Mechanics and Function of DNA Looping and Supercoiling.

DNA is an essential molecule that enables the storage and retrieval of genetic information. Since the discovery of its structure (double helix), the relationship between the molecule's structure and function has been studied extensively. Here we extend beyond the static structure and consider how the mechanical properties and dynamics influence its function. To do so, we exercise an elasto-dynamic rod model for DNA. By exercising this model, we study two biologically relevant systems. First, we study DNA looping by Lac repressor. Although this is a classic gene regulatory system, the mechanics of the DNA loop remain largely unknown. Therefore, we compute the effects of inter-operator length, intrinsic curvature, and protein flexibility on the energetics and topology these loops. We calculate that anti-parallel loops are energetically preferred, the elastic energy of a family of intrinsically curved DNA loops spans 5-12 kT, and identify the sensitivity of elastic energy to protein flexibility. Our computations compare favorably with published experimental data and motivate experimental work in the Kahn lab at the University of Maryland. Furthermore, we contribute an efficient method to analyze a large family of intrinsically curved DNA molecules and a method to account for Lac repressor flexibility in our rod model. In addition, we analyze cryo-EM images (obtained by the Stasiak lab at the Université de Lausanne) of DNA minicircles with similar lengths to the Lac repressor DNA loops. Second, we study the relaxation of DNA supercoils by topoisomerase. In doing so, we make advancements to the rod model and perform the first multi-scale model of supercoil relaxation by topoisomerase. Specifically, we contribute an efficient method to account for self contact and electrostatics in our elastic rod model. In our multi-scale simulation we couple our rod model with recent data (from MD simulations by the Andricioaei lab at the University of California - Irvine) that characterizes the the mechanics of topoisomerase. In doing so we gain insight into the dynamics of supercoil relaxation and make a first prediction of the relaxation time (0.1-1.0 μs).Ph.D.Mechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/75861/1/tlillian_1.pd

Robotic Versus Laparoscopic Ventral Hernia Repair: Two-Year Results From a Prospective, Multicenter, Blinded Randomized Clinical Trial

OBJECTIVE: Report the 2-year outcomes of a multicenter randomized controlled trial comparing robotic versus laparoscopic intraperitoneal onlay mesh ventral hernia repair. BACKGROUND: Ventral hernia repair is one of the most common operations performed by general surgeons. To our knowledge, no studies have been published to date comparing long-term outcomes of laparoscopic versus robotic ventral hernia repair. METHODS: The trial was registered at clinicaltrials.gov (NCT03490266). Clinical outcomes included surgical site infection, surgical site occurrence, hernia occurrence, readmission, reoperation, and mortality. RESULTS: A total of 175 consecutive patients were approached that were deemed eligible for elective minimally invasive ventral hernia repair. In all, 124 were randomized and 101 completed follow-up at 2 years. Two-year follow-up was completed in 54 patients (83%) in the robotic arm and 47 patients (80%) in the laparoscopic arm. No differences were seen in surgical site infection or surgical site occurrence. Hernia recurrence occurred in 2 patients (4%) receiving robotic repair versus in 6 patients (13%) receiving laparoscopic repair (relative risk: 0.3, 95% CI: 0.06-1.39; P =0.12). No patients (0%) required reoperation in the robotic arm whereas 5 patients (11%) underwent reoperation in the laparoscopic arm ( P =0.019, relative risk not calculatable due to null outcome). CONCLUSIONS: Robotic ventral hernia repair demonstrated at least similar if not improved outcomes at 2 years compared with laparoscopy. There is potential benefit with robotic repair; however, additional multi-center trials and longer follow-up are needed to validate the hypothesis-generating findings of this study

FRET studies of a landscape of Lac repressor-mediated DNA loops

DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacI•DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Förster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-β-d-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility

Polyantigenic Interferon-γ Responses Are Associated with Protection from TB among HIV-Infected Adults with Childhood BCG Immunization

Surrogate immunologic markers for natural and vaccine-mediated protection against tuberculosis (TB) have not been identified. HIV-infected adults with childhood BCG immunization entering the placebo arm of the DarDar TB vaccine trial in Dar es Salaam, Tanzania, were assessed for interferon gamma (IFN-γ) responses to three mycobacterial antigen preparations--secreted Mycobacterium tuberculosis antigens 85 (Ag85), early secretory antigenic target 6 (ESAT-6) and polyantigenic whole cell lysate (WCL). We investigated the association between the number of detectable IFN-γ responses at baseline and the subsequent risk of HIV-associated TB. During a median follow-up of 3.3 years, 92 (9.4%) of 979 placebo recipients developed TB. The incidence of TB was 14% in subjects with no detectable baseline IFN-γ responses vs. 8% in subjects with response to polyantigenic WCL (P = 0.028). Concomitant responses to secreted antigens were associated with further reduction in the incidence of HIV-associated TB. Overall the percentage of subjects with 0, 1, 2 and 3 baseline IFN-γ responses to mycobacterial preparations who developed HIV-associated TB was 14%, 8%, 7% and 4%, respectively (P = 0.004). In a multivariate Cox regression model, the hazard of developing HIV-associated TB was 46% lower with each increment in the number of detectable baseline IFN-γ responses (P<0.001). Among HIV-infected adults who received BCG in childhood and live in a TB-endemic country, polyantigenic IFN-γ responses are associated with decreased risk of subsequent HIV-associated TB. ClinicalTrials.gov NCT0052195

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment