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Transcription through chromatin under torsion represents a fundamental problem in biology. Pol II must overcome nucleosome obstacles and, because of the DNA helical structure, must also rotate relative to the DNA, generating torsional stress. However, there is a limited understanding of how Pol II transcribes through nucleosomes while supercoiling DNA. In this work, we developed methods to visualize Pol II rotation of DNA during transcription and determine how torsion slows down the transcription rate. We found that Pol II stalls at ± 9 pN·nm torque, nearly sufficient to melt DNA. The stalling is due to extensive backtracking, and the presence of TFIIS increases the stall torque to + 13 pN·nm, making Pol II a powerful rotary motor. This increased torsional capacity greatly enhances Pol II's ability to transcribe through a nucleosome. Intriguingly, when Pol II encounters a nucleosome, nucleosome passage becomes more efficient on a chromatin substrate than on a single-nucleosome substrate, demonstrating that chromatin efficiently buffers torsional stress via its torsional mechanical properties. Furthermore, topoisomerase II relaxation of torsional stress significantly enhances transcription, allowing Pol II to elongate through multiple nucleosomes. Our results demonstrate that chromatin greatly reduces torsional stress on transcription, revealing a novel role of chromatin beyond the more conventional view of it being just a roadblock to transcription., Competing Interests: COMPETING INTERESTS The authors declare no competing financial interests.

DNA replication and transcription occur simultaneously on the same DNA template, leading to inevitable conflicts between the replisome and RNA polymerase. These conflicts can stall the replication fork and threaten genome stability. Although numerous studies show that head-on conflicts are more detrimental and more prone to promoting R-loop formation than co-directional conflicts, the fundamental cause for the RNA polymerase roadblock polarity remains unclear, and the structure of these R-loops is speculative. In this work, we use a simple model system to address this complex question by examining the Pol II roadblock to a DNA fork advanced via mechanical unzipping to mimic the replisome progression. We found that the Pol II binds more stably to resist removal in the head-on configuration, even with minimal transcript size, demonstrating that the Pol II roadblock has an inherent polarity. However, an elongating Pol II with a long RNA transcript becomes an even more potent and persistent roadblock while retaining the polarity, and the formation of an RNA-DNA hybrid mediates this enhancement. Surprisingly, we discovered that when a Pol II collides with the DNA fork head-on and becomes backtracked, an RNA-DNA hybrid can form on the lagging strand in front of Pol II, creating a topological lock that traps Pol II at the fork. TFIIS facilitates RNA-DNA hybrid removal by severing the connection of Pol II with the hybrid. We further demonstrate that this RNA-DNA hybrid can prime lagging strand replication by T7 DNA polymerase while Pol II is still bound to DNA. Our findings capture basal properties of the interactions of Pol II with a DNA fork, revealing significant implications for transcription-replication conflicts., Competing Interests: COMPETING INTERESTS The authors declare no competing financial interests.

In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1-linker-mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kay, Aplin, Simonet, Beenken, Miller, Libal, Boersma, Sheets and Heikal.)

