Isothermal microwave biology : catalysis and fermentation

This thesis looks directly into the controversial subject of the microwave field effect by the production of a versatile prototype isothermal microwave reactor for the investigation of enzymatic and microbiological reactions. The observed results from the prototype reactor and experiments conducted conclude that there is a nonthermal, nonlinear response between the exposure microwave power and rate and yield of cellulose saccharification. The nature of the nonthermal response is controversial and may be dependent on the definition of "nonthermal,' leading to ambiguity of exact mechanism. Enzymatic and microbial conversion of cellulosic material to ethanol is a highly desirable industrial process. Whether the demand is for the mitigation of climate change, political obligations or energy independence, the use of arable land for energy crops limits the available glucose carbon sources for conversion to bioproducts. To prevent this limitation, cellulose (~-l,4-linked glucose polymers) are touted as the "silver bullet" to prevent carbon exhaustion or impinging on food crops. The technical constraint for the industrialization of cellulose based processing is the rate limitation in the cellulase enzymatic action on cellulose. The enzyme rate is limited by feedback cycles and limited mechanical freedom, therefore a relatively high enzyme concentration is required to speed up the process. To date, the associated enzyme production costs and infrastructure prevents bulk volume exploitation. Biomolecular advances (amino acid substitutions, recombination of expression vectors etc) have gone some way to increase either enzymatic rate or enzyme concentration. The work presented in this thesis differs by increasing the rate of the enzyme without molecular modification. Using a microwave field, the work presented shows that by separating the system into its base units, irradiation of the enzyme/substrate complex in an aqueous environment can increase both the initial enzyme rate and the saccharification yield without alteration of the temperature set point. This study shows that the rate increase is not proportional to the microwave field power. An optimal power in each study is either found or suggested. The results cited show that in the three systems (Endoglucanase and cellobiohydrolase with cellulose, endoglucanase and cellobiohydrolase and ~- glucosidase with cellulose, and ~-glucosidase with cellobiose) the initial rates can be increased by 201 %, 65.5% and 69% respectively. In the total hydrolytic process (endoglucanase and cellobiohydrolase and ~-glucosidase on a cellulose substrate) the final glucose yield was increased by 43% in comparison to the conventional thermal control reaction. This is shown in Figure 1. 10.000 1 9.000 1 8.000 j 7.000 6.000 o 20 40 60 80 100 120 140 160 180 I I 1 I U 5.000 r:: o u 4.000 3.000 2.000 j i t t , f 1.000 0.000 Time (hours) =->=OOOW Glucose' ?012W Glucose ?p025W Glucose ~050W Glucose ?075W Glucose Figure 1. Microwave irradiated "cellulase" enzymes with cellulose substrate I For development into an industrial system and looking towards simultaneous saccharification and fermentation (SSF), the yeast Saccharomyces cerevisiae was subjected to irradiated microwave fermentations on a glucose substrate. Although inconclusive in terms of rate increase, cell density 1 was comparable across the power range showing that the irradiation does not have a derogatory effect. ! The natural evolution of the conclusions drawn would be development of the system into a SSF or SSCF configuration for bio-product formation is possible with irradiation up to SOW. ii The novelty of the experiments conducted is twofold. Firstly, the reactor has been designed to ensure that the microwave irradiation is independent of the bulk temperature therefore allowing the exploration of the microwave field effect independently to the thermal effect. Secondly, the microwave source is a continuous microwave irradiation (none pulse irradiation) ensuring that the reaction is subjected to the microwave field for the entire reaction

Natural history of prostate cancer on active surveillance: stratification by MRI using the PRECISE recommendations in a UK cohort

Objectives: The PRECISE recommendations for magnetic resonance imaging (MRI) in patients on active surveillance (AS) for prostate cancer (PCa) include repeated measurement of each lesion, and attribution of a PRECISE radiological progression score for the likelihood of clinically significant change over time. We aimed to compare the PRECISE score with clinical progression in patients who are managed using an MRI-led AS protocol. Methods: A total of 553 patients on AS for low- and intermediate-risk PCa (up to Gleason score 3 + 4) who had two or more MRI scans performed between December 2005 and January 2020 were included. Overall, 2161 scans were retrospectively re-reported by a dedicated radiologist to give a PI-RADS v2 score for each scan and assess the PRECISE score for each follow-up scan. Clinical progression was defined by histological progression to ≥ Gleason score 4 + 3 (Gleason Grade Group 3) and/or initiation of active treatment. Progression-free survival was assessed using Kaplan-Meier curves and log-rank test was used to assess differences between curves. Results: Overall, 165/553 (30%) patients experienced the primary outcome of clinical progression (median follow-up, 74.5 months; interquartile ranges, 53–98). Of all patients, 313/553 (57%) did not show radiological progression on MRI (PRECISE 1–3), of which 296/313 (95%) had also no clinical progression. Of the remaining 240/553 patients (43%) with radiological progression on MRI (PRECISE 4–5), 146/240 (61%) experienced clinical progression (p < 0.0001). Patients with radiological progression on MRI (PRECISE 4-5) showed a trend to an increase in PSA density. Conclusions: Patients without radiological progression on MRI (PRECISE 1-3) during AS had a very low likelihood of clinical progression and many could avoid routine re-biopsy. Key Points: • Patients without radiological progression on MRI (PRECISE 1–3) during AS had a very low likelihood of clinical progression and many could avoid routine re-biopsy. • Clinical progression was almost always detectable in patients with radiological progression on MRI (PRECISE 4–5) during AS. • Patients with radiological progression on MRI (PRECISE 4–5) during AS showed a trend to an increase in PSA density

