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Lipid rafts are known as highly ordered lipid domains that are enriched in saturated lipids such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have never been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here we report that microscopic GM1-enriched domains can form, without lipid cross-linking, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains, but only partially co-localize with them. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains. By measuring dissociation constants, we demonstrate that EphA2 oligomerization is the same in EphA2-enriched and -depleted domains. However, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biology of lipid rafts.

The three members of the PEAK family of pseudokinases (PEAK1, PEAK2, and PEAK3) are molecular scaffolds that have recently emerged as important regulatory nodes in signaling pathways that control cell migration, morphology, and proliferation, and they are increasingly found to be mis-regulated in human cancers. While no structures of PEAK3 have been solved to date, crystal structures of the PEAK1 and PEAK2 pseudokinase domains revealed their dimeric organization. It remains unclear how dimerization plays a role in PEAK scaffolding functions, as no structures of PEAK family members in complex with their binding partners have been solved. Here, we report the cryo-EM structure of the PEAK3 pseudokinase, also adopting a dimeric state, and in complex with an endogenous 14-3-3 heterodimer purified from mammalian cells. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the Split HElical Dimerization (SHED) domain of the PEAK3 dimer. The binding interface is comprised of a canonical primary interaction involving two phosphorylated 14-3-3 consensus binding sites located in the N-terminal domains of the PEAK3 monomers docked in the conserved amphipathic grooves of the 14-3-3 dimer, and a unique secondary interaction between 14-3-3 and PEAK3 that has not been observed in any previous structures of 14-3-3/client complexes. Disruption of these interactions results in the relocation of PEAK3 to the nucleus and changes its cellular interactome. Lastly, we identify Protein Kinase D as the regulator of the PEAK3/14-3-3 interaction, providing a mechanism by which the diverse functions of the PEAK3 scaffold might be fine-tuned in cells.

We introduce concentration-dependent Number and Brightness (cdN&B), a fluorescence fluctuation technique which can be implemented on a standard confocal microscope and can report on the thermodynamics of membrane protein association in the native plasma membrane. It uses transient transfection to enable measurements of oligomer size as a function of receptor concentration over a broad range, yielding the association constant. We discuss artifacts in cdN&B that are concentration-dependent and can distort the oligomerization curves, and we outline procedures that can correct for them. Using cdN&B, we characterize the association of neuropilin 1 (NRP1), a protein which plays a critical role in the development of the embryonic cardiovascular and nervous systems. We show that NRP1 associates into a tetramer in a concentration-dependent manner, and we quantify the strength of the association. This work demonstrates the utility of cdN&B as a powerful tool in biophysical chemistry.

Ligand bias is the ability of ligands to differentially activate certain receptor signaling responses compared with others. It reflects differences in the responses of a receptor to specific ligands and has implications for the development of highly specific therapeutics. Whereas ligand bias has been studied primarily for G protein–coupled receptors (GPCRs), there are also reports of ligand bias for receptor tyrosine kinases (RTKs). However, the understanding of RTK ligand bias is lagging behind the knowledge of GPCR ligand bias. In this review, we highlight how protocols that were developed to study GPCR signaling can be used to identify and quantify RTK ligand bias. We also introduce an operational model that can provide insights into the biophysical basis of RTK activation and ligand bias. Finally, we discuss possible mechanisms underpinning RTK ligand bias. Thus, this review serves as a primer for researchers interested in investigating ligand bias in RTK signaling.

Here, we discuss the transition model of receptor tyrosine kinase (RTK) activation, which is derived from biophysical investigations of RTK interactions and signaling. The model postulates that (1) RTKs can interact laterally to form dimers even in the absence of ligand, (2) different unliganded RTK dimers have different stabilities, (3) ligand binding stabilizes the RTK dimers, and (4) ligand binding causes structural changes in the RTK dimer. The model is grounded in the principles of physical chemistry and provides a framework to understand RTK activity and to make predictions in quantitative terms. It can guide basic research aimed at uncovering the mechanism of RTK activation and, in the long run, can empower the search for modulators of RTK function.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

Tropomyosin receptor kinase B (Trk-B) belongs to the second largest family of membrane receptors, Receptor Tyrosine Kinases (RTKs). Trk-B is known to interact with three different neurotrophins: Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-4 (NT-4), and Neurotrophin-3 (NT-3). All three neurotrophins are involved in survival and proliferation of neuronal cells, but each induces distinct signaling through Trk-B. We hypothesize that the different biological effects correlate with differences in the interactions between the Trk-B receptors, when bound to different ligands, in the plasma membrane. To test this hypothesis, we use quantitative FRET to characterize Trk-B dimerization in response to NT-3 and NT-4 in live cells, and compare it to the previously published data for Trk-B in the absence and presence of BDNF. Our study reveals that the distinct Trk-B signaling outcomes are underpinned by both different configurations and different stabilities of the three ligand-bound Trk-B dimers in the plasma membrane.

