Using E. coli NfsA as a model to improve our understanding of enzyme engineering

There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to the difficulty for step-wise evolutionary approaches in effectively retaining mutations that are beneficial in combination with one another, but on an individual basis are neutral or deleterious (i.e., exhibit positive epistasis). We sought to determine whether a superior enzyme identified using a simultaneous mass site directed mutagenesis approach could have been identified using a step-wise approach. We conducted simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 394 million unique variants. We then applied a powerful positive selection using chloramphenicol which is toxic to E. coli but can be detoxified via nitro-reduction. This has enabled us to recover a diverse range of highly active nitroreductase variants. For two of the most active variants, we have created all possible combinations of single mutations. This allowed us to examine whether a step-wise mutagenesis pathway could have also yielded these enzymes. As anticipated, we identified complex epistatic interactions between residues in these enzyme variants. We have also investigated the “black-box” effect of enzyme engineering, examining the consequences that evolving NfsA towards one specialist activity had on the other promiscuous activities of NfsA. Variants generated in this study have also had practical applications, in particular for targeted cell ablation in zebrafish. We have identified NfsA variants that are highly active with nil-bystander prodrugs that can selectively ablate nitroreductase expressing cells without harm to adjacent cells. In ongoing work, our lead variants are being evaluated for their utility in transgenic zebrafish models of degenerative disease

Simultaneous randomisation of eight key active site residues in E. coli NfsA to generate superior nitroreductases for prodrug activation

There is a substantial gap between the levels of enzyme activity Nature can evolve and those that scientists can engineer in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to an inability of step-wise evolutionary approaches to effectively retain mutations that are beneficial in combination with one another, but on an individual basis are neutral or even slightly deleterious (i.e., exhibit positive epistasis). To overcome this limitation, we are seeking to “jump” straight to an enzyme with peak activity by conducting simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has several diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 425 million unique variants. We then applied a powerful selection system using either or both of two recently identified positive selection compounds, which has enabled us to recover a diverse range of highly active nitroreductase variants. These have been screened against a panel of prodrug substrates to identify variants that are improved with specific prodrug substrates of interest. A primary focus has been developing nitroreductases as tools for targeted cell ablation in zebrafish. The basic system involves co-expression of a nitroreductase and fluorescent reporter under the control of a cell type specific promoter in a transgenic fish. Expression of the nitroreductase selectively sensitises target cells to a prodrug which, following nitroreduction, yields a cytotoxic compound that causes precise targeted cell ablation. We have identified several nil-bystander prodrugs that are able to selectively ablate nitroreductase expressing cells with no harm to nearby cells, and have paired these with highly specialised NfsA variants to improve the efficacy and accuracy of cell ablation. We have also screened our mass-randomisation libraries to recover nitroreductases that have non-overlapping prodrug specificities, to be used in a multiplex cell ablation system. This expands upon the previous system, by using pairs of selective nitroreductases and two different prodrugs to facilitate independent ablation of multiple cell types. For example, we have identified a specialist NfsA variant that has activity for tinidazole and not for metronidazole, achieved by including metronidazole as a simultaneous counter-selection during the initial positive selection process. This elegant positive/negative selection eliminated activity with metronidazole, while still ensuring that some level of nitroreductase activity was retained overall

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The role of networks to overcome large-scale challenges in tomography : the non-clinical tomography users research network

Our ability to visualize and quantify the internal structures of objects via computed tomography (CT) has fundamentally transformed science. As tomographic tools have become more broadly accessible, researchers across diverse disciplines have embraced the ability to investigate the 3D structure-function relationships of an enormous array of items. Whether studying organismal biology, animal models for human health, iterative manufacturing techniques, experimental medical devices, engineering structures, geological and planetary samples, prehistoric artifacts, or fossilized organisms, computed tomography has led to extensive methodological and basic sciences advances and is now a core element in science, technology, engineering, and mathematics (STEM) research and outreach toolkits. Tomorrow's scientific progress is built upon today's innovations. In our data-rich world, this requires access not only to publications but also to supporting data. Reliance on proprietary technologies, combined with the varied objectives of diverse research groups, has resulted in a fragmented tomography-imaging landscape, one that is functional at the individual lab level yet lacks the standardization needed to support efficient and equitable exchange and reuse of data. Developing standards and pipelines for the creation of new and future data, which can also be applied to existing datasets is a challenge that becomes increasingly difficult as the amount and diversity of legacy data grows. Global networks of CT users have proved an effective approach to addressing this kind of multifaceted challenge across a range of fields. Here we describe ongoing efforts to address barriers to recently proposed FAIR (Findability, Accessibility, Interoperability, Reuse) and open science principles by assembling interested parties from research and education communities, industry, publishers, and data repositories to approach these issues jointly in a focused, efficient, and practical way. By outlining the benefits of networks, generally, and drawing on examples from efforts by the Non-Clinical Tomography Users Research Network (NoCTURN), specifically, we illustrate how standardization of data and metadata for reuse can foster interdisciplinary collaborations and create new opportunities for future-looking, large-scale data initiatives

Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues

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NTR 2.0: a rationally engineered prodrug-converting enzyme with substantially enhanced efficacy for targeted cell ablation.

