Ultrahigh-Throughput Microfluidic Droplet Screening of Metagenomic Libraries for Esterases and Kemp Eliminases

In the search for new enzymes, functional metagenomic libraries are particularly interesting since they give access to the genomes of the 99% of microorganisms which cannot be cultured in the laboratory. However, using classical biochemical assays to screen such libraries is impeded by the fact that enzymes for a given reaction are incredibly rare (1 in 105 on average). Ultrahigh-throughput droplet microfluidics has emerged as an effective technology to overcome this limitation, with the ability to screen 107 reactions per day. However, fewer than ten enzyme assays have been implemented in droplets to date and only once has droplet microfluidics been applied to functional metagenomics. Therefore, for the wider adoption of this technology, it is critical to develop more enzyme assays in droplets. To address this need, first I built an ultrahigh-throughput droplet sorting instrument and then set out to establish two new enzyme assays in droplets: one for the industrially important esterase reaction and another for the Kemp elimination, an artificial reaction. In Chapter 2, I describe the state-of-the-art fluorescence-activated droplet sorter (FADS) I developed for use with fluorogenic enzyme substrates. I improved this instrument incrementally based on the needs of collaborative projects, which ensured the success of these projects in screening enzyme libraries. In Chapter 3, I established an esterase assay and performed the first reported functional metagenomic screen for esterases in droplets. Over 30 million droplets were sorted, amounting to 10× coverage of a metagenomic library consisting of over one million members; making this the largest esterase screen performed to date. Twelve clones encoding thirteen novel esterases were isolated. The majority were members of the α/β-hydrolase super-family of proteins. Four came from small families taking up less than 1% of the sequence space within the super-family. Therefore, functional screening at ultrahigh-throughput provided access to thinly populated sequence-space. It is unlikely that these sequences would have been explored using prediction-based methods. Eight out of eleven enzymes had detectable thioesterase activity, three had β-lactamase activity, one had β-galactosidase activity, and one had Kemp eliminase activity. This finding corroborates the idea that the ability of enzymes to catalyse more than one reaction, a property called enzyme promiscuity, is widespread. In Chapter 4, I established a Kemp eliminase assay in droplets with the aim of exploring how widespread promiscuous enzymes catalysing this non-natural reaction are. Using the substrate 5-nitro-1,2-benzisoxazole in combination with absorbance-activated droplet sorting (AADS). I describe the enrichment of a previously reported Kemp eliminase, HG3.17, over a negative control using droplet microfluidics and use this method to screen substitution, insertion and deletion libraries of HG3.17. Active library variants were enriched and the variants with improved soluble expression isolated. Five locations were identified that tolerate insertions and deletions and, in one investigated case, have improved soluble expression. These variants may serve as starting points to explore previously inaccessible mutational trajectories to improve the catalytic parameters of HG3.17. Due to the limited sensitivity of the assay, functional metagenomic screening was not possible using this substrate. In Chapter 5, I report the newly-discovered fluorogenic Kemp substrate 5-azido-1,2-benzisoxazole, reported and characterised here for the first time. I established this substrate in droplets and, using the FADS instrument, enriched the Kemp eliminase HG3.17 over a negative control. This assay was sufficiently sensitive to detect Kemp eliminase activity in a metagenomic library. The apparent hit rate was comparable to that of the esterases, suggesting that promiscuous enzymes capable of catalysing this reaction are commonplace. A large number of false positives impeded the straightforward isolation of the responsible library members. This constraint is likely to be overcome in future by using a bias-free metagenomic library. Here, a combination of functional re-screening and sequencing allowed the identification of one lead: a predicted class IV adenylyl cyclase. In conclusion, I have built a highly sensitive droplet sorting instrument using which I isolated thirteen new esterases, established the first Kemp eliminase assay in droplets, used both for mutant library and metagenomic screening, and additionally enabled the improvement of numerous enzymes through library screenings in collaboration with others. This work contributes to the success of ultrahigh-throughput droplet microfluidics in furthering our understanding of enzymes in nature and our ability to tailor them for green chemistry applications in industry.EPSRC Centre for Doctoral Training in Sensor Technologies and Application Grant Number: EP/L015889/

Deep learning guided image-based droplet sorting for on-demand selection and analysis of single cells and 3D cell cultures.

