Living lab modelling as a pilot study assessing the potential psychological health benefits of forest environment for cancer survivors.

OBJECTIVE: To evaluate the physiological and psychological changes in cancer survivors who engage in repeated forest therapy in a living environment. METHODS: This study included stay-based forest therapy for female cancer survivors aged ≥40 years. The program was conducted in two cycles, each spanning 3 weeks and consisting of a 2-night, 3-day stay, followed by daily life integration. The cycles were repeated from July 2, 2022, to August 18, 2022. Participant assessment included standard physical health parameters and a questionnaire on general characteristics, lifestyle habits, stress levels, and health status. RESULTS: Thirty-seven female cancer survivors participated in the forest healing program, 56.8% of whom had a history of breast cancer. The median body mass index (BMI) was 23.80 kg/m2 (range, 21.00-25.60). More than half of the patients reported mild-to-moderate fatigue, chronic pain, and mild-to-moderate depression (81%, 65%, and 73%, respectively). After two cycles of forest therapy, no significant differences were observed in terms of fatigue, pain, or BMI levels. However, significant improvements were found in quality of life measures, particularly the psychological quality of life (mean score 12.54 at baseline vs. 13.48 after cycle 2; P=0.007). Positive improvements were also observed in terms of stress (mean score 17.03 vs. 13.76; P=0.002) and depression (mean score 8.35 vs. 6.11; P=0.002) levels. CONCLUSION: Our forest-healing program demonstrated that nature-based therapies improve the mental health and quality of life of female cancer survivors, suggesting the need for further research on nature-based interventions to better support cancer survivors.

Viscosity of Al-Ni-Co-Nd(Sm) glass-forming melts at high temperatures.

Obtaining amorphous alloys with good mechanical and anticorrosion properties is an important problem of modern condensed matter physics. Since the preparation of amorphous alloys involves casting them from liquid state, information on the properties of the melts is needed. Viscosity is one of the most informative structure-sensitive property of melts. In this paper viscosity of some glass-forming Al-Ni-Co-Nd(Sm) melts with different ratio of transition metals was studied using damped oscillation method in a wide temperature range up to 1550 K. Activation energies of the viscous flow were calculated from the experimental data. The hysteresis of viscosity temperature dependences during heating and subsequent cooling was found. It can be associated with a melt transition to a more homogeneous state. The repeated heating and cooling of the melts without crystallization lead to Arrhenius type of viscosity temperature dependences.

Effects of broadband music and audible band music on relaxation states and cognitive function in young adults: a randomized controlled trial.

BACKGROUND: Although broadband music with inaudible high-frequency components may benefit human well-being, this research area is largely unexplored and lacks sufficient studies on the topic. This study aimed to investigate and compare the effects of broadband and audible band music on relaxation states and cognitive function in young adults. METHODS: A single-blind randomized controlled trial was conducted in a professional soundproof laboratory from December 22, 2022, to January 18, 2023 with 32 participants randomly assigned to two groups, "Day 1 broadband + Day 2 audible band" (n = 16) and "Day 1 audible band + Day 2 broadband" (n = 16), listening to either broadband or audible band music (the same music piece played on the piano and harp) for two sessions of 15 min each on two consecutive days. Cognitive function was measured using CNS Vital Signs at pre-listening, after the 1st session, and after the 2nd session, while heart rate was monitored throughout the experiment. Visual Analog Scale was also administered for self-reported arousal, stress, thinking ability, and attention following each listening session. RESULTS: No significant differences were found in heart rate, cognitive flexibility, and executive function between the broadband listening group and the audible band-listening group (p > 0.05). However, the broadband group exhibited significant differences in mean heart rate at several time points, as well as a significant improvement in VAS stress level during the 2nd listening session compared to the 1st (p < 0.05). On the other hand, significant improvements in cognitive flexibility and executive function were observed in the audible band group across different time points (p < 0.05). CONCLUSION: Comparative analysis showed that broadband and audible band music influenced cognitive function differently. Short-term audible band music listening significantly improved cognitive flexibility and executive function, while short-term broadband music listening significantly reduced reaction time in cognitive tests. Additionally, broadband music consistently resulted in lower mean heart rates compared to audible band music at all time points, suggesting that it may be more effective in promoting relaxation and reducing stress, although these differences were not statistically significant. Since the cognitive enhancing effects of broadband music may be counteracted by the drowsy effect of the selected relaxing music, using different types of music may be necessary to confirm its effects in future studies.

