Fully in vitro iterative construction of a 24 kb-long artificial DNA sequence to store digital information.

In the absence of a DNA template, the ab initio production of long double-stranded DNA molecules of predefined sequences is particularly challenging. The DNA synthesis step remains a bottleneck for many applications such as functional assessment of ancestral genes, analysis of alternative splicing or DNA-based data storage. In this report we propose a fully in vitro protocol to generate very long double-stranded DNA molecules starting from commercially available short DNA blocks in less than 3 days using Golden Gate assembly. This innovative application allowed us to streamline the process to produce a 24 kb-long DNA molecule storing part of the Declaration of the Rights of Man and of the Citizen of 1789 . The DNA molecule produced can be readily cloned into a suitable host/vector system for amplification and selection.

Association of preoperative anaesthesia consultation prior to elective noncardiac surgery with patient and health system outcomes: a population-based study.

BACKGROUND: Surgical volumes and use of preoperative anaesthesia consultations are increasing. However, contemporary data estimating the association between preoperative anaesthesia consultation and patient (days alive and at home [DAH30], mortality) and system (costs, length of stay, and readmissions) outcomes are not available. METHODS: We conducted a population-based comparative effectiveness study using linked health administrative data among patients aged ≥40 yr who underwent intermediate-risk to high-risk elective, inpatient, noncardiac surgery in Ontario, Canada (2009-17). Our primary outcome was DAH30. Secondary outcomes included DAH90, 30-day and 1-yr mortality, 30-day health system costs, length of index admission, and 30-day readmissions. Propensity score overlap weights were used to adjust for confounders. Prespecified effect modifier analyses focused on high-risk subgroups. RESULTS: Among 364 149 patients, 274 365 (75.3%) received a preoperative anaesthesia consultation. No adjusted association was found (22.5 days vs 22.5 days; adjusted ratio of means 1.00, 95% CI 1.00-1.00) between consultation and DAH30 in the full population. We identified significant effect modification (significantly more DAH30) among patients with ischaemic heart disease, ASA physical status ≥4, frailty index score ≥0.21, and who underwent vascular surgery. Secondary outcomes were associated with preoperative consultation, including greater DAH90, decreased length of stay, lower 30-day and 1-yr mortality, and reduced 30-day costs. CONCLUSIONS: Preoperative anaesthesia consultation was not associated with greater DAH30 across the overall study population. However, important potential benefits were observed among high-risk subgroups. Research is needed to identify optimal patient populations and consultation processes.

Optical nanotopography of fluorescent surfaces by axial position modulation.

We present an optical method that combines confocal microscopy with position modulation to perform axial tracking and topographic imaging of fluorescent surfaces. Using a remote focusing system, the confocal observation volume is oscillated in the axial direction. The resulting modulation of the detected signal is used as a feedback to precisely control the distance to an object of interest. The accuracy of this method is theoretically analyzed and the axial-locking accuracy is experimentally evaluated. Topographic imaging is demonstrated on fluorescently coated beads and fixed cells. This microscope allows for nanometric topography or tracking of dynamic fluorescent surfaces.

Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells.

Real-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues.