Characterization of a pathway of genomic instability induced by R-loops and its regulation by topoisomerases in E. coli.

The prototype enzymes of the ubiquitous type IA topoisomerases (topos) family are Escherichia coli topo I (topA) and topo III (topB). Topo I shows preference for relaxation of negative supercoiling and topo III for decatenation. However, as they could act as backups for each other or even share functions, strains lacking both enzymes must be used to reveal the roles of type IA enzymes in genome maintenance. Recently, marker frequency analysis (MFA) of genomic DNA from topA topB null mutants revealed a major RNase HI-sensitive DNA peak bordered by Ter/Tus barriers, sites of replication fork fusion and termination in the chromosome terminus region (Ter). Here, flow cytometry for R-loop-dependent replication (RLDR), MFA, R-loop detection with S9.6 antibodies, and microscopy were used to further characterize the mechanism and consequences of over-replication in Ter. It is shown that the Ter peak is not due to the presence of a strong origin for RLDR in Ter region; instead RLDR, which is partly inhibited by the backtracking-resistant rpoB*35 mutation, appears to contribute indirectly to Ter over-replication. The data suggest that RLDR from multiple sites on the chromosome increases the number of replication forks trapped at Ter/Tus barriers which leads to RecA-dependent DNA amplification in Ter and to a chromosome segregation defect. Overproducing topo IV, the main cellular decatenase, does not inhibit RLDR or Ter over-replication but corrects the chromosome segregation defect. Furthermore, our data suggest that the inhibition of RLDR by topo I does not require its C-terminal-mediated interaction with RNA polymerase. Overall, our data reveal a pathway of genomic instability triggered by R-loops and its regulation by various topos activities at different steps.

Topoisomerases I and III inhibit R-loop formation to prevent unregulated replication in the chromosomal Ter region of Escherichia coli.

Type 1A topoisomerases (topos) are the only ubiquitous topos. E. coli has two type 1A topos, topo I (topA) and topo III (topB). Topo I relaxes negative supercoiling in part to inhibit R-loop formation. To grow, topA mutants acquire compensatory mutations, base substitutions in gyrA or gyrB (gyrase) or amplifications of a DNA region including parC and parE (topo IV). topB mutants grow normally and topo III binds tightly to single-stranded DNA. What functions topo I and III share in vivo and how cells lacking these important enzymes can survive is unclear. Previously, a gyrB(Ts) compensatory mutation was used to construct topA topB null mutants. These mutants form very long filaments and accumulate diffuse DNA, phenotypes that appears to be related to replication from R-loops. Here, next generation sequencing and qPCR for marker frequency analysis were used to further define the functions of type 1A topos. The results reveal the presence of a RNase HI-sensitive origin of replication in the terminus (Ter) region of the chromosome that is more active in topA topB cells than in topA and rnhA (RNase HI) null cells. The S9.6 antibodies specific to DNA:RNA hybrids were used in dot-blot experiments to show the accumulation of R-loops in rnhA, topA and topA topB null cells. Moreover topA topB gyrB(Ts) strains, but not a topA gyrB(Ts) strain, were found to carry a parC parE amplification. When a topA gyrB(Ts) mutant carried a plasmid producing topo IV, topB null transductants did not have parC parE amplifications. Altogether, the data indicate that in E. coli type 1A topos are required to inhibit R-loop formation/accumulation mostly to prevent unregulated replication in Ter, and that they are essential to prevent excess negative supercoiling and its detrimental effects on cell growth and survival.

An Emergency Medical Technician Administered Falls-Assessment Protocol to Safely Identify Elderly Adults with Non-Urgent Conditions that may Avoid Transport to Emergency Department.

Background: Approximately two-thirds of patients transported to emergency departments (ED) for a fall are discharged from the ED without urgent treatment. This pilot study tests the feasibility of implementing a pre-hospital falls-assessment protocol performed by emergency medical technicians (EMTs) to determine whether a patient who fell needs an ED assessment or could be referred safely to a community resource. Methods: The protocol was administered by trained EMTs to adults aged ≥ 65 after a fall between October 2019 and March 2020 in Sherbrooke (QC). All patients were transported to ED regardless of protocol outcome (transport recommended/not recommended). The objective was to assess if EMTs could complete the protocol and make the appropriate decision concerning the transport to ED. Secondary objectives aimed to assess the accuracy in identifying patients who do not require transport, and to measure the impact on avoidable ambulance transports. Results: A total of 125 EMTs interventions were carried out: 17 patients were in the transport not recommended group, representing 14% of transport to hospital for falls-related EMTs calls that could be possibly avoided. Of these, 110 were transported to ED. Mean duration of on-site EMTs interventions was of 31 minutes. Forty-seven patients were admitted, mostly for infections and fractures, including four in the transport not recommended group. Conclusions: This study showed that EMTs can administer a falls-assessment protocol aimed at identifying patients that need an ED evaluation. Results permitted to amend the protocol before the second phase of the project evaluating the safety of the protocol.

