Harnessing DNA Nanotechnology and Chemistry for Applications in Photonics and Electronics.

Many photonic and electronic devices rely on nanotechnology and nanofabrication, but DNA-based approaches have yet to make a significant commercial impact in these fields even though DNA molecules are now well-established as versatile building blocks for nanostructures. As we describe here, DNA molecules can be chemically modified with a wide variety of functional groups enabling nanocargoes to be attached at precisely determined locations. DNA nanostructures can also be used as templates for the growth of inorganic structures. Together, these factors enable the use of DNA nanotechnology for the construction of many novel devices and systems. In this topical review, we discuss four case studies of potential applications in photonics and electronics: carbon nanotube transistors, devices for quantum computing, artificial electromagnetic materials, and enzymatic fuel cells. We conclude by speculating about the barriers to the exploitation of these technologies in real-world settings.

Advanced preclinical models for evaluation of drug-induced liver injury - consensus statement by the European Drug-Induced Liver Injury Network [PRO-EURO-DILI-NET].

Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF) and one of the leading indications for liver transplantation in Western societies. Given the wide use of both prescribed and over the counter drugs, DILI has become a major health issue for which there is a pressing need to find novel and effective therapies. Although significant progress has been made in understanding the molecular mechanisms underlying DILI, our incomplete knowledge of its pathogenesis and inability to predict DILI is largely due to both discordance between human and animal DILI in preclinical drug development and a lack of models that faithfully recapitulate complex pathophysiological features of human DILI. This is exemplified by the hepatotoxicity of acetaminophen (APAP) overdose, a major cause of ALF because of its extensive worldwide use as an analgesic. Despite intensive efforts utilising current animal and in vitro models, the mechanisms involved in the hepatotoxicity of APAP are still not fully understood. In this expert Consensus Statement, which is endorsed by the European Drug-Induced Liver Injury Network, we aim to facilitate and outline clinically impactful discoveries by detailing the requirements for more realistic human-based systems to assess hepatotoxicity and guide future drug safety testing. We present novel insights and discuss major players in APAP pathophysiology, and describe emerging in vitro and in vivo pre-clinical models, as well as advanced imaging and in silico technologies, which may improve prediction of clinical outcomes of DILI.

Detection of acquired radioresistance in breast cancer cell lines using Raman spectroscopy and machine learning.

Radioresistance-a living cell's response to, and development of resistance to ionising radiation-can lead to radiotherapy failure and/or tumour recurrence. We used Raman spectroscopy and machine learning to characterise biochemical changes that occur in acquired radioresistance for breast cancer cells. We were able to distinguish between wild-type and acquired radioresistant cells by changes in chemical composition using Raman spectroscopy and machine learning with 100% accuracy. In studying both hormone receptor positive and negative cells, we found similar changes in chemical composition that occur with the development of acquired radioresistance; these radioresistant cells contained less lipids and proteins compared to their parental counterparts. As well as characterising acquired radioresistance in vitro, this approach has the potential to be translated into a clinical setting, to look for Raman signals of radioresistance in tumours or biopsies; that would lead to tailored clinical treatments.

An Engineered E.†coli Strain for Direct in Vivo Fluorination.

Selectively fluorinated compounds are found frequently in pharmaceutical and agrochemical products where currently 25-30 % of optimised compounds emerge from development containing at least one fluorine atom. There are many methods for the site-specific introduction of fluorine, but all are chemical and they often use environmentally challenging reagents. Biochemical processes for C-F bond formation are attractive, but they are extremely rare. In this work, the fluorinase enzyme, originally identified from the actinomycete bacterium Streptomyces cattleya, is engineered into Escherichia coli in such a manner that the organism is able to produce 5'-fluorodeoxyadenosine (5'-FDA) from S-adenosyl-l-methionine (SAM) and fluoride in live E. coli cells. Success required the introduction of a SAM transporter and deletion of the endogenous fluoride efflux capacity in order to generate an E. coli host that has the potential for future engineering of more elaborate fluorometabolites.

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.

Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.

Toward Better Understanding of EBPR Systems via Linking Raman-Based Phenotypic Profiling with Phylogenetic Diversity.

This study reports a proof-of concept study to demonstrate the novel approach of phenotyping microbial communities in enhanced biological phosphorus removal (EBPR) systems using single cell Raman microspectroscopy and link it with phylogentic structures. We use hierarchical clustering analysis (HCA) of single-cell Raman spectral fingerprints and intracellular polymer signatures to separate and classify the functionally relevant populations in EBPR systems, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), as well as other microbial populations. We then investigated the link between Raman-based community phenotyping and 16S rRNA gene-based phylogenetic characterization of four lab-scale EBPR systems with varying solid retention time (SRT) to gain insights into possible genotype-function relationships. Combined and simultaneous phylogenetic and phenotypic evaluation of EBPR ecosystems revealed SRT-dependent phylogenetic and phenotypic characteristics of the PAOs and GAOs, and their association with EBPR performance. The phenotypic diversity and plasticity of PAO populations, which otherwise could not be obtained with phylogenetic analysis alone, showed complex but potentially crucial association with EBPR process stability.

