Rapid Prototyping Platform for Saccharomyces cerevisiae Using Computer-Aided Genetic Design Enabled by Parallel Software and Workcell Platform Development.

Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.

Synthetic biology in the UK - An outline of plans and progress.

Synthetic biology is capable of delivering new solutions to key challenges spanning the bioeconomy, both nationally and internationally. Recognising this significant potential and the associated need to facilitate its translation and commercialisation the UK government commissioned the production of a national Synthetic Biology Roadmap in 2011, and subsequently provided crucial support to assist its implementation. Critical infrastructural investments have been made, and important strides made towards the development of an effectively connected community of practitioners and interest groups. A number of Synthetic Biology Research Centres, DNA Synthesis Foundries, a Centre for Doctoral Training, and an Innovation Knowledge Centre have been established, creating a nationally distributed and integrated network of complementary facilities and expertise. The UK Synthetic Biology Leadership Council published a UK Synthetic Biology Strategic Plan in 2016, increasing focus on the processes of translation and commercialisation. Over 50 start-ups, SMEs and larger companies are actively engaged in synthetic biology in the UK, and inward investments are starting to flow. Together these initiatives provide an important foundation for stimulating innovation, actively contributing to international research and development partnerships, and helping deliver useful benefits from synthetic biology in response to local and global needs and challenges.

Cartilage thickness visualization using 2D WearMaps and TrackBack.

Osteoarthritis (OA) has a significant impact in terms of morbidity, quality of life, economic and social cost. It is the most prevalent form of arthritis - affecting a large proportion of the population, internationally. The use of Magnetic Resonance (MR) Imaging (MRI) has gained significant support. MRI allows detailed, multi-planar analysis of the joint anatomy, as well as cartilage and underlying bone status; with the ability to view articular surfaces at any angle. In this paper we describe a user interface to visualize the articular cartilage thickness using 2D WearMap derived using MR knee images. The user interface comprises an interactive function (TrackBack) which allows to the Clinician to easily and rapidly refer to the radiological information (e.g. MR images), while maintaining the geometric integrity between the WearMap and the MR image.

An MRI derived articular cartilage visualization framework.

OBJECTIVE: We present a multi-dimensional framework for the visualization of femoral articular cartilage. The framework comprises methods for visualizing and quantifying changes in cartilage thickness and surface morphology derived from MRI based cartilage segmentation. Adequate visualization of cartilage allows accurate and clinically meaningful assessment of cartilage surface morphology and thickness. In current practice the routine use of conventional 2D MR images provides limited qualitative information and is inconvenient because the imaged volume has to be reviewed slice by slice. METHOD: A Graphical User Interface (GUI) that encapsulates the framework described above was developed. In the first stage of the analysis MR images of the knee are segmented to delineate cartilage boundaries. Cartilage thicknesses are subsequently measured. The detected points and corresponding thickness data are utilized to produce a visualization framework. RESULTS: The system was tested using data from six example patients. The spatial distribution of cartilage on the articular surface was visualized using a 3D WearMap. The 2D WearMap allowed the entire cartilage surface to be studied at once. Quantitative interaction with the 2D WearMap was assisted by the ability to ascertain cartilage surface dimensions and TrackBack from a point of interest to the original MR image. As a result, the detection of wear patterns and lesions was efficiently carried out. CONCLUSION: A means of quantitatively visualizing cartilage defects non-invasively is presented. This stands to reduce clinician reporting times, as well as allowing quantitative follow-up that facilitates osteoarthritis (OA) screening and planning/evaluating interventions.

Geometric Framework linking different levels of the Biological Continuum.

With the increasing importance of molecular and cellular biology, a new type of medicine, molecular based medicine, is now developing. This will significantly alter the way in which medicine is practiced. Central to these developments is the concept of the Biological Continuum (BC). Medicine today is often practiced at one or two of these levels, i.e. there is generally no vertically integrated approach. In any area of application there will be a wide range of data (both 2-D and 3-D) across the BC. Hence, there is a need to readily access and view the full range of data. In this paper we describe a web-based interface which allows the user to view images and other data, and to navigate seamlessly from one level of the BC to another level (e.g. from Visceral to Tissue). We present a geometric framework to link images from these two levels. The interface was developed using SVG and Javascript. An example case study, which focuses on the knee, is presented MR images of knee at the visceral level and histology images of cartilage at the tissue level. We have shown that with such an interface it is possible to view images from different levels of the BC in a vertically integrated manner.

Miniature ultrasonic probe construction for minimal access surgery.