Application of a New Device for Vision Relaxation in Computer Users.

This study aims to explore the potential of a novel EYE ROLL device designed to facilitate guided vision relaxation exercises in an open space. A prospective study was performed on 89 participants who perform screenwork for at least four hours daily. All participants were randomly divided into three groups: a Control group with no exercising, a Manual group undertook manual vision relax ation exercises, and an Eyeroll group engaged in EYE ROLL device-assisted vision relaxation exercises. Each participant underwent three evaluations (an initial baseline assessment, a 4-week follow-up, and an 8-week follow-up) with four assessment tools: a comprehensive vision examination, an in-depth questionnaire, saccadic eye movement recordings, and objective accommodation measurements. There was a statistically significant decrease (35% and above) in complaint scores at the 4-week follow-up in both training groups. Although statistically insignificant, complaints continued to decrease after an 8-week period. No significant changes were observed in clinical or objective accommodative parameters. Some variation of visual functions was observed in all groups due to repeated measures. Vision relaxation exercises combined with proper vision ergonomics and working habits can reduce asthenopic complaints. The EYE ROLL device presents a promising tool for integrating these exercises into the working environment.

Gelatin-Based Scaffolds with Carrageenan and Chitosan for Soft Tissue Regeneration.

Motivated by the enormous potential of hydrogels in regenerative medicine, new biocompatible gelatin-based hybrid hydrogels were developed through a green process using poly(ethylene glycol) diglycidyl ether as a cross-linking agent, adding carrageenan and chitosan polysaccharides to the network to better mimic the hybrid composition of native extracellular matrix. Overall, the hydrogels show suitable structural stability, high porosity and pore interconnectivity, good swellability, and finally, biocompatibility. Their mechanical behavior, investigated by tensile and compression tests, appears to be characterized by nonlinear elasticity with high compliance values, fast stress-relaxation, and good strain reversibility with no sign of mechanical failure for compressive loading-unloading cycles at relatively high deformation levels of 50%. Degradation tests confirm the hydrogel bioresorbability by gradual hydrolysis, during which the structural integrity of both materials is maintained, while their mechanical behavior becomes more and more compliant. Human Umbilical Cord-derived Mesenchymal Stem Cells (hUC-MSCs) were used to test the hydrogels as potential carriers for cell delivery in tissue engineering. hUC-MSCs cultured inside the hydrogels show a homogenous distribution and maintain their growth and viability for at least 21 days of culture, with an increasing proliferation trend. Hence, this study contributes to a further understanding of the potential use of hybrid hydrogels and hUC-MSCs for a wide range of biomedical applications, particularly in soft tissue engineering.

Polydimethylsiloxane enabled triple-action water-resistant coating with desirable relaxation rate in clear aligner.

Clear aligners undergo rapid stress relaxation in warm, moist oral environments, compromising therapeutic effectiveness and longevity of treatment. To develop an innovative multilayer composite material with improved stability and reduced stress release, we have engineered an innovative coating characterized by the surface aggregation of polydimethylsiloxane (PDMS), which imparts a pronounced hydrophobic effect. In addition, the chemically and physically cross-linked structure of the coating reduces the free volume created by molecular chain rearrangement owing to the presence of water molecules, thereby minimizing water penetration into the coating. Concurrently, the coating's internal structure is enriched with numerous polar functional groups to capture water molecules that penetrate into the inside of the coating. Through combination of these mechanisms, water molecules are effectively sequestered, thereby impeding their penetration into the polyethylene terephthalate glycol (PETG) substrate. The impact of the polydimethylsiloxane content on the triple-action water-resistance mechanisms was thoroughly examined using attenuated total reflection (ATR)-Fourier transform infrared (FTIR), water absorption rate, water swelling rate, and X-ray photoelectron spectroscopy. The low surface energy cross-linked polyurethane coating is applied to the polyethylene terephthalate glycol (PETG) substrate to create a novel composite material with specific mechanical properties and reduced stress relaxation. The composite material remains stable in simulated oral environment with linear swelling rate of 0.58 % upon water absorption. Additionally, the stress release rate of the composite material within 336 h is notably lower (23.64 %) than that of PETG (62.29 %).

Differential Back Muscle Flexion-Relaxation Phenomenon in Constrained versus Unconstrained Leg Postures.

