Publications
87 results found
Martin A, Tempra C, Yu Y, et al., 2024, Exposure to Aldehyde Cherry e-Liquid Flavoring and Its Vaping Byproduct Disrupt Pulmonary Surfactant Biophysical Function., Environ Sci Technol, Vol: 58, Pages: 1495-1508
Over the past decade, there has been a significant rise in the use of vaping devices, particularly among adolescents, raising concerns for effects on respiratory health. Pressingly, many recent vaping-related lung injuries are unexplained by current knowledge, and the overall implications of vaping for respiratory health are poorly understood. This study investigates the effect of hydrophobic vaping liquid chemicals on the pulmonary surfactant biophysical function. We focus on the commonly used flavoring benzaldehyde and its vaping byproduct, benzaldehyde propylene glycol acetal. The study involves rigorous testing of the surfactant biophysical function in Langmuir trough and constrained sessile drop surfactometer experiments with both protein-free synthetic surfactant and hydrophobic protein-containing clinical surfactant models. The study reveals that exposure to these vaping chemicals significantly interferes with the synthetic and clinical surfactant biophysical function. Further atomistic simulations reveal preferential interactions with SP-B and SP-C surfactant proteins. Additionally, data show surfactant lipid-vaping chemical interactions and suggest significant transfer of vaping chemicals to the experimental subphase, indicating a toxicological mechanism for the alveolar epithelium. Our study, therefore, reveals novel mechanisms for the inhalational toxicity of vaping. This highlights the need to reassess the safety of vaping liquids for respiratory health, particularly the use of aldehyde chemicals as vaping flavorings.
Bernardino de la Serna J, 2024, The potential impacts of micro-and-nano plastics on various organ systems in humans, EBioMedicine, Vol: 99, ISSN: 2352-3964
Humans are exposed to micro-and-nano plastics (MNPs) through various routes, but the adverse health effects of MNPs on different organ systems are not yet fully understood. This review aims to provide an overview of the potential impacts of MNPs on various organ systems and identify knowledge gaps in current research. The summarized results suggest that exposure to MNPs can lead to health effects through oxidative stress, inflammation, immune dysfunction, altered biochemical and energy metabolism, impaired cell proliferation, disrupted microbial metabolic pathways, abnormal organ development, and carcinogenicity. There is limited human data on the health effects of MNPs, despite evidence from animal and cellular studies. Most of the published research has focused on specific types of MNPs to assess their toxicity, while other types of plastic particles commonly found in the environment remain unstudied. Future studies should investigate MNPs exposure by considering realistic concentrations, dose-dependent effects, individual susceptibility, and confounding factors.
Ali N, Xavier J, Engur M, et al., 2023, The impact of e-cigarette exposure on different organ systems: a review of recent evidence and future perspectives, Journal of Hazardous Materials, Vol: 457, ISSN: 0304-3894
The use of electronic cigarettes (e-cigs) is rapidly increasing worldwide and is promoted as a smoking cessation tool. The impact of traditional cigs on human health has been well-defined in both animal and human studies. In contrast, little is known about the adverse effects of e-cigs exposure on human health. This review summarizes the impact of e-cigs exposure on different organ systems based on the rapidly expanding recent evidence from experimental and human studies. A number of growing studies have shown the adverse effects of e-cigs exposure on various organ systems. The summarized data in this review indicate that while e-cigs use causes less adverse effects on different organs compared to traditional cigs, its long-term exposure may lead to serious health effects. Data on short-term organ effects are limited and there is no sufficient evidence on long-term organ effects. Moreover, the adverse effects of secondhand and third hand e-cigs vapour exposure have not been thoroughly investigated in previous studies. Although some studies demonstrated e-cigs used as a smoking cessation tool, there is a lack of strong evidence to support it. While some researchers suggested e-cigs as a safer alternative to tobacco smoking, their long-term exposure health effects remain largely unknown. Therefore, more epidemiological and prospective studies including mechanistic studies are needed to address the potential adverse health effects of e-cigs to draw a firm conclusion about their safe use. A wide variation in e-cigs products and the lack of standardized testing methods are the major barriers to evaluating the existing data. Specific regulatory guidelines for both e-cigs components and the manufacturing process may be effective to protect consumer health.
