DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate.

Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme-DNA complex by depleting Mg(2+)-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event

ENIGMA and global neuroscience: A decade of large-scale studies of the brain in health and disease across more than 40 countries.

This review summarizes the last decade of work by the ENIGMA (Enhancing NeuroImaging Genetics through Meta Analysis) Consortium, a global alliance of over 1400 scientists across 43 countries, studying the human brain in health and disease. Building on large-scale genetic studies that discovered the first robustly replicated genetic loci associated with brain metrics, ENIGMA has diversified into over 50 working groups (WGs), pooling worldwide data and expertise to answer fundamental questions in neuroscience, psychiatry, neurology, and genetics. Most ENIGMA WGs focus on specific psychiatric and neurological conditions, other WGs study normal variation due to sex and gender differences, or development and aging; still other WGs develop methodological pipelines and tools to facilitate harmonized analyses of "big data" (i.e., genetic and epigenetic data, multimodal MRI, and electroencephalography data). These international efforts have yielded the largest neuroimaging studies to date in schizophrenia, bipolar disorder, major depressive disorder, post-traumatic stress disorder, substance use disorders, obsessive-compulsive disorder, attention-deficit/hyperactivity disorder, autism spectrum disorders, epilepsy, and 22q11.2 deletion syndrome. More recent ENIGMA WGs have formed to study anxiety disorders, suicidal thoughts and behavior, sleep and insomnia, eating disorders, irritability, brain injury, antisocial personality and conduct disorder, and dissociative identity disorder. Here, we summarize the first decade of ENIGMA's activities and ongoing projects, and describe the successes and challenges encountered along the way. We highlight the advantages of collaborative large-scale coordinated data analyses for testing reproducibility and robustness of findings, offering the opportunity to identify brain systems involved in clinical syndromes across diverse samples and associated genetic, environmental, demographic, cognitive, and psychosocial factors

Long-term outcome after anterior cervical discectomy without fusion

To retrospectively study the long-term outcome of patients after anterior cervical discectomy without fusion (ACD) compared to results published on the long-term outcome after ACD with fusion (ACDF). We reviewed the charts of all patients receiving ACD surgery between 1985 and 2000 to analyze the direct post-operative results as well as complications of the surgery. Moreover, 102 patients, randomly selected, were interviewed with the neck disability index to study possible persisting complaints up to 18 years after ACD surgery. A total of 551 Patients were identified. Two months post-operative follow up at the outpatient clinic revealed that 90.1% of patients were satisfied with the result of ACD surgery. At the time of the survey, this percentage had dropped to 67.6%. In addition, 20.6% and 11.8% had obtained moderate to severe complaints, respectively, in daily-life activities. Complaints were mainly localized in the neck region and occasionally provoked radiating pain in the arm. On the short term, ACD leads to a satisfied outcome. Over the longer term, patients report increasing complaints. The increase in complaints at the time of the survey may be the result of ongoing degenerative effects. Compared to published data on ACDF, there is no superiority of any fusion technique compared to ACD alone

ENIGMA and global neuroscience: A decade of large-scale studies of the brain in health and disease across more than 40 countries

This review summarizes the last decade of work by the ENIGMA (Enhancing NeuroImaging Genetics through Meta Analysis) Consortium, a global alliance of over 1400 scientists across 43 countries, studying the human brain in health and disease. Building on large-scale genetic studies that discovered the first robustly replicated genetic loci associated with brain metrics, ENIGMA has diversified into over 50 working groups (WGs), pooling worldwide data and expertise to answer fundamental questions in neuroscience, psychiatry, neurology, and genetics. Most ENIGMA WGs focus on specific psychiatric and neurological conditions, other WGs study normal variation due to sex and gender differences, or development and aging; still other WGs develop methodological pipelines and tools to facilitate harmonized analyses of "big data" (i.e., genetic and epigenetic data, multimodal MRI, and electroencephalography data). These international efforts have yielded the largest neuroimaging studies to date in schizophrenia, bipolar disorder, major depressive disorder, post-traumatic stress disorder, substance use disorders, obsessive-compulsive disorder, attention-deficit/hyperactivity disorder, autism spectrum disorders, epilepsy, and 22q11.2 deletion syndrome. More recent ENIGMA WGs have formed to study anxiety disorders, suicidal thoughts and behavior, sleep and insomnia, eating disorders, irritability, brain injury, antisocial personality and conduct disorder, and dissociative identity disorder. Here, we summarize the first decade of ENIGMA's activities and ongoing projects, and describe the successes and challenges encountered along the way. We highlight the advantages of collaborative large-scale coordinated data analyses for testing reproducibility and robustness of findings, offering the opportunity to identify brain systems involved in clinical syndromes across diverse samples and associated genetic, environmental, demographic, cognitive, and psychosocial factors

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40-60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase