DNA unzipped under a constant force exhibits multiple metastable intermediates

Single molecule studies, at constant force, of the separation of double-stranded DNA into two separated single strands may provide information relevant to the dynamics of DNA replication. At constant applied force, theory predicts that the unzipped length as a function of time is characterized by jumps during which the strands separate rapidly, followed by long pauses where the number of separated base pairs remains constant. Here, we report previously uncharacterized observations of this striking behavior carried out on a number of identical single molecules simultaneously. When several single lphage molecules are subject to the same applied force, the pause positions are reproducible in each. This reproducibility shows that the positions and durations of the pauses in unzipping provide a sequence-dependent molecular fingerprint. For small forces, the DNA remains in a partially unzipped state for at least several hours. For larger forces, the separation is still characterized by jumps and pauses, but the double-stranded DNA will completely unzip in less than 30 min

Theory of biopolymer stretching at high forces

We provide a unified theory for the high force elasticity of biopolymers solely in terms of the persistence length, $\xi_p$, and the monomer spacing, $a$. When the force f>\fh \sim k_BT\xi_p/a^2 the biopolymers behave as Freely Jointed Chains (FJCs) while in the range \fl \sim k_BT/\xi_p < f < \fh the Worm-like Chain (WLC) is a better model. We show that $\xi_p$ can be estimated from the force extension curve (FEC) at the extension $x\approx 1/2$ (normalized by the contour length of the biopolymer). After validating the theory using simulations, we provide a quantitative analysis of the FECs for a diverse set of biopolymers (dsDNA, ssRNA, ssDNA, polysaccharides, and unstructured PEVK domain of titin) for $x \ge 1/2$. The success of a specific polymer model (FJC or WLC) to describe the FEC of a given biopolymer is naturally explained by the theory. Only by probing the response of biopolymers over a wide range of forces can the $f$-dependent elasticity be fully described.Comment: 20 pages, 4 figure

Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA-DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA-DNA interactions that we report here may play a role in the chromosome organization and gene regulationopen

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Rol de la figura de enlace en el modelo de transición en endocrinología: experiencia en el ámbito público

Introducción: Un proceso de transición planificado entre los sistemas de salud pediátricos y de adultos es necesario para poder garantizar una continuidad en la atención de los adolescentes. El objetivo del trabajo fue evaluar la población de pacientes del Servicio de Endocrinología del Hospital Garrahan en fase de transición y sus familias, desarrollar un protocolo para la transición de los adolescentes con patología endocrinológica crónica al Hospital de Clínicas José de San Martin y evaluar el rol de la “figura de enlace” en este proceso. Materiales y Métodos: Estudio observacional, transversal/prospectivo. Se obtuvieron datos sobre la consulta ambulatoria de 72 adolescentes mayores a 15 años con patología endocrinológica a los cuales se los acompañó en el proceso de transición. Se realizaron entrevistas y encuestas a los adolescentes, sus familias y a 16 endocrinólogos intervinientes en el seguimiento (9 pediátricos- 7 adultos). Resultados: La mayoría de los adolescentes evidenciaron falta de autonomía general, con mayor afectación en el área de “seguimiento de los problemas de salud”. Esto, junto al paternalismo del pediatra y la sobreprotección familiar representaron inconvenientes para la transición. La mitad de los adultos entrevistados consideraron falta de autonomía o preparación en sus hijos considerando la edad ideal para la transición entre los 18-21 años. Las sensaciones referidas por los pacientes como sus acompañantes incluyen principalmente el miedo y ansiedad, y llamativamente en los pacientes la vergüenza. La creación de un consultorio de transición en el centro de adultos y el acompañamiento de la “figura de enlace”, permitieron una mejor articulación y continuidad en el cuidado de la salud.A planned transition process between pediatric and adult health systems is necessary to ensure continuity of care for adolescents. The aim of this study was to evaluate the patient population of the Endocrinology Service at Garrahan Hospital during the transition phase, along with their families, to develop a protocol for transitioning adolescents with chronic endocrinological disorders to Hospital de Clínicas José de San Martín, and to evaluate the role of the “liaison person” in this process. Materials and Methods: This observational, cross-sectional/ prospective study obtained data from outpatient consultations of 72 adolescents over 15 years of age with endocrinological disorders who were accompanied during the transition process. Interviews and surveys were conducted with the adolescents, their families, and 16 endocrinologists involved in the follow-up (9 pediatricians and 7 adult physicians). Results: Most of the adolescents showed a general lack of autonomy, with greater challenges in the area of “follow-up of health problems.” This, combined with the paternalism of the pediatrician and the overprotection of the family, represented obstacles to the transition. Half of the parents interviewed perceived a lack of autonomy or preparation in their children, considering the ideal age for transition to be between 18-21 years old. The primary feelings reported by the patients and those who accompanied them included fear and anxiety, with patients also feeling embarrassment. The creation of a transition clinic in the adult center and the support of a “liaison person” allowed for better coordination and continuity in health care.Fil: Soto, M. F.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Mocarbel, Y.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Rosso, D.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Danilowicz, Karina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Ciaccio, Marta Graciela Cristina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Guercio, Gabriela Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentin

