Nonquantal transmission at the vestibular hair cell-calyx synapse: KLV currents modulate fast electrical and slow K+ potentials.

Vestibular hair cells transmit information about head position and motion across synapses to primary afferent neurons. At some of these synapses, the afferent neuron envelopes the hair cell, forming an enlarged synaptic terminal called a calyx. The vestibular hair cell-calyx synapse supports a mysterious form of electrical transmission that does not involve gap junctions, termed nonquantal transmission (NQT). The NQT mechanism is thought to involve the flow of ions from the presynaptic hair cell to the postsynaptic calyx through low-voltage-activated channels driven by changes in cleft [K+] as K+ exits the hair cell. However, this hypothesis has not been tested with a quantitative model and the possible role of an electrical potential in the cleft has remained speculative. Here, we present a computational model that captures experimental observations of NQT and identifies features that support the existence of an electrical potential (ϕ) in the synaptic cleft. We show that changes in cleft ϕ reduce transmission latency and illustrate the relative contributions of both cleft [K+] and ϕ to the gain and phase of NQT. We further demonstrate that the magnitude and speed of NQT depend on calyx morphology and that increasing calyx height reduces action potential latency in the calyx afferent. These predictions are consistent with the idea that the calyx evolved to enhance NQT and speed up vestibular signals that drive neural circuits controlling gaze, balance, and orientation.

Subsequent Renal Cancer Among Childhood Cancer Survivors: Analysis of Surveillance, Epidemiology, and End Results.

Renal cancer, although still rare among individuals under 45 years of age, is on the rise in the general population. The risk and timing of subsequent renal cancer in survivors of childhood cancer is not well established. Using the SEER registry, we reported the incidence of subsequent malignant renal neoplasms after treatment for primary malignancy diagnosed under 20 years of age. We evaluated clinical characteristics, standardized incidence ratio (SIR), and Kaplan-Meier survival estimates. Fifty-three survivors developed subsequent renal cancer (54 total cases). Of these, 54.7% were female, 88.7% were white, and 13.2% were Hispanic. Mean ages at primary malignancy and subsequent renal cancer were 10.1 and 31.1 years, respectively. Forty-seven cases were second cancers, 6 were third, and 1 was fourth. For survivors of childhood cancer, the overall SIR for renal cancer was 4.52 (95% CI: 3.39-5.89). The 5-year overall survival rate after development of subsequent renal cancer was 73% (95% CI: 58%-83%). Renal cancer occurs 4.5 times more frequently in childhood cancer survivors than in the general population, necessitating long-term care considerations.

Utilization of Thiopurine Metabolites and Allopurinol in Pediatric Acute Lymphoblastic Leukemia: Consideration for an Algorithmic Approach.

Persistently elevated absolute neutrophil counts during maintenance for acute lymphoblastic leukemia is a risk factor for relapse and may be related to wild-type thiopurine methyltransferase activity and overly efficient shunting of 6-mercaptopurine to hepatotoxic metabolites (6-methylmercaptopurine nucleotides), leading to low 6-thioguanine nucleotides. 6-mercaptopurine is also metabolized by xanthine oxidase, and therefore allopurinol, an inhibitor of xanthine oxidase, allows for increased 6-thioguanine nucleotides and decreased 6-methylmercaptopurine nucleotide. Here, we report our experience with allopurinol for persistently elevated absolute neutrophil count or hepatotoxicity and suggest an algorithmic approach for checking thiopurine metabolites and initiating allopurinol in acute lymphoblastic leukemia maintenance.

SDF-1α stiffens myeloma bone marrow mesenchymal stromal cells through the activation of RhoA-ROCK-Myosin II.