Saeed HK, Jarman PJ, Sreedharan S, et al., 2023, From chemotherapy to phototherapy - changing the therapeutic action of a metallo-intercalating Ruⁱⁱ -Reⁱ luminescent system by switching its sub-cellular location, Chemistry: A European Journal, Vol: 29, ISSN: 0947-6539
The synthesis of a new heterodinuclear ReI RuII metallointercalator containing RuII (dppz) and ReI (dppn) moieties is reported. Cell-free studies reveal that the complex has similar photophysical properties to its homoleptic M(dppz) analogue and it also binds to DNA with a similar affinity. However, the newly reported complex has very different in-cell properties to its parent. In complete contrast to the homoleptic system, the RuII (dppz)/ReI (dppn) complex is not intrinsically cytotoxic but displays appreciable phototoxic, despite both complexes displaying very similar quantum yields for singlet oxygen sensitization. Optical microscopy suggests that the reason for these contrasting biological effects is that whereas the homoleptic complex localises in the nuclei of cells, the RuII (dppz)/ReI (dppn) complex preferentially accumulates in mitochondria. These observations illustrate how even small structural changes in metal based therapeutic leads can modulate their mechanism of action.
Kobiela A, Hovhannisyan L, Jurkowska P, et al., 2023, Excess filaggrin in keratinocytes is removed by extracellular vesicles to prevent premature death and this mechanism can be hijacked by Staphylococcus aureus in a TLR2-dependent fashion, Journal of Extracellular Vesicles, Vol: 12, ISSN: 2001-3078
Filaggrin (FLG) protein is indispensable for multiple aspects of the epidermal barrier function but its accumulation in a monomeric filaggrin form may initiate premature keratinocytes death; it is unclear how filaggrin levels are controlled before the formation of storing keratohyalin granules. Here we show that keratinocyte-secreted small extracellular vesicles (sEVs) may contain filaggrin-related cargo providing a route of eliminating excess filaggrin from keratinocytes; blocking of sEV release has cytotoxic effects on those cells. Filaggrin-containing sEVs are found in plasma in both healthy individuals and atopic dermatitis patients. Staphylococcus aureus (S. aureus) enhances packaging and secretion of filaggrin-relevant products within the sEVs for enhanced export via a TLR2-mediated mechanism which is also linked to the ubiquitination process. This filaggrin removal system, preventing premature keratinocyte death and epidermal barrier dysfunction, is exploited by S. aureus which promotes filaggrin elimination from the skin that could help safeguard bacterial growth.
Manchanda Y, Bitsi S, Chen S, et al., 2023, Enhanced endosomal signaling and desensitization of GLP-1R versus GIPR in pancreatic beta cells, Endocrinology, Vol: 164, Pages: 1-15, ISSN: 0013-7227
The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are prime therapeutic targets for the treatment of type 2 diabetes (T2D) and obesity. They are expressed in pancreatic beta cells where they potentiate insulin release in response to food intake. Despite GIP being the main incretin in healthy individuals, GLP-1R has been favored as a therapeutic target due to blunted GIPR responses in T2D patients and conflicting effects of GIPR agonists and antagonists in improving glucose tolerance and preventing weight gain. There is, however, a recently renewed interest in GIPR biology following the realization that GIPR responses can be restored after an initial period of blood glucose normalization and the recent development of dual GLP-1R/GIPR agonists with superior capacity for controlling blood glucose levels and weight. The importance of GLP-1R trafficking and subcellular signaling in the control of receptor outputs is well established, but little is known about the pattern of spatiotemporal signaling from the GIPR in beta cells. Here, we have directly compared surface expression, trafficking and signaling characteristics of both incretin receptors in pancreatic beta cells to identify potential differences that might underlie distinct pharmacological responses associated with each receptor. Our results indicate increased cell surface levels, internalization, degradation, and endosomal versus plasma membrane activity for the GLP-1R, while the GIPR is instead associated with increased plasma membrane recycling, reduced desensitization, and enhanced downstream signal amplification. These differences might have potential implications for the capacity of each incretin receptor to control beta cell function.