RNA Unwinding by NS3 Helicase: A Statistical Approach

The study of double-stranded RNA unwinding by helicases is a problem of basic scientific interest. One such example is provided by studies on the hepatitis C virus (HCV) NS3 helicase using single molecule mechanical experiments. HCV currently infects nearly 3% of the world population and NS3 is a protein essential for viral genome replication. The objective of this study is to model the RNA unwinding mechanism based on previously published data and study its characteristics and their dependence on force, ATP and NS3 protein concentration. In this work, RNA unwinding by NS3 helicase is hypothesized to occur in a series of discrete steps and the steps themselves occurring in accordance with an underlying point process. A point process driven change point model is employed to model the RNA unwinding mechanism. The results are in large agreement with findings in previous studies. A gamma distribution based renewal process was found to model well the point process that drives the unwinding mechanism. The analysis suggests that the periods of constant extension observed during NS3 activity can indeed be classified into pauses and subpauses and that each depend on the ATP concentration. The step size is independent of external factors and seems to have a median value of 11.37 base pairs. The steps themselves are composed of a number of substeps with an average of about 4 substeps per step and an average substep size of about 3.7 base pairs. An interesting finding pertains to the stepping velocity. Our analysis indicates that stepping velocity may be of two kinds- a low and a high velocity

Pegvisomant in acromegaly: a multicenter real-life study in Argentina

OBJECTIVE: To describe the long term safety and efficacy of pegvisomant (PEGV), and the predictors of treatment response in patients with acromegaly in the real life setting. SUBJECTS AND METHODS: We retrospectively reviewed the clinical, hormonal and radiological data of acromegalic patients treated with PEGV in 17 Argentine centers. RESULTS: Seventy-five patients (age range 22-77, 51 females) with acromegaly have been treated with PEGV for up to 118 months (median 27 months). Before PEGV, 97.3% of patients had been treated with medical therapy, surgery and/or radiotherapy, two patients had no previous treatment. At that time, all patients had an IGF-1 above the upper normal limit (ULN) (mean 2.4 x ULN ± 0.98, range 1.25-7). At diagnosis of acromegaly 84% presented macroadenomas, prior to PEGV only 23,5% of patients remained with tumor remnant > 1 cm, the remaining showed normal or less than 1 cm images. Disease control (IGF-1 ≤ 1.2 x ULN) was achieved in 62.9% of patients with a mean dose of 11.8 mg/day. Thirty-four patients (45%) received PEGV monotherapy, while 41 (55%) received combined therapy with either somatostatin analogues and/or cabergoline. Adverse events related to PEGV were: local injection site reaction in 5.3%, elevated liver enzymes in 9.3%, and tumor size growth in 9.8%. Pre-PEGV IGF-I level was the only predictor of treatment response: 2.1 x ULN vs 2.8 x ULN in controlled and uncontrolled patients respectively (p < 0.001). CONCLUSION: this long term experience indicates PEGV treatment was highly effective and safe in our series of Argentine patients with acromegaly refractory to standard therapies. Arch Endocrinol Metab. 