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community

Space telescope and optical reverberation mapping project. IV. Anomalous behavior of the broad ultraviolet emission lines in NGC 5548

During an intensive Hubble Space Telescope (HST) Cosmic Origins Spectrograph (COS) UV monitoring campaign of the Seyfert 1 galaxy NGC 5548 performed from 2014 February to July, the normally highly correlated far UV continuum and broad emission line variations decorrelated for 60–70 days, starting 75 days after the first HST/ COS observation. Following this anomalous state, the flux and variability of the broad emission lines returned to a more normal state. This transient behavior, characterized by significant deficits in flux and equivalent width of the strong broad UV emission lines, is the first of its kind to be unambiguously identified in an active galactic nucleus reverberation mapping campaign. The largest corresponding emission line flux deficits occurred for the high ionization, collisionally excited lines C IV and Si IV(+O IV]), and also He II(+O III]), while the anomaly in Lyα was substantially smaller. This pattern of behavior indicates a depletion in the flux of photons with Eph > 54 eV relative to those near 13.6 eV. We suggest two plausible mechanisms for the observed behavior: (i) temporary obscuration of the ionizing continuum incident upon broad line region (BLR) clouds by a moving veil of material lying between the inner accretion disk and inner (BLR), perhaps resulting from an episodic ejection of material from the disk, or (ii) a temporary change in the intrinsic ionizing continuum spectral energy distribution resulting in a deficit of ionizing photons with energies >54 eV, possibly due to a transient restructuring of the Comptonizing atmosphere above the disk. Current evidence appears to favor the latter explanation

Space telescope and optical reverberation mapping project : III. optical continuum emission and broadband time delays in NGC 5548

We present ground-based optical photometric monitoring data for NGC 5548, part of an extended multiwavelength reverberation mapping campaign. The light curves have nearly daily cadence from 2014 January to July in nine filters (BVRI and ugriz). Combined with ultraviolet data from the Hubble Space Telescope and Swift, we confirm significant time delays between the continuum bands as a function of wavelength, extending the wavelength coverage from 1158 Å to the z band (~9160 Å). We find that the lags at wavelengths longer than the V band are equal to or greater than the lags of high-ionization-state emission lines (such as He II l1640 and l4686), suggesting that the continuum-emitting source is of a physical size comparable to the inner broad-line region (BLR). The trend of lag with wavelength is broadly consistent with the prediction for continuum reprocessing by an accretion disk with t μ l4 3. However, the lags also imply a disk radius that is 3 times larger than the prediction from standard thin-disk theory, assuming that the bolometric luminosity is 10% of the Eddington luminosity (L = 0.1LEdd). Using optical spectra from the Large Binocular Telescope, we estimate the bias of the interband continuum lags due to BLR emission observed in the filters. We find that the bias for filters with high levels of BLR contamination (~20%) can be important for the shortest continuum lagsand likely has a significant impact on the u and U bands owing to Balmer continuum emission

Space telescope and optical reverberation mapping project : III. optical continuum emission and broadband time delays in NGC 5548

We present ground-based optical photometric monitoring data for NGC 5548, part of an extended multiwavelength reverberation mapping campaign. The light curves have nearly daily cadence from 2014 January to July in nine filters (BVRI and ugriz). Combined with ultraviolet data from the Hubble Space Telescope and Swift, we confirm significant time delays between the continuum bands as a function of wavelength, extending the wavelength coverage from 1158 Å to the z band (~9160 Å). We find that the lags at wavelengths longer than the V band are equal to or greater than the lags of high-ionization-state emission lines (such as He II l1640 and l4686), suggesting that the continuum-emitting source is of a physical size comparable to the inner broad-line region (BLR). The trend of lag with wavelength is broadly consistent with the prediction for continuum reprocessing by an accretion disk with t μ l4 3. However, the lags also imply a disk radius that is 3 times larger than the prediction from standard thin-disk theory, assuming that the bolometric luminosity is 10% of the Eddington luminosity (L = 0.1LEdd). Using optical spectra from the Large Binocular Telescope, we estimate the bias of the interband continuum lags due to BLR emission observed in the filters. We find that the bias for filters with high levels of BLR contamination (~20%) can be important for the shortest continuum lagsand likely has a significant impact on the u and U bands owing to Balmer continuum emission

Space telescope and optical reverberation mapping project. IV. Anomalous behavior of the broad ultraviolet emission lines in NGC 5548

During an intensive Hubble Space Telescope (HST) Cosmic Origins Spectrograph (COS) UV monitoring campaign of the Seyfert 1 galaxy NGC 5548 performed from 2014 February to July, the normally highly correlated far UV continuum and broad emission line variations decorrelated for 60–70 days, starting 75 days after the first HST/ COS observation. Following this anomalous state, the flux and variability of the broad emission lines returned to a more normal state. This transient behavior, characterized by significant deficits in flux and equivalent width of the strong broad UV emission lines, is the first of its kind to be unambiguously identified in an active galactic nucleus reverberation mapping campaign. The largest corresponding emission line flux deficits occurred for the high ionization, collisionally excited lines C IV and Si IV(+O IV]), and also He II(+O III]), while the anomaly in Lyα was substantially smaller. This pattern of behavior indicates a depletion in the flux of photons with Eph > 54 eV relative to those near 13.6 eV. We suggest two plausible mechanisms for the observed behavior: (i) temporary obscuration of the ionizing continuum incident upon broad line region (BLR) clouds by a moving veil of material lying between the inner accretion disk and inner (BLR), perhaps resulting from an episodic ejection of material from the disk, or (ii) a temporary change in the intrinsic ionizing continuum spectral energy distribution resulting in a deficit of ionizing photons with energies >54 eV, possibly due to a transient restructuring of the Comptonizing atmosphere above the disk. Current evidence appears to favor the latter explanation