Image quality assessment for machine learning tasks using meta-reinforcement learning

In this paper, we consider image quality assessment (IQA) as a measure of how images are amenable with respect to a given downstream task, or task amenability. When the task is performed using machine learning algorithms, such as a neural-network-based task predictor for image classification or segmentation, the performance of the task predictor provides an objective estimate of task amenability. In this work, we use an IQA controller to predict the task amenability which, itself being parameterised by neural networks, can be trained simultaneously with the task predictor. We further develop a meta-reinforcement learning framework to improve the adaptability for both IQA controllers and task predictors, such that they can be fine-tuned efficiently on new datasets or meta-tasks. We demonstrate the efficacy of the proposed task-specific, adaptable IQA approach, using two clinical applications for ultrasound-guided prostate intervention and pneumonia detection on X-ray images

Linking farmer and beekeeper preferences with ecological knowledge to improve crop pollination

1. Pollination by insects is a key input into many crops, with managed honeybees often being hired to support pollination services. Despite substantial research into pollination management, no European studies have yet explored how and why farmers managed pollination services and few have explored why beekeepers use certain crops. 2. Using paired surveys of beekeepers and farmers in 10 European countries, this study examines beekeeper and farmer perceptions and motivations surrounding crop pollination. 3. Almost half of the farmers surveyed believed they had pollination service deficits in one or more of their crops. 4. Less than a third of farmers hired managed pollinators, however most undertook at least one form of agri-environment management known to benefit pollinators, although few did so to promote pollinators. 5. Beekeepers were ambivalent towards many mass flowering crops, with some beekeepers using crops for their honey that other beekeepers avoid because of perceived pesticide risks. 6. The findings highlight a number of largely overlooked knowledge gaps that will affect knowledge exchange and co-operation between the two groups

Microvawe pyrolysis of biomass: control of process parameters for high pyrolysis oil yields and enhanced oil quality

The oil yield and quality of pyrolysis oil from microwave heating of biomass was established by studying the behaviour of Larch in microwave processing. This is the first study in biomass pyrolysis to use a microwave processing technique and methodology that is fundamentally scalable, from which the basis of design for a continuous processing system can be derived to maximise oil yield and quality. It is shown systematically that sample size is a vital parameter that has been overlooked by previous work in this field. When sample size is controlled the liquid product yield is comparable to conventional pyrolysis, and can be achieved at an energy input of around 600 kWh/t. The quality of the liquid product is significantly improved compared to conventional pyrolysis processes, which results from the very rapid heating and quenching that can be achieved with microwave processing. The yields of Levoglucosan and phenolic compounds were found to be an order of magnitude higher in microwave pyrolysis when compared with conventional fast pyrolysis. Geometry is a key consideration for the development of a process at scale, and the opportunities and challenges for scale-up are discussed within this paper

Evaluation of PSA and PSA Density in a Multiparametric Magnetic Resonance Imaging-Directed Diagnostic Pathway for Suspected Prostate Cancer: The INNOVATE Trial

