Accurate and rapid antibiotic susceptibility testing using a machine learning-assisted nanomotion technology platform.

Antimicrobial resistance (AMR) is a major public health threat, reducing treatment options for infected patients. AMR is promoted by a lack of access to rapid antibiotic susceptibility tests (ASTs). Accelerated ASTs can identify effective antibiotics for treatment in a timely and informed manner. We describe a rapid growth-independent phenotypic AST that uses a nanomotion technology platform to measure bacterial vibrations. Machine learning techniques are applied to analyze a large dataset encompassing 2762 individual nanomotion recordings from 1180 spiked positive blood culture samples covering 364 Escherichia coli and Klebsiella pneumoniae isolates exposed to cephalosporins and fluoroquinolones. The training performances of the different classification models achieve between 90.5 and 100% accuracy. Independent testing of the AST on 223 strains, including in clinical setting, correctly predict susceptibility and resistance with accuracies between 89.5% and 98.9%. The study shows the potential of this nanomotion platform for future bacterial phenotype delineation.

Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers.

Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions.

Calsyntenin-1 regulates targeting of dendritic NMDA receptors and dendritic spine maturation in CA1 hippocampal pyramidal cells during postnatal development.

Calsyntenin-1 is a transmembrane cargo-docking protein important for kinesin-1-mediated fast transport of membrane-bound organelles that exhibits peak expression levels at postnatal day 7. However, its neuronal function during postnatal development remains unknown. We generated a knock-out mouse to characterize calsyntenin-1 function in juvenile mice. In the absence of calsyntenin-1, synaptic transmission was depressed. To address the mechanism, evoked EPSPs were analyzed revealing a greater proportion of synaptic GluN2B subunit-containing receptors typical for less mature synapses. This imbalance was due to a disruption in calsyntenin-1-mediated dendritic transport of NMDA receptor subunits. As a consequence of increased expression of GluN2B subunits, NMDA receptor-dependent LTP was enhanced at Schaffer collateral-CA1 pyramidal cell synapses. Interestingly, these defects were accompanied by a decrease in dendritic arborization and increased proportions of immature filopodia-like dendritic protrusions at the expense of thin-type dendritic spines in CA1 pyramidal cells. Thus, these results highlight a key role for calsyntenin-1 in the transport of NMDA receptors to synaptic targets, which is necessary for the maturation of neuronal circuits during early development.

Perisynaptic chondroitin sulfate proteoglycans restrict structural plasticity in an integrin-dependent manner.

During early postnatal development of the CNS, neuronal networks are configured through the formation, elimination, and remodeling of dendritic spines, the sites of most excitatory synaptic connections. The closure of this critical period for plasticity correlates with the maturation of the extracellular matrix (ECM) and results in reduced dendritic spine dynamics. Chondroitin sulfate proteoglycans (CSPGs) are thought to be the active components of the mature ECM that inhibit functional plasticity in the adult CNS. These molecules are diffusely expressed in the extracellular space or aggregated as perineuronal nets around specific classes of neurons. We used organotypic hippocampal slices prepared from 6-d-old Thy1-YFP mice and maintained in culture for 4 weeks to allow ECM maturation. We performed live imaging of CA1 pyramidal cells to assess the effect of chondroitinase ABC (ChABC)-mediated digestion of CSPGs on dendritic spine dynamics. We found that CSPG digestion enhanced the motility of dendritic spines and induced the appearance of spine head protrusions in a glutamate receptor-independent manner. These changes were paralleled by the activation of ß1-integrins and phosphorylation of focal adhesion kinase at synaptic sites, and were prevented by preincubation with a ß1-integrin blocking antibody. Interestingly, microinjection of ChABC close to dendritic segments was sufficient to induce spine remodeling, demonstrating that CSPGs located around dendritic spines modulate their dynamics independently of perineuronal nets. This restrictive action of perisynaptic CSPGs in mature neural tissue may account for the therapeutic effects of ChABC in promoting functional recovery in impaired neural circuits.

Magnetically guided lentiviral-mediated transduction of airway epithelial cells.

BACKGROUND: Lentiviral (LV) vectors are able to only slowly and inefficiently transduce nondividing cells such as those of the airway epithelium. To address this issue, we have exploited the magnetofection technique in in vitro models of airway epithelium. METHODS: Magnetofectins were formed by noncovalent interaction between LV particles and polycation-coated iron oxide nanoparticles. Efficiency of LV-mediated transduction (as evaluated through green fluorescent protein (GFP) expression by cytofluorimetric analysis) was measured in bronchial epithelial cells in the presence or absence of a magnetic field. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) release; cell monolayer integrity by measurement of transepithelial resistance (TER) and evaluation of correct zonula occludens-1 (ZO-1) localization at tight junctions (TJs) by immunofluorescence and confocal microscopy. RESULTS: In nonpolarized cells, magnetofectins enhanced LV-mediated transduction at multiplicity of infection (MOI) of 50 up to 3.9-fold upon a 24-h incubation, to levels that approached those achieved at MOI of 200 for LV alone, in the presence or absence of the magnetic field. Magnetofection significantly increased the percentage of transduced cells up to 186-fold already after 15 min of incubation. In polarized cells, magnetofection increased GFP+ cells up to 24-fold compared to LV alone. Magnetofection did not enhance LDH release and slightly altered TER but not ZO-1 localization at the TJs. CONCLUSIONS: We conclude that magnetofection can facilitate in vitro LV-mediated transduction of airway epithelial cells, in the absence of overt cytotoxicity and maintaining epithelial integrity, by lowering the necessary vector dose and reducing the incubation time required to achieve efficient transduction.