GeneMill: A 21st century platform for innovation.

GeneMill officially launched on 4th February 2016 and is an open access academic facility located at The University of Liverpool that has been established for the high-throughput construction and testing of synthetic DNA constructs. GeneMill provides end-to-end design, construction and phenotypic characterization of small to large gene constructs or genetic circuits/pathways for academic and industrial applications. Thus, GeneMill is equipping the scientific community with easy access to the validated tools required to explore the possibilities of Synthetic Biology.

Targeted omics analyses, and metabolic enzyme activity assays demonstrate maintenance of key mucociliary characteristics in long term cultures of reconstituted human airway epithelia.

3D reconstituted respiratory epithelia have emerged as better in vitro models for toxicological testing compared to cell lines due to the conservation of key morphological features and functions. MucilAir™ is a commercially available human airway epithelia system that can potentially maintain functional attributes for up to a year, however, detailed mucociliary characteristics and xenobiotic metabolism relevant to inhaled pro-toxicant bioactivation is lacking. Here, we assessed in MucilAir™ some key biomarkers that are characteristic of the respiratory epithelia including morphology, function and xenobiotics metabolism. The end points that were measured included targeted proteomics using a panel of 243 airway surface liquid (ASL) proteins, cilia beat frequency (CBF), a qRT-PCR screen of xenobiotic metabolizing enzymes, and CYP2A6/13, CYP1A1/1B1 activity. Comparison of ASL proteomics with human sputum identified key proteins common to both matrices, but present at different levels. Xenobiotic metabolism gene profiling demonstrated strong similarities with the normal human lung and did not reveal any consistent changes when assessed over a 6 month period. Inducibility and activity of CYP1A1/1B1 and activity of CYP2A6/2A13 were present at one month in culture and maintained in one tested MucilAir™ donor for several months. In conclusion, MucilAir™ presented important morphological and metabolic characteristics of a mucociliary epithelium in short and long term culture. MucilAir™ is therefore a potentially useful model to test repeated sub-cytotoxic doses of toxicants.

Metabolite profile shifts in the heathland lichen Cladonia portentosa in response to N deposition reveal novel biomarkers.

The heathland lichen Cladonia portentosa was collected from sites in mainland Britain differing either in rates of wet N deposition or in annual mean N concentration in rainfall based on a modelled data set. Methanolic extracts of thalli were analyzed by liquid chromatography time-of-flight mass spectrometry to yield metabolic profiles. Differences between sites in metabolite concentration were quantified using multivariate statistical tools and used to identify potential biomarker molecules. The abundances of three structurally related betaine lipids showed an increase with increasing modelled N deposition to a threshold of 22.3 kg ha(-1) year(-1) after which they remained constant. In contrast, the abundance of a phosphatidylcholine (PC) lipid showed concomitant decrease. Correlations of the identified biomarkers with N deposition and precipitation were stronger than those with N concentrations. The results presented in this study clearly show that N enrichment associated with tissue P limitation changes lipid composition, leading to shifts from PCs to betaine lipids, and that these lipids identified have the potential to be used as biomarkers for nitrogen enrichment.

Alterations in the metabolic fingerprint of Cladonia portentosa in response to atmospheric nitrogen deposition.

Nitrogen availability has profound ecological consequences in nutrient-limited systems. In terrestrial settings these would include the upland heaths, sand dunes and blanket bogs of temperate latitudes. Understanding the physiological consequences of nitrogen enrichment is a first critical step in predicting possible consequences. Results are presented from a metabolic fingerprinting study using Fourier transform-infrared spectroscopy (FTIR) to detect biochemical differences in the lichen Cladonia portentosa collected from 25 sites across mainland Britain varying in their nitrogen input. Partial least-squares regression analysis of the FTIR data demonstrated that changes in broad biochemical classes were consistently correlated with mean annual wet inorganic nitrogen deposition loads. These results demonstrated a direct coupling of a broad range of metabolic processes in C. portentosa to nitrogen deposition.

FT-IR as an alternative method for measuring chemical properties during composting.

