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.

Phrenic nerve stimulation in an ovine model with temporary removable pacing leads.

Background: The objective of this study was to assess the feasibility and safety of a novel, removable, surgically implanted, temporary neurostimulation approach involving the distal portion of the phrenic nerve. Methods: Temporary phrenic nerve pacing electrodes were implanted surgically using an ovine model (4 animals). The primary endpoint was the ability to successfully match the animal's minute-ventilation upon implantation of both phrenic nerve pacers on day 1. Secondary endpoints were successful phrenic neurostimulation by both electrodes 15 and 30 days after initial implantation. We also assessed safe removal of the electrodes at 15 and 30 days after implementation. Results: In 3 of 4 animals, electrodes were successfully implanted in both right and left phrenic nerves. On day 1, median ventilation-minute induced by neurostimulation was not significantly different from baseline ventilation-minute [4.9 L·min-1 (4.4-5.5) vs. 4.4 L·min-1 (4.3-5.2); P=0.4] after 15 minutes. Neurostimulation was still possible 15 and 30 days after implementation in all left side phrenic nerves. On the right side, stimulation was possible at all times in 1 animal but not in the remaining 3 animals for at least one time point, possibly due to lead displacement. Analysis of pathology after percutaneous electrode removal showed integrity of the distal portion of all phrenic nerves. Conclusions: Efficient temporary neurostimulation through the distal portion of the phrenic nerve was possible at baseline. The main complication was the displacement of electrodes on the right phrenic nerve on two occasions, which was due to the anatomy of the ovine model. It compromised diaphragm pacing on day 15 and day 30. The electrodes could be safely removed percutaneously without damage to the phrenic nerves.

The terminal segment of the human phrenic nerve as a novel implantation site for diaphragm pacing electrodes: Anatomical and clinical description.

BACKGROUND: Diaphragm pacing allows certain ventilator-dependent patients to achieve weaning from mechanical ventilation. The reference method consists in implanting intrathoracic contact electrodes around the phrenic nerve during video-assisted thoracic surgery, which involves time-consuming phrenic nerve dissection with a risk of nerve damage. Identifying a phrenic segment suitable for dissection-free implantation of electrodes would constitute progress. STUDY DESIGN: This study characterizes a free terminal phrenic segment never fully described before. We conducted a cadaver study (n = 14) and a clinical observational study during thoracic procedures (n = 54). RESULTS: A free terminal phrenic segment was observed on both sides in 100% of cases, "jumping" from the pericardium to the diaphragm and measuring 60 mm [95% confidence interval; 48-63] and 72.5 mm [65-82] (right left, respectively; p = 0.0038; cadaver study). This segment rolled up on itself at end-expiration and became unravelled and elongated with diaphragm descent (clinical study). Three categories of fat pads were defined (type 1: pericardiophrenic bundle free of surrounding fat; type 2: single fatty fringe leaving the phrenic nerve visible until diaphragmatic entry; type 3: multiple fatty fringes masking the site of penetration of the phrenic nerve) that depended on body mass index (p = 0.001, clinical study). Hematoxylin-eosin and toluidine blue staining (cadaver study) showed that all of the phrenic fibers in the distal, pre-branching part of the terminal segment were contained within a single epineurium containing a variable number of fascicles (right: 1 [95%CI 0.65-4.01]; left 5 [3.37-7.63]; p = 0.03). CONCLUSION: Diaphragm pacing through periphrenic electrodes positioned on the terminal phrenic segment should be tested.

Stimulation of angiogenesis by Cyr61 gene: a new therapeutic candidate.

Cyr61 is a secreted, cysteine-rich heparin-binding protein that is associated with extracellular matrix and cell surface, and has been demonstrated to be proangiogenic in vitro. In the present study we evaluated the angiogenic effect of human Cyr61 in an adenoviral context in the rabbit ischemic hindlimb model. For this purpose, three randomized groups of New Zealand White rabbits received intramuscular injections of 5 x 10(8) infectious units of an adenovirus carrying either the Cyr61 gene (Ad-Cyr61), the vascular endothelial growth factor gene (Ad-VEGF(165)) used as the angiogenic gene of reference, or no transgene (Ad-Null), 10 days after femoral artery excision in one limb. Perfusion of the ischemic limb was evaluated before adenoviral treatment (day 10) and 30 days postinjection (day 40). Angiographic, hemodynamic, and histologic parameters indicated that animals in the Ad-Cyr61 group had significantly better perfusion than in the Ad-Null group. Interestingly, this improvement exceeded that achieved with Ad-VEGF(165). In conclusion, Cyr61 gene transfer appears potent in stimulating limb revascularization, thereby promoting great improvement in tissue perfusion in the ischemic limb. These findings indicate that Cyr61 could be a promising therapeutic candidate for treating severe peripheral ischemic diseases.

Bmcc1s, a novel brain-isoform of Bmcc1, affects cell morphology by regulating MAP6/STOP functions.

The BCH (BNIP2 and Cdc42GAP Homology) domain-containing protein Bmcc1/Prune2 is highly enriched in the brain and is involved in the regulation of cytoskeleton dynamics and cell survival. However, the molecular mechanisms accounting for these functions are poorly defined. Here, we have identified Bmcc1s, a novel isoform of Bmcc1 predominantly expressed in the mouse brain. In primary cultures of astrocytes and neurons, Bmcc1s localized on intermediate filaments and microtubules and interacted directly with MAP6/STOP, a microtubule-binding protein responsible for microtubule cold stability. Bmcc1s overexpression inhibited MAP6-induced microtubule cold stability by displacing MAP6 away from microtubules. It also resulted in the formation of membrane protrusions for which MAP6 was a necessary cofactor of Bmcc1s. This study identifies Bmcc1s as a new MAP6 interacting protein able to modulate MAP6-induced microtubule cold stability. Moreover, it illustrates a novel mechanism by which Bmcc1 regulates cell morphology.