Displacement and dissociation of oligonucleotides during DNA hairpin closure under strain.

The hybridization kinetic of an oligonucleotide to its template is a fundamental step in many biological processes such as replication arrest, CRISPR recognition, DNA sequencing, DNA origami, etc. Although single kinetic descriptions exist for special cases of this problem, there are no simple general prediction schemes. In this work, we have measured experimentally, with no fluorescent labelling, the displacement of an oligonucleotide from its substrate in two situations: one corresponding to oligonucleotide binding/unbinding on ssDNA and one in which the oligonucleotide is displaced by the refolding of a dsDNA fork. In this second situation, the fork is expelling the oligonucleotide thus significantly reducing its residence time. To account for our data in these two situations, we have constructed a mathematical model, based on the known nearest neighbour dinucleotide free energies, and provided a good estimate of the residence times of different oligonucleotides (DNA, RNA, LNA) of various lengths in different experimental conditions (force, temperature, buffer conditions, presence of mismatches, etc.). This study provides a foundation for the dynamics of oligonucleotide displacement, a process of importance in numerous biological and bioengineering contexts.

Strand switching mechanism of Pif1 helicase induced by its collision with a G-quadruplex embedded in dsDNA.

G-rich sequences found at multiple sites throughout all genomes may form secondary structures called G-quadruplexes (G4), which act as roadblocks for molecular motors. Among the enzymes thought to process these structures, the Pif1 DNA helicase is considered as an archetypical G4-resolvase and its absence has been linked to G4-related genomic instabilities in yeast. Here we developed a single-molecule assay to observe Pif1 opening a DNA duplex and resolving the G4 in real time. In support of former enzymological studies, we show that the helicase reduces the lifetime of G4 from hours to seconds. However, we observe that in the presence of a G4, Pif1 exhibits a strong strand switching behavior, which can lead to Pif1 escaping G4 resolution, depending on the structural context surrounding the substrate. This behavior is also detected in the presence of other roadblocks (LNA or RNA). We propose that the efficiency of Pif1 to remove a roadblock (G4 or other) is affected by its strand switching behavior and depends on the context surrounding the obstacle. We discuss how this switching behavior may explain several aspects of Pif1 substrate preference and affect its activity as a G4 resolvase in vivo.

Nucleosome Positioning on Large Tandem DNA Repeats of the '601' Sequence Engineered in Saccharomyces cerevisiae.

The artificial 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, application of this property in vivo has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro, analysis of nucleosome occupancy in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.

The mindfulness trajectories of addressing suicidal behaviour: A systematic review.

BACKGROUND AND OBJECTIVES: Suicidal behaviour has been a persistent concern in medical as well as general settings. Many psychotherapeutic approaches have tried to address suicidal behaviour in different ways. Mindfulness-based interventions (MBIs) have garnered much attention in the last decade because of their treatment efficacy. This systematic review aimed to examine evidence-based research regarding the effectiveness of MBIs as a psychotherapy intervention on suicidality and to deliver suggestions that might help future research. METHOD: The identification of literature was made through an extensive search of the electronic databases, to extract studies relating to the efficacy of MBIs on addressing suicidal behaviour. Additional researches based on library sources were searched manually. The studies' selection was based on a pre-determined inclusion and exclusion criteria as well as the quality of the studies. RESULTS: The present review helped us identify 13 studies, including six randomised controlled trials, two controlled studies and five pre-post observational studies. The findings reported in the studies were mostly favourable to MBIs as an effective intervention strategy for suicidal behaviour. CONCLUSION: MBIs show promising effects as an intervention for suicidal behaviour. However, large scale, high-quality trials with active control, and long term intervention efficacy studies are needed to understand the mechanisms through which MBIs reduce suicidal behaviour.

Theoretical Mapping of Suicidal Risk Factors During the COVID-19 Pandemic: A Mini-Review.

Suicide prevention in times of COVID-19 pandemic has become more challenging than ever due to unusual circumstances. The common risk factors identified with regard to suicidal behavior are fear of COVID-19, economic instability, poor access to healthcare facilities, pre-existing psychiatric disorders, and social disconnect. The studies done so far have reported either case studies or have made an effort to understand the risk factors. An understanding of the underlying causal pattern from existing theories, behind these risks, will enable adopting appropriate prevention mechanisms. Hence, this review examines evidence related to risk factors of suicides that occurred during COVID 19 and discusses it in the light of three major theoretical approaches: interpersonal model, stress diathesis model, and cognitive model. The insights obtained from the three viewpoints reveal that perceived burdensomeness, thwarted belongingness, stress sensitivity, cognitive errors such as magnification, catastrophic thinking, arbitrary inference, and mind-reading are likely reasons behind these risk factors for suicide. It is suggested that awareness regarding COVID-19 stressors, use of community-based approaches like gatekeeper training, and brief online psychotherapy by using techniques of mindfulness, interpersonal psychotherapy, and cognitive behavior therapy can be useful in reducing suicide risk during COVID-19.