Previous studies examining the flexion-relaxation phenomenon (FRP) in back muscles through trunk forward flexion tests have yielded inconsistent findings, primarily due to variations in leg posture control. This study aimed to explore the influence of leg posture control and individual flexibility on FRP in back and low limb muscles. Thirty-two male participants, evenly distributed into high- and low-flexibility groups, were recruited. Activities of the erector spinae, biceps femoris, and gastrocnemius muscles, alongside the lumbosacral angle (LSA), were recorded as participants executed trunk flexion from 0° to 90° in 15° increments, enabling an analysis of FRP and its correlation with the investigated variables. The findings highlighted significant effects of all examined factors on the measured responses. At a trunk flexion angle of 60°, the influence of leg posture and flexibility on erector spinae activities was particularly pronounced. Participants with limited flexibility exhibited the most prominent FRP under constrained leg posture, while those with greater flexibility and unconstrained leg posture displayed the least FRP, indicated by their relatively larger LSAs. Under constrained leg posture conditions, participants experienced an approximate 1/3 to 1/2 increase in gastrocnemius activity throughout trunk flexion from 30° to 90°, while biceps femoris activity remained relatively constant. Using an inappropriate leg posture during back muscle FRP assessments can overestimate FRP. These findings offer guidance for designing future FRP research protocols.

Clinical validation of fully automated laminar knee cartilage transverse relaxation time (T2) analysis in anterior cruciate ligament (ACL)-injured knees- on behalf of the osteoarthritis (OA)-Bio consortium.

Background: Magnetic resonance imaging (MRI) cartilage transverse relaxation time (T2) reflects cartilage composition, mechanical properties, and early osteoarthritis (OA). T2 analysis requires cartilage segmentation. In this study, we clinically validate fully automated T2 analysis at 1.5 Tesla (T) in anterior cruciate ligament (ACL)-injured and healthy knees. Methods: We studied 71 participants: 20 ACL-injured patients with, and 22 without dynamic knee instability, 13 with surgical reconstruction, and 16 healthy controls. Sagittal multi-echo-spin-echo (MESE) MRIs were acquired at baseline and 1-year follow-up. Femorotibial cartilage was segmented manually; a convolutional neural network (CNN) algorithm was trained on MRI data from the same scanner. Results: Dice similarity coefficients (DSCs) of automated versus manual segmentation in the 71 participants were 0.83 (femora) and 0.89 (tibiae). Deep femorotibial T2 was similar between automated (45.7±2.6 ms) and manual (45.7±2.7 ms) segmentation (P=0.828), whereas superficial layer T2 was slightly overestimated by automated analysis (53.2±2.2 vs. 52.1±2.1 ms for manual; P<0.001). T2 correlations were r=0.91-0.99 for deep and r=0.86-0.97 for superficial layers across regions. The only statistically significant T2 increase over 1 year was observed in the deep layer of the lateral femur [standardized response mean (SRM) =0.58 for automated vs. 0.52 for manual analysis; P<0.001]. There was no relevant difference in baseline/longitudinal T2 values/changes between the ACL-injured groups and healthy participants, with either segmentation method. Conclusions: This clinical validation study suggests that automated cartilage T2 analysis from MESE at 1.5T is technically feasible and accurate. More efficient 3D sequences and longer observation intervals may be required to detect the impact of ACL injury induced joint instability on cartilage composition (T2).

Vibrational spectroscopy of dynamic interactions and relaxation processes in ionic systems.

In recent years, there has been a boom in the publication of research results on ionic (electrolyte) systems used in the creation of electrochemical energy systems (batteries, supercapacitors, etc.). One of the alternative ways to obtain dynamic information in ionic systems is spectroscopic experiments, and the most valuable information on the behavior of condensed systems at picosecond and adjacent time intervals is provided by vibrational spectroscopy. The source of this information is the analysis of the shape and width of the lines in the vibrational spectra of ion systems. This review aims to systematize the theoretical and methodological aspects of the study of the dynamics of ion systems by vibrational spectroscopy.

Glycerol-slaved 1H-1H NMR cross-relaxation in quasi-native lysozyme.