The increasing use of minimal access techniques for surgery has produced a need for imaging technologies that can be used during such interventions. Ultrasound imaging has the advantage that the probe itself can be interventional. Interventional ultrasound probes must be sufficiently small to gain access to the surgical site, and any rigid portion must be limited in length to permit adequate flexibility. In practice this means the ultrasound probes have to operate at high frequencies, and a set of design curves have been produced which relate the number of elements and the ultrasound frequency to the probe dimensions for both linear and cylindrical array configurations. Constructing high-frequency sub-miniature probes presents a number of technical challenges, in particular relating to interconnects and packaging. Solutions to these challenges are discussed using the fabrication of a 1 mm diameter intravascular probe as an example.

Time-varying threshold integral pulse frequency modulation.

Several methods have been proposed so far for the analysis of the integral pulse frequency modulation (IPFM) model and detecting its corresponding physiological information. Most of these methods rely on the low-pass filtering method to extract the modulating signal of the model. In this paper, we present an entirely new approach based on vector space theory. The new method is developed for a more comprehensive form of the IPFM model, namely the time-varying threshold integral pulse frequency modulation (TVTIPFM) model. The new method decomposes the driving signals of the TVTIPFM model into a series of orthogonal basis functions and constructs a matrix identity through which the input signals can be obtained by a parametric solution. As a particular case, we apply this method to R-R intervals of the SA node to discriminate between its autonomic nervous modulation and the stretch induced effect.

Investigation of acoustic noise on 15 MRI scanners from 0.2 T to 3 T.

Acoustic noise levels for fast MRI pulse sequences were surveyed on 14 systems with field strengths ranging from 0.2 T to 3 T. A microphone insensitive to the magnetic environment was placed close to the magnet isocenter and connected via an extension cable to a sound level meter outside the scan room. Measured noise levels varied from 82.5 +/- 0.1 dB(A) for a 0.23 T system to 118.4 +/- 1.3 dB(A) for a 3 T system. Further measurements on four of the closed-bore systems surveyed showed that: 1) pulse sequence parameters (particularly FOV and TR) were more influential in determining noise level than field strength, 2) the noise level was found to vary along the z-direction with a maximum near the bore entrance, and 3) in one of two systems tested there was a significant increase in noise with a volunteer present instead of a test object. The results underline the importance of hearing protection for patients and for staff spending extended periods in the scan room.

Transient phase locking patterns among respiration, heart rate and blood pressure during cardiorespiratory synchronisation in humans.

The interactions between respiration, heart rate and blood pressure variability (HRV, BPV), are considered to be of paramount importance for the study of the functional organisation of the autonomic nervous system (ANS). The aim of the reported study is to detect and classify the intermittent phase locking (PL) phenomena between respiration, HRV and BPV during cardiorespiratory synchronisation experiments, by using the following time-domain techniques: Poincaré maps, recurrence plots, time-space separation plots and frequency tracking locus. The experimental protocol consists of three stages, with normal subjects in paced breathing at 15, 12 and 8 breaths min-1. Transient phenomena of coordination between respiration and the major rhythms of HRV and BPV (low and high frequency, LF and HF) have been detected and classified: no interaction between LF and HF rhythms at 15 breaths min-1; short time intervals of stable 1:2 frequency and phase synchronisation during the 12 breaths min-1 stage; 1:1 PL during the 8 breaths min-1 stage. 1:1 and 1:2 PL phenomena occurred when the respiration frequency was quite close to the LF frequency or when it was about twice the LF frequency, respectively. The complex organisation of the ANS seems to provoke transient rather than permanent PL phenomena between the co-ordinating components of respiration and cardiovascular variability series.

Real-time heart rate variability extraction using the Kaiser window.

A new method for real-time heart rate variability (HRV) detection from the R-wave signal, based on the integral pulse frequency modulation (IPFM) model and its similarity to pulse position modulation, is presented. The proposed method exerts low-pass filtering with a Kaiser window. It can also be used for off-line HRV analysis in both the time and frequency domains. Real-time bandpass filtering as a new HRV investigation method and as a by-product of the proposed algorithm is also introduced. Furthermore, the discrete time domain version of the French-Holden algorithm is developed, and it is thoroughly proved that low-pass filtering is an ideal method for detection of HRV.

Interactive algorithms for the segmentation and quantitation of 3-D MRI brain scans.

Interactive algorithms are an attractive approach to the accurate segmentation of 3D brain scans as they potentially improve the reliability of fully automated segmentation while avoiding the labour intensiveness and inaccuracies of manual segmentation. We present a 3D image analysis package (MIDAS) with a novel architecture enabling highly interactive segmentation algorithms to be implemented as add on modules. Interactive methods based on intensity thresholding, region growing and the constrained application of morphological operators are also presented. The methods involve the application of constraints and freedoms on the algorithms coupled with real time visualisation of the effect. This methodology has been applied to the segmentation, visualisation and measurement of the whole brain and a small irregular neuroanatomical structure, the hippocampus. We demonstrate reproducible and anatomically accurate segmentations of these structures. The efficacy of one method in measuring volume loss (atrophy) of the hippocampus in Alzheimer's disease is shown and is compared to conventional methods.