Eukaryotic cells exploit dynamic and compartmentalized ionic strength to impact a myriad of biological functions such as enzyme activities, protein-protein interactions, and catalytic functions. Herein, we investigated the fluorescence depolarization dynamics of recently developed ionic strength biosensors (mCerulean3-linker-mCitrine) in Hofmeister salt (KCl, NaCl, NaI, and Na 2 SO 4 ) solutions. The mCerulean3-mCitrine acts as a Förster resonance energy transfer (FRET) pair, tethered together by two oppositely charged α-helices in the linker region. We developed a time-resolved fluorescence depolarization anisotropy approach for FRET analyses, in which the donor (mCerulean3) is excited by 425-nm laser pulses, followed by fluorescence depolarization analysis of the acceptor (mCitrine) in KE (lysine-glutamate), arginine-aspartate, and arginine-glutamate ionic strength sensors with variable amino acid sequences. Similar experiments were carried out on the cleaved sensors as well as an E6G2 construct, which has neutral α-helices in the linker region, as a control. Our results show distinct dynamics of the intact and cleaved sensors. Importantly, the FRET efficiency decreases and the donor-acceptor distance increases as the environmental ionic strength increases. Our chemical equilibrium analyses of the collapsed-to-stretched conformational state transition of KE reveal that the corresponding equilibrium constant and standard Gibbs free energy changes are ionic strength dependent. We also tested the existing theoretical models for FRET analyses using steady-state anisotropy, which reveal that the angle between the dipole moments of the donor and acceptor in the KE sensor are sensitive to the ionic strength. These results help establish the time-resolved depolarization dynamics of these genetically encoded donor-acceptor pairs as a quantitative means for FRET analysis, which complement traditional methods such as time-resolved fluorescence for future in vivo studies., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Living cells are complex, crowded, and dynamic and continually respond to environmental and intracellular stimuli. They also have heterogeneous ionic strength with compartmentalized variations in both intracellular concentrations and types of ions. These challenges would benefit from the development of quantitative, noninvasive approaches for mapping the heterogeneous ionic strength fluctuations in living cells. Here, we investigated a class of recently developed ionic strength sensors that consists of mCerulean3 (a cyan fluorescent protein) and mCitrine (a yellow fluorescent protein) tethered via a linker made of two charged α-helices and a flexible loop. The two helices are designed to bear opposite charges, which is hypothesized to increase the ionic screening and therefore a larger intermolecular distance. In these protein constructs, mCerulean3 and mCitrine act as a donor-acceptor pair undergoing Förster resonance energy transfer (FRET) that is dependent on both the linker amino acids and the environmental ionic strength. Using time-resolved fluorescence of the donor (mCerulean3), we determined the sensitivity of the energy transfer efficiencies and the donor-acceptor distances of these sensors at variable concentrations of the Hofmeister series of salts (KCl, LiCl, NaCl, NaBr, NaI, Na 2 SO 4 ). As controls, similar measurements were carried out on the FRET-incapable, enzymatically cleaved counterparts of these sensors as well as a construct designed with two electrostatically neutral α-helices (E6G2). Our results show that the energy transfer efficiencies of these sensors are sensitive to both the linker amino acid sequence and the environmental ionic strength, whereas the sensitivity of these sensors to the identity of the dissolved ions of the Hofmeister series of salts seems limited. We also developed a theoretical framework to explain the observed trends as a function of the ionic strength in terms of the Debye screening of the electrostatic interaction between the two charged α-helices in the linker region. These controlled solution studies represent an important step toward the development of rationally designed FRET-based environmental sensors while offering different models for calculating the energy transfer efficiency using time-resolved fluorescence that is compatible with future in vivo studies.

Although common in biological systems, synthetic self-assembly routes to complex 3D photonic structures with tailored degrees of disorder remain elusive. Here we show how liquids can be used to finely control disorder in porous 3D photonic crystals, leading to complex and hierarchical geometries. In these optofluidic crystals, dynamically tunable disorder is superimposed onto the periodic optical structure through partial wetting or evaporation. In both cases, macroscopic symmetry breaking is driven by subtle sub-wavelength variations in the pore geometry. These variations direct site-selective infiltration of liquids through capillary interactions. Incorporating cross-linkable resins into our liquids, we developed methods to freeze in place the filling patterns at arbitrary degrees of partial wetting and intermediate stages of drying. These percolation lithography techniques produced permanent photonic structures with adjustable disorder. By coupling strong changes in optical properties to subtle differences in fluid behavior, optofluidic crystals may also prove useful in rapid analysis of liquids.