1H-1H nuclear cross-relaxation experiments have been carried out with lysozyme in variable glycerol viscosity to study intramolecular motion, self-diffusion, and isotropic rigid-body rotational tumbling at 298 K, pH 3.8. Dynamics of intramolecular 1H-1H cross-relaxation rates, the increase in internuclear spatial distances, and lateral and rotational diffusion coefficients all show fractional viscosity dependence with a power law exponent κ in the 0.17-0.83 range. The diffusion coefficient of glycerol Ds with the bulk viscosity itself is non-Stokesian, having a fractional viscosity dependence on the medium viscosity (Ds âˆ¼ Î·-κ, κ ≈ 0.71). The concurrence and close similarity of the fractional viscosity dependence of glycerol diffusion on the one hand, and diffusion and intramolecular cross-relaxation rates of the protein on the other lead to infer that relaxation of glycerol slaves protein relaxations. Glycerol-transformed native lysozyme to a quasi-native state does not affect the conclusion that both global and internal fluctuations are slaved to glycerol relaxation.

Poor Self-Reported Sleep is Associated with Prolonged White Matter T2 Relaxation in Psychotic Disorders.

Background: Schizophrenia (SZ) and bipolar disorder (BD) are characterized by white matter (WM) abnormalities, however, their relationship with illness presentation is not clear. Sleep disturbances are common in both disorders, and recent evidence suggests that sleep plays a critical role in WM physiology. Therefore, it is plausible that sleep disturbances are associated with impaired WM integrity in these disorders. To test this hypothesis, we examined the association of self-reported sleep disturbances with WM transverse (T2) relaxation times in patients with SZ spectrum disorders and BD with psychotic features. Methods: 28 patients with psychosis (17 BD-I, with psychotic features and 11 SZ spectrum disorders) were included. Metabolite and water T2 relaxation times were measured in the anterior corona radiata at 4T. Sleep was evaluated using the Pittsburgh Sleep Quality Index. Results: PSQI total score showed a moderate to strong positive correlation with water T2 (r = 0.64, p<0.001). Linear regressions showed that this association was specific to sleep disturbance but was not a byproduct of exacerbation in depressive, manic, or psychotic symptoms. In our exploratory analysis, sleep disturbance was correlated with free water percentage, suggesting that increased extracellular water may be a mechanism underlying the association of disturbed sleep and prolonged water T2 relaxation. Conclusion: Our results highlight the connection between poor sleep and WM abnormalities in psychotic disorders. Future research using objective sleep measures and neuroimaging techniques suitable to probe free water is needed to further our insight into this relationship.

Quantitative muscle MRI in sporadic inclusion body myositis (sIBM): A prospective cohort study.

Background: Sporadic inclusion body myositis (sIBM) is the predominant idiopathic inflammatory myopathy (IIM) in older people. Limitations of classical clinical assessments have been discussed as possible explanations for failed clinical trials, underlining the need for more sensitive outcome measures. Quantitative muscle MRI (qMRI) is a promising candidate for evaluating and monitoring sIBM. Objective: Longitudinal assessment of qMRI in sIBM patients. Methods: We evaluated fifteen lower extremity muscles of 12 sIBM patients (5 females, mean age 69.6, BMI 27.8) and 12 healthy age- and gender-matched controls. Seven patients and matched controls underwent a follow-up evaluation after one year. Clinical assessment included testing for muscle strength with Quick Motor Function Measure (QMFM), IBM functional rating scale (IBM-FRS), and gait analysis (6-minute walking distance). 3T-MRI scans of the lower extremities were performed, including a Dixon-based sequence, T2 mapping and Diffusion Tensor Imaging. The qMRI-values fat-fraction (FF), water T2 relaxation time (wT2), fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (λ1), and radial diffusivity (RD) were analysed. Results: Compared to healthy controls, significant differences for all qMRI parameters averaged over all muscles were found in sIBM using a MANOVA (p < 0.001). In low-fat muscles (FF < 10% ), a significant increase of wT2 and FA with an accompanying decrease of MD, λ1, and RD was observed (p≤0.020). The highest correlation with clinical assessments was found for wT2 values in thigh muscles (r≤-0.634). Significant changes of FF (+3.0% ), wT2 (+0.6 ms), MD (-0.04 10 - 3mm2/s), λ1 (-0.05 10 - 3mm2/s), and RD (-0.03 10 - 3mm2/s) were observed in the longitudinal evaluation of sIBM patients (p≤0.001). FA showed no significant change (p = 0.242). Conclusion: qMRI metrics correlate with clinical findings and can reflect different ongoing pathophysiological mechanisms. While wT2 is an emerging marker of disease activity, the role of diffusion metrics, possibly reflecting changes in fibre size and intracellular deposits, remains subject to further investigations.

Reliability and changes in knee cartilage T2 relaxation time from 6 to 24 months after anatomic anterior cruciate ligament reconstruction.

