Correction to: Atomistic Simulations Modify Interpretation of Spin-Label Oximetry Data. Part 1: Intensified Water-Lipid Interfacial Resistances.

[This corrects the article PMC10249954.].

Simulation Study of Breast Cancer Lipid Changes Affecting Membrane Oxygen Permeability: Effects of Chain Length and Cholesterol.

Tumor radiotherapy relies on intracellular oxygen (O2) to generate reactive species that trigger cell death, yet hypoxia is common in cancers of the breast. De novo lipid synthesis in tumors supports cell proliferation but also may lead to unusually high levels of the 16:1 palmitoleoyl (Y) phospholipid tail, which is two carbons shorter than the 18:1 oleoyl (O) tail abundant in normal breast tissue. Here, we use atomic resolution molecular dynamics simulations to test two hypotheses: (1) the shorter, 16:1 Y, tail of the de novo lipid biosynthesis product 1-palmitoyl,2-palmitoleoyl-phosphatidylcholine (PYPC) promotes lower membrane permeability relative to the more common lipid 1-palmitoyl,2-oleoylphosphatidylcholine (POPC), by reducing oxygen solubility in the interleaflet region, and (2) cholesterol further lessens the permeability of PYPC by reducing overall O2 solubility and promoting PYPC tail order adjacent to the rigid cholesterol ring system. The simulations conducted here indicate that PYPC has a permeability of 14 ± 1 cm/s at 37 °C, comparable to 15.4 ± 0.4 cm/s for POPC. Inclusion of cholesterol in a 1:1 ratio with phospholipid intensifies the effect of chain length, giving permeabilities of 10.2 ± 0.2 cm/s for PYPC/cholesterol and 11.0 ± 0.6 cm/s for POPC/cholesterol. These findings indicate that PYPC may not substantially influence membrane-level oxygen flux and is unlikely to hinder breast tissue oxygenation.

Updated Evaluation of Cholesterol's Influence on Membrane Oxygen Permeability.

There is a surprising gap in knowledge regarding the mechanism of oxygen (O2) diffusional delivery at the level of tissues and cells. Yet, the effectiveness of tumor radiotherapy, the success of tissue engineering, and healthy metabolism all require ample intracellular oxygen. Tissue-level diffusion takes place in a complex and crowded macromolecular environment. Cholesterol-rich cellular membranes have been thought to reduce oxygen flux. Here, we use atomistic molecular dynamics simulations to update prior estimates of bilayer permeability and related parameters for 1-palmitoyl,2-oleoylphosphatidylcholine (POPC) and POPC/cholesterol bilayers, using a modified O2 model with improved membrane-water partitioning behavior. This work estimates an oxygen permeability coefficient of 15 ± 1 cm/s for POPC and 11.5 ± 0.4 cm/s for POPC/cholesterol (1:1 molecular ratio) at 37 °C. The permeability of POPC is found to be ~1/3 that of a water layer of similar thickness, and the permeability of POPC/cholesterol is estimated to be 20-30% below that of POPC. Void pathway visualization and free energy data support channeling of oxygen toward the center of cholesterol-incorporating membranes, while partition coefficient data suggest reduced membrane solubility of oxygen due to cholesterol. Further study is needed to understand whether diffusion pathway changes due to cholesterol and other molecular compositional factors influence oxygen availability within tissue.

Atomistic simulations modify interpretation of spin-label oximetry data. Part 1: intensified water-lipid interfacial resistances.

