The LDL receptor is regulated by membrane cholesterol as revealed by fluorescence fluctuation analysis.

Membrane cholesterol-rich domains have been shown to be important for regulating a range of membrane protein activities. Low-density lipoprotein receptor (LDLR)-mediated internalization of cholesterol-rich LDL particles is tightly regulated by feedback mechanisms involving intracellular sterol sensors. Since LDLR plays a role in maintaining cellular cholesterol homeostasis, we explore the role that membrane domains may have in regulating LDLR activity. We expressed a fluorescent LDLR-mEGFP construct in HEK293T cells and imaged the unligated receptor or bound to an LDL/DiI fluorescent ligand using total internal reflection fluorescence microscopy. We studied the receptor's spatiotemporal dynamics using fluorescence fluctuation analysis methods. Image cross correlation spectroscopy reveals a lower LDL-to-LDLR binding fraction when membrane cholesterol concentrations are augmented using cholesterol esterase, and a higher binding fraction when the cells are treated with methyl-ß-cyclodextrin) to lower membrane cholesterol. This suggests that LDLR's ability to metabolize LDL particles is negatively correlated to membrane cholesterol concentrations. We then tested if a change in activity is accompanied by a change in membrane localization. Image mean-square displacement analysis reveals that unligated LDLR-mEGFP and ligated LDLR-mEGFP/LDL-DiI constructs are transiently confined on the cell membrane, and the size of their confinement domains increases with augmented cholesterol concentrations. Receptor diffusion within the domains and their domain-escape probabilities decrease upon treatment with methyl-ß-cyclodextrin, consistent with a change in receptor populations to more confined domains, likely clathrin-coated pits. We propose a feedback model to account for regulation of LDLR within the cell membrane: when membrane cholesterol concentrations are high, LDLR is sequestered in cholesterol-rich domains. These LDLR populations are attenuated in their efficacy to bind and internalize LDL. However, when membrane cholesterol levels drop, LDL has a higher binding affinity to its receptor and the LDLR transits to nascent clathrin-coated domains, where it diffuses at a slower rate while awaiting internalization.

Awake endotracheal intubation using a hyperangulated video laryngoscope with a Total Control Introducer in a patient with a history of difficult intubation.

We report the first use of a fully articulating introducer called the Total Control Introducer (TCI) in combination with a hyperangulated video laryngoscope (VL) to perform an awake intubation in a patient with a history of difficult intubation. After appropriate airway topicalisation, a VL with a hyperangulated blade was inserted to visualise the glottis. A TCI articulating introducer was then used to dynamically navigate through the oropharynx into the trachea. Under indirect visualisation, an endotracheal tube was then passed over the TCI. The TCI was removed and the endotracheal tube was secured. General anaesthesia was induced after confirmation of intubation with capnography and auscultation. The patient was successfully intubated on the first attempt without complications.

Combined use of a Total Control Introducer and a hyperangulated video laryngoscope to place a left-sided double lumen endotracheal tube in a patient with a history of difficult laryngoscopy.

We describe the use of a Total Control Introducer (TCI) in combination with video laryngoscopy (VL) to place a left-sided double-lumen endotracheal tube (DLT) in a patient with a history of difficult laryngoscopy undergoing video-assisted thoracoscopic surgery (VATS). VL was used to obtain visualisation of the glottis and a TCI articulating introducer was used to dynamically navigate the airway and access the trachea. A 39 French DLT was subsequently passed over the TCI shaft and into the trachea under indirect visualisation. The TCI shaft was removed and the DLT was gently guided into the left main bronchus. Successful endobronchial intubation was confirmed with capnography, auscultation and fibreoptic bronchoscopy. We propose that the combined use of VL and a TCI can facilitate placement of a DLT in a patient with a known difficult airway who may otherwise be limited to a bronchial blocker placement for lung isolation during VATS.

First Case Report of Intubation With a Total Control Introducer and a Hyperangulated Video Laryngoscope.

We describe the first use of an articulating Total Control Introducer (TCI) and video laryngoscope (VL) to guide intubation of a patient with known difficult airway. The patient was an 84-year-old woman with a Mallampati IV airway assessment, small mouth opening, limited neck extension, and micrognathia. We utilized a VL to visualize the glottis and an articulating TCI for endotracheal tube placement. Anteflexion and retroflexion of the introducer tip allowed dynamic navigation through the upper airway and into the trachea. Intubation was performed on first attempt without complications.

Employment of a promoter-swapping technique shows that PhoU modulates the activity of the PstSCAB2 ABC transporter in Escherichia coli.

Expression of the Pho regulon in Escherichia coli is induced in response to low levels of environmental phosphate (P(i)). Under these conditions, the high-affinity PstSCAB(2) protein (i.e., with two PstB proteins) is the primary P(i) transporter. Expression from the pstSCAB-phoU operon is regulated by the PhoB/PhoR two-component regulatory system. PhoU is a negative regulator of the Pho regulon; however, the mechanism by which PhoU accomplishes this is currently unknown. Genetic studies of phoU have proven to be difficult because deletion of the phoU gene leads to a severe growth defect and creates strong selection for compensatory mutations resulting in confounding data. To overcome the instability of phoU deletions, we employed a promoter-swapping technique that places expression of the phoBR two-component system under control of the P(tac) promoter and the lacO(ID) regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes in E. coli. Here we utilized P(phoB)::P(tac) and P(pstS)::P(tac) strains to characterize phenotypes resulting from various DeltaphoU mutations. Our results indicate that PhoU controls the activity of the PstSCAB(2) transporter, as well as its abundance within the cell. In addition, we used the P(phoB)::P(tac) DeltaphoU strain as a platform to begin characterizing new phoU mutations in plasmids.