R-Loop Detection in Bacteria.

Early evidence for R-loop formation in vivo came from the study of Escherichia coli topA (topoisomerase I; topo I) null mutants. Assays with plasmids to detect RNase HI-sensitive hypernegative supercoiling or R-looped DNA were used in vitro and in vivo to demonstrate R-loop formation. In addition, these R-loop-dependent topological modifications of plasmid DNA were shown to correlate with severe growth and gene expression inhibition in topA null mutants that could be corrected by RNase HI overproduction. However, direct evidence for R-loop formation on chromosomal DNA from E. coli cells was only obtained recently by using the S9.6 antibody to detect RNA-DNA hybrids in dot-blot experiments. Here, we present a protocol for such experiments with a special emphasis on the procedure used for bacterial genomic DNA extraction and preparation including treatment with appropriate ribonucleases to eliminate RNA-RNA hybrids (that are also recognized by S9.6) as well as single-stranded RNA (ssRNA), in order to obtain a signal that is specific to stable RNA-DNA hybrids generated. Furthermore, we recommend that the results of such experiments be correlated with RNase HI-sensitive phenotypes.

Supercoiling, R-loops, Replication and the Functions of Bacterial Type 1A Topoisomerases.

Type 1A topoisomerases (topos) are the only topos that bind single-stranded DNA and the only ones found in all cells of the three domains of life. Two subfamilies, topo I and topo III, are present in bacteria. Topo I, found in all of them, relaxes negative supercoiling, while topo III acts as a decatenase in replication. However, recent results suggest that they can also act as back-up for each other. Because they are ubiquitous, type 1A enzymes are expected to be essential for cell viability. Single topA (topo I) and topB (topo III) null mutants of Escherichia coli are viable, but for topA only with compensatory mutations. Double topA topB null mutants were initially believed to be non-viable. However, in two independent studies, results of next generation sequencing (NGS) have recently shown that double topA topB null mutants of Bacillus subtilis and E. coli are viable when they carry parC parE gene amplifications. These genes encode the two subunits of topo IV, the main cellular decatenase. Here, we discuss the essential functions of bacterial type 1A topos in the context of this observation and new results showing their involvement in preventing unregulated replication from R-loops.

Criterion validity study of the cervical range of motion (CROM) device for rotational range of motion on healthy adults.

STUDY DESIGN: This study compared range of motion (ROM) measurements using a cervical range of motion device (CROM) and an optoelectronic system (OPTOTRAK). OBJECTIVES: To examine the criterion validity of the CROM for the measurement of cervical ROM on healthy adults. BACKGROUND: Whereas measurements of cervical ROM are recognized as part of the assessment of patients with neck pain, few devices are available in clinical settings. Two papers published previously showed excellent criterion validity for measurements of cervical flexion/extension and lateral flexion using the CROM. METHODS AND MEASURES: Subjects performed neck rotation, flexion/extension, and lateral flexion while sitting on a wooden chair. The ROM values were measured by the CROM as well as the OPTOTRAK. RESULTS: The cervical rotational ROM values using the CROM demonstrated a good to excellent linear relationship with those using the OPTOTRAK: right rotation, r = 0.89 (95% confidence interval, 0.81-0.94), and left rotation, r = 0.94 (95% confidence interval, 0.90-0.97). Similar results were also obtained for flexion/extension and lateral flexion ROM values. CONCLUSION: The CROM showed excellent criterion validity for measurements of cervical rotation. We propose using ROM values measured by the CROM as outcome measures for patients with neck pain.

Gender-specific associations between functional autonomy and physical capacities in independent older adults: results from the NuAge study.

BACKGROUND: Even with healthy and active aging, many older adults will experience a decrease in physical capacities. This decrease might be associated with diminished functional autonomy. However, little is known about the physical capacities associated with functional autonomy in older women and men. OBJECTIVE: This study aimed to examine gender-specific associations between functional autonomy and physical capacities in independent older women and men. METHODS: Secondary analyses were carried out using cross-sectional data from 652 women and 613 men who participated in the NuAge longitudinal study. The "functional autonomy measurement system" (SMAF) was used to evaluate functional autonomy. The physical capacities measured (tests used) were: biceps and quadriceps strength (Microfet dynamometer), grip strength (Martin vigorimeter), unipodal balance, changing position & walking (timed up and go), normal & fast walking (four-meter walking speed) and changing position (chair stand). Correlation and multiple linear regression analyses adjusted for age, depressive symptoms and body composition were performed. RESULTS: On average, participants were aged 73 years and had mild to moderate functional autonomy loss. In women, after controlling for age, depressive symptoms and body composition, greater functional autonomy was best explained by faster changing position & walking skills and superior biceps strength (R(2)=0.46; p<0.001). After controlling for depressive symptoms, faster changing position & walking skills and better unipodal balance best explained greater functional autonomy in men (R(2)=0.21; p<0.001). CONCLUSION: According to these results, physical capacities are moderately associated with functional autonomy among independent older adults, especially women.