The role of membrane cholesterol in cellular function and dysfunction has been the subject of much inquiry. A few studies have suggested that cholesterol may slow oxygen diffusive transport, altering membrane physical properties and reducing oxygen permeability. The primary experimental technique used in recent years to study membrane oxygen transport is saturation-recovery electron paramagnetic resonance (EPR) oximetry, using spin-label probes targeted to specific regions of a lipid bilayer. The technique has been used, in particular, to assess the influence of cholesterol on oxygen transport and membrane permeability. The reliability of such EPR recordings at the water-lipid interface near the phospholipid headgroups has been challenged by all-atom molecular dynamics (MD) simulation data that show substantive agreement with spin-label probe measurements throughout much of the bilayer. This work uses further MD simulations, with an updated oxygen model, to determine the location of the maximum resistance to permeation and the rate-limiting barrier to oxygen permeation in 1-palmitoyl,2-oleoylphosphatidylcholine (POPC) and POPC/cholesterol bilayers at 25 and 35°C. The current simulations show a spike of resistance to permeation in the headgroup region that was not detected by EPR but was predicted in early theoretical work by Diamond and Katz. Published experimental nuclear magnetic resonance (NMR) oxygen measurements provide key validation of the MD models and indicate that the positions and relative magnitudes of the phosphatidylcholine resistance peaks are accurate. Consideration of the headgroup-region resistances predicts bilayer permeability coefficients lower than estimated in EPR studies, giving permeabilities lower than the permeability of unstirred water layers of the same thickness. Here, the permeability of POPC at 35°C is estimated to be 13 cm/s, compared with 10 cm/s for POPC/cholesterol and 118 cm/s for simulation water layers of similar thickness. The value for POPC is 12 times lower than estimated from EPR measurements, while the value for POPC/cholesterol is 5 times lower. These findings underscore the value of atomic resolution models for guiding the interpretation of experimental probe-based measurements.

Phospholipid prodrug conjugates of insoluble chemotherapeutic agents for ultrasound targeted drug delivery.

The hydrophobicity and high potency of many therapeutic agents makes them difficult to use effectively in clinical practice. This work focuses on conjugating phospholipid tails (2T) onto podophyllotoxin (P) and its analogue (N) using a linker and characterizing the effects of their incorporation into lipid-based drug delivery vehicles for triggered ultrasound delivery. Differential Scanning Calorimetry results show that successfully synthesized lipophilic prodrugs, 2T-P (~28 % yield) and 2T-N(~26 % yield), incorporate within the lipid membranes of liposomes. As a result of this, increased stability and incorporation are observed in 2T-P and 2T-N in comparison to the parent compounds P and N. Molecular dynamic simulation results support that prodrugs remain within the lipid membrane over a relevant range of concentrations. 2T-N's (IC50: 20 nM) biological activity was retained in HeLa cells (cervical cancer), whereas 2T-P's (IC50: ~4 µM) suffered, presumably due to steric hindrance. Proof-of-concept studies using ultrasound in vitro microbubble and nanodroplet delivery vehicles establish that these prodrugs are capable of localized drug delivery. This study provides useful information about the synthesis of double tail analogues of insoluble chemotherapeutic agents to facilitate incorporation into drug delivery vehicles. The phospholipid attachment strategy presented here could be applied to other well suited drugs such as gemcitabine, commonly known for its treatment of pancreatic cancer.

Reduced Oxygen Permeability upon Protein Incorporation Within Phospholipid Bilayers.

Intracellular oxygenation is key to energy metabolism as well as tumor radiation therapy. Although integral proteins are ubiquitous in membranes, few studies have considered their effects on molecular oxygen permeability. Published experimental work with rhodopsin and bacteriorhodopsin has led to the hypothesis that integral proteins lessen membrane oxygen permeability, as well as the permeability of the lipid region. The current work uses atomistic molecular dynamics simulations to test the influence of an ungated potassium channel protein on the oxygen permeability of palmitoyloleoylphosphatidylcholine (POPC) bilayers with and without cholesterol. Consistent with experiment, whole-membrane oxygen permeability is cut in half upon adding 30 wt% potassium channel protein to POPC, and the apparent permeability of the lipid portion of the membrane decreases by 40%. Unexpectedly, oxygen is found to interact directly with the protein surface, accompanied by a 40% reduction of the apparent whole-membrane diffusion coefficient. Similar effects are seen in systems combining the potassium channel with 1:1 POPC/cholesterol, but the magnitude of permeability reduction is smaller by ~30%. Overall, the simulations indicate that integral proteins can reduce oxygen permeability by altering the diffusional path and the local diffusivity. This effect may be especially important in the protein-dense membranes of mitochondria.

Predicted Decrease in Membrane Oxygen Permeability with Addition of Cholesterol.

