The hydrophobic surfactant proteins, SP-B and SP-C, promote rapid adsorption by the surfactant lipids to the surface of the liquid that lines the alveolar air sacks of the lungs. To gain insights into the mechanisms of their function, we used X-ray diffuse scattering (XDS) and molecular dynamics (MD) simulations to determine the location of SP-B and SP-C within phospholipid bilayers. Initial samples contained the surfactant lipids from extracted calf surfactant with increasing doses of the proteins. XDS located protein density near the phospholipid headgroup and in the hydrocarbon core, presumed to be SP-B and SP-C, respectively. Measurements on dioleoyl­phosphatidylcholine (DOPC) with the proteins produced similar results. MD simulations of the proteins with DOPC provided molecular detail and allowed direct comparison of the experimental and simulated results. Simulations used conformations of SP-B based on other members of the saposin-like family, which form either open or closed V-shaped structures. For SP-C, the amino acid sequence suggests a partial α-helix. Simulations fit best with measurements of XDS for closed SP-B, which occurred at the membrane surface, and SP-C oriented along the hydrophobic interior. Our results provide the most definitive evidence yet concerning the location and orientation of the hydrophobic surfactant proteins.

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To establish how the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of lipids to an air/water interface, we used x-ray diffuse scattering (XDS) to determine an order parameter of...

IntroductionMyocardial ischemia disrupts the cardio-spinal neural network that controls the cardiac sympathetic preganglionic neurons, leading to sympathoexcitation and ventricular tachyarrhythmias (VTs). Spinal cord stimulation (SCS) is capable of suppressing the sympathoexcitation caused by myocardial ischemia. However, how SCS modulates the spinal neural network is not fully known.MethodsIn this pre-clinical study, we investigated the impact of SCS on the spinal neural network in mitigating myocardial ischemia-induced sympathoexcitation and arrhythmogenicity. Ten Yorkshire pigs with left circumflex coronary artery (LCX) occlusion-induced chronic myocardial infarction (MI) were anesthetized and underwent laminectomy and a sternotomy at 4−5 weeks post-MI. The activation recovery interval (ARI) and dispersion of repolarization (DOR) were analyzed to evaluate the extent of sympathoexcitation and arrhythmogenicity during the left anterior descending coronary artery (LAD) ischemia. Extracellular in vivo and in situ spinal dorsal horn (DH) and intermediolateral column (IML) neural recordings were performed using a multichannel microelectrode array inserted at the T2-T3 segment of the spinal cord. SCS was performed for 30 min at 1 kHz, 0.03 ms, 90% motor threshold. LAD ischemia was induced pre- and 1 min post-SCS to investigate how SCS modulates spinal neural network processing of myocardial ischemia. DH and IML neural interactions, including neuronal synchrony as well as cardiac sympathoexcitation and arrhythmogenicity markers were evaluated during myocardial ischemia pre- vs. post-SCS.ResultsARI shortening in the ischemic region and global DOR augmentation due to LAD ischemia was mitigated by SCS. Neural firing response of ischemia-sensitive neurons during LAD ischemia and reperfusion was blunted by SCS. Further, SCS showed a similar effect in suppressing the firing response of IML and DH neurons during LAD ischemia. SCS exhibited a similar suppressive impact on the mechanical, nociceptive and multimodal ischemia sensitive neurons. The LAD ischemia and reperfusion-induced augmentation in neuronal synchrony between DH-DH and DH-IML pairs of neurons were mitigated by the SCS.DiscussionThese results suggest that SCS is decreasing the sympathoexcitation and arrhythmogenicity by suppressing the interactions between the spinal DH and IML neurons and activity of IML preganglionic sympathetic neurons.

Targeting protein-protein interactions (PPI) within the cell is an appealing prospect for cancer research and therapy. Methods to inhibit PPIs have been limited though due to the ineffectiveness of small molecules for this task, and to the inability to get macromolecule cargoes -such as antibodies -into the cytosol. Because of this, targets such as RAS and MYC are sometimes considered ''undruggable'' even though they have been studied for decades. To address this, we are developing pH-sensitive pore-forming peptides to enable endosomal escape of endocytosed protein cargo. One such peptide, pHD108, is a pHtriggered gain-of-function variant of melittin, a membrane permeabilizing peptide found in bee venom. In synthetic unilamellar vesicles, pHD108 has no poration activity at pH 7; at pH % 5.5 it releases macromolecules from lipid vesicles at very low peptide concentration. In this work, we have shown that, with a low pH pulse, pHD108 allows 150 kDa proteins (IgGs) through the plasma membrane. High concentrations of peptide are needed, however, so steps to increase potency has yielded an optimal acylation strategy of a 6-carbon acyl chain at the N-terminus. To assess endosomolytic activity, a novel assay was developed using domain III of Pseudomonas Exotoxin A. When given to cells alone, the exotoxin does not induce apoptosis but when delivered to the cytosol it will induce apoptosis. pHD108 shows activity here. To further increase potency we plan to screen a focused library of peptides with rationally modified sequence variations while employing the optimal acylation strategy. Hits found there will be characterized further. Our findings here will ultimately allow researchers to target intracellular PPIs in living cells using exogenous antibodies.