Multiple myeloma (MM) is a B lymphocyte malignancy that remains incurable despite extensive research efforts. This is due, in part, to frequent disease recurrences associated with the persistence of myeloma cancer stem cells (mCSCs). Bone marrow mesenchymal stromal cells (BMSCs) play critical roles in supporting mCSCs through genetic or biochemical alterations. Previously, we identified mechanical distinctions between BMSCs isolated from MM patients (mBMSCs) and those present in the BM of healthy individuals (nBMSCs). These properties of mBMSC contributed to their ability to preferentially support mCSCs. To further illustrate mechanisms underlying the differences between mBMSCs and nBMSCs, here we report that (i) mBMSCs express an abnormal, constitutively high level of phosphorylated Myosin II, which leads to stiffer membrane mechanics, (ii) mBMSCs are more sensitive to SDF-1α-induced activation of MYL2 through the G(i./o)-PI3K-RhoA-ROCK-Myosin II signaling pathway, affecting Young's modulus in BMSCs and (iii) activated Myosin II confers increased cell contractile potential, leading to enhanced collagen matrix remodeling and promoting the cell-cell interaction between mCSCs and mBMSCs. Together, our findings suggest that interfering with SDF-1α signaling may serve as a new therapeutic approach for eliminating mCSCs by disrupting their interaction with mBMSCs.

Caregiver Experiences With Oral Bilingualism in Children Who Are Deaf and Hard of Hearing in the United States: Impact on Child Language Proficiency.

PURPOSE: Best practices recommend promoting the use of the home language and allowing caregivers to choose the language(s) that they want to use with their child who is deaf or hard of hearing (DHH). We examined whether Spanish-speaking caregivers of children who are DHH receive professional recommendations on oral bilingualism that follow best practices. We also assessed whether professional recommendations, caregiver beliefs, and language practices had an impact on child language(s) proficiency. METHOD: Sixty caregivers completed a questionnaire on demographic questions, language(s) use and recommendations, beliefs on bilingualism, and child language proficiency measures in English, Spanish, and American Sign Language (ASL). Professional recommendations on oral bilingualism were reported descriptively, and linear regression was used to identify the predictors of child language(s) proficiency. RESULTS: We found that only 23.3% of the caregivers were actively encouraged to raise their child orally bilingual. Language practices predicted child proficiency in each language (English, Spanish, and ASL), but professional recommendations and caregiver beliefs did not. CONCLUSIONS: Our results revealed that most caregivers received recommendations that do not follow current best practices. Professional training is still needed to promote bilingualism and increase cultural competence when providing services to caregivers who speak languages different from English. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.21644846.

The Remarkable Outer Hair Cell: Proceedings of a Symposium in Honour of W. E. Brownell.

In 1985, Bill Brownell and colleagues published the remarkable observation that cochlear outer hair cells (OHCs) express voltage-driven mechanical motion: electromotility. They proposed OHC electromotility as the mechanism for the elusive "cochlear amplifier" required to explain the sensitivity of mammalian hearing. The finding and hypothesis stimulated an explosion of experiments that have transformed our understanding of cochlear mechanics and physiology, the evolution of hair cell structure and function, and audiology. Here, we bring together examples of current research that illustrate the continuing impact of the discovery of OHC electromotility.

Outer hair cell electromechanics as a problem in soft matter physics: Prestin, the membrane and the cytoskeleton.

The electromechanical coupling exhibited by cochlear outer hair cells is a remarkable biophysical phenomenon. These specialized cells generate forces at acoustic frequencies and enable high-frequency hearing in mammals. While there has been significant progress since the discovery of electromotility - including the discovery of the motor protein prestin - we still do not have a clear picture of how electromotility works. A particularly vexing problem is how forces, generated by a membrane-based motor, are rapidly transmitted to the underlying cytoskeleton to enable force generation on the microsecond time scales required for amplification of acoustic signals. Here we approach the problem of electromotility from the perspective of soft matter physics in light of recent ultrastructural findings from 3D electron tomography studies on outer hair cells immobilized by high-pressure freezing. We then survey our understanding of prestin-membrane and prestin-cytoskeletal interactions in the context recently published cryoelectron microscopy (cryo-EM) structures of prestin. This will lead to the proposal of a new conceptual model of electromotility consistent with conformational states observed in the pillar proteins and actin filaments. This article is part of the Special Issue Outer hair cell Edited by Joseph Santos-Sacchi and Kumar Navaratnam.

Structural and functional diversity of mitochondria in vestibular/cochlear hair cells and vestibular calyx afferents.