Electronic pacemakers can treat electrical conduction disorders in hearts; however, they are invasive, bulky, and linked to increased incidence of infection at the tissue-device interface. Thus, researchers have looked to other more biocompatible methods for cardiac pacing or resynchronization, such as femtosecond infrared light pulsing, optogenetics, and polymer-based cardiac patches integrated with metal electrodes. Here we develop a biocompatible nongenetic approach for the optical modulation of cardiac cells and tissues. We demonstrate that a polymer-silicon nanowire composite mesh can be used to convert fast moving, low-radiance optical inputs into stimulatory signals in target cardiac cells. Our method allows for the stimulation of the cultured cardiomyocytes or ex vivo heart to beat at a higher target frequency.

Receptor tyrosine kinases (RTKs) are single-pass membrane proteins that control vital cell processes such as cell growth, survival, and differentiation. There is a growing body of evidence that RTKs from different subfamilies can interact and that these diverse interactions can have important biological consequences. However, these heterointeractions are often ignored, and their strengths are unknown. In this work, we studied the heterointeractions of nine RTK pairs, epidermal growth factor receptor (EGFR)–EPH receptor A2 (EPHA2), EGFR–vascular endothelial growth factor receptor 2 (VEGFR2), EPHA2–VEGFR2, EPHA2–fibroblast growth factor receptor 1 (FGFR1), EPHA2–FGFR2, EPHA2–FGFR3, VEGFR2–FGFR1, VEGFR2–FGFR2, and VEGFR2–FGFR3, using a FRET-based method. Surprisingly, we found that RTK heterodimerization and homodimerization strengths can be similar, underscoring the significance of RTK heterointeractions in signaling. We discuss how these heterointeractions can contribute to the complexity of RTK signal transduction, and we highlight the utility of quantitative FRET for probing multiple interactions in the plasma membrane.

In this work, we put forward the provocative hypothesis that the active, ligand-bound RTK dimers from unrelated subfamilies can associate into heterooligomers with novel signaling properties. This hypothesis is based on a quantitative FRET study that monitors the interactions between EGFR and VEGFR2 in the plasma membrane of live cells in the absence of ligand, in the presence of either EGF or VEGF, and in the presence of both ligands. We show that direct interactions occur between EGFR and VEGFR2 in the absence of ligand and in the presence of the two cognate ligands. However, there are not significant heterointeractions between EGFR and VEGFR2 when only one of the ligands is present. Since RTK dimers and RTK oligomers are believed to signal differently, this finding suggests a novel mechanism for signal diversification.

All members of the Eph receptor family of tyrosine kinases contain a SAM domain near the C terminus, which has been proposed to play a role in receptor homotypic interactions and/or interactions with binding partners. The SAM domain of EphA2 is known to be important for receptor function, but its contribution to EphA2 lateral interactions in the plasma membrane has not been determined. Here we use a FRET-based approach to directly measure the effect of the SAM domain on the stability of EphA2 dimers on the cell surface in the absence of ligand binding. We also investigate the functional consequences of EphA2 SAM domain deletion. Surprisingly, we find that the EphA2 SAM domain inhibits receptor dimerization and decreases EphA2 tyrosine phosphorylation. This role is dramatically different from the role of the SAM domain of the related EphA3 receptor, which we previously found to stabilize EphA3 dimers and increase EphA3 tyrosine phosphorylation in cells in the absence of ligand. Thus, the EphA2 SAM domain likely contributes to a unique mode of EphA2 interaction that leads to distinct signaling outputs.

DNA is rapidly cleaved under mild alkaline conditions at apyrimidinic/apurinic sites, but the half-life is several weeks in phosphate buffer (pH 7.5). However, abasic sites are ~100-fold more reactive within nucleosome core particles (NCPs). Histone proteins catalyze the strand scission, and at superhelical location 1.5, the histone H4 tail is largely responsible for the accelerated cleavage. The rate constant for strand scission at an abasic site is enhanced further in a nucleosome core particle when it is part of a bistranded lesion containing a proximal strand break. Cleavage of this form results in a highly deleterious double-strand break. This acceleration is dependent upon the position of the abasic lesion in the NCP and its structure. The enhancement in cleavage rate at an apurinic/apyrimidinic site rapidly drops off as the distance between the strand break and abasic site increases and is negligible once the two forms of damage are separated by 7 bp. However, the enhancement of the rate of double-strand break formation increases when the size of the gap is increased from one to two nucleotides. In contrast, the cleavage rate enhancement at 2-deoxyribonolactone within bistranded lesions is more modest, and it is similar in free DNA and nucleosome core particles. We postulate that the enhanced rate of double-strand break formation at bistranded lesions containing apurinic/apyrimidinic sites within nucleosome core particles is a general phenomenon and is due to increased DNA flexibility.

Cell-penetrating peptides (CPP) are a potential drug delivery vehicle into cells and across biological barriers. We discovered that a CPP, called the CL peptide, delivered a conjugated fluorophore into different cell lines and enhanced ten-fold the permeability of the fluorophore across an epithelium. We synthesized CL peptide variants to identify the important parameters that determine the cell- and barrier-penetrating properties. We also used Fully-quantified Spectral Imaging (FSI) method to investigate the binding of the CL-fluorophore conjugate and its variants to live cell membranes and correlate it to the intracellular delivery efficiency.

