Isolating residents including wandering residents in care and group homes: Medical ethics and English law in the context of Covid-19.

This article investigates the lawfulness of isolating residents of care and group homes during the COVID-19 pandemic. Many residents are mobile, and their freedom to move is a central ethical tenet and human right. It is not however an absolute right and trade-offs between autonomy, liberty and health need to be made since COVID-19 is highly infectious and poses serious risks of critical illness and death. People living in care and group homes may be particularly vulnerable because recommended hygiene practices are difficult for them and many residents are elderly, and/or have co-morbidities. In some circumstances, the trade-offs can be made easily with the agreement of the resident and for short periods of time. However challenging cases arise, in particular for residents and occupants with dementia who 'wander', meaning they have a strong need to walk, sometimes due to agitation, as may also be the case for some people with developmental disability (e.g. autism), or as a consequence of mental illness. This article addresses three central questions: (1) in what circumstances is it lawful to isolate residents of social care homes to prevent transmission of COVID-19, in particular where the resident has a strong compulsion to walk and will not, or cannot, remain still and isolated? (2) what types of strategies are lawful to curtail walking and achieve isolation and social distancing? (3) is law reform required to ensure any action to restrict freedoms is lawful and not excessive? These questions emerged during the first wave of the COVID-19 pandemic and are still relevant. Although focussed on COVID-19, the results are also relevant to other future outbreaks of infectious diseases in care and group homes. Likewise, while we concentrate on the law in England and Wales, the analysis and implications have international significance.

Ethical principles and a practical approach to support policy making through the next phases of the COVID-19 pandemic and beyond.

There is an urgent need for an ethical framework to help us address the local and national challenges that we face as clinicians during the COVID-19 pandemic. We propose four key commitments from which a practical and consistent ethical approach can be derived. These commitments are to articulate the needs, rights and interests of the different stakeholders affected by any policy; to be accountable and transparent, recognising that people are autonomous individuals with values and concerns of their own; to consider the impact of our actions on the sustainability of the NHS, infrastructure, service demands and staff welfare; and to treat everybody equitably, with all deserving of consideration and care. Implementing these commitments will require a number of specific actions. We must put in place frameworks enabling clear advocacy for each competing objective; communicate policy and practice effectively to the public; promote integration of decision-making among social, primary, secondary and tertiary care and reduce or stop unnecessary or inefficient interventions; minimise health inequalities; and build spare capacity into the system.In this article, we expand on these actions, and note the legal context in which this would be delivered.

What do the British public think of inequality in health, wealth, and power?

Despite the importance of public opinion for policy formation and the political salience of inequality, the public's views about the desirability of equality, not only in health but also in economics and politics, has attracted little attention. We report the results of an on-line survey administered in late 2016 in Great Britain (N = 1667 with a response rate of 35-50%). The survey allowed for testing the sensitivity of public opinion across two other variables: absolute versus relative (everyone should have the same versus inequality should be reduced) and bivariate versus univariate (inequality in one domain is judged in relation to inequality in another versus inequality in a domain is judged independently of other domains). It also allowed examination of how far support for equality in one domain overlaps with support for equality in another. We find that for health, economic and political equality a relative conception of equality attracts more support than an absolute conception, and that for health and political equality a bivariate conception attracts more support than a univariate conception. We also find that conceptions of equality affect how much overlap exists between support for different forms of equality, with a bivariate and relative conception resulting in more overlap than a univariate and absolute conception. We also find evidence for Walzer's 'complex equality' theory in which people tolerate inequality in one domain if it does not control inequality in another.