Exploring Synthetic and Systems Biology at the University of Edinburgh.

The Centre for Synthetic and Systems Biology ('SynthSys') was originally established in 2007 as the Centre for Integrative Systems Biology, funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and the Engineering and Physical Sciences Research Council (EPSRC). Today, SynthSys embraces an extensive multidisciplinary community of more than 200 researchers from across the University with a common interest in synthetic and systems biology. Our research is broad and deep, addressing a diversity of scientific questions, with wide ranging impact. We bring together the power of synthetic biology and systems approaches to focus on three core thematic areas: industrial biotechnology, agriculture and the environment, and medicine and healthcare. In October 2015, we opened a newly refurbished building as a physical hub for our new U.K. Centre for Mammalian Synthetic Biology funded by the BBSRC/EPSRC/MRC as part of the U.K. Research Councils' Synthetic Biology for Growth programme.

Label-free biomarkers of human embryonic stem cell differentiation to hepatocytes.

Four different label-free, minimally invasive, live single cell analysis techniques were applied in a quantitative comparison, to characterize embryonic stem cells and the hepatocytes into which they were differentiated. Atomic force microscopy measures the cell's mechanical properties, Raman spectroscopy measures its chemical properties, and dielectrophoresis measures the membrane's capacitance. They were able to assign cell type of individual cells with accuracies of 91% (atomic force microscopy), 95.5% (Raman spectroscopy), and 72% (dielectrophoresis). In addition, stimulated Raman scattering (SRS) microscopy was able to easily identify hepatocytes in images by the presence of lipid droplets. These techniques, used either independently or in combination, offer label-free methods to study individual living cells. Although these minimally invasive biomarkers can be applied to sense phenotypical or environmental changes to cells, these techniques have most potential in human stem cell therapies where the use of traditional biomarkers is best avoided. Destructive assays consume valuable stem cells and do not characterize the cells which go on to be used in therapies; whereas immunolabeling risks altering cell behavior. It was suggested how these four minimally invasive methods could be applied to cell culture, and how they could in future be combined into one microfluidic chip for cell sorting. © 2016 International Society for Advancement of Cytometry.

PaperClip: rapid multi-part DNA assembly from existing libraries.

Assembly of DNA 'parts' to create larger constructs is an essential enabling technique for bioengineering and synthetic biology. Here we describe a simple method, PaperClip, which allows flexible assembly of multiple DNA parts from currently existing libraries cloned in any vector. No restriction enzymes, mutagenesis of internal restriction sites, or reamplification to add end homology are required. Order of assembly is directed by double stranded oligonucleotides-'Clips'. Clips are formed by ligation of pairs of oligonucleotides corresponding to the ends of each part. PaperClip assembly can be performed by polymerase chain reaction or by cell extract-mediated recombination. Once multi-use Clips have been prepared, assembly of at least six DNA parts in any order can be accomplished with high efficiency within several hours.

Label-free identification and characterization of living human primary and secondary tumour cells.

We used three label-free minimally invasive methods to characterize individual cells derived from primary and secondary tumours from the same patient, and of the same type ­ colorectal. Raman spectroscopy distinguished cells by their biochemical 'fingerprint' in a vibrational spectrum with 100% accuracy, and revealed that the primary cell line contains more lipids and alpha-helix proteins, whereas the secondary cell line contains more porphyrins and beta-sheet proteins. Stimulated Raman scattering (SRS) microscopy distinguished cells in chemically-specific images of CH2 bonds which revealed lipid droplets in secondary tumour cells. Atomic force microscopy (AFM) was used to distinguish cells with 80% accuracy by measuring their elasticity ­ secondary tumour cells (SW620) are around 3 times softer than primary ones (SW480). As well as characterizing the physical and biochemical differences between cell lines in vitro, these techniques offer three novel methods which could potentially be used for diagnosis ­ to assign a tumour as primary or secondary.

Exploring the cellular and tissue uptake of nanomaterials in a range of biological samples using multimodal nonlinear optical microscopy.

