The consequences of replicating in the wrong orientation: Bacterial chromosome duplication without an active replication origin

Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid meta¬bolism. In cells lacking RNase HI replication initiates indepen¬dently of DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis in cells lacking RecG. However, recently we suggested that this synthesis initiates at intermediates resulting from replication fork fusions. Here we present data suggesting that in cells lacking RecG or RNase HI origin-independent synthesis arises by different mechanisms, indicative of these two proteins having different roles in vivo. Our data support the idea that RNase HI processes R-loops, while RecG is required to process replication fork fusion intermediates. However, regardless of how origin-independent synthesis is initiated, a fraction of forks will proceed in an orientation opposite to normal. We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly-transcribed areas. Thus, despite their different functions, RecG and RNase HI are both important factors for maintaining replication control and orientation. Their absence causes severe replication problems, highlighting the advantages of the normal chromosome arrangement, which exploits a single origin to control the number of forks and their orientation relative to transcription, and a defined termination area to contain fork fusions. Any changes to this arrangement endanger cell cycle control, chromosome dynamics and, ultimately, cell viability.This work was supported by the Royal Society (RG110414 to C.J.R.) and The Biotechnology and Biological Sciences Research Council (BB/K015729/1 to C.J.R.)

The effect of infusions of adrenaline, noradrenaline and dopamine on cerebral autoregulation under isoflurane anaesthesia in an ovine model

Publisher's copy made available with the permission of the publisher © Australian Society of AnaesthetistsThe effects of infusions of adrenaline, noradrenaline and dopamine on cerebral autoregulation under steady-state isoflurane anaesthesia were compared with the awake state. Six studies each were conducted in two cohorts of adult ewes: awake sheep and those anaesthetized with 2% isoflurane anaesthesia. In random order, each animal received ramped infusions of adrenaline, noradrenaline (0-40 µg/min) and dopamine (0-40 µg/kg/min). Cerebral blood flow was measured continuously from changes in Doppler velocities in the sagittal sinus. Autoregulation was determined by linear regression analysis between cerebral blood flow and mean arterial pressure. Isoflurane did not significantly alter cerebral blood flow relative to pre-anaesthesia values (P>0.05). All three catecholamines significantly and equivalently increased MAP from baseline in a dose dependent manner in both the awake and isoflurane cohorts. Although adrenaline significantly increased cerebral blood flow from baseline in the awake cohort (P0.05). Over a specific dose range, systemic hypertension induced by adrenaline, noradrenaline and dopamine did not significantly increase cerebral blood flow under 2% isoflurane anaesthesia. The concomitant administration of isoflurane and the catecholamines was not associated with altered autoregulatory function compared to the awake state.http://www.aaic.net.au/Article.asp?D=200236

Identifying Video Game Preferences Among Adults Interested in Quitting Smoking Cigarettes: Survey Study.

BACKGROUND: Smoking is the most prevalent cause of morbidity and mortality in the United States. Although most individuals who smoke express a desire to quit smoking, only a small percentage are successful. Serious games have become popular in health sectors as a potential avenue for delivering a scalable treatment that is both accessible and engaging for the smoking population. Several smoking cessation games have already been developed, but these games feature a broad range of gameplay elements and are not necessarily based on existing video game preferences in the general or smoking population. OBJECTIVE: To better inform treatment development, this study aims to evaluate video game genre preferences among treatment-seeking individuals who smoke (N=473). METHODS: Participants responded to a screening survey to enroll in a larger, serious game intervention for smoking cessation. During this screening survey, participants were asked to disclose their favorite video games, which resulted in 277 unique game titles. These titles were coded for genre categories based on publisher listings and game features. The genres were then analyzed for the frequency of reporting overall and across age groups. RESULTS: Action, Role-Playing, and Action-Adventure were the most reported genres among adults aged ≤34 years; Action, Action-Adventure, and Logic were the most reported genres among adults aged 35-44 years; and Logic and Action were the most reported genres among adults aged ≥45 years. CONCLUSIONS: These data indicate that treatment-seeking individuals who smoke have different game preferences across age groups, and the data provide novel information to inform the development of future serious games targeting the smoking population that are tailored to the preferences of their age group. TRIAL REGISTRATION: ClinicalTrials.gov NCT03929003; https://clinicaltrials.gov/ct2/show/NCT03929003

Structure and functional motifs of GCR1, the only plant protein with a GPCR fold?

Whether GPCRs exist in plants is a fundamental biological question. Interest in deorphanizing new G protein coupled receptors (GPCRs), arises because of their importance in signaling. Within plants, this is controversial as genome analysis has identified 56 putative GPCRs, including GCR1 which is reportedly a remote homologue to class A, B and E GPCRs. Of these, GCR2, is not a GPCR; more recently it has been proposed that none are, not even GCR1. We have addressed this disparity between genome analysis and biological evidence through a structural bioinformatics study, involving fold recognition methods, from which only GCR1 emerges as a strong candidate. To further probe GCR1, we have developed a novel helix alignment method, which has been benchmarked against the the class A – class B - class F GPCR alignments. In addition, we have presented a mutually consistent set of alignments of GCR1 homologues to class A, class B and class F GPCRs, and shown that GCR1 is closer to class A and /or class B GPCRs than class A, class B or class F GPCRs are to each other. To further probe GCR1, we have aligned transmembrane helix 3 of GCR1 to each of the 6 GPCR classes. Variability comparisons provide additional evidence that GCR1 homologues have the GPCR fold. From the alignments and a GCR1 comparative model we have identified motifs that are common to GCR1, class A, B and E GPCRs. We discuss the possibilities that emerge from this controversial evidence that GCR1 has a GPCR fol

