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

Haematozoa of wild catfishes in northern Australia

Very little is known about the diversity, prevalence, or pathogenicity of haematozoa in Australian freshwater fishes. Blood smears from 189 native catfishes, of six different species, from northern Australia were examined for haematozoa. Haematozoan infections were observed only in fishes from Queensland, at an overall prevalence of 0.191 (95% CI = 0.134-0.265). Intraerythrocytic haemogregarines were present in Neoarius graeffei from the Brisbane River at a prevalence of 0.35 (0.181-0.567). Trypanosomes were present in Tandanus species from four rivers, at prevalences ranging from 0.111 (0.020-0.330) to 1 (0.635-1), and in N. graeffei from one river in Queensland, at a prevalence of 0.063 (0.003-0.305). The haematozoans observed appeared to have little impact on their hosts. Tandanus spp. were significantly more likely to be infected with trypanosomes, suggesting a high parasite-host specificity. This is the first widespread survey of wild Australian freshwater catfishes for haematozoa, resulting in the first report of haemogregarines from Australian freshwater fish, and the first report of trypanosomes from Neoarius graeffei and Tandanus tropicanus

The Impact of Iterative Reconstruction on Computed Tomography Radiation Dosimetry: Evaluation in a Routine Clinical Setting

PURPOSE: To evaluate the effect of introduction of iterative reconstruction as a mandated software upgrade on radiation dosimetry in routine clinical practice over a range of computed tomography examinations. METHODS: Random samples of scanning data were extracted from a centralised Picture Archiving Communication System pertaining to 10 commonly performed computed tomography examination types undertaken at two hospitals in Western Australia, before and after the introduction of iterative reconstruction. Changes in the mean dose length product and effective dose were evaluated along with estimations of associated changes to annual cancer incidence. RESULTS: We observed statistically significant reductions in the effective radiation dose for head computed tomography (22-27%) consistent with those reported in the literature. In contrast the reductions observed for non-contrast chest (37-47%); chest pulmonary embolism study (28%), chest/abdominal/pelvic study (16%) and thoracic spine (39%) computed tomography. Statistically significant reductions in radiation dose were not identified in angiographic computed tomography. Dose reductions translated to substantial lowering of the lifetime attributable risk, especially for younger females, and estimated numbers of incident cancers. CONCLUSION: Reduction of CT dose is a priority Iterative reconstruction algorithms have the potential to significantly assist with dose reduction across a range of protocols. However, this reduction in dose is achieved via reductions in image noise. Fully realising the potential dose reduction of iterative reconstruction requires the adjustment of image factors and forgoing the noise reduction potential of the iterative algorithm. Our study has demonstrated a reduction in radiation dose for some scanning protocols, but not to the extent experimental studies had previously shown or in all protocols expected, raising questions about the extent to which iterative reconstruction achieves dose reduction in real world clinical practice

A higher order control volume based finite element method to prodict the deformation of heterogeneous materials

Materials with obvious internal structure can exhibit behaviour, under loading, that cannot be described by classical elasticity. It is therefore important to develop computational tools incorporating appropriate constitutive theories that can capture their unconventional behaviour. One such theory is micropolar elasticity. This paper presents a linear strain control volume finite element formulation incorporating micropolar elasticity. Verification results from a micropolar element patch test as well as convergence results for a stress concentration problem are included. The element will be shown to pass the patch test and also exhibit accuracy that is at least equivalent to its finite element counterpart

Cryptosporidium in fish: alternative sequencing approaches and analyses at multiple loci to resolve mixed infections

Currently, the systematics, biology and epidemiology of piscine Cryptosporidium species are poorly understood. Here, we compared Sanger ‒ and next-generation ‒ sequencing (NGS), of piscine Cryptosporidium, at the 18S rRNA and actin genes. The hosts comprised 11 ornamental fish species, spanning four orders and eight families. The objectives were: to (i) confirm the rich genetic diversity of the parasite and the high frequency of mixed infections; and (ii) explore the potential of NGS in the presence of complex genetic mixtures. By Sanger sequencing, four main genotypes were obtained at the actin locus, while for the 18S locus, seven genotypes were identified. At both loci, NGS revealed frequent mixed infections, consisting of one highly dominant variant plus substantially rarer genotypes. Both sequencing methods detected novel Cryptosporidium genotypes at both loci, including a novel and highly abundant actin genotype that was identified by both Sanger sequencing and NGS. Importantly, this genotype accounted for 68·9% of all NGS reads from all samples (249 585/362 372). The present study confirms that aquarium fish can harbour a large and unexplored Cryptosporidium genetic diversity. Although commonly used in molecular parasitology studies, nested PCR prevents quantitative comparisons and thwarts the advantages of NGS, when this latter approach is used to investigate multiple infections

The influence of void size on the micropolar constitutive properties of model heterogeneous materials

In this paper the mechanical behaviour of model heterogeneous materials consisting of regular periodic arrays of circular voids within a polymeric matrix is investigated. Circular ring samples of the materials were fabricated by machining the voids into commercially available polymer sheet. Ring samples of differing sizes but similar geometries were loaded using mechanical testing equipment. Sample stiffness was found to depend on sample size with stiffness increasing as size reduced. The periodic nature of the void arrays also facilitated detailed finite element analysis of each sample. The results obtained by analysis substantiate the observed dependence of stiffness on size. Classical elasticity theory does not acknowledge this size effect but more generalized elasticity theories do predict it. Micropolar elasticity theory has therefore been used to interpret the sample stiffness data and identify constitutive properties. Modulus values for the model materials have been quantified. Values of two additional constitutive properties, the characteristic length and the coupling number, which are present within micropolar elasticity but absent from its classic counterpart have also been determined. The dependence of these additional properties on void size has been investigated and characteristic length values compared to the length scales inherent within the structure of the model materials