Uncovering the heterogeneity of cellular populations and multicellular constructs is a long-standing goal in fields ranging from antimicrobial resistance to cancer research. Emerging technology platforms such as droplet microfluidics hold the promise to decipher such heterogeneities at ultra-high-throughput. However, there is a lack of methods able to rapidly identify and isolate single cells or 3D cell cultures. Here we demonstrate that deep neural networks can accurately classify single droplet images in real-time based on the presence and number of micro-objects including single mammalian cells and multicellular spheroids. This approach also enables the identification of specific objects within mixtures of objects of different types and sizes. The training sets for the neural networks consisted of a few hundred images manually picked and augmented to up to thousands of images per training class. Training required less than 10 minutes using a single GPU, and yielded accuracies of over 90% for single mammalian cell identification. Crucially, the same model could be used to classify different types of objects such as polystyrene spheres, polyacrylamide beads and MCF-7 cells. We applied the developed method for the selection of 3D cell cultures generated with Hek293FT cells encapsulated in agarose gel beads, highlighting the potential of the technology for the selection of objects with a high diversity of visual appearances. The real-time sorting of single droplets was in-line with droplet generation and occurred at rates up to 40 per second independently of image size up to 480 × 480 pixels. The presented microfluidic device also enabled storage of sorted droplets to allow for downstream analyses

Bis(monoacylglycero)phosphate Forms Stable Small Lamellar Vesicle Structures: Insights into Vesicular Body Formation in Endosomes

AbstractBis(monoacylglycero)phosphate (BMP) is an unusually shaped lipid found in relatively high percentage in the late endosome. Here, we report the characterization of the morphology and molecular organization of dioleoyl-BMP (DOBMP) with dynamic light scattering, transmission electron microscopy, nuclear magnetic resonance (NMR) spectroscopy, and electron paramagnetic resonance spectroscopy. The morphology of hydrated DOBMP dispersions varies with pH and ionic strength, and DOBMP vesicles are significantly smaller in diameter than phosphatidylcholine dispersions. At neutral pH, DOBMP forms highly structured, clustered dispersions 500 nm in size. On the other hand, at acidic pH, spherically shaped vesicles are formed. NMR and spin-labeled electron paramagnetic resonance demonstrate that DOBMP forms a lamellar mesophase with acyl-chain packing similar to that of other unsaturated phospholipids. 31P NMR reveals an orientation of the phosphate group in DOBMP that differs significantly from that of other phospholipids. These macroscopic and microscopic structural characterizations suggest that the biosynthesis of BMP on the inner luminal membrane of maturing endosomes may possibly produce budded vesicles high in BMP content, which form small vesicular structures stabilized by the physical properties of the BMP lipid

Programming Light-Harvesting Efficiency Using DNA Origami.