Background: Neck pain is common, disabling and costly. Exercise is one treatment approach., Objectives: To assess the effectiveness of exercises to improve pain, disability, function, patient satisfaction, quality of life and global perceived effect in adults with neck pain., Search Methods: We searched MEDLINE, MANTIS, ClinicalTrials.gov and three other computerized databases up to between January and May 2014 plus additional sources (reference checking, citation searching, contact with authors)., Selection Criteria: We included randomized controlled trials (RCTs) comparing single therapeutic exercise with a control for adults suffering from neck pain with or without cervicogenic headache or radiculopathy., Data Collection and Analysis: Two review authors independently conducted trial selection, data extraction, 'Risk of bias' assessment and clinical relevance. The quality of the evidence was assessed using GRADE. Meta-analyses were performed for relative risk and standardized mean differences (SMD) with 95% confidence intervals (CIs) after judging clinical and statistical heterogeneity., Main Results: Twenty-seven trials (2485 analyzed /3005 randomized participants) met our inclusion criteria.For acute neck pain only, no evidence was found.For chronic neck pain, moderate quality evidence supports 1) cervico-scapulothoracic and upper extremity strength training to improve pain of a moderate to large amount immediately post treatment [pooled SMD (SMDp) -0.71 (95% CI: -1.33 to -0.10)] and at short-term follow-up; 2) scapulothoracic and upper extremity endurance training for slight beneficial effect on pain at immediate post treatment and short-term follow-up; 3) combined cervical, shoulder and scapulothoracic strengthening and stretching exercises varied from a small to large magnitude of beneficial effect on pain at immediate post treatment [SMDp -0.33 (95% CI: -0.55 to -0.10)] and up to long-term follow-up and a medium magnitude of effect improving function at both immediate post treatment and at short-term follow-up [SMDp -0.45 (95%CI: -0.72 to -0.18)]; 4) cervico-scapulothoracic strengthening/stabilization exercises to improve pain and function at intermediate term [SMDp -14.90 (95% CI:-22.40 to -7.39)]; 5) Mindfulness exercises (Qigong) minimally improved function but not global perceived effect at short term. Low evidence suggests 1) breathing exercises; 2) general fitness training; 3) stretching alone; and 4) feedback exercises combined with pattern synchronization may not change pain or function at immediate post treatment to short-term follow-up. Very low evidence suggests neuromuscular eye-neck co-ordination/proprioceptive exercises may improve pain and function at short-term follow-up.For chronic cervicogenic headache, moderate quality evidence supports static-dynamic cervico-scapulothoracic strengthening/endurance exercises including pressure biofeedback immediate post treatment and probably improves pain, function and global perceived effect at long-term follow-up. Low grade evidence supports sustained natural apophyseal glides (SNAG) exercises.For acute radiculopathy, low quality evidence suggests a small benefit for pain reduction at immediate post treatment with cervical stretch/strengthening/stabilization exercises., Authors' Conclusions: No high quality evidence was found, indicating that there is still uncertainty about the effectiveness of exercise for neck pain. Using specific strengthening exercises as a part of routine practice for chronic neck pain, cervicogenic headache and radiculopathy may be beneficial. Research showed the use of strengthening and endurance exercises for the cervico-scapulothoracic and shoulder may be beneficial in reducing pain and improving function. However, when only stretching exercises were used no beneficial effects may be expected. Future research should explore optimal dosage.

We present a simple one-pot co-assembly method for the synthesis of hierarchically structured pigment particles consisting of silica inverse-opal bricks that are doped with plasmonic absorbers. We study the interplay between the plasmonic and photonic resonances and their effect on the visual appearance of macroscopic collections of photonic bricks that are distributed in randomized orientations. Manipulating the pore geometry tunes the wavelength- and angle-dependence of the scattering profile, which can be engineered to produce angle-dependent Bragg resonances that can either enhance or contrast with the color produced by the plasmonic absorber. By controlling the overall dimensions of the photonic bricks and their aspect ratios, their preferential alignment can either be encouraged or suppressed. This causes the Bragg resonance to appear either as uniform color travel in the former case or as sparse iridescent sparkle in the latter case. By manipulating the surface chemistry of these photonic bricks, which introduces a fourth length-scale (molecular) of independent tuning into our design, we can further engineer interactions between liquids and the pores. This allows the structural color to be maintained in oil-based formulations, and enables the creation of dynamic liquid-responsive images from the pigment.