Chemical properties have been used as a way of following the composting process and compost maturity, however, their analysis is very time consuming as each must be separately determined. By developing a more rapid method to predict these properties, time and cost would be saved. This study investigates the use of Fourier Transform mid-Infrared Spectroscopy (FT-IR) for this purpose. FT-IR spectra and measured values of several chemical properties from a variety of compost mixtures were used to produce calibrated models using partial least-squares regression analysis which predicted the known chemical properties. These models displayed a range of accuracies that for most properties was more than sufficient to follow at least broad dynamic changes associated with maturation. The best calibrations were achieved for total C, total N, LOI, lignin, and cellulose with r(2) values within the range 56-77%. Some degree of calibration was achieved for available-P and NH(4)(+)-N, with r(2) values of between 40% and 57%. No useful calibration could be achieved for NO(3)(-) or pH.

Quantification of hydroxycinnamic acids and lignin in perennial forage and energy grasses by Fourier-transform infrared spectroscopy and partial least squares regression.

Levels of lignin and hydroxycinnamic acid wall components in three genera of forage grasses (Lolium,Festuca and Dactylis) have been accurately predicted by Fourier-transform infrared spectroscopy using partial least squares models correlated to analytical measurements. Different models were derived that predicted the concentrations of acid detergent lignin, total hydroxycinnamic acids, total ferulate monomers plus dimers, p-coumarate and ferulate dimers in independent spectral test data from methanol extracted samples of perennial forage grass with accuracies of 92.8%, 86.5%, 86.1%, 59.7% and 84.7% respectively, and analysis of model projection scores showed that the models relied generally on spectral features that are known absorptions of these compounds. Acid detergent lignin was predicted in samples of two species of energy grass, (Phalaris arundinacea and Pancium virgatum) with an accuracy of 84.5%.

Circadian rhythms of ethylene emission in Arabidopsis.

Ethylene controls multiple physiological processes in plants, including cell elongation. Consequently, ethylene synthesis is regulated by internal and external signals. We show that a light-entrained circadian clock regulates ethylene release from unstressed, wild-type Arabidopsis (Arabidopsis thaliana) seedlings, with a peak in the mid-subjective day. The circadian clock drives the expression of multiple ACC SYNTHASE genes, resulting in peak RNA levels at the phase of maximal ethylene synthesis. Ethylene production levels are tightly correlated with ACC SYNTHASE 8 steady-state transcript levels. The expression of this gene is controlled by light, by the circadian clock, and by negative feedback regulation through ethylene signaling. In addition, ethylene production is controlled by the TIMING OF CAB EXPRESSION 1 and CIRCADIAN CLOCK ASSOCIATED 1 genes, which are critical for all circadian rhythms yet tested in Arabidopsis. Mutation of ethylene signaling pathways did not alter the phase or period of circadian rhythms. Mutants with altered ethylene production or signaling also retained normal rhythmicity of leaf movement. We conclude that circadian rhythms of ethylene production are not critical for rhythmic growth.

The circadian clock that controls gene expression in Arabidopsis is tissue specific.

The expression of CHALCONE SYNTHASE (CHS) expression is an important control step in the biosynthesis of flavonoids, which are major photoprotectants in plants. CHS transcription is regulated by endogenous programs and in response to environmental signals. Luciferase reporter gene fusions showed that the CHS promoter is controlled by the circadian clock both in roots and in aerial organs of transgenic Arabidopsis plants. The period of rhythmic CHS expression differs from the previously described rhythm of chlorophyll a/b-binding protein (CAB) gene expression, indicating that CHS is controlled by a distinct circadian clock. The difference in period is maintained in the wild-type Arabidopsis accessions tested and in the de-etiolated 1 and timing of CAB expression 1 mutants. These clock-affecting mutations alter the rhythms of both CAB and CHS markers, indicating that a similar (if not identical) circadian clock mechanism controls these rhythms. The distinct tissue distribution of CAB and CHS expression suggests that the properties of the circadian clock differ among plant tissues. Several animal organs also exhibit heterogeneous circadian properties in culture but are believed to be synchronized in vivo. The fact that differing periods are manifest in intact plants supports our proposal that spatially separated copies of the plant circadian clock are at most weakly coupled, if not functionally independent. This autonomy has apparently permitted tissue-specific specialization of circadian timing.