The uptake of nanomaterials (NMs) by cells is critical in determining their potential biological impact, whether beneficial or detrimental. Thus, investigation of NM internalization by cells is a common consideration in hazard and efficacy studies. There are currently a number of approaches that are routinely used to investigate NM-cell interactions, each of which have their own advantages and limitations. Ideally, imaging modalities used to investigate NM uptake by cells should not require the NM to be labelled (e.g. with fluorophores) to facilitate its detection. We present a multimodal imaging approach employing a combination of label-free microscopies that can be used to investigate NM-cell interactions. Coherent anti-Stokes Raman scattering microscopy was used in combination with either two-photon photoluminescence or four-wave mixing (FWM) to visualize the uptake of gold or titanium dioxide NMs respectively. Live and fixed cell imaging revealed that NMs were internalized by J774 macrophage and C3A hepatocyte cell lines (15-31 µg ml(-1)). Sprague Dawley rats were exposed to NMs (intratracheal instillation, 62 µg) and NMs were detected in blood and lung leucocytes, lung and liver tissue, demonstrating that NMs could translocate from the exposure site. Obtained data illustrate that multimodal nonlinear optical microscopy may help overcome current challenges in the assessment of NM cellular uptake and biodistribution. It is therefore a powerful tool that can be used to investigate unlabelled NM cellular and tissue uptake in three dimensions, requires minimal sample preparation, and is applicable to live and fixed cells.

Non-invasive methods of monitoring Fe3O4 magnetic nanoparticle toxicity in human liver HepaRG cells using impedance biosensing and Coherent anti-Stokes Raman spectroscopic (CARS) microscopy.

Functionalized nanoparticles have been developed for use in nanomedicines for treating life threatening diseases including various cancers. To ensure safe use of these new nanoscale reagents, various assays for biocompatibility or cytotoxicity in vitro using cell lines often serve as preliminary assessments prior to in vivo animal testing. However, many of these assays were designed for soluble, colourless materials and may not be suitable for coloured, non-transparent nanoparticles. Moreover, cell lines are not always representative of mammalian organs in vivo. In this work, we use non-invasive impedance sensing methods with organotypic human liver HepaRG cells as a model to test the toxicity of PEG-Fe3O4 magnetic nanoparticles. We also use Coherent anti-Stokes Raman Spectroscopic (CARS) microscopy to monitor the formation of lipid droplets as a parameter to the adverse effect on the HepaRG cell model. The results were also compared with two commercial testing kits (PrestoBlue and ATP) for cytotoxicity. The results suggested that the HepaRG cell model can be a more realistic model than commercial cell lines while use of impedance monitoring of Fe3O4 nanoparticles circumventing the uncertainties due to colour assays. These methods can play important roles for scientists driving towards the 3Rs principle - Replacement, Reduction and Refinement.

Exploring standards for multicellular mammalian synthetic biology.

Synthetic biology is moving towards bioengineering multicellular mammalian systems that are poised to advance tissue engineering, biomedicine, and the food industry. Despite progress, the field lacks a framework of standards that could greatly accelerate further development. Here, we explore the landscape of standards for multicellular mammalian synthetic biology. We discuss the limits of current technical standards and categorise unaddressed parameters into an abstraction hierarchy. We then define the concept of a 'synthetic multicellular mammalian system' and apply our standard hierarchy framework to illustrate how it could aid bioengineering endeavours. We conclude with promising areas that could shape the future of the field, flagging the need for a critical and holistic consideration of standards that requires cross-disciplinary dialogue.

Using ultraviolet absorption spectroscopy to study nanoswitches based on non-canonical DNA structures.

Non-canonical forms of DNA are attracting increasing interest for applications in nanotechnology. It is frequently convenient to characterize DNA molecules using a label-free approach such as ultraviolet absorption spectroscopy. In this paper we present the results of our investigation into the use of this technique to probe the folding of quadruplex and triplex nanoswitches. We confirmed that four G-quartets were necessary for folding at sub-mM concentrations of potassium and found that the wrong choice of sequence for the linker between G-tracts could dramatically disrupt folding, presumably due to the presence of kinetic traps in the folding landscape. In the case of the triplex nanoswitch we examined, we found that the UV spectrum showed a small change in absorbance when a triplex was formed. We anticipate that our results will be of interest to researchers seeking to design DNA nanoswitches based on quadruplexes and triplexes.

Standardization of Synthetic Biology Tools and Assembly Methods for Saccharomyces cerevisiae and Emerging Yeast Species.

As redesigning organisms using engineering principles is one of the purposes of synthetic biology (SynBio), the standardization of experimental methods and DNA parts is becoming increasingly a necessity. The synthetic biology community focusing on the engineering of Saccharomyces cerevisiae has been in the foreground in this area, conceiving several well-characterized SynBio toolkits widely adopted by the community. In this review, the molecular methods and toolkits developed for S. cerevisiae are discussed in terms of their contributions to the required standardization efforts. In addition, the toolkits designed for emerging nonconventional yeast species including Yarrowia lipolytica, Komagataella phaffii, and Kluyveromyces marxianus are also reviewed. Without a doubt, the characterized DNA parts combined with the standardized assembly strategies highlighted in these toolkits have greatly contributed to the rapid development of many metabolic engineering and diagnostics applications among others. Despite the growing capacity in deploying synthetic biology for common yeast genome engineering works, the yeast community has a long journey to go to exploit it in more sophisticated and delicate applications like bioautomation.