Mechanistic characterization of the DEAD-box RNA helicase Ded1 from yeast as revealed by a novel technique using single-molecule magnetic tweezers.

DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.

Effectiveness of mindfulness based cognitive behavior therapy on life satisfaction, and life orientation of adolescents with depression and suicidal ideation.

Suicide and depression are among the most alarming phenomena prevalent throughout the world. Various approaches have tried to explain the intricacies in depression and suicide, as a consequence of faulty psychological adjustment of the individual. Several therapeutic approaches have been developed to strengthen one's coping process, among which cognitive behaviour therapy has shown promising results. Also, mindfulness-based approaches to cognitive behavioural therapy have further accelerated the well-being of such individuals. This study was conducted with an aim to see the effect of mindfulness-based cognitive behaviour therapy on life satisfaction and life orientation in adolescents with depression and suicidal behaviour. A sample of 30 adolescents who scored high on scales of depression and suicidal tendencies were administered pre-test measures on life satisfaction and life orientation. After that they were exposed to an eight weeks programme on mindfulness-based cognitive behaviour therapy, followed by a post-assessment on the same measures. The analysis of pre and post test revealed a significant enhancement in life satisfaction, life orientation, and family functioning as well as a reduction in depressive symptoms and suicidal ideation. It is concluded that mindfulness-based cognitive behaviour therapy serves as an effective medium to enhance the psychological functioning of depressive and suicidal adolescents.

UPF1-like helicase grip on nucleic acids dictates processivity.

Helicases are molecular engines which translocate along nucleic acids (NA) to unwind double-strands or remodel NA-protein complexes. While they have an essential role in genome structure and expression, the rules dictating their processivity remain elusive. Here, we developed single-molecule methods to investigate helicase binding lifetime on DNA. We found that UPF1, a highly processive helicase central to nonsense-mediated mRNA decay (NMD), tightly holds onto NA, allowing long lasting action. Conversely, the structurally similar IGHMBP2 helicase has a short residence time. UPF1 mutants with variable grip on DNA show that grip tightness dictates helicase residence time and processivity. In addition, we discovered via functional studies that a decrease in UPF1 grip impairs NMD efficiency in vivo. Finally, we propose a three-state model with bound, sliding and unbound molecular clips, that can accurately predict the modulation of helicase processivity.

A conserved structural element in the RNA helicase UPF1 regulates its catalytic activity in an isoform-specific manner.

The RNA helicase UPF1 is a key component of the nonsense mediated mRNA decay (NMD) pathway. Previous X-ray crystal structures of UPF1 elucidated the molecular mechanisms of its catalytic activity and regulation. In this study, we examine features of the UPF1 core and identify a structural element that adopts different conformations in the various nucleotide- and RNA-bound states of UPF1. We demonstrate, using biochemical and single molecule assays, that this structural element modulates UPF1 catalytic activity and thereby refer to it as the regulatory loop. Interestingly, there are two alternatively spliced isoforms of UPF1 in mammals which differ only in the lengths of their regulatory loops. The loop in isoform 1 (UPF11) is 11 residues longer than that of isoform 2. We find that this small insertion in UPF11 leads to a two-fold increase in its translocation and ATPase activities. To determine the mechanistic basis of this differential catalytic activity, we have determined the X-ray crystal structure of the helicase core of UPF11 in its apo-state. Our results point toward a novel mechanism of regulation of RNA helicases, wherein alternative splicing leads to subtle structural rearrangements within the protein that are critical to modulate enzyme movements and catalytic activity.

A mechanistic study of helicases with magnetic traps.

Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA, or RNA/RNA) and thus are essential to DNA replication and the maintenance of nucleic acid integrity. We review the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins. Many features have been uncovered by these studies that were obscured by bulk studies, such as DNA strands switching, mechanical (rather than biochemical) coupling between helicases and polymerases, helicase-induced re-hybridization and stalled fork rescue.

Single molecule studies of helicases with magnetic tweezers.

Helicases are a broad family of enzymes that perform crucial functions in DNA replication and in the maintenance of DNA and RNA integrity. A detailed mechanical study of helicases on DNA and RNA is possible using single molecule manipulation methods. Among those, magnetic tweezers (or traps) present a convenient, moderate throughput assay (tens of enzymes can be monitored simultaneously) that allow for high resolution (single base-pair) studies of these enzymes in various conditions and on various substrates (double and single stranded DNA and RNA). Here we discuss various implementation of the basic assay relevant for these studies.

Nonequilibrium fluctuations of mechanically stretched single red blood cells detected by optical tweezers.