Identification of multiple genomic DNA sequences which form i-motif structures at neutral pH.

i-Motifs are alternative DNA secondary structures formed in cytosine-rich sequences. Particular examples of these structures, traditionally assumed to be stable only at acidic pH, have been found to form under near-physiological conditions. To determine the potential impact of these structures on physiological processes, investigation of sequences with the capacity to fold under physiological conditions is required. Here we describe a systematic study of cytosine-rich DNA sequences, with varying numbers of consecutive cytosines, to gain insights into i-motif DNA sequence and structure stability. i-Motif formation was assessed using ultraviolet spectroscopy, circular dichroism and native gel electrophoresis. We found that increasing cytosine tract lengths resulted in increased thermal stability; sequences with at least five cytosines per tract folded into i-motif at room temperature and neutral pH. Using these results, we postulated a folding rule for i-motif formation, analogous to (but different from) that for G-quadruplexes. This indicated that thousands of cytosine-rich sequences in the human genome may fold into i-motif structures under physiological conditions. Many of these were found in locations where structure formation is likely to influence gene expression. Characterization of a selection of these identified i-motif forming sequences uncovered 17 genomic i-motif forming sequence examples which were stable at neutral pH.

Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences.

G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions.

Stability and structure of long intramolecular G-quadruplexes.

G-quadruplexes are formed from guanine-rich sequences of DNA and RNA. They consist of stacks of square arrangements of guanines called G-quartets. Increasing evidence suggests that these structures are involved in cellular processes such as transcription or translation. Knowing their structure and their stability in vitro should help to predict their formation in vivo and to understand their biological functions. Many studies have been performed on isolated G-quadruplexes, but little attention has been given to their interactions. Here, we present non-denaturing gel electrophoresis, UV melting, and circular dichroism data obtained for long sequences of DNA which are capable of forming two simultaneous G-quadruplexes, namely, d(TG(3)T(3)G(3)T(3)G(3)T(3)G(3)T(n)G(3)T(3)G(3)T(3)G(3)T(3)G(3)T), with n varying from one to seven. These sequences can form up to two separate G-quadruplexes. We also study mutated versions of these sequences designed to form one G-quadruplex at specific positions on the strand. Comparing results from the original sequences and their mutated versions, we show that for the former different folded states coexist: either with six stacked G-quartets or only three, in various combinations. Which ones are favored depends on n. Moreover, for n greater than three, the thermodynamic stability stays constant, contrary to an expected decrease in stability if the six G-quartets were stacked together in a single structure. This result agrees with a beads-on-a-string folding model for long sequences of G-quadruplexes, where two adjacent G-quadruplexes fold independently.

A toolbox for predicting g-quadruplex formation and stability.

G-quadruplexes are four stranded nucleic acid structures formed around a core of guanines, arranged in squares with mutual hydrogen bonding. Many of these structures are highly thermally stable, especially in the presence of monovalent cations, such as those found under physiological conditions. Understanding of their physiological roles is expanding rapidly, and they have been implicated in regulating gene transcription and translation among other functions. We have built a community-focused website to act as a repository for the information that is now being developed. At its core, this site has a detailed database (QuadDB) of predicted G-quadruplexes in the human and other genomes, together with the predictive algorithm used to identify them. We also provide a QuadPredict server, which predicts thermal stability and acts as a repository for experimental data from all researchers. There are also a number of other data sources with computational predictions. We anticipate that the wide availability of this information will be of use both to researchers already active in this exciting field and to those who wish to investigate a particular gene hypothesis.

Structure, location and interactions of G-quadruplexes.

Four-stranded G-rich DNA structures called G-quadruplexes have been the subject of increasing interest recently. Experimental and computational techniques have been used to implicate them in important biological processes such as transcription and translation. In this minireview, I discuss how they form, what structures they adopt and with what stability. I then discuss the computational approaches used to predict them on a genomic scale and how the information derived can be combined with experiments to understand their biological functions. Other minireviews in this series deal with G-quadruplex nucleic acids and human disease [Wu Y & Brosh RM Jr (2010) FEBS J] and making sense of G-quadruplex and i-motif function in oncogene promoters [Brooks TA et al. (2010) FEBS J].

Seven essential questions on G-quadruplexes.