Cerebrovascular carbon dioxide reactivity in sheep: Effect of propofol or isoflurane anaesthesia

Publisher's copy made available with the permission of the publisher © Australian Society of AnaesthetistsPropofol and isoflurane are commonly used in neuroanaesthesia. Some published data suggest that the use of these agents is associated with impaired cerebral blood flow/carbon dioxide (CO₂) reactivity. Cerebrovascular CO₂ reactivity was therefore measured in three cohorts of adult merino sheep: awake (n=6), anaesthetized with steady-state propofol (15 mg/min; n=6) and anaesthetized with 2% isoflurane (n=6). Changes in cerebral blood flow were measured continuously from changes in velocities of blood in the sagittal sinus via a Doppler probe. Alterations in the partial pressure of carbon dioxide in arterial blood (PaCO₂) over the range 18-63 mmHg were achieved by altering either the inspired CO₂ concentration or the rate of mechanical ventilation. Cerebral blood flow/CO₂ relationships were determined by linear regression analysis, with changes in cerebral blood flow expressed as a percentage of the value for a PaCO₂ of 35 mmHg. Propofol decreased cerebral blood flow by 55% relative to pre-anaesthesia values (P=0.0001), while isoflurane did not significantly alter cerebral blood flow (88.45% of baseline, P=0.39). Significant linear relationships between cerebral blood flow and CO₂ tension were determined in all individual studies (r2 ranged from 0.72 to 0.99). The slopes of the lines were highly variable between individuals for the awake cohort (mean 4.73, 1.42-7.12, 95% CI). The slopes for the propofol (mean 2.67, 2.06-3.28, 95% CI) and isoflurane (mean 2.82, 2.19-3.45, 95% CI) cohorts were more predictable. However, there was no significant difference between these anaesthetic agents with respect to the CO₂ reactivity of cerebral blood flow.J. A. Myburgh, R. N. Upton, G. L. Ludbrook, A. Martinez, C. Granthttp://www.aaic.net.au/Article.asp?D=200137

Assessing the effect of dynamics on the closed-loop protein-folding hypothesis

The closed-loop (loop-n-lock) hypothesis of protein folding suggests that loops of about 25 residues, closed through interactions between the loop ends (locks), play an important role in protein structure. Coarse-grain elastic network simulations, and examination of loop lengths in a diverse set of proteins, each supports a bias towards loops of close to 25 residues in length between residues of high stability. Previous studies have established a correlation between total contact distance (TCD), a metric of sequence distances between contacting residues (cf. contact order), and the log-folding rate of a protein. In a set of 43 proteins, we identify an improved correlation ( r 2 = 0.76), when the metric is restricted to residues contacting the locks, compared to the equivalent result when all residues are considered ( r 2 = 0.65). This provides qualified support for the hypothesis, albeit with an increased emphasis upon the importance of a much larger set of residues surrounding the locks. Evidence of a similar-sized protein core/extended nucleus (with significant overlap) was obtained from TCD calculations in which residues were successively eliminated according to their hydrophobicity and connectivity, and from molecular dynamics simulations. Our results suggest that while folding is determined by a subset of residues that can be predicted by application of the closed-loop hypothesis, the original hypothesis is too simplistic; efficient protein folding is dependent on a considerably larger subset of residues than those involved in lock formation. </jats:p

Use of modified U1 snRNAs to inhibit HIV-1 replication

Control of RNA processing plays a central role in regulating the replication of HIV-1, in particular the 3′ polyadenylation of viral RNA. Based on the demonstration that polyadenylation of mRNAs can be disrupted by the targeted binding of modified U1 snRNA, we examined whether binding of U1 snRNAs to conserved 10 nt regions within the terminal exon of HIV-1 was able to inhibit viral structural protein expression. In this report, we demonstrate that U1 snRNAs complementary to 5 of the 15 regions targeted result in significant suppression of HIV-1 protein expression and viral replication coincident with loss of viral RNA. Suppression of viral gene expression is dependent upon appropriate assembly of a U1 snRNP particle as mutations of U1 snRNA that affect binding of U1 70K or Sm proteins significantly reduced efficacy. However, constructs lacking U1A binding sites retained significant anti-viral activity. This finding suggests a role for these mutants in situations where the wild-type constructs cause toxic effects. The conserved nature of the sequences targeted and the high efficacy of the constructs suggests that this strategy has significant potential as an HIV therapeutic

The Adoption and Implementation of Evidence-Based Practice (EBP) Among Allied Health Professions.

Background and aims: Evidence-based practice (EBP) is widely accepted within patient care as it ensures health care professionals remain informed of recent evidence and research relating to their clinical practice. However, the particular characteristics detrimental to the successful implementation of EBP within Allied Health Professionals' (AHP) clinical practice are unknown. The purpose of this study was to assess and characterise adoption of EBP within AHP's clinical practice. Methods: Questionnaires comprising the Evidence-Based Practice Questionnaire (EBPQ; Upton and Upton, 2006a) were administered to 154 (response rate=27.3%) newly qualified practitioners (NQPs) from NHSScotland. Data were analysed to determine attitudes, knowledge and skill of EBP; K-means cluster and chi-square analyses were conducted in order to differentiate profiles of NPQs within high-, medium- and low- categories on the EBPQ practice and knowledge/skills sub-sections. Findings: Moderate scores were recorded for NQP's implementation, knowledge, and attitudes toward EBP. Chi-square analysis performed on the high-, moderate- and low- practice and skills' profiles revealed no significant results for NQP's year qualified, age, or year of clinical practice. Conclusions: The findings illustrate that the majority of NQPs have a good understanding of the application and importance of EPB, and suggests the improvement in NQPs training with regards to EBP enables them to successfully transfer acquired knowledge within their clinical practice