OBJECTIVES: To assess the clinical outcomes of mpMRI before biopsy and evaluate the space remaining for novel biomarkers. METHODS: The INNOVATE study was set up to evaluate the validity of novel fluidic biomarkers in men with suspected prostate cancer who undergo pre-biopsy mpMRI. We report the characteristics of this clinical cohort, the distribution of clinical serum biomarkers, PSA and PSA density (PSAD), and compare the mpMRI Likert scoring system to the Prostate Imaging–Reporting and Data System v2.1 (PI-RADS) in men undergoing biopsy. RESULTS: 340 men underwent mpMRI to evaluate suspected prostate cancer. 193/340 (57%) men had subsequent MRI-targeted prostate biopsy. Clinically significant prostate cancer (csigPCa), i.e., overall Gleason ≥ 3 + 4 of any length OR maximum cancer core length (MCCL) ≥4 mm of any grade including any 3 + 3, was found in 96/195 (49%) of biopsied patients. Median PSA (and PSAD) was 4.7 (0.20), 8.0 (0.17), and 9.7 (0.31) ng/mL (ng/mL/mL) in mpMRI scored Likert 3,4,5 respectively for men with csigPCa on biopsy. The space for novel biomarkers was shown to be within the group of men with mpMRI scored Likert3 (178/340) and 4 (70/350), in whom an additional of 40% (70/178) men with mpMRI-scored Likert3, and 37% (26/70) Likert4 could have been spared biopsy. PSAD is already considered clinically in this cohort to risk stratify patients for biopsy, despite this 67% (55/82) of men with mpMRI-scored Likert3, and 55% (36/65) Likert4, who underwent prostate biopsy had a PSAD below a clinical threshold of 0.15 (or 0.12 for men aged <50 years). Different thresholds of PSA and PSAD were assessed in mpMRI-scored Likert4 to predict csigPCa on biopsy, to achieve false negative levels of ≤5% the proportion of patients whom who test as above the threshold were unsuitably high at 86 and 92% of patients for PSAD and PSA respectively. When PSA was re tested in a sub cohort of men repeated PSAD showed its poor reproducibility with 43% (41/95) of patients being reclassified. After PI-RADS rescoring of the biopsied lesions, 66% (54/82) of the Likert3 lesions received a different PI-RADS score. CONCLUSIONS: The addition of simple biochemical and radiological markers (Likert and PSAD) facilitate the streamlining of the mpMRI-diagnostic pathway for suspected prostate cancer but there remains scope for improvement, in the introduction of novel biomarkers for risk assessment in Likert3 and 4 patients, future application of novel biomarkers tested in a Likert cohort would also require re-optimization around Likert3/PI-RADS2, as well as reproducibility testing

Dynamic Evolution of Pathogenicity Revealed by Sequencing and Comparative Genomics of 19 Pseudomonas syringae Isolates

Closely related pathogens may differ dramatically in host range, but the molecular, genetic, and evolutionary basis for these differences remains unclear. In many Gram- negative bacteria, including the phytopathogen Pseudomonas syringae, type III effectors (TTEs) are essential for pathogenicity, instrumental in structuring host range, and exhibit wide diversity between strains. To capture the dynamic nature of virulence gene repertoires across P. syringae, we screened 11 diverse strains for novel TTE families and coupled this nearly saturating screen with the sequencing and assembly of 14 phylogenetically diverse isolates from a broad collection of diseased host plants. TTE repertoires vary dramatically in size and content across all P. syringae clades; surprisingly few TTEs are conserved and present in all strains. Those that are likely provide basal requirements for pathogenicity. We demonstrate that functional divergence within one conserved locus, hopM1, leads to dramatic differences in pathogenicity, and we demonstrate that phylogenetics-informed mutagenesis can be used to identify functionally critical residues of TTEs. The dynamism of the TTE repertoire is mirrored by diversity in pathways affecting the synthesis of secreted phytotoxins, highlighting the likely role of both types of virulence factors in determination of host range. We used these 14 draft genome sequences, plus five additional genome sequences previously reported, to identify the core genome for P. syringae and we compared this core to that of two closely related non-pathogenic pseudomonad species. These data revealed the recent acquisition of a 1 Mb megaplasmid by a sub-clade of cucumber pathogens. This megaplasmid encodes a type IV secretion system and a diverse set of unknown proteins, which dramatically increases both the genomic content of these strains and the pan-genome of the species

National records of 3000 European bee and hoverfly species: A contribution to pollinator conservation

Pollinators play a crucial role in ecosystems globally, ensuring the seed production of most flowering plants. They are threatened by global changes and knowledge of their distribution at the national and continental levels is needed to implement efficient conservation actions, but this knowledge is still fragmented and/or difficult to access. As a step forward, we provide an updated list of around 3000 European bee and hoverfly species, reflecting their current distributional status at the national level (in the form of present, absent, regionally extinct, possibly extinct or non-native). This work was attainable by incorporating both published and unpublished data, as well as knowledge from a large set of taxonomists and ecologists in both groups. After providing the first National species lists for bees and hoverflies for many countries, we examine the current distributional patterns of these species and designate the countries with highest levels of species richness. We also show that many species are recorded in a single European country, highlighting the importance of articulating European and national conservation strategies. Finally, we discuss how the data provided here can be combined with future trait and Red List data to implement research that will further advance pollinator conservation