Background: Neck disorders are common, disabling and costly. The effectiveness of exercise as a physiotherapy intervention remains unclear., Objectives: To improve pain, disability, function, patient satisfaction, quality of life and global perceived effect in adults with neck pain., Search Methods: Computerized searches were conducted up to February 2012., Selection Criteria: We included single therapeutic exercise randomized controlled trials for adults with neck pain with or without cervicogenic headache or radiculopathy., Data Collection and Analysis: Two review authors independently conducted selection, data extraction, 'Risk of bias' assessment, and clinical relevance. The quality of the body of evidence was assessed using GRADE. Relative risk and standardized mean differences (SMD) were calculated.  After judging clinical and statistical heterogeneity, we performed meta-analyses., Main Results: Six of the 21 selected trials had low risk of bias. Moderate quality evidence shows that combined cervical, scapulothoracic stretching and strengthening are beneficial for pain relief post treatment (pooled SMD -0.35, 95% confidence interval (CI): -0.60, -0.10) and at intermediate follow-up (pooled SMD -0.31, 95% CI: -0.57, -0.06), and improved function short term and intermediate term (pooled SMD -0.45, 95% CI: -0.72, -0.18) for chronic neck pain. Moderate quality evidence demonstrates patients are very satisfied with their care when treated with therapeutic exercise. Low quality evidence shows exercise is of benefit for pain in the short term and for function up to long-term follow-up for chronic neck pain. Low to moderate quality evidence shows that chronic neck pain does not respond to upper extremity stretching and strengthening or a general exercise program.Low to moderate quality evidence supports self-mobilization, craniocervical endurance and low load cervical-scapular endurance exercises in reducing pain, improving function and global perceived effect in the long term for subacute/chronic cervicogenic headache. Low quality evidence supports neck strengthening exercise in acute cervical radiculopathy for pain relief in the short term., Authors' Conclusions: Low to moderate quality evidence supports the use of specific cervical and scapular stretching and strengthening exercise for chronic neck pain immediately post treatment and intermediate term, and cervicogenic headaches in the long term. Low to moderate evidence suggests no benefit for some upper extremity stretching and strengthening exercises or a general exercise program.  Future trials should consider using an exercise classification system to establish similarity between protocols and adequate sample sizes. Factorial trials would help determine the active treatment agent within a treatment regimen where a standardized representation of dosage is essential. Standardized reporting of adverse events is needed for balancing the likelihood of treatment benefits over potential harms.

Background: There is a lack of standardized mobility measures specific to the long-term care (LTC) population. Therefore, the Continuing Care Activity Measure (CCAM) was developed., Objective: This study determined levels of reliability, validity for clinical utilization, and sensitivity to change of this measure., Design: This was a prospective longitudinal cohort study among elderly people with primarily physical or medical impairments who were residing in LTC institutions that provide nursing home and more-complex care, with access to physical therapy services., Method: The CCAM, the Clinical Outcome Variables Scale (COVS), the Social Engagement Scale (SES) of the Resident Assessment Instrument-Minimum Data Set (RAI-MDS) 2.0 instrument, and the Resource Utilization Groups, version 3, (RUG-III) were administered by clinical and research physical therapists, with timing dictated by the study purpose., Results: The participants were 136 residents of LTC institutions and 21 physical therapists. The CCAM interrater reliability (intraclass correlation coefficient [ICC]) was .97 (95% confidence interval=.91-1.00), and test-retest reliability (ICC) over a period of 1 week was .99 (95% confidence interval=.93-1.00). Over 6 months, the absolute change in total score was 5.88 for the CCAM and 4.26 for the COVS; the CCAM was 28% more responsive across all participants (n=105) and 68% more responsive for those scoring in the lower half (n=49). The minimal detectable difference of the CCAM was 8.6 across all participants. The CCAM correlated with the COVS, nursing care hours inferred from the RUG-III, and the SES., Limitations: Some participants were lost to follow-up., Conclusions: The CCAM is a reliable and valid tool to measure gross motor function and physical mobility for elderly people in LTC institutions. It discriminates among functional levels, measures individual functional change, and can contribute to clinical decision making.