Purpose The critical management decision in pediatric pancreatic injuries involves whether or not to operate on patients with grade II or III injuries. Because of the rarity of these injuries, no one hospital cares for enough patients to determine the outcome of this decision. Given this, the American Pediatric Surgical Association accrued a series of patients with pancreatic injuries from the members of its Trauma Committee. Methods A retrospective review of concurrent pancreatic injuries from 9 level 1 pediatric trauma centers was performed. Results Data on 131 children were submitted. Forty-three patients suffered grade II or grade III injuries. Twenty patients underwent an operation, and 23 were observed. Patients who underwent an operation had an average length of stay of 16.1 days compared with 14.2 days. Two in the operative group received total parenteral nutrition compared with 12 in the nonoperative group. Eight in the nonoperative group developed a pseudocyst compared with 3 in the operative group. Conclusions Children with grade II or grade III pancreatic injuries managed nonoperatively had a higher rate of pseudocyst, lower rate of reoperation, and a comparable length of stay compared with those who underwent surgery. These data will be used to help design a prospective study of pancreatic injury management.

Eph-A2 is a member of the largest Receptor Tyrosine Kinases (RTKs) family and hence it is activated through lateral dimerization. The dimerization of Eph-A2 receptor triggers signaling in the membrane plane that regulates many physiological processes in humans. This protein is very often overexpressed in a variety of cancers. EphA2 receptor is auto phosphorylated on Tyr through lateral dimerization and Serine phosphorylated by Akt. The Serine phosphorylation of EphA2 receptor is linked to tumorigenic behavior of EphA2 receptor. We asked if Serine phosphorylation and tyrosine phosphorylation can affect the dimerization of EphA2 receptor. We generated the kinase dead (K646M mutant) EphA2 and S897A EphA2 mutant. We studied the dimerization of these mutants using FRET based assay in live cells and compared with the wild type data. Out data suggest that both mutations affect dimerization.

Following an alcohol binge, cyclosporine A (CyA) levels rose by 100% in a 51-year-old transplant recipient treated with CyA. As CyA and ethanol are both metabolized by the cytochrome P-450 enzyme system, ethanol could theoretically interfere with CyA metabolism. Therefore, eight male renal transplant recipients were assessed in a crossover study to determine the effects of acute ethanol ingestion on CyA serum concentrations. CyA serum concentrations did not rise following 50 mL of 100% alcohol. We conclude that heavy alcohol intake may increase CyA levels but that acute moderate intake does not.

A 54 year old woman had a Bentley DiaTAP button implanted in her thigh for hemodialysis access. She had been started on continuous ambulatory peritoneal dialysis (CAPD) because of intractable vascular access problems. Unfortunately, CAPD had to be discontinued because of fungal peritonitis. Transplantation had not been possible because of circulating cytotoxic antibodies. The prosthetic complex soon became infected with Staphylococcus epidermidis and blood cultures were intermittently positive for 11 months. However, with continuing vancomycin therapy she remained in her usual state of health, without side effects from vancomycin, until venous thrombosis resolved and it became possible to remove the infected prosthetic complex and implant a button in an arm. As a last resort, it may be possible to maintain a patient on dialysis in reasonable health with a DiaTAP button graft complex infected with Staphylococcus epidermidis and intermittent positive blood cultures using long term vancomycin therapy. Such management probably would not be appropriate for any other organism.

The outcome and complications which developed in 8 hemodialysis patients who received 12 DiaTAP bioCarbon button vascular-access devices were reviewed. All patients had a poor vascular access history. Three of twelve devices have been replaced because of thrombosis and two because of infection. Four patients have had 10 episodes of reduced blood flow. Streptokinase infusion into the DiaTAP button led to improved blood flow in 8 of 10 episodes. The 6-month survival rate of the DiaTAP access device was 67% and the average functioning life was 9.4 months. It was a valuable form of access when others had failed.

In many studies, idiopathic calcium-phosphate and calcium-oxalate stones are grouped together as calcium stones, yet calcium-phosphate stones may be the second commonest type submitted for analysis in some areas. In 12 reports from five countries, calcium-phosphate stones averaged 9% of 40,000 stones and in Newfoundland, 12% of 4014 stones analyzed (1). Based on prevalence studies (2), we estimate that our Renal Laboratory receives for analysis from the Newfoundland population 15–20% of stones passed spontaneously or removed.

To determine the risk of nephrotoxicity induced by the infusion of radiographic contrast material, we undertook a prospective study of consecutive patients undergoing radiographic procedures with intravascular contrast material. There were three study groups: patients with diabetes mellitus and normal renal function (n = 85), patients with preexisting renal insufficiency (serum creatinine level, ≥150 μmol per liter) without diabetes (n = 101), and patients with both diabetes and renal insufficiency (n = 34). The control group consisted of patients undergoing CT scanning or abdominal imaging procedures without the infusion of contrast material who had diabetes mellitus (n = 59), preexisting renal insufficiency (n = 145), or both (n = 64). Clinically important acute renal failure (defined as an increase of >50 percent in the serum creatinine level) attributable to the contrast material did not occur in nondiabetic patients with preexisting renal insufficiency or in diabetics with normal renal funct...