers to critically compare and choose a suitable DAT for their research question, leading to improvement of nutritional epidemiology research

Electric current circuits in astrophysics

Cosmic magnetic structures have in common that they are anchored in a dynamo, that an external driver converts kinetic energy into internal magnetic energy, that this magnetic energy is transported as Poynting fl ux across the magnetically dominated structure, and that the magnetic energy is released in the form of particle acceleration, heating, bulk motion, MHD waves, and radiation. The investigation of the electric current system is particularly illuminating as to the course of events and the physics involved. We demonstrate this for the radio pulsar wind, the solar flare, and terrestrial magnetic storms