Background: Neck disorders are common, limit function, and are costly to individuals and society. Exercise therapy is a commonly used treatment for neck pain. The effectiveness of exercise therapy remains unclear., Objectives: To assess the effectiveness of exercise therapy to relieve pain, or improve function, disability, patient satisfaction, and global perceived effect in adults with mechanical neck disorders (MND)., Search Strategy: Computerised bibliographic databases including CENTRAL, MEDLINE, EMBASE, MANTIS, CINAHL, and ICL were searched, without language restrictions, from their beginning up to March 2004, and reference lists of articles were scanned., Selection Criteria: Selected studies were randomised [RCTs] or quasi-randomised trials and investigated the use of exercise therapy as a treatment in adults with MND with or without headache or radicular signs and symptoms., Data Collection and Analysis: Two reviewers independently conducted citation identification, study selection, data abstraction, and methodological quality assessment. Using a random effects model, relative risk and standardized mean differences were calculated. The reasonableness of combining studies was assessed on clinical and statistical grounds. In the absence of heterogeneity, pooled effect measures were calculated. When trials were considered homogenous, results were summarised using a rating system of five levels of evidence., Main Results: Thirty-one trials were selected, 19% (van Tulder criteria) to 35% (Jadad scale) had high quality. There is limited evidence of benefit that acute range of motion (AROM) may reduce pain in acute MND (whiplash associated disorder (WAD)) in the short term. There is moderate evidence of benefit that neck strengthening exercises reduce pain, improve function and global perceived effect for chronic neck disorder with headache in the short and long term. There is unclear evidence regarding the impact of a stretching and strengthening program on pain, function and global perceived effect for MND. However, when this stretching and strengthening program focuses on the cervical or cervical and shoulder/thoracic region, there is moderate evidence of benefit on pain in chronic MND [pooled SMD -0.42 (95%CI: -0.83 to -0.01)] and neck disorder plus headache, in the short and long term. There is strong evidence of benefit favouring a multimodal care approach of exercise combined with mobilisation or manipulation for subacute and chronic MND with or without headache, in the short and long term. A program of eye fixation or proprioception exercises imbedded in a more complete program shows moderate evidence of benefit for pain [pooled SMD -0.72 (95% CI:-1.12 to -0.32)], function, and global perceived for chronic MND in the short term, and on pain and function for acute and subacute MND with headache or WAD in the long term. There is limited evidence of benefit on pain relief in the short term for a home mobilisation program with other physical modalities over a program of rest then gradual mobilisation for acute MND or WAD. There was evidence of no difference between the different exercise approaches., Authors' Conclusions: The evidence summarised in this systematic review indicates that specific exercises may be effective for the treatment of acute and chronic MND, with or without headache. To be of benefit, a stretching and strengthening exercise program should concentrate on the musculature of the cervical, shoulder-thoracic area, or both. A multimodal care approach of exercise, combined with mobilisation or manipulation for subacute and chronic MND with or without headache, reduced pain, improved function, and global perceived effect in the short and long term. The relative benefit of other treatments (such as physical modalities) compared with exercise or between different exercise programs needs to be explored. The quality of future trials should improve through more effective 'blinding' procedures and better control of compliance and co-intervention. Phase II trials would help identify the most effective treatment characteristics and dosages.