Ceragenins: cholic acid-based mimics of antimicrobial peptides.

The prevalence of drug-resistant bacteria drives the quest for new antimicrobials, including those that are not expected to readily engender resistance. One option is to mimic Nature's most ubiquitous means of controlling bacterial growth, antimicrobial peptides, which have evolved over eons. In general, bacteria remain susceptible to these peptides. Human antimicrobial peptides play a central role in innate immunity, and deficiencies in these peptides have been tied to increased rates of infection. However, clinical use of antimicrobial peptides is hampered by issues of cost and stability. The development of nonpeptide mimics of antimicrobial peptides may provide the best of both worlds: a means of using the same mechanism chosen by Nature to control bacterial growth without the problems associated with peptide therapeutics. The ceragenins were developed to mimic the cationic, facially amphiphilic structures of most antimicrobial peptides. These compounds reproduce the required morphology using a bile-acid scaffolding and appended amine groups. The resulting compounds are actively bactericidal against both gram-positive and gram-negative organisms, including drug-resistant bacteria. This antimicrobial activity originates from selective association of the ceragenins with negatively charged bacterial membrane components. Association has been studied with synthetic models of bacterial membrane components, with bacterial lipopolysaccharide, with vesicles derived from bacterial phospholipids, and with whole cells. Comparisons of the antimicrobial activities of ceragenins and representative antimicrobial peptides suggest that these classes of compounds share a mechanism of action. Rapid membrane depolarization is caused by both classes as well as blebbing of bacterial membranes. Bacteria express the same genes in response to both classes of compounds. On the basis of the antibacterial activities of ceragenins and preliminary in vivo studies, we expect these compounds to find use in augmenting or replacing antimicrobial peptides in treating human disease.

Randomized controlled trial of a simplified adductor canal block performed for analgesia following total knee arthroplasty.

BACKGROUND AND OBJECTIVES: The objective of the study was to determine if injection of local anesthetic into the vastus medialis and sartorius muscles adjacent to the adductor canal produces sensory changes comparable with adductor canal block (ACB). This could result in a technically easier and potentially safer alternative to ACB. METHODS: In this randomized controlled trial, patients received either ACB (n=20) or a simplified adductor canal (SAC) block performed using a new fenestrated nerve block needle (n=20). The time to perform each block as well as the number of attempts to position the needle were evaluated. A non-inferiority test was used to compare pain scores and opioid requirements for the ACB and the SAC block. RESULTS: The SAC block was performed more rapidly, with fewer needle passes, and had a higher success rate than the ACB. Three block failures and two vessel punctures were observed in the ACB group, while none of these events occurred in SAC block patients. Analgesia and opioid consumption for patients treated with the SAC block were not inferior to ACB. CONCLUSION: The SAC block is technically easier to perform and potentially safer than ACB. This procedure can be performed using easily visible ultrasound landmarks and has the potential for use among a wide range of healthcare providers. TRIAL REGISTRATION NUMBER: NCT02786888.

Optimization of ceragenins for prevention of bacterial colonization of hydrogel contact lenses.

PURPOSE: We provided contact lens hydrogels with an antibacterial innate immune function using nonpeptide mimics of endogenous antimicrobial peptides. METHODS: Antimicrobial peptide mimics, ceragenins, were prepared for either covalent attachment to hydrogels or for controlled elution from lenses. The lipophilicity of the ceragenins was varied incrementally to provide differing levels of association with hydrophobic domains in lenses. Ceragenin-containing lenses were challenged repeatedly with Staphylococcus aureus or Pseudomonas aeruginosa in nutrient media. Bacterial growth and biofilm formation on lenses were quantified. RESULTS: A ceragenin covalently fixed in lenses effectively inhibited S. aureus biofilm formation on lenses in 10% tryptic soy broth (approximately 3-log reduction), but did not reduce biofilm formation in 100% tryptic soy broth. Ceragenins designed to elute from lenses were incorporated at 1% relative to the dry weight of the lenses. The ceragenin with the optimal lipid content, CSA-138, prevented bacterial colonization of lenses for 15 days with P. aeruginosa and for 30 days with S. aureus (daily exchange of growth media and reinoculation with 106 CFU). Measurement of CSA-138 elution showed that concentrations of the ceragenin never exceeded 5 µg/mL in a 24-hour period and that after 4 days of elution, concentrations dropped to <0.5 µg/mL, while maintaining antibacterial activity. CONCLUSIONS: Ceragenin CSA-138 appears well suited for providing an innate immune-like function to abiotic hydrogel contact lenses for extended periods of time. Elution of even low concentrations of CSA-138 (<0.5 µg) is sufficient to eliminate inocula of 106 CFU of S. aureus and P. aeruginosa.