Intracellular polyphosphate length characterization in polyphosphate accumulating microorganisms (PAOs): Implications in PAO phenotypic diversity and enhanced biological phosphorus removal performance.

Polyphosphate (polyP) accumulating organisms (PAOs) are the key agent to perform enhanced biological phosphorus removal (EBPR) activity, and intracellular polyP plays a key role in this process. Potential associations between EBPR performance and the polyP structure have been suggested, but are yet to be extensively investigated, mainly due to the lack of established methods for polyP characterization in the EBPR system. In this study, we explored and demonstrated that single-cell Raman spectroscopy (SCRS) can be employed for characterizing intracellular polyPs of PAOs in complex environmental samples such as EBPR systems. The results, for the first time, revealed distinct distribution patterns of polyP length (as Raman peak position) in PAOs in lab-scale EBPR reactors that were dominated with different PAO types, as well as among different full-scale EBPR systems with varying configurations. Furthermore, SCRS revealed distinctive polyP composition/features among PAO phenotypic sub-groups, which are likely associated with phylogenetic and/or phenotypic diversity in EBPR communities, highlighting the possible resolving power of SCRS at the microdiversity level. To validate the observed polyP length variations via SCRS, we also performed and compared bulk polyP length characteristics in EBPR biomass using conventional polyacrylamide gel electrophoresis (PAGE) and solution 31P nuclear magnetic resonance (31P-NMR) methods. The results are consistent with the SCRS findings and confirmed the variations in the polyP lengths among different EBPR systems. Compared to conventional methods, SCRS exhibited advantages as compared to conventional methods, including the ability to characterize in situ the intracellular polyPs at subcellular resolution in a label-free and non-destructive way, and the capability to capture subtle and detailed biochemical fingerprints of cells for phenotypic classification. SCRS also has recognized limitations in comparison with 31P-NMR and PAGE, such as the inability to quantitatively detect the average polyP chain length and its distribution. The results provided initial evidence for the potential of SCRS-enabled polyP characterization as an alternative and complementary microbial community phenotyping method to facilitate the phenotype-function (performance) relationship deduction in EBPR systems.

Optical spectroscopy for noninvasive monitoring of stem cell differentiation.

There is a requirement for a noninvasive technique to monitor stem cell differentiation. Several candidates based on optical spectroscopy are discussed in this review: Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and coherent anti-Stokes Raman scattering (CARS) microscopy. These techniques are briefly described, and the ability of each to distinguish undifferentiated from differentiated cells is discussed. FTIR spectroscopy has demonstrated its ability to distinguish between stem cells and their derivatives. Raman spectroscopy shows a clear reduction in DNA and RNA concentrations during embryonic stem cell differentiation (agreeing with the well-known reduction in the nucleus to cytoplasm ratio) and also shows clear increases in mineral content during differentiation of mesenchymal stem cells. CARS microscopy can map these DNA, RNA, and mineral concentrations at high speed, and Mutliplex CARS spectroscopy/microscopy is highlighted as the technique with most promise for future applications.

Development of tip-enhanced optical spectroscopy for biological applications: a review.

Tip-enhanced optical spectroscopy is an approach that holds a good deal of promise for the nanoscale characterisation of matter. Tip-enhanced Raman spectroscopy (TERS) has been demonstrated on a variety of samples: inorganic, organic and biological. Imaging using TERS has been shown for carbon nanotubes due to their high scattering efficiency. There are a number of compelling motivations to consider alternative approaches for biological samples; most importantly, the potential for heat damage of biomolecules and long acquisition times. These issues may be addressed through the development of tip-enhanced coherent anti-Stokes Raman scattering microscopy.

Raman spectroscopy and related techniques in biomedicine.

In this review we describe label-free optical spectroscopy techniques which are able to non-invasively measure the (bio)chemistry in biological systems. Raman spectroscopy uses visible or near-infrared light to measure a spectrum of vibrational bonds in seconds. Coherent anti-Stokes Raman (CARS) microscopy and stimulated Raman loss (SRL) microscopy are orders of magnitude more efficient than Raman spectroscopy, and are able to acquire high quality chemically-specific images in seconds. We discuss the benefits and limitations of all techniques, with particular emphasis on applications in biomedicine--both in vivo (using fiber endoscopes) and in vitro (in optical microscopes).