We study the thermal and out-of-equilibrium mechanical dynamics of single, living human red blood cells (RBCs) by combining two-probe passive and active microrheology techniques. Both experiments were performed quasisimultaneously on the same cell using two identical polystyrene probes, biochemically attached to the cell membrane. We obtained compelling evidence of nonequilibrium fluctuations in the RBCs under physiological condition and without the influence of any external chemicals. The spectral distributions of metabolically driven forces and viscoelastic response were evaluated in the relaxed and stretched states, intended to simulate the varying natural environment of the cells during blood circulation. We found that the internally generated forces are more pronounced in the stretched state, suggesting a stress-dependent RBC activity.

Direct observation of single DNA structural alterations at low forces with surface-enhanced Raman scattering.

DNA experiences numerous mechanical events, necessitating single-molecule force spectroscopy techniques to provide insight into DNA mechanics as a whole system. Inherent Brownian motion limits current force spectroscopy methods from observing possible bond level structural changes. We combine optical trapping and surface-enhanced Raman scattering to establish a direct relationship between DNA's extension and structure in the low force, entropic regime. A DNA molecule is trapped close to a surface-enhanced Raman scattering substrate to facilitate a detectable Raman signal. DNA Raman modes shift in response to applied force, indicating phosphodiester mechanical alterations. Molecular dynamic simulations confirm the local structural alterations and the Raman sensitive band identified experimentally. The combined Raman and force spectroscopy technique, to our knowledge, is a novel methodology that can be generalized to all single-molecule studies.

Studying single red blood cells under a tunable external force by combining passive microrheology with Raman spectroscopy.

The dynamic micromechanical and structural properties of single human red blood cells are studied using a combination of dual trap optical tweezers and confocal Raman spectroscopy. Such a combination permits us to show a direct relationship between the rheological properties and chemical structure conformation. The frequency dependence of the complex stiffness of the cells was measured using both one and two probe response functions under identical experimental conditions. Both the microrheology and Raman measurements were performed at different stretching forces applied to the cell. A detailed analysis of the auto- and cross-correlated probe motions allows exploring the local and overall viscoelastic properties of the cells over a controlled range of the deformations. The observed growth of the cell viscoelasticity with stretching was associated with structural changes in the cell membrane monitored via the Raman spectroscopy.

Absorption spectroscopy of single red blood cells in the presence of mechanical deformations induced by optical traps.

The electronic properties of single human red blood cells under mechanical deformations were investigated using a combination of dual beam optical tweezers and UV-vis absorption spectroscopy. The mechanical deformations were induced by two near-infrared optical traps with different trapping powers and trap configurations. The deformations were applied in two ways: locally, due to the mechanical forces around the traps, and by stretching the cell by moving the traps in opposite directions. In the presence of local deformations, the single cell undergoes a transition from an oxygenated state to a partially deoxygenated state. This process was found to be reversible and strongly power-dependent. Stretching the cell caused an opposite effect, indicating that the electronic response of the whole cell is dominated by the local interaction with the trapping beams. Results are discussed considering light-induced local heating, the Stark effect, and biochemical alterations due to mechanical forces, and are compared with reports of previous Raman spectroscopy studies. The information gained by the analysis of a single red blood cell's electronic response facilitates the understanding of fundamental physiological processes and sheds further light on the cell's mechanochemistry. This information may offer new opportunities for the diagnosis and treatment of blood diseases.

Mechanochemistry of single red blood cells monitored using Raman tweezers.

Two microparticles were biochemically attached to a red blood cell at diametrically opposite parts and held by optical traps allowing to impose deformations. The cell deformation was monitored from the microscopy images. Raman spectra of the cell under tunable deformations were studied. Vibrational spectra analysis at different stretching states was supported with two statistical methods. Principal Component Analysis distinguishes the most prominent changes in spectra while 2D correlation technique monitors the evolution of Raman bands during stretching. The measurements show significant changes in the cell chemical structure with stretching however the changes saturate above 20% of cell deformation. Mechanical deformation of the cell mainly affects the bands corresponding to hemoglobin but contributions from spectrin and membrane proteins can not be excluded. The saturation of bands at higher deformations suggests some structural relaxation that RBC has to undergo to bear extra load. The results confirm widely accepted belief that spectrin released from membrane proteins allows for significant shape changes of the cells. We therefore tentatively suggest that interaction between membrane and cytoskeleton during deformation can be efficiently probed by confocal Raman spectroscopy, in particular via the peak around 1035 cm(-1).

Colored noise in the fluctuations of an extended DNA molecule detected by optical trapping.

We studied fluctuations of an optically trapped bead connected to a single DNA molecule anchored between the bead and a cover glass or between two optically trapped beads. Power spectral densities of the bead position for different extensions of the molecule were compared with the power spectral density of the position fluctuations of the same bead without the molecule attached. Experiments showed that the fluctuations of the DNA molecule extended up to 80% by a force of 3 pN include the colored noise contribution with spectral dependence 1/f (α) with α ~ 0.75.