The remarkable performance and quantum efficiency of biological light-harvesting complexes has prompted a multidisciplinary interest in engineering biologically inspired antenna systems as a possible route to novel solar cell technologies. Key to the effectiveness of biological "nanomachines" in light capture and energy transport is their highly ordered nanoscale architecture of photoactive molecules. Recently, DNA origami has emerged as a powerful tool for organizing multiple chromophores with base-pair accuracy and full geometric freedom. Here, we present a programmable antenna array on a DNA origami platform that enables the implementation of rationally designed antenna structures. We systematically analyze the light-harvesting efficiency with respect to number of donors and interdye distances of a ring-like antenna using ensemble and single-molecule fluorescence spectroscopy and detailed Förster modeling. This comprehensive study demonstrates exquisite and reliable structural control over multichromophoric geometries and points to DNA origami as highly versatile platform for testing design concepts in artificial light-harvesting networks.A. W. C. acknowledges support from the Winton Programme for the Physics of Sustainability. U. F. K. was partly supported by an ERC starting grant (PassMembrane, EY 261101). E. A.H. acknowledges support from Janggen-Pöhn Stiftung and the Schweizerischer Nationalfonds (SNF). P. T. acknowledges support by a starting grant (SiMBA, EU 261162) of the European Research Council (ERC). B. W. gratefully acknowledges support by the Braunschweig International Graduate School of Metrology B-IGSM and the DFG Research Training Group GrK1952/1 ‘Metrology for Complex Nanosystems’. P. M. thankfully acknowledges the support of the EPSRC Centre for Doctoral Training in Sensor Technologies and Applications EP/L015889/1.This is the final version of the article. It first appeared from ACS via https://doi.org/10.1021/acs.nanolett.5b0513

Integrated transcriptomic and proteomic analyses ofP. falciparumgametocytes: molecular insight into sex-specific processes and translational repression

Sexual differentiation of malaria parasites into gametocytes in the vertebrate host and subsequent gamete fertilization in mosquitoes is essential for the spreading of the disease. The molecular processes orchestrating these transitions are far from fully understood. Here, we report the first transcriptome analysis of male and female Plasmodium falciparum gametocytes coupled with a comprehensive proteome analysis. In male gametocytes there is an enrichment of proteins involved in the formation of flagellated gametes; proteins involved in DNA replication, chromatin organization and axoneme formation. On the other hand, female gametocytes are enriched in proteins required for zygote formation and functions after fertilization; protein-, lipid- and energy-metabolism. Integration of transcriptome and proteome data revealed 512 highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time. Despite a high degree of conservation between Plasmodium species, 260 of these 'repressed transcripts' have not been previously described. Moreover, for some of these genes, protein expression is only reported in oocysts and sporozoites indicating that repressed transcripts can be partitioned into short- and long-term storage. Finally, these data sets provide an essential resource for identification of vaccine/drug targets and for further mechanistic studies

Prevalence of chronic pain in LTCs and multimorbidity : a cross-sectional study using UK Biobank

This study was funded by Versus Arthritis (grant number 21970). This research has been conducted using the UK Biobank Resource, approved project number 14151.Objectives : Chronic pain is often experienced alongside other long-term conditions (LTCs), yet our understanding of this, particularly in relation to multimorbidity (≥2 LTCs) is poor. We aimed to examine associations between the presence/extent of chronic pain with type/number of LTCs experienced. Methods : We examined the relationship between number/type of LTCs (N = 45) in UK Biobank participants (n = 500,295) who self-reported chronic pain lasting ≥3 months in seven body sites or widespread. Relative risk ratios (RRR) for presence/extent of chronic pain sites were compared using logistic regression adjusted for sociodemographic (sex/age/socioeconomic status) and lifestyle factors (smoking/alcohol intake/BMI/physical activity). Results : 218,648 participants self-reported chronic pain. Of these, 69.1% reported ≥1 LTC and 36.2% reported ≥2 LTCs. In 31/45 LTCs examined, >50% of participants experienced chronic pain. Chronic pain was common with migraine/headache and irritable bowel syndrome where pain is a primary symptom, but also with mental health conditions and diseases of the digestive system. Participants with >4 LTCs were over three times as likely to have chronic pain (RRR 3.56, 95% confidence intervals (CIs) 3.44–3.68) and 20 times as likely to have widespread chronic pain (RRR 20.13, 95% CI 18.26–22.19) as those with no LTCs. Conclusions: Chronic pain is extremely common across a wide range of LTCs. People with multimorbidity were at higher risk of having a greater extent of chronic pain. These results show that chronic pain is a key factor for consideration in the management of patients with LTCs or multimorbidity.Publisher PDFPeer reviewe