Mitochondria supply energy in the form of ATP to drive a plethora of cellular processes. In heart and liver cells, mitochondria occupy over 20% of the cellular volume and the major need for ATP is easily identifiable - i.e., to drive cross-bridge recycling in cardiac cells or biosynthetic machinery in liver cells. In vestibular and cochlear hair cells the overall cellular mitochondrial volume is much less, and mitochondria structure varies dramatically in different regions of the cell. The regional demands for ATP and cellular forces that govern mitochondrial structure and localization are not well understood. Below we review our current understanding of the heterogeneity of form and function in hair cell mitochondria. A particular focus of this review will be on regional specialization in vestibular hair cells, where large mitochondria are found beneath the cuticular plate in close association with the striated organelle. Recent findings on the role of mitochondria in hair cell death and aging are covered along with potential therapeutic approaches. Potential avenues for future research are discussed, including the need for integrated computational modeling of mitochondrial function in hair cells and the vestibular afferent calyx.

Cysteine mutagenesis reveals transmembrane residues associated with charge translocation in prestin.

The solute carrier transmembrane protein prestin (SLC26A5) drives an active electromechanical transduction process in cochlear outer hair cells that increases hearing sensitivity and frequency discrimination in mammals. A large intramembraneous charge movement, the nonlinear capacitance (NLC), is the electrical signature of prestin function. The transmembrane domain (TMD) helices and residues involved in the intramembrane charge displacement remain unknown. We have performed cysteine-scanning mutagenesis with serine or valine replacement to investigate the importance of cysteine residues to prestin structure and function. The distribution of oligomeric states and membrane abundance of prestin was also probed to investigate whether cysteine residues participate in prestin oligomerization and/or NLC. Our results reveal that 1) Cys-196 (TMD 4) and Cys-415 (TMD 10) do not tolerate serine replacement, and thus maintaining hydrophobicity at these locations is important for the mechanism of charge movement; 2) Cys-260 (TMD 6) and Cys-381 (TMD 9) tolerate serine replacement and are probably water-exposed; and 3) if disulfide bonds are present, they do not serve a functional role as measured via NLC. These novel findings are consistent with a recent structural model, which proposes that prestin contains an occluded aqueous pore, and we posit that the orientations of transmembrane domain helices 4 and 10 are essential for proper prestin function.

Selective cell-surface labeling of the molecular motor protein prestin.

Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

Supporting Equity and Inclusion of Deaf and Hard-of-Hearing Individuals in Professional Organizations.

Disability is an important and often overlooked component of diversity. Individuals with disabilities bring a rare perspective to science, technology, engineering, mathematics, and medicine (STEMM) because of their unique experiences approaching complex issues related to health and disability, navigating the healthcare system, creatively solving problems unfamiliar to many individuals without disabilities, managing time and resources that are limited by physical or mental constraints, and advocating for themselves and others in the disabled community. Yet, individuals with disabilities are underrepresented in STEMM. Professional organizations can address this underrepresentation by recruiting individuals with disabilities for leadership opportunities, easing financial burdens, providing equal access, fostering peer-mentor groups, and establishing a culture of equity and inclusion spanning all facets of diversity. We are a group of deaf and hard-of-hearing (D/HH) engineers, scientists, and clinicians, most of whom are active in clinical practice and/or auditory research. We have worked within our professional societies to improve access and inclusion for D/HH individuals and others with disabilities. We describe how different models of disability inform our understanding of disability as a form of diversity. We address heterogeneity within disabled communities, including intersectionality between disability and other forms of diversity. We highlight how the Association for Research in Otolaryngology has supported our efforts to reduce ableism and promote access and inclusion for D/HH individuals. We also discuss future directions and challenges. The tools and approaches discussed here can be applied by other professional organizations to include individuals with all forms of diversity in STEMM.

A high throughput microelectroporation device to introduce a chimeric antigen receptor to redirect the specificity of human T cells.