Object-based three-dimensional X-ray imaging.

A form of three-dimensional X-ray imaging, called Object 3-D, is introduced, where the relevant subject material is represented as discrete 'objects'. The surface of each such object is derived accurately from the projections of its outline, and of its other discontinuities, in about ten conventional X-ray views, distributed in solid angle. This technique is suitable for many applications, and permits dramatic savings in radiation exposure and in data acquisition and manipulation. It is well matched to user-friendly interactive displays.

Use of the frequency-tracking locus in estimating the degree of respiratory entrainment in preterm infants.

In order to define the complex interactions between external stimuli and non-linear physiological systems, a technique (the frequency-tracking locus, FTL) was devised that describes the cycle-by-cycle changes in phase angle and amplitude between two signals. Qualitative assessment of the nature of interactions between the signals can be made by examining the FTL. Quantitation of the extent of entrainment of the spontaneous physiological rhythm is possible after deriving a numerical index (the path-length index, PLI) describing the departure of the system from a fully entrained state. The FTL was applied to the study of interactions between spontaneous respiratory effort and mechanical inflation in preterm newborn babies undergoing mechanical ventilation. Stable and unstable states of 1:1 interaction were noted while integer-ratio relationships were seen at low rates of mechanical ventilation. Stable states of entrainment corresponded to a PLI value near unity, and the value of PLI increased rapidly as interactions became unstable. The FTL may be used to describe complex interactions in physiological systems, and may be used as a guide to baby-ventilator matching during mechanical ventilation of the newborn.

Study of the relationship between estimates of enzyme kinetic parameters.

Previous indications of an intrinsic relationship between estimates of Km and Vmax calculated from the Michaelis-Menten equation have been explored further. A mathematically linear relationship could be established for the estimates of the two parameters. The relationship--the trend line--holds whether or not the experimental error is linked to the rate of reaction, the substrate concentration or both, provided that the distribution of errors is symmetrical. The practical implication is that enzyme variants with low values of Km and Vmax may not be distinguishable from those with high values of Km and Vmax.

Advanced spectral estimators for detailed blood flow studies.

Recent publications have emphasized the relationship between the spectrum of the backscattered acoustic signal, beam geometry, and flow patterns in the measurement of blood flow by Doppler ultrasound. On this basis, we believe that in the future more importance will be placed on analyzing various characteristics of the spectral shape rather than absolute parameters of measurement, such as the mean frequency. The potential of this approach for extracting more information from the raw Doppler signal is introduced by considering the Spectral Broadening Index (SBI). We explain the use of the SBI parameter for measuring flow angle under restricted flow conditions. This is done by using an analytic/computational model for prediction of the spectral broadening effect. By simulation study, the performance of various spectral estimators for determining the SBI from finite Doppler signal segments is evaluated.

Biomedical signal processing (in four parts). Part 3. The power spectrum and coherence function.

This is the third in a series of four tutorial papers on biomedical signal processing and concerns the estimation of the power spectrum (PS) and coherence function (CF) od biomedical data. The PS is introduced and its estimation by means of the discrete Fourier transform is considered in terms of the problem of resolution in the frequency domain. The periodogram is introduced and its variance, bias and the effects of windowing and smoothing are considered. The use of the autocovariance function as a stage in power spectral estimation is described and the effects of windows in the autocorrelation domain are compared with the related effects of windows in the original time domain. The concept of coherence is introduced and the many ways in which coherence functions might be estimated are considered.

Biomedical signal processing (in four parts). Part 2. The frequency transforms and their inter-relationships.

This is the second in a series of four tutorial papers on biomedical signal processing, and it concerns the relationships between commonly used frequency transforms. It begins with the Fourier series and Fourier transform for continuous time signals and extends these concepts for aperiodic discrete time data and then periodic discrete time data. The Laplace transform is discussed as an extension of the Fourier transform. The z-transform is introduced and the ideas behind the chirp-z transform are described. The equivalence between the time and frequency domains is described in terms of Parseval's theorem and the theory of convolution. The use of the FFT for fast convolution and fast correlation is described for both short recordings and long recordings that must be processed in sections.

Biomedical signal processing (in four parts). Part 1. Time-domain methods.

This is the first of a series of four tutorial papers on biomedical signal processing. It provides an introduction to terminology and basic ideas for testing for randomness and trend, and for the determination of basic signal properties in the time domain, given the uncertainties associated with the estimation process. Techniques outlined in the paper are: the coherent average, cross-correlation and covariance, autocorrelation and phase-shift averaging.