Liekkinen J, Olzynska A, Cwiklik L, et al., 2023, Surfactant Proteins SP-B and SP-C in Pulmonary Surfactant Monolayers: Physical Properties Controlled by Specific Protein-Lipid Interactions, LANGMUIR, Vol: 39, Pages: 4338-4350, ISSN: 0743-7463
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Manchanda Y, Bitsi S, Chen S, et al., 2022, An examination of the divergent spatiotemporal signaling of GLP-1R <i>versus</i> GIPR in pancreatic beta cells
<jats:title>Abstract</jats:title><jats:p>The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are class B GPCRs and prime therapeutic targets for the treatment of type 2 diabetes (T2D) and obesity. They are expressed in pancreatic beta cells where they potentiate insulin release in response to food intake. Despite GIP being the main incretin in healthy individuals, GLP-1R has been favoured <jats:italic>versus</jats:italic> GIPR as a therapeutic target due to GIPR responses being blunted in T2D patients and the conflicting effects of GIPR agonists and antagonists in improving glucose tolerance and preventing weight gain. There is, however, a recently renewed interest in GIPR biology following the realisation that GIPR responses can be restored after an initial period of blood glucose normalization and the recent development of dual GLP-1R-GIPR agonists with superior capacity for the control of blood glucose levels and weight. The importance of GLP-1R trafficking and subcellular signaling in the control of receptor outputs is well established, but little is known about the pattern of spatiotemporal signaling from the GIPR in beta cells. Here we have directly compared the main trafficking and signaling characteristics of both receptors in pancreatic beta cells, finding striking differences in their propensities for internalization, recycling, and degradation, as well as plasma membrane <jats:italic>versus</jats:italic> endosomal activity, with potential implications for receptor-specific control of beta cell function.</jats:p>
Gardeta SR, García-Cuesta EM, D'Agostino G, et al., 2022, Sphingomyelin depletion inhibits CXCR4 dynamics and CXCL12-mediated directed cell migration in human T cells, Frontiers in Immunology, Vol: 13, ISSN: 1664-3224
Sphingolipids, ceramides and cholesterol are integral components of cellular membranes, and they also play important roles in signal transduction by regulating the dynamics of membrane receptors through their effects on membrane fluidity. Here, we combined biochemical and functional assays with single-particle tracking analysis of diffusion in the plasma membrane to demonstrate that the local lipid environment regulates CXCR4 organization and function and modulates chemokine-triggered directed cell migration. Prolonged treatment of T cells with bacterial sphingomyelinase promoted the complete and sustained breakdown of sphingomyelins and the accumulation of the corresponding ceramides, which altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under these conditions CXCR4 retained some CXCL12-mediated signaling activity but failed to promote efficient directed cell migration. Our data underscore a critical role for the local lipid composition at the cell membrane in regulating the lateral mobility of chemokine receptors, and their ability to dynamically increase receptor density at the leading edge to promote efficient cell migration.
Morana O, Nieto-Garai JA, Björkholm P, et al., 2022, Identification of a new cholesterol-binding site within the IFN-γ receptor that is required for signal transduction., Advanced Science, Vol: 9, ISSN: 2198-3844
The cytokine interferon-gamma (IFN-γ) is a master regulator of innate and adaptive immunity involved in a broad array of human diseases that range from atherosclerosis to cancer. IFN-γ exerts it signaling action by binding to a specific cell surface receptor, the IFN-γ receptor (IFN-γR), whose activation critically depends on its partition into lipid nanodomains. However, little is known about the impact of specific lipids on IFN-γR signal transduction activity. Here, a new conserved cholesterol (chol) binding motif localized within its single transmembrane domain is identified. Through direct binding, chol drives the partition of IFN-γR2 chains into plasma membrane lipid nanodomains, orchestrating IFN-γR oligomerization and transmembrane signaling. Bioinformatics studies show that the signature sequence stands for a conserved chol-binding motif presented in many mammalian membrane proteins. The discovery of chol as the molecular switch governing IFN-γR transmembrane signaling represents a significant advance for understanding the mechanism of lipid selectivity by membrane proteins, but also for figuring out the role of lipids in modulating cell surface receptor function. Finally, this study suggests that inhibition of the chol-IFNγR2 interaction may represent a potential therapeutic strategy for various IFN-γ-dependent diseases.