The objectives of this study were to evaluate the reliability of cartilage T2 relaxation time measurements and to identify focal changes in T2 relaxation on the affected knee from 6 to 24 months after anatomic anterior cruciate ligament reconstruction (ACLR). Data from 41 patients who received anatomic ACLR were analyzed. A bilateral 3.0-T MRI was acquired 6 and 24 months after ACLR. T2 relaxation time was measured in subregions of the femoral condyle and the tibial plateau. The root-mean-square coefficient of variation (RMSCV) was calculated to evaluate the reliability of T2 relaxation time in the contralateral knee. Subregion changes in the affected knee T2 relaxation time were identified using the contralateral knee as a reference. The superficial and full thickness layers of the central and inner regions showed good reliability. Conversely, the outer regions on the femoral side and regions in the deep layers showed poor reliability. T2 relaxation time increased in only 3 regions on the affected knee when controlling for changes in the contralateral knee, while changes in T2 relaxation time were identified in 14 regions when not using the contralateral knee as a reference. In conclusion, evaluation of cartilage degeneration by T2 relaxation time after ACLR is most reliable for central and inner cartilage regions. Cartilage degeneration occurs in the central and outer regions of the lateral femoral condyle from 6 to 24 months after anatomic ACLR.

Encapsulated Atomic Hydrogen in Octamethyl-POSS cages: a Pulsed EPR Study.

Encapsulated atomic hydrogen in polyhedral oligomeric silsesquioxane (POSS) cages is a promising candidate for spin-based quantum technologies. Key parameters such as spin relaxation times and magnetic interactions with surrounding electron and nuclear spins can be typically probed with advanced electron paramagnetic resonance (EPR) methods. Here we present a detailed pulsed EPR study of the species H@Si8O12R8 with R=CH3, namely encapsulated atomic hydrogen in the octamethyl POSS derivative. The temperature dependence of the spin-lattice relaxation rate 1/T1 is analyzed in terms of a Raman process with a Debye temperature of ΘD=145 K and a thermally activated process with Ea=794 K (552 cm-1), whereas, the phase memory time TM shows the typical shortening behaviour at T<150 K observed for all methyl-containing derivatives. The hyperfine coupling of the cage 29Si nuclei is measured by hyperfine sublevel correlation (HYSCORE) spectroscopy and is found to fulfil the so-called "matching condition" at the low-field EPR transition. The potential of this paramagnetic molecule to perform one-qubit quantum operations is probed by room-temperature Rabi oscillations.

Water rotational relaxation time measurement by shortwave infrared micro spectroscopy(SWIR) at sub-zero temperatures.

The shortwave infrared spectroscopy (SWIR) is the noble method which allows to evaluate the rotational relaxation time of water (RRTW) in a sample. Because SWIR requires the reference sample of pure water, the measurement temperature is limited only at above 0 °C. In this study, we expanded this temperature limitation of SWIR by using alternative reference solutions with freezing points below 0 °C, including sugar and glycerol solutions. The results showed that some reference sample solutions are useable for evaluating RRTW in samples below 0 °C. It was found that RRTW in solution measured by newly proposed SWIR agrees with RRTW measured by dielectric spectroscopy in 10% accuracy when it is shorter than 100psec.

Tailoring component incorporation for homogenized perovskite solar cells.

Deep-level traps at the buried interface of perovskite and energy mismatch problems between the perovskite layer and heterogeneous interfaces restrict the development of ideal homogenized films and efficient perovskite solar cells (PSCs) using the one-step spin-coating method. Here, we strategically employed sparingly soluble germanium iodide as a homogenized bulk in-situ reconstruction inducing material preferentially aggregated at the perovskite buried interface with gradient doping, markedly reducing deep-level traps and withstanding local lattice strain, while minimizing non-radiative recombination losses and enhancing the charge carrier lifetime over 9 µs. Furthermore, this gradient doping assisted in modifying the band diagram at the buried interface into a desirable flattened alignment, substantially mitigating the energy loss of charge carriers within perovskite films and improving the carrier extraction equilibrium. As a result, the optimized device achieved a champion power conversion efficiency of 25.24% with a fill factor of up to 84.65%, and the unencapsulated device also demonstrated excellent light stability and humidity stability. This work provides a straightforward and reliable homogenization strategy of perovskite components for obtaining efficient and stable PSCs.