It has been demonstrated that a chimeric antigen receptor (CAR) can directly recognize the CD19 molecule expressed on the cell surface of B-cell malignancies independent of major histocompatibility complex (MHC). Although T-cell therapy of tumors using CD19-specific CAR is promising, this approach relies on using expression vectors that stably integrate the CAR into T-cell chromosomes. To circumvent the potential genotoxicity that may occur from expressing integrating transgenes, we have expressed the CD19-specific CAR transgene from mRNA using a high throughput microelectroporation device. This research was accomplished using a microelectroporator to achieve efficient and high throughput non-viral gene transfer of in vitro transcribed CAR mRNA into human T cells that had been numerically expanded ex vivo. Electro-transfer of mRNA avoids the potential genotoxicity associated with vector and transgene integration and the high throughput capacity overcomes the expected transient CAR expression, as repeated rounds of electroporation can replace T cells that have lost transgene expression. We fabricated and tested a high throughput microelectroporator that can electroporate a stream of 2 x 10(8) primary T cells within 10 min. After electroporation, up to 80% of the passaged T cells expressed the CD19-specific CAR. Video time-lapse microscopy (VTLM) demonstrated the redirected effector function of the genetically manipulated T cells to specifically lyse CD19+ tumor cells. Our biomedical microdevice, in which T cells are transiently and safely modified to be tumor-specific and then can be re-infused, offers a method for redirecting T-cell specificity, that has implications for the development of adoptive immunotherapy.

Altering amine basicities in biodegradable branched polycationic polymers for nonviral gene delivery.

In this work, biodegradable branched polycationic polymers were synthesized by Michael addition polymerization from different amine monomers and the triacrylate monomer trimethylolpropane triacrylate. The polymers varied in the number of amines that dissociate in different pH ranges, which are considered to be beneficial to different parts of the gene delivery process. P-DED, a polymer synthesized from trimethylolpropane triacrylate and dimethylethylenediamine, had the highest number of protonated amines that are available for plasmid DNA (pDNA) complexation at pH 7.4 of all polymers synthesized. P-DED formed a positive polyplex (13.9 +/- 0.5 mV) at a polymer/pDNA weight ratio of 10:1 in contrast with the other polymers synthesized, which formed positive polyplexes only at higher weight ratios. Polyplexes formed with the synthesized polymers at the highest polymer/pDNA weight ratio tested (300:1) resulted in higher transfection with enhanced green fluorescent protein reporter gene (5.3 +/- 1.0 to 30.6 +/- 6.6%) compared with naked pDNA (0.8 +/- 0.4%), as quantified by flow cytometry. Polyplexes formed with P-DED (weight ratio of 300:1) also showed higher transfection (30.6 +/- 6.6%) as compared with polyplexes formed with branched polyethylenimine (weight ratio of 2:1, 25.5 +/- 2.7%). The results from this study demonstrated that polymers with amines that dissociate above pH 7.4, which are available as positively charged groups for pDNA complexation at pH 7.4, can be synthesized to produce stable polyplexes with increased zeta potential and decreased hydrodynamic size that efficiently transfect cells. This work indicated that polymers containing varying amine functionalities with different buffering capabilities can be synthesized by using different amine monomers and used as effective gene delivery vectors.

Amphipath-induced nanoscale changes in outer hair cell plasma membrane curvature.

Outer hair cell (OHC) electromotility enables frequency selectivity and sensitivity in mammalian audition. Electromotility is generated by the transmembrane protein prestin and is sensitive to amphipathic compounds including salicylate, chlorpromazine (CPZ), and trinitrophenol (TNP). Although these compounds induce observable membrane curvature changes in erythrocytes, their effects on OHC membrane curvature are unknown. In this work, fluorescence polarization microscopy was applied to investigate the effects of salicylate, CPZ, and TNP on di-8-ANEPPS orientation in the OHC plasma membrane. Our results demonstrate the ability of fluorescence polarization microscopy to measure amphipath-induced changes in di-8-ANEPPS orientation, consistent with nanoscale changes in membrane curvature between regularly spaced proteins connecting the OHC plasma membrane and cytoskeleton. Simultaneous application of oppositely charged amphipaths generally results in no net membrane bending, consistent with predictions of the bilayer couple hypothesis; however, the application of salicylate (10 mM), which inhibits electromotility, is not reversed by the addition of CPZ. This result supports other findings that suggest salicylate primarily influences electromotiliy and OHC nonlinear capacitance via a direct interaction with prestin. In contrast, we find that CPZ and TNP influence the voltage sensitivity of prestin via membrane bending, demonstrating the mechanosensitivity of this unique membrane motor protein.