The helical duplex architecture of DNA was discovered by Francis Crick and James Watson in 1951 and is well known and understood. However, nucleic acids can also adopt alternative structural conformations that are less familiar, although no less biologically relevant, such as the G-quadruplex. G-quadruplexes continue to be the subject of a rapidly expanding area of research, owing to their significant potential as therapeutic targets and their unique biophysical properties. This review begins by focusing on G-quadruplex structure, elucidating the intermolecular and intramolecular interactions underlying its formation and highlighting several substructural variants. A variety of methods used to characterize these structures are also outlined. The current state of G-quadruplex research is then addressed by proffering seven pertinent questions for discussion. This review concludes with an overview of possible directions for future research trajectories in this exciting and relevant field.

Genome-wide analysis of a G-quadruplex-specific single-chain antibody that regulates gene expression.

G-quadruplex nucleic acids have been proposed to play a role in a number of fundamental biological processes that include transcription and translation. We have developed a single-chain antibody that is selective for G-quadruplex DNA over double-stranded DNA, and here show that when it is expressed in human cells, it significantly affects the expression of a wide variety of genes, in a manner that correlates with the presence of predicted G-quadruplexes. We observe cases where gene expression is increased or decreased, and that there are apparent interactions with G-quadruplex motifs at the beginning and end of the genes, and on either strand. The outcomes of this genome-wide study demonstrate that G-quadruplex recognition by the antibody has physiological consequences, and provides insights into some of the complexity associated with G-quadruplex-based regulation.

Direct visualization of G-quadruplexes in DNA using atomic force microscopy.

The formation of G-quadruplexes in G-rich regions of DNA is believed to affect DNA transcription and replication. However, it is currently unclear how this formation occurs in the presence of a complementary strand. We have used atomic force microscopy (AFM) to image stable RNA/DNA hybrid loops generated by transcription of the plasmid pPH600, which contains a 604-bp fragment of the murine immunoglobulin Sgamma3 switch region. We show that the non-RNA-containing portion folds into G-quadruplexes, consistent with computational predictions. We also show that hybrid formation prevents further transcription from occurring, implying a regulatory role. After in vitro transcription, almost all (93%) of the plasmids had an asymmetric loop, a large asymmetric blob or a spur-like projection at the appropriate position on the DNA contour. The loops disappeared following treatment of the transcribed plasmid with RNase H, which removes mRNA hybridized with the template strand. Replacement of K+ in the transcription buffer with either Na+ or Li+ caused a reduction in the percentage of plasmids containing loops, blobs or spurs, consistent with the known effects of monovalent cations on G-quadruplex stability. The minimal sample preparation required for AFM imaging has permitted direct observation of the structural changes resulting from G-quadruplex formation.

Stable G-quadruplexes are found outside nucleosome-bound regions.

Unusual DNA structures such as four-stranded G-quadruplexes have been found to form in vivo and to play regulatory roles. However, since DNA is normally present as chromatin, with the double helix wrapped around histone proteins, it is unclear how a G-quadruplex could form in such a region. Here, we show that regulatory G-quadruplexes found upstream of transcription start sites are in fact located in a region depleted of nucleosomes, and that in general in both Caenorhabditis elegans and humans, stable G-quadruplexes are located outside nucleosome-bound regions, hence making it easier for them to form in vivo.

Interfacing systems biology and synthetic biology.

A report of BioSysBio 2009, the IET conference on Synthetic Biology, Systems Biology and Bioinformatics, Cambridge, UK, 23-25 March 2009.

Meeting report: second international meeting on quadruplex DNA.

A two and a half day meeting on G-quadruplexes was held in Louisville, KY, USA (April 18-21, 2009). A specific goal of this conference was to promote discussion on the biology of G-quadruplexes. In practice this was represented in four main ways, namely in biophysics, bio/nanotechnology, therapeutics, and what might be termed "intrinsic biology". Research into the basic biophysical and structural properties of G-quadruplexes continues to be important for understanding biology, and for optimizing aptamers for therapeutic and bio/technological purposes. The meeting comprised two Keynote lectures, twenty-three invited talks, and forty-two posters covering various aspects of these topics using a wide variety of technologies.