Aberrations in cholesterol homeostasis are associated with several diseases that can be linked to changes in cellular oxygen usage. Prior biological and physical studies have suggested that membrane cholesterol content can modulate oxygen delivery, but questions of magnitude and biological significance remain open for further investigation. Here, we use molecular dynamics simulations in a first step toward reexamining the rate impact of cholesterol on the permeation of oxygen through phospholipid bilayers. The simulation models are closely compared with published electron paramagnetic resonance (EPR) oximetry measurements. The simulations predict an oxygen permeability reduction due to cholesterol but also suggest that the EPR experiments may have underestimated resistance to oxygen permeation in the phospholipid headgroup region.

Influence of Cholesterol on the Oxygen Permeability of Membranes: Insight from Atomistic Simulations.

Cholesterol is widely known to alter the physical properties and permeability of membranes. Several prior works have implicated cell membrane cholesterol as a barrier to tissue oxygenation, yet a good deal remains to be explained with regard to the mechanism and magnitude of the effect. We use molecular dynamics simulations to provide atomic-resolution insight into the influence of cholesterol on oxygen diffusion across and within the membrane. Our simulations show strong overall agreement with published experimental data, reproducing the shapes of experimental oximetry curves with high accuracy. We calculate the upper-limit transmembrane oxygen permeability of a 1-palmitoyl,2-oleoylphosphatidylcholine phospholipid bilayer to be 52 ± 2 cm/s, close to the permeability of a water layer of the same thickness. With addition of cholesterol, the permeability decreases somewhat, reaching 40 ± 2 cm/s at the near-saturating level of 62.5 mol % cholesterol and 10 ± 2 cm/s in a 100% cholesterol mimic of the experimentally observed noncrystalline cholesterol bilayer domain. These reductions in permeability can only be biologically consequential in contexts where the diffusional path of oxygen is not water dominated. In our simulations, cholesterol reduces the overall solubility of oxygen within the membrane but enhances the oxygen transport parameter (solubility-diffusion product) near the membrane center. Given relatively low barriers to passing from membrane to membrane, our findings support hydrophobic channeling within membranes as a means of cellular and tissue-level oxygen transport. In such a membrane-dominated diffusional scheme, the influence of cholesterol on oxygen permeability is large enough to warrant further attention.

Repression of inflammasome by Francisella tularensis during early stages of infection.

Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1ß and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing a mutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1ß and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1ß expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1ß observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1ß by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect.

Identification of a novel Francisella tularensis factor required for intramacrophage survival and subversion of innate immune response.

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpA-like protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium.

Protein C depletion early after trauma increases the risk of ventilator-associated pneumonia.

INTRODUCTION: Mechanically ventilated trauma patients have a high risk for the development of ventilator-associated pneumonia (VAP). We have recently reported that reduced plasma protein C (PC) levels early after trauma/shock are associated with coagulopathy and mortality. Furthermore, trauma patients with tissue injury and shock are at higher risk for the development of VAP. OBJECTIVE: We hypothesized that low PC levels early after trauma are associated with an increased susceptibility to VAP in trauma patients. METHODS: Fifty-nine acutely injured, intubated trauma patients were admitted to the critical care unit. Serial blood samples were drawn and coagulation factors were measured. VAP was diagnosed by presence of bacteria on bronchial alveolar lavage specimen, bilateral infiltrates on chest roentgenogram, and fever or elevated white blood cell count. RESULTS: There were no differences in demographic or injury characteristics between patients who developed VAP and those who did not. As expected, patients who developed VAP had more ventilator days, hospital days, intensive care unit days, and greater mortality (all p < 0.05). Patients in both groups had lower mean PC levels at 6 hours compared with baseline. Noninfected patients' PC subsequently returned to near baseline levels, whereas those patients who eventually acquired VAP had significantly lower PC levels at both 12 and 24 hours (12 hours: 79 vs. 96%, p = 0.05; 24 hours: 75 vs. 97% p = 0.02). Soluble endothelial PC receptor (sEPCR) levels were also lower at 24 hours (82 vs. 99% in the noninfected group, p = 0.04). DISCUSSION: The activation of PC pathway early after trauma may protect the vascular endothelium by both its anticoagulant and cytoprotective effects. However, trauma patients who later developed VAP have significantly lower plasma levels of PC within 24 hours after injury, suggesting a possible consumption of this vitamin K-dependent protein and an inhibition of its activation by inflammatory mediators. EPCR is involved in the activation of PC and is also a mediator of its cytoprotective effects. CONCLUSION: Critically ill trauma patients have an early activation of the PC pathway, associated with a rapid decrease in the plasma levels of this protein and increase in EPCR. Plasma levels of PC return to normal levels within 24 hours in most patients. However, patients who go on to acquire VAP have persistently low plasma levels of PC in the immediate period after trauma. Whether PC could play a mechanistic role in the host response against nosocomial lung infection warrants further study.