Biodegradable branched polycationic polymers with varying hydrophilic spacers for nonviral gene delivery.

Biodegradable branched polycationic polymers with varying hydrophilic spacer lengths were synthesized from different triacrylate monomers and the amine monomer 1-(2-aminoethyl)piperazine by Michael addition polymerization. The hydrophilic spacers were varied by the number of ethyleneoxy groups in the triacrylate monomer (E/M) that ranged from 0 to 14. The polymer degradation depended on the spacer length and pH; the amount of ester degraded as determined by (1)H NMR after 14 days was 43.4 +/- 2.1% (pH 5.0) and 89.7 +/- 1.3% (pH 7.4) for the polymer with 0 E/M compared to 55.7 +/- 2.6% (pH 5.0) and 98.5 +/- 1.6% (pH 7.4) for the polymer with 14 E/M. Cell viability of rat fibroblasts after exposure to polymer solutions of concentrations up to 1000 microg/mL remained high (above 66.9 +/- 12.1% compared to below 7.6 +/- 1.1% for polyethylenimine at a concentration of 50 microg/mL or higher) and increased with the spacer length. The polyplexes made with all the synthesized polymers showed higher transfection efficiency (4.5 +/- 1.7% to 9.4 +/- 2.0%, dependent on the polymer/pDNA weight ratio) with an enhanced green fluorescent protein reporter gene compared to naked pDNA (0.8 +/- 0.4%) as quantified by flow cytometry. This study demonstrates that hydrophilic spacers can be incorporated into polycationic polymers to reduce their cytotoxicity and enhance their degradability for nonviral gene delivery.

3D Ultrastructure of the Cochlear Outer Hair Cell Lateral Wall Revealed By Electron Tomography.

Outer Hair Cells (OHCs) in the mammalian cochlea display a unique type of voltage-induced mechanical movement termed electromotility, which amplifies auditory signals and contributes to the sensitivity and frequency selectivity of mammalian hearing. Electromotility occurs in the OHC lateral wall, but it is not fully understood how the supramolecular architecture of the lateral wall enables this unique form of cellular motility. Employing electron tomography of high-pressure frozen and freeze-substituted OHCs, we visualized the 3D structure and organization of the membrane and cytoskeletal components of the OHC lateral wall. The subsurface cisterna (SSC) is a highly prominent feature, and we report that the SSC membranes and lumen possess hexagonally ordered arrays of particles. We also find the SSC is tightly connected to adjacent actin filaments by short filamentous protein connections. Pillar proteins that join the plasma membrane to the cytoskeleton appear as variable structures considerably thinner than actin filaments and significantly more flexible than actin-SSC links. The structurally rich organization and rigidity of the SSC coupled with apparently weaker mechanical connections between the plasma membrane (PM) and cytoskeleton reveal that the membrane-cytoskeletal architecture of the OHC lateral wall is more complex than previously appreciated. These observations are important for our understanding of OHC mechanics and need to be considered in computational models of OHC electromotility that incorporate subcellular features.

Generation of the endocochlear potential: a biophysical model.

The generation and maintenance of the endocochlear potential (EP) by the stria vascularis is essential for proper function of the cochlea. We present a mathematical model that captures the critical biophysical interactions between the distinct cellular layers that generate the EP. By describing the relationship between the K(+) concentration in the intrastrial space and the intermediate cell transmembrane potential, we rationalize the presence of a large intermediate cell K(+) conductance and predict that the intrastrial [K(+)] is approximately 4 mM at steady state. The model also predicts that the stria vascularis is capable of buffering the EP against external perturbations in a manner modulated by changes in intrastrial [K(+)], thus facilitating hearing sensitivity across the broad dynamic range of the auditory system.

The influence of an in vitro generated bone-like extracellular matrix on osteoblastic gene expression of marrow stromal cells.