Function and targeting of G-quadruplexes.

Guanine-rich sequences of DNA and RNA may fold in vitro and in vivo into G-quadruplexes, four-stranded helical structures held together by a guanine core. G-quadruplexes have various biological functions, including inhibition of telomerase and the regulation of gene transcription and translation, and have become an active target for drug development, particularly for novel anticancer therapies. The physiological functions of G-quadruplexes are discussed in this review and the current knowledge of G-quadruplex ligand and drug development is outlined.

G-quadruplexes: the beginning and end of UTRs.

Molecular mechanisms that regulate gene expression can occur either before or after transcription. The information for post-transcriptional regulation can lie within the sequence or structure of the RNA transcript and it has been proposed that G-quadruplex nucleic acid sequence motifs may regulate translation as well as transcription. Here, we have explored the incidence of G-quadruplex motifs in and around the untranslated regions (UTRs) of mRNA. We observed a significant strand asymmetry, consistent with a general depletion of G-quadruplex-forming RNA. We also observed a positional bias in two distinct regions, each suggestive of a specific function. We observed an excess of G-quadruplex motifs towards the 5'-ends of 5'-UTRs, supportive of a hypothesis linking 5'-UTR RNA G-quadruplexes to translational control. We then analysed the vicinity of 3'-UTRs and observed an over-representation of G-quadruplex motifs immediately after the 3'-end of genes, especially in those cases where another gene is in close proximity, suggesting that G-quadruplexes may be involved in the termination of gene transcription.

Four-stranded nucleic acids: structure, function and targeting of G-quadruplexes.

There are many structures that can be adopted by nucleic acids other than the famous Watson-Crick duplex form. This tutorial review describes the guanine rich G-quadruplex structure, highlighting the chemical interactions governing its formation, and the topological variants that exist. The methods that are used to study G-quadruplex structures are described, with examples of the information that may be derived from these different methods. Next, the proposed biological functions of G-quadruplexes are discussed, highlighting especially their presence in telomeric regions and gene promoters. G-quadruplex structures are the subject of considerable interest for the development of small-molecule ligands, and are also the targets of a wide variety of natural proteins.

Thermodynamic prediction of RNA-DNA duplex-forming regions in the human genome.

RNA-DNA hybrids can form in a physiological context, especially as a consequence of transcription, accompanied by the separation of the second strand of DNA. These structures seem to be important in regulating some aspects of transcription, and also form in the immunoglobulin switch domains. In some cases they are sufficiently large and stable to be directly visualised. I present a thermodynamic analysis of their formation, based on known experimental data. I then use this analysis to predict 28 700 regions in the genome likely to form RNA-DNA hybrids when the RNA strand is present, producing a list of regions for experimental analysis, as well as rationalizing the formation of RNA-DNA hybrids in previously known regions.

Four-stranded DNA: cancer, gene regulation and drug development.

DNA can form many structures other than the famous double helix. In particular, guanine-rich DNA of particular sequences can form four-stranded structures, called G-quadruplexes. This article describes the structural form of these sequences, techniques for predicting which sequences can fold up in this manner and efforts towards stability prediction. It then discusses the biological significance of these structures, focusing on their importance in telomeric regions at the end of chromosomes, and their existence in gene promoters and mRNA, where they may be involved with regulating transcription and translation, respectively. Ligands that are capable of selectively binding to these structures are introduced and described, as are DNA aptamers that form G-quadruplex structures; both of these classes of compound have been investigated as anticancer agents in clinical trials. The growing use of G-quadruplexes in the nanotechnology field is also outlined. The article concludes with an analysis of future directions the field may take, with some proposals for further important studies.