Droge F, Noakes FF, Archer SA, et al., 2021, A dinuclear Osmium(II) complex near-infrared nanoscopy probe for nuclear DNA, Journal of the American Chemical Society, Vol: 143, Pages: 20442-20453, ISSN: 0002-7863
With the aim of developing photostable near-infrared cell imaging probes, a convenient route to the synthesis of heteroleptic OsII complexes containing the Os(TAP)2 fragment is reported. This method was used to synthesize the dinuclear OsII complex, [{Os(TAP)2}2tpphz]4+ (where tpphz = tetrapyrido[3,2-a:2′,3′-c:3″,2′′-h:2‴,3′′′-j]phenazine and TAP = 1,4,5,8- tetraazaphenanthrene). Using a combination of resonance Raman and time-resolved absorption spectroscopy, as well as computational studies, the excited state dynamics of the new complex were dissected. These studies revealed that, although the complex has several close lying excited states, its near-infrared, NIR, emission (λmax = 780 nm) is due to a low-lying Os → TAP based 3MCLT state. Cell-based studies revealed that unlike its RuII analogue, the new complex is neither cytotoxic nor photocytotoxic. However, as it is highly photostable as well as live-cell permeant and displays NIR luminescence within the biological optical window, its properties make it an ideal probe for optical microscopy, demonstrated by its use as a super-resolution NIR STED probe for nuclear DNA.
Lorizate M, Terrones O, NietoGarai JA, et al., 2021, Super‐resolution microscopy using a bioorthogonal‐based cholesterol probe provides unprecedented capabilities for imaging nanoscale lipid heterogeneity in living cells, Small Methods, Vol: 5, Pages: 1-15, ISSN: 2366-9608
Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal-based cholesterol probe (chol-N3) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol-N3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live-cell super-resolution microscopy, chol-N3 demonstrates the existence of cholesterol-rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.
Bernabé-Rubio M, Bosch-Fortea M, Alonso MA, et al., 2021, Multi-dimensional and spatiotemporal correlative imaging at the plasma membrane of live cells to determine the continuum nano-to-micro scale lipid adaptation and collective motion., Methods, ISSN: 1046-2023
The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant-associated membrane patch (RAMP), once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of the spatiotemporal lipid collective assembly at the plasma membrane as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.
Bernabé-Rubio M, Bosch-Fortea M, Alonso MA, et al., 2021, Multi-dimensional and spatiotemporal correlative imaging at the plasma membrane of live cells to determine the continuum nano-to-micro scale lipid adaptation and collective motion
<jats:title>Abstract</jats:title><jats:p>The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant associated membrane patch (RAMP) once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of lipid collective assembly at the plasma membrane spatiotemporally as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.</jats:p>
Bernabe-Rubio M, Bosch-Fortea M, Garcia E, et al., 2021, Adaptive lipid immiscibility and Membrane remodeling are active functional Determinants of primary ciliogenesis, Small Methods, Vol: 5, ISSN: 2366-9608
Small Methods published by Wiley-VCH GmbH Lipid liquid–liquid immiscibility and its consequent lateral heterogeneity have been observed under thermodynamic equilibrium in model and native membranes. However, cholesterol-rich membrane domains, sometimes referred to as lipid rafts, are difficult to observe spatiotemporally in live cells. Despite their importance in many biological processes, robust evidence for their existence remains elusive. This is mainly due to the difficulty in simultaneously determining their chemical composition and physicochemical nature, whilst spatiotemporally resolving their nanodomain lifetime and molecular dynamics. In this study, a bespoke method based on super-resolution stimulated emission depletion (STED) microscopy and raster imaging correlation spectroscopy (RICS) is used to overcome this issue. This methodology, laser interleaved confocal RICS and STED-RICS (LICSR), enables simultaneous tracking of lipid lateral packing and dynamics at the nanoscale. Previous work indicated that, in polarized epithelial cells, the midbody remnant licenses primary cilium formation through an unidentified mechanism. LICSR shows that lipid immiscibility and its adaptive collective nanoscale self-assembly are crucial for the midbody remnant to supply condensed membranes to the centrosome for the biogenesis of the ciliary membrane. Hence, this work poses a breakthrough in the field of lipid biology by providing compelling evidence of a functional role for liquid ordered-like membranes in primary ciliogenesis.
