Correlating fluorescence microscopy, optical and magnetic tweezers to study single chiral biopolymers such as DNA.

Biopolymer topology is critical for determining interactions inside cell environments, exemplified by DNA where its response to mechanical perturbation is as important as biochemical properties to its cellular roles. The dynamic structures of chiral biopolymers exhibit complex dependence with extension and torsion, however the physical mechanisms underpinning the emergence of structural motifs upon physiological twisting and stretching are poorly understood due to technological limitations in correlating force, torque and spatial localization information. We present COMBI-Tweez (Combined Optical and Magnetic BIomolecule TWEEZers), a transformative tool that overcomes these challenges by integrating optical trapping, time-resolved electromagnetic tweezers, and fluorescence microscopy, demonstrated on single DNA molecules, that can controllably form and visualise higher order structural motifs including plectonemes. This technology combined with cutting-edge MD simulations provides quantitative insight into complex dynamic structures relevant to DNA cellular processes and can be adapted to study a range of filamentous biopolymers.

Rolling circle RNA synthesis catalyzed by RNA.

RNA-catalyzed RNA replication is widely considered a key step in the emergence of life's first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here, we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. We observe sustained RCS by a triplet polymerase ribozyme beyond full-length circle synthesis with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularization. Thus, all steps of a viroid-like RNA replication pathway can be catalyzed by RNA alone. Finally, we explore potential RCS mechanisms by molecular dynamics simulations, which indicate a progressive build-up of conformational strain upon RCS with destabilization of nascent strand 5'- and 3'-ends. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

Many organisms today rely on a trio of molecules for their survival: DNA, to store their genetic information; proteins, to conduct the biological processes required for growth or replication; and RNA, to mainly act as an intermediary between DNA and proteins. Yet, how these inanimate molecules first came together to form a living system remains unclear. Circumstantial evidence suggests that the first lifeforms relied to a much greater exrtent on RNA to conduct all necessary biological processes. There is no trace of this 'RNA world' today, but molecular 'fossils' may exist in current biology. Viroids, for example, are agents which can infect and replicate inside plant cells. They are formed of nothing but a circular strand of RNA that serves not only as genetic storage but also as ribozymes (RNA-based enzymes). Viroids need proteins from the host plant to replicate, but scientists have been able to engineer ribozymes that can copy complex RNA strands. This suggests that viroid-like replication could be achieved using only RNA. Kristoffersen et al. put this idea to the test and showed that it is possible to use RNA enzymatic activity alone to carry out all the steps of a viroid-like copying mechanism. This process included copying a viroid-like RNA circle with RNA, followed by trimming the copy to the right size and reforming the circle. These two latter steps could be carried out by a ribozyme that could itself be encoded on the RNA circle. A computer simulation indicated that RNA synthesis on the circle caused increasing tension that could ease some of the barriers to replication. These results increase our understanding of how RNA copying by RNA could be possible. This may lead to developing molecular models of a primordial RNA-based replication, which could be used to investigate early genetic systems and may have potential applications in synthetic biology.

SerraNA: a program to determine nucleic acids elasticity from simulation data.

The resistance of DNA to stretch, twist and bend is broadly well estimated by experiments and is important for gene regulation and chromosome packing. However, their sequence-dependence and how bulk elastic constants emerge from local fluctuations is less understood. Here, we present SerraNA, which is an open software that calculates elastic parameters of double-stranded nucleic acids from dinucleotide length up to the whole molecule using ensembles from numerical simulations. The program reveals that global bendability emerge from local periodic bending angles in phase with the DNA helicoidal shape. We apply SerraNA to the whole set of 136 tetra-bp combinations and we observe a high degree of sequence-dependence with differences over 200% for all elastic parameters. Tetramers with TA and CA base-pair steps are especially flexible, while the ones containing AA and AT tend to be the most rigid. Thus, AT-rich motifs can generate extreme mechanical properties, which are critical for creating strong global bends when phased properly. Our results also indicate base mismatches would make DNA more flexible, while protein binding would make it more rigid. SerraNA is a tool to be applied in the next generation of interdisciplinary investigations to further understand what determines the elasticity of DNA.

Extraspinal articular tuberculosis: An 11-year retrospective study of demographic features and clinical outcomes in East London.

OBJECTIVES: To describe demographic features, clinical outcomes and diagnostic delay amongst patients with extra-spinal articular tuberculosis (TB) in a low-incidence setting. METHODS: Cases of TB treated at our institution between 2004 and 2014 were identified via the London TB register (LTBR). Demographic features of extra-spinal articular TB cases were compared to controls with TB at all other sites. For articular cases (excluding individuals <16 years or with spinal TB without peripheral joint involvement) clinical data were retrospectively collected. RESULTS: 6,146 TB patients were identified over the study period; 146 (2.4%) cases had extra-spinal articular infection. There was no difference in median age between extra-spinal articular TB cases and controls with TB at other sites (31 vs 32 years, p = 0.57). Articular cases were more likely to be male (70.6% vs 59.5%, p = 0.007), Bangladeshi (28.7% vs 18.0%) or Pakistani (24.0% vs 16.1%) and were less likely to be Black-African (9.5% vs 19.8%) (p < 0.001). 93 cases were included in the case series; 85 (88.5%) were migrants and 83 (89.2%) were South Asian. Knee and elbow joints were affected in 22 (23.7%) and 18 (19.4%) cases respectively. The median durations of pre-healthcare and healthcare associated delay were 16 and 6 weeks respectively. Where mycobacterial culture was performed, 57/75 (76%) were positive for Mycobacterium tuberculosis. 86 (92.5%) cases received standard quadruple therapy for a median of 6 months (IQR 6-9). Recurrence of TB infection occurred in 4 (4.3%) cases and there were no TB related deaths. Seven (7.6%) cases required surgical intervention. CONCLUSIONS: Extra-spinal articular TB more commonly affected men and people of South Asian ethnicity. Significant diagnostic delays were identified, including avoidable healthcare-associated delays.

National roll-out of latent tuberculosis testing and treatment for new migrants in England: a retrospective evaluation in a high-incidence area.

Latent tuberculosis infection (LTBI) screening is an important intervention for tuberculosis (TB) elimination in low-incidence countries and is, therefore, a key component of England's TB control strategy. This study describes outcomes from a LTBI screening programme in a high-incidence area to inform national LTBI screening in England and other low-incidence countries.We conducted a retrospective cohort study of LTBI screening among eligible migrants (from high-incidence countries and entered the UK within the last 5 years), who were identified at primary-care clinics in Newham, London between August 2014 and August 2015. Multivariable logistic regression was used to identify factors associated with LTBI testing uptake, interferon-γ release assay (IGRA) positivity and treatment uptake.40% of individuals offered LTBI screening received an IGRA test. The majority of individuals tested were 16-35 years old, male and born in India, Bangladesh or Pakistan. Country of birth, smoking status and co-morbidities were associated with LTBI testing uptake. IGRA positivity was 32% among those tested and was significantly associated with country of birth, age, sex and co-morbidities.This study identifies factors associated with screening uptake, IGRA positivity and treatment uptake, and improves understanding of groups that should be supported to increase acceptability of LTBI testing and treatment in the community.