Increased mortality of ventilated patients with endotracheal Pseudomonas aeruginosa without clinical signs of infection.

OBJECTIVE: To investigate the frequency and outcomes of ventilated patients with newly acquired large burdens of Pseudomonas aeruginosa and to test the hypothesis that large quantities of bacteria are associated with adverse patient outcomes. DESIGN: A prospective, single-center, observational, cohort study. SETTING: Medical-surgical intensive care units in a tertiary care university hospital. PATIENTS: All adult patients requiring > or = 48 hrs of mechanical ventilation and identified as having newly acquired P. aeruginosa in their lower respiratory tracts between October 2002 and April 2006. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Daily surveillance cultures of endotracheal aspirates were performed on patients intubated > or = 48 hrs; 69 patients with newly acquired P. aeruginosa were enrolled. Daily P. aeruginosa quantification of endotracheal aspirates was performed; clinical signs of infection were noted. Of 45 patients with high P. aeruginosa burdens (> or = 1,000,000 colony-forming units/mL in endotracheal aspirates; > or = 10,000 colony-forming units/mL in bronchoalveolar-lavage), 17 (37.8%) patients did not meet clinical criteria for ventilator-associated pneumonia and had a statistically significant higher risk of death (adjusted hazard ratio, 37.53; 95% confidence interval, 3.79-371.96; p = 0.002) when compared with the patients who had P. aeruginosa ventilator-associated pneumonia. When excluding the ten patients who had ventilator-associated pneumonia attributed to bacteria other than P. aeruginosa or attributed to multiple bacteria including P. aeruginosa, the risk of death remained statistically significant (adjusted hazard ratio, 23.98; 95% confidence interval: 2.49-230.53; p = 0.006). Furthermore, more patients with high P. aeruginosa burdens secreted the type III secretion facilitator protein, PcrV (p = 0.01). CONCLUSIONS: A group of patients with large burdens of P. aeruginosa who did not meet clinical criteria for ventilator-associated pneumonia had an increased risk of death when compared with patients who had high P. aeruginosa burdens and met ventilator-associated pneumonia criteria. Patients with high P. aeruginosa burden seemed to possess more virulent strains.

Increased plasminogen activator inhibitor-1 concentrations in bronchoalveolar lavage fluids are associated with increased mortality in a cohort of patients with Pseudomonas aeruginosa.

BACKGROUND: Increased plasminogen activator inhibitor-1 (PAI-1) concentrations are found in bronchoalveolar lavage (BAL) fluids from patients with ventilator-associated pneumonia or acute respiratory distress syndrome. The authors hypothesized that PAI-1 concentrations were associated with increased mortality in patients with either Pseudomonas aeruginosa-induced ventilator-associated pneumonia or tracheobronchial colonization. METHODS: In a prospective cohort study, daily aspirates from intubated patients were cultured for P. aeruginosa. Positive patients had blind BAL (bBAL) that was processed for biomarker concentrations. Secretion of type III secretion cytotoxins were also analyzed from the P. aeruginosa strains. RESULTS: Thirty-three patients were enrolled. Ten of the 33 patients died. bBAL PAI-1 concentrations were significantly increased in nonsurvivors compared with survivors (31.7 vs. 3.4 ng/ml, P = 0.001 for hospital mortality; 35.9 vs. 4.7 ng/ml, P = 0.02 for 28-day mortality). Even when acute respiratory distress syndrome patients were excluded, there was a significant difference between the survivors and nonsurvivors for bBAL PAI-1 concentrations (P = 0.005). Eighty-three percent of P. aeruginosa strains isolated from patients with high concentrations of bBAL PAI-1 also had strains that secreted cytotoxins. CONCLUSIONS: PAI-1 concentrations in bBALs correlated with mortality in ventilated patients with positive cultures for P. aeruginosa. Elevated bBAL PAI-1 concentrations also correlated with the secretion of type III exotoxins by P. aeruginosa.