Contera S, de la Serna JB, Tetley TD, 2020, Biotechnology, nanotechnology and medicine, Emerging Topics in Life Sciences, Vol: 4, Pages: 551-554, ISSN: 2397-8554
The 1980s mark the starting point of nanotechnology: the capacity to synthesise, manipulate and visualise matter at the nanometre scale. New powers to reach the nanoscale brought us the unprecedented possibility to directly target at the scale of biomolecular interactions, and the motivation to create smart nanostructures that could circumvent the hurdles hindering the success of traditional pharmacological approaches. Forty years on, the progressive integration of bio- and nanotechnologies is starting to produce a transformation of the way we detect, treat and monitor diseases and unresolved medical problems [ 1]. While much of the work remains in research laboratories, the first nano-based treatments, vaccines, drugs, and diagnostic devices, are now receiving approval for commercialisation and clinical use. In this special issue we review recent advances of nanomedical approaches to combat antibiotic resistance, treatment and detection of cancers, targeting neurodegerative diseases, and applications as diverse as dentistry and the treatment of tuberculosis. We also examine the use of advanced smart nanostructured materials in areas such as regenerative medicine, and the controlled release of drugs and treatments. The latter is currently poised to bring ground-breaking changes in immunotherapy: the advent of ‘vaccine implants’ that continuously control and improve immune responses over time. With the increasingly likely prospect of ending the COVID 19 pandemic with the aid of a nanomedicine-based vaccine (both Moderna and BioNTech/Pfizer vaccines are based on lipid nanoparticle formulations), we are witnessing the coming of age of nanomedicine. This makes it more important than ever to concentrate on safety: in parallel to pursuing the benefits of nanomedine, we must strengthen the continuous focus on nanotoxicology and safety regulation of nanomedicines that can deliver the medical revolution that is within our grasp.
Dushianthan A, Clark H, Madsen J, et al., 2020, Nebulised surfactant for the treatment of severe COVID-19 in adults (COV-Surf): A structured summary of a study protocol for a randomized controlled trial, Trials, Vol: 21, Pages: 1-3, ISSN: 1745-6215
Intervention and comparator: Intervention: The study is based on an investigational drug/device combinationproduct. The surfactant product is Bovactant (Alveofact®), a natural animal derived (bovine) lung surfactantformulated as a lyophilized powder in 108 mg vials and reconstituted to 45 mg/mL in buffer supplied in a prefilledsyringe. It is isolated by lung lavage and, by weight, is a mixture of: phospholipid (75% phosphatidylcholine, 13%phosphatidylglycerol, 3% phosphatidylethanolamine, 1% phosphatidylinositol and 1% sphingomyelin), 5%cholesterol, 1% lipid-soluble surfactant-associated proteins (SP-B and SP-C), very low levels of free fatty acid, lysophosphatidylcholine, water and 0.3% calcium. The Drug Delivery Device is the AeroFact-COVID™ nebulizer, aninvestigational device based on the Aerogen® Solo vibrating mesh nebulizer.The timing and escalation dosing plans for the surfactant are as follows.Cohort 1: Three patients will receive 10 vials (1080 mg) each of surfactant at dosing times of 0 hours, 8 hours and24 hours. 2 controls with no placebo intervention.Cohort 2: Three patients will receive 10 vials (1080 mg) of surfactant at dosing times of 0 hours and 8 hours, and30 vials (3240 mg) at a dosing time of 24 hours. 2 controls with no placebo intervention.Cohort 3: Three patients will receive 10 vials (1080 mg) of surfactant at a dosing time of 0 hours, and 30 vials (3240mg) at dosing times of 8 hours and 24 hours. 2 controls with no placebo intervention.Cohort 4: Three patients will receive 30 (3240 mg) vials each of surfactant at dosing times of 0 hours, 8 hours and24 hours. 2 controls. 2 controls with no placebo intervention.The trial steering committee, advised by the data monitoring committee, will review trial progression and doseescalation/maintenance/reduction after each cohort is completed (48-hour primary outcome timepoint reached)based on available feasibility, adverse event, safety and efficacy data. The trial will not be discontinue
Liekkinen J, de Santos Moreno B, Paananen R, et al., 2020, Understanding the functional properties