2019;63(4):320-7.FIl: Garcia Basavilbaso, Natalia Ximena. Hospital Carlos G. Durand. Departamento de Endocrinología; Argentina.FIl: Ballarino, Maria Carolina. Hospital Militar Central. Servicio de Endocrinología; Argentina.FIl: Bruera, Darío. Clínica Caraffa. Servicio de Endocrinología; Argentina.FIl: Bruno, Oscar D. Hospital de Clínicas "José de San Martín". Servicio de Endocrinología; Argentina.FIl: Chervin, Alberto B. Hospital Santa Lucía. Servicio de Endocrinología; Argentina.FIl: Danilowicz, Karina. Hospital de Clínicas José de San Martín. Servicio de Endocrinología; Argentina.FIl: Fainstein Day, Patricia. Hospital Italiano. Servicio de Endocrinología; Argentina.FIl: Fidalgo, Silvina Gabriela. Hospital Churruca. Servicio de Endocrinología; Argentina.FIl: Frigeri, Adriana. Hospital Teodoro Alvarez. Servicio de Endocrinología; Argentina.FIl: Glerean, Mariela. Hospital Italiano. Servicio de Endocrinología; Argentina.FIl: Guelman, Rodolfo. Hospital Italiano. Servicio de Endocrinología; Argentina.FIl: Isaac, Gabriel. Hospital Privado de la Comunidad. Servicio de Endocrinología; Argentina.FIl: Knoblovits, Pablo. Hospital Italiano. Servicio de Endocrinología; Argentina.FIl: Librandi, Fabiana. Hospital Rivadavia. Servicio de Endocrinología; Argentina.FIl: López Montes, Monica. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas “José de San Martín”. Servicio de Endocrinología; Argentina.FIl: Mallea-Gil, María Susana. Hospital Militar Central. Servicio de Endocrinología; Argentina.FIl: Manavela, Marcos. Hospital de Clínicas “José de San Martín”. Servicio de Endocrinología; Argentina.FIl: Mereshian, Paula. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas “José de San Martín”. Servicio de Endocrinología; Argentina.FIl: Moncet, Daniel. Hospital Privado de la Comunidad. Servicio de Endocrinología; Argentina.FIl: Katz, Debora Adela. Fleni. Departamento de Neurología. Servicio de Neuroendocrinología; Argentina.FIl: Pignatta, Analia. Hospital Interzonal San Juan Bautista. Servicio de Endocrinología; Argentina.FIl: Rogozinsky, Amelia. Hospital Ramos Mejía. Servicio de Endocrinología; Argentina.FIl: Sago, Laura R. Hospital Italiano de La Plata. Servicio de Endocrinología; Argentina.FIl: Servidio, Marisa. Hospital Teodoro Alvarez. Servicio de Endocrinología; Argentina.FIl: Spezzi, Monica. Instituto Médico Platense. Servicio de Endocrinología; Argentina.FIl: Stalldecker, Graciela. Hospital Pirovano; Argentina.FIl: Tkatch, Julieta. Hospital Carlos G. Durand. Departamento de Endocrinología; Argentina.FIl: Vitale, Nicolas Marcelo. Hospital Santa Lucía. Servicio de Endocrinología; Argentina.FIl: Guitelman, Mirtha. Hospital Carlos G. Durand. Departamento de Endocrinología; Argentina

Signatures of DNA flexibility, interactions and sequence-related structural variations in classical X-ray diffraction patterns

The theory of X-ray diffraction from ideal, rigid helices allowed Watson and Crick to unravel the DNA structure, thereby elucidating functions encoded in it. Yet, as we know now, the DNA double helix is neither ideal nor rigid. Its structure varies with the base pair sequence. Its flexibility leads to thermal fluctuations and allows molecules to adapt their structure to optimize their intermolecular interactions. In addition to the double helix symmetry revealed by Watson and Crick, classical X-ray diffraction patterns of DNA contain information about the flexibility, interactions and sequence-related variations encoded within the helical structure. To extract this information, we have developed a new diffraction theory that accounts for these effects. We show how double helix non-ideality and fluctuations broaden the diffraction peaks. Meridional intensity profiles of the peaks at the first three helical layer lines reveal information about structural adaptation and intermolecular interactions. The meridional width of the fifth layer line peaks is inversely proportional to the helical coherence length that characterizes sequence-related and thermal variations in the double helix structure. Analysis of measured fiber diffraction patterns based on this theory yields important parameters that control DNA structure, packing and function