[Airway management in neonates and infants : Recommendations according to the ESAIC/BJA guidelines]. / Atemwegssicherung bei Neugeborenen und Säuglingen : Handlungsempfehlung gemäß der BJA/ESAIC-Leitlinie.

Securing an airway enables the oxygenation and ventilation of the lungs and is a potentially life-saving medical procedure. Adverse and critical events are common during airway management, particularly in neonates and infants. The multifactorial reasons for this include patient-dependent, user-dependent and also external factors. The recently published joint ESAIC/BJA international guidelines on airway management in neonates and infants are summarized with a focus on the clinical application. The original publication of the guidelines focussed on naming formal recommendations based on systematically documented evidence, whereas this summary focusses particularly on the practicability of their implementation.

The relationship between challenge-hindrance stressors and innovative behavior among medical postgraduates in China: the mediation role of academic engagement and the moderating effect of relaxation.

This study investigated the relationship between challenge-hindrance stressors and innovative behavior of medical postgraduates in China, examining the mediating role of academic engagement and the moderating effect of relaxation. Drawing from a sample of 437 medical postgraduates from three Chinese universities, our findings revealed that challenge stressors positively correlated with innovative behavior, while the direct relationship between hindrance stressors and innovative behavior was not statistically significant. Furthermore, academic engagement mediated the relationship between two types of stressors and innovative behavior. Challenge stressors enhanced academic engagement, which in turn fostered innovative behavior. Conversely, hindrance stressors were found to diminish academic engagement, which in turn indirectly limited innovative behavior. Additionally, relaxation was identified as a moderating factor that helped mitigate the negative effects of hindrance stressors on academic engagement and indirectly on innovative behavior. These results suggested that academic engagement as a mechanism played a pivotal role in determining how different stressors influenced innovative behavior, underscoring the need for stress management, particularly through relaxation techniques, to maintain high levels of academic engagement and innovative behavior. This study offers practical insights for medical education policymakers and educators in China, emphasizing the importance of balancing stressors and incorporating relaxation practices to enhance the innovative capabilities of medical postgraduates in demanding academic environments.

Substrate stress relaxation regulates neural stem cell fate commitment.

Adult neural stem cells (NSCs) reside in the dentate gyrus of the hippocampus, and their capacity to generate neurons and glia plays a role in learning and memory. In addition, neurodegenerative diseases are known to be caused by a loss of neurons and glial cells, resulting in a need to better understand stem cell fate commitment processes. We previously showed that NSC fate commitment toward a neuronal or glial lineage is strongly influenced by extracellular matrix stiffness, a property of elastic materials. However, tissues in vivo are not purely elastic and have varying degrees of viscous character. Relatively little is known about how the viscoelastic properties of the substrate impact NSC fate commitment. Here, we introduce a polyacrylamide-based cell culture platform that incorporates mismatched DNA oligonucleotide-based cross-links as well as covalent cross-links. This platform allows for tunable viscous stress relaxation properties via variation in the number of mismatched base pairs. We find that NSCs exhibit increased astrocytic differentiation as the degree of stress relaxation is increased. Furthermore, culturing NSCs on increasingly stress-relaxing substrates impacts cytoskeletal dynamics by decreasing intracellular actin flow rates and stimulating cyclic activation of the mechanosensitive protein RhoA. Additionally, inhibition of motor-clutch model components such as myosin II and focal adhesion kinase partially or completely reverts cells to lineage distributions observed on elastic substrates. Collectively, our results introduce a unique system for controlling matrix stress relaxation properties and offer insight into how NSCs integrate viscoelastic cues to direct fate commitment.

Methods to Quantitatively Measure Topological Changes Induced by DNA-Binding Proteins In Vivo and In Vitro.

Agarose gel electrophoresis in the presence of chloroquine (an intercalating agent) can be used to resolve and characterize the population of topoisomers present in supercoiled plasmid DNA. Here, we describe how chloroquine gel electrophoresis can capture changes in the topoisomer distribution of plasmid DNA that bears a recognition site for a given protein, if that plasmid is isolated from cells producing the protein of interest. We also describe two complementary in vitro assays, which can be used to capture transient changes in DNA supercoiling caused when the purified protein of interest engages its recognition site. These are the topoisomerase I-mediated relaxation assay (TMRA) and the ligase-mediated supercoiling assay (LMSA). Together, these in vivo and in vitro methods allow the capture and measurement of changes in DNA topology that are triggered by DNA-binding proteins, especially those that multimerize on or spread along DNA.