The function and development of cells rely heavily on the signaling interactions with the surrounding extracellular matrix (ECM). Therefore, a tissue engineering scaffold should mimic native ECM to recreate the in vivo environment. Previously, we have shown that an in vitro generated ECM secreted by cultured cells enhances the mineralized matrix deposition of marrow stromal cells (MSCs). In this study, MSC expression of 45 bone-related genes using real-time reverse transcriptase polymerase chain reaction (RT-PCR) was determined. Upregulation of osteoblastic markers such as collagen type I, matrix extracellular phosphoglycoprotein with ASARM motif, parathyroid hormone receptor, and osteocalcin, indicated that the MSCs on plain titanium scaffolds differentiated down the osteoblastic lineage and deposited a mineralized matrix on day 12. Significant mineralized matrix deposition was observed as early as day 4 on ECM-containing scaffolds and was associated with the enhancement in expression of a subset of osteoblast-specific genes that included a 2-fold increase in osteopontin expression at day 1 and a 6.5-fold increase in osteocalcin expression at day 4 as well as downregulation of chondrogenic gene markers. These results were attributed to the cellular interactions with growth factors and matrix molecules that are likely present in the in vitro generated ECM since the genes for insulin-like growth factor 1, insulin-like growth factor 2, vascular endothelial growth factor, dentin matrix protein, collagen type IV, cartilage oligomeric protein, and matrix metalloproteinase 13 were significantly upregulated during ECM construct generation. Overall, the data demonstrate that modulation of MSC differentiation occurs at the transcriptional level and gene expression of bone-related proteins is differentially regulated by the ECM. This study presents enormous implications for tissue engineering strategies, as it demonstrates that modification of a biomaterial with an in vitro generated ECM containing cell-generated bioactive signaling molecules can effectively direct gene expression and differentiation of seeded progenitor cell populations.

Interactions of ibuprofen with hybrid lipid bilayers probed by complementary surface-enhanced vibrational spectroscopies.

The incorporation of small molecules into lipid bilayers is a process of biological importance and clinical relevance that can change the material properties of cell membranes and cause deleterious side effects for certain drugs. Here we report the direct observation, using surface-enhanced Raman and IR spectroscopies (SERS, SEIRA), of the insertion of ibuprofen molecules into hybrid lipid bilayers. The alkanethiol-phospholipid hybrid bilayers were formed onto gold nanoshells by self-assembly, where the underlying nanoshell substrates provided the necessary enhancements for SERS and SEIRA. The spectroscopic data reveal specific interactions between ibuprofen and phospholipid moieties and indicate that the overall hydrophobicity of ibuprofen plays an important role in its intercalation in these membrane mimics.

Peroxidation of polyunsaturated phosphatidyl-choline lipids during electroformation.

Giant unilamellar vesicles (GUVs) have been utilized both as model systems to study the physico-chemical properties of biomembranes and as host materials for investigating biological processes in microbioreactors. GUVs are commonly formed by an electroformation technique. However, there is a concern that the electric fields applied during electroformation can peroxidize lipid acyl chains, thereby altering the phospholipid composition and material properties of the synthesized vesicles. Here in this paper, we report the effect of electroformation on the extent of peroxidation of a number of polyunsaturated phosphatidyl-choline lipids (PULs). Specifically, we detected peroxidation byproducts (malonaldehydes and conjugated dienes) of the following lipids utilizing UV/Vis spectroscopy: dilinoleoyl phosphatidyl-choline (DLPC) (di-18:2 PC), dilinolenoyl phosphatidyl-choline (DNPC) (di-18:3 PC), diarachidonoyl phosphatidyl-choline (DAPC) (di-20:4 PC), and didocosaheexaenoyl phosphatidyl-choline (DHA) (di-22:6 PC). The results indicate that PC PULs lipids are prone to peroxidation, with increasing unsaturation levels leading to higher levels of peroxidation byproducts. The levels of peroxidation byproducts of DAPC were found to depend linearly on the strength of the electric field, indicating that the observed effects were due to the applied electric field. Lipid peroxidation can affect a number of important membrane properties, including domain formation and mechanical stability. Thus, alteration of the chemical composition of polyunsaturated lipids (PULs) by the electroformation technique can potentially complicate the interpretation of experimental studies that utilize GUVs composed of PULs.