Identifying opportunities for improving the coherence of global agreements for species conservation

The current global biodiversity governance system is failing to adequately protect species and halt extinctions. This raises concerns that a lack of coherence among conventions has hindered their effective implementation. We assessed the possibility for improved convention coherence by identifying overlaps among four major international biodiversity conventions; Conservation of Wetlands of International Importance especially as a Waterfowl Habitat (Ramsar), Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), Convention on the Conservation of Migratory Species of Wild Animals (CMS), and Convention on Biological Diversity (CBD). We applied topic modeling to convention texts to identify overlaps in treaty implementation and purpose. We assessed overlap among species listed under CITES and CMS, and threatened species, which are targeted by CBD's Aichi Target 12. We found that convention texts shared similar articles on their implementation, but differed in articles relating to their purpose. We identified 137 threatened species that are also migratory and threatened by unsustainable international trade. The geographic distribution of species common to two or more conventions showed a concentration in parts of Asia. Our analysis suggests that implementation mechanisms are already well aligned to support increased cooperation across conventions, and that cooperation would provide complementarity rather than result in redundancies. We demonstrate that it is possible to identify where co-operation could have a disproportionately positive impact on alleviating the complex of pressures affecting species

Real-world Independent Testing of e-ASPECTS Software (RITeS): statistical analysis plan

Background: Artificial intelligence-based software may automatically detect ischaemic stroke lesions and provide an Alberta Stroke Program Early CT score (ASPECTS) on CT, and identify arterial occlusion and provide a collateral score on CTA. Large-scale independent testing will inform clinical use, but is lacking. We aim to test e-ASPECTS and e-CTA (Brainomix, Oxford UK) using CT scans obtained from a range of clinical studies.Methods: Using prospectively collected baseline CT and CTA scans from 10 national/international clinical stroke trials or registries (total >6600 patients), we will select a large clinically representative sample for testing e-ASPECTS and e-CTA compared to previously acquired independent expert human interpretation (reference standard). Our primary aims are to test agreement between software-derived and masked human expert ASPECTS, and the diagnostic accuracy of e-ASPECTS for identifying all causes of stroke symptoms using follow-up imaging and final clinical opinion as diagnostic ground truth. Our secondary aims are to test when and why e-ASPECTS is more or less accurate, or succeeds/fails to produce results, agreement between e-CTA and human expert CTA interpretation, and repeatability of e-ASPECTS/e-CTA results. All testing will be conducted on an intention-to-analyse basis. We will assess agreement between software and expert-human ratings and test the diagnostic accuracy of software. Conclusions: RITeS will provide comprehensive, robust and representative testing of e-ASPECTS and e-CTA against the current gold-standard, expert-human interpretation

Photocatalytic Lime Render for Indoor and Outdoor Air Quality Improvement

This article reports a novel photocatalytic lime render for indoor and outdoor air quality improvement that is composed of a lime binder and doped TiO2 (KRONOClean 7000®) nanoparticles. These nanoparticles were distributed throughout the bulk of the finishing render, instead of as a thin coating, thus ensuring the durability of the photocatalytic properties upon superficial damage. The physical properties of these renders were not affected by the addition of nanoparticles except in the case of surface area, which increased significantly. In terms of their photocatalytic activity, these novel lime renders were shown to degrade up to 12% NOx under UV light and up to 11% formaldehyde under visible light.This research was funded by the European Union’s Seventh Framework Programme for research, technological development, and demonstration under the Grant Agreement No. 609234 related to the ECO-SEE project: “Eco-innovative, Safe and Energy Efficient wall panels and materials for a healthier indoor environment” This work was partly developed within the scope of the project CICECO–Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. David Maria Tobaldi is overly grateful to Portuguese national funds (OE), through FCT, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of 29 August, changed by Law 57/2017, of 19 July