Characterisation of the material and mechanical properties of atomic force microscope cantilevers with a plan-view trapezoidal geometry

Cantilever devices have found applications in numerous scientific fields and instruments, including the atomic force microscope (AFM), and as sensors to detect a wide range of chemical and biological species. The mechanical properties, in particular, the spring constant of these devices is crucial when quantifying adhesive forces, material properties of surfaces, and in determining deposited mass for sensing applications. A key component in the spring constant of a cantilever is the plan-view shape. In recent years, the trapezoidal plan-view shape has become available since it offers certain advantages to fast-scanning AFM and can improve sensor performance in fluid environments. Euler beam equations relating cantilever stiffness to the cantilever dimensions and Young’s modulus have been proven useful and are used extensively to model cantilever mechanical behaviour and calibrate the spring constant. In this work, we derive a simple correction factor to the Euler beam equation for a beam-shaped cantilever that is applicable to any cantilever with a trapezoidal plan-view shape. This correction factor is based upon previous analytical work and simplifies the application of the previous researchers formula. A correction factor to the spring constant of an AFM cantilever is also required to calculate the torque produced by the tip when it contacts the sample surface, which is also dependent on the plan-view shape. In this work, we also derive a simple expression for the torque for triangular plan-view shaped cantilevers and show that for the current generation of trapezoidal plan-view shaped AFM cantilevers, this will be a good approximation. We shall apply both these correction factors to determine Young’s modulus for a range of trapezoidal-shaped AFM cantilevers, which are specially designed for fast-scanning. These types of AFM probes are much smaller in size when compared to standard AFM probes. In the process of analysing the mechanical properties of these cantilevers, important insights are also gained into their spring constant calibration and dimensional factors that contribute to the variability in their spring constant.Ashley D. Slattery, Adam J. Blanch, Cameron J. Shearer, Andrew J. Stapleton, Renee V. Goreham, Sarah L. Harmer, Jamie S. Quinton, and Christopher T. Gibso

Fingertip whole blood as an indicator of omega-3 long-chain polyunsaturated fatty acid changes during dose-response supplementation in women: comparison with plasma and erythrocyte fatty acids

The sensitivity of fingertip whole blood to reflect habitual dietary and dose-dependent supplemental omega-3 long-chain polyunsaturated fatty acid (n-3 LCPUFA) intake in premenopausal women was compared to that of venous erythrocytes and plasma fatty acids. Samples were obtained from women in a randomised, double-blind, placebo-controlled trial in which premenopausal women (n = 53) were supplemented with DHA-rich tuna oil capsules and/or placebo (Sunola oil) capsules (6 capsules per day) for 8 weeks to achieve doses of either 0, 0.35, 0.7 or 1.05 g/day n-3 LCPUFA. All blood biomarkers were very similar in their ability to reflect dietary n-3 LCPUFA intake (r = 0.38–0.46 for EPA and DHA intake), and in their dose-dependent increases in n-3 LCPUFA levels after supplementation (R2 = 0.41–0.51 for dose effect on biomarker EPA and DHA levels (mol %)). Fingertip whole blood is an effective alternative to erythrocytes and plasma as a biomarker n-3 LCPUFA intake in premenopausal women.Barbara J. Meyer, Cassandra Sparkes, Andrew J. Sinclair, Robert A. Gibson and Paul L. Els

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

Validation of the DECAF score to predict hospital mortality in acute exacerbations of COPD

Background Hospitalisation due to acute exacerbations of COPD (AECOPD) is common, and subsequent mortality high. The DECAF score was derived for accurate prediction of mortality and risk strati fi cation to inform patient care. We aimed to validate the DECAF score, internally and externally, and to compare its performance to other predictive tools. Methods The study took place in the two hospitals within the derivation study (internal validation) and in four additional hospitals (external validation) between January 2012 and May 2014. Consecutive admissions were identi fi ed by screening admissions and searching coding records. Admission clinical data, including DECAF indices, and mortality were recorded. The prognostic value of DECAF and other scores were assessed by the area under the receiver operator characteristic (AUROC) curve. Results In the internal and external validation cohorts, 880 and 845 patients were recruited. Mean age was 73.1 (SD 10.3) years, 54.3% were female, and mean (SD) FEV 1 45.5 (18.3) per cent predicted. Overall mortality was 7.7%. The DECAF AUROC curve for inhospital mortality was 0.83 (95% CI 0.78 to 0.87) in the internal cohort and 0.82 (95% CI 0.77 to 0.87) in the external cohort, and was superior to other prognostic scores for inhospital or 30-day mortality. Conclusions DECAF is a robust predictor of mortality, using indices routinely available on admission. Its generalisability is supported by consistent strong performance; it can identify low-risk patients (DECAF 0 – 1) potentially suitable for Hospital at Home or early supported discharge services, and high-risk patients (DECAF 3 – 6) for escalation planning or appropriate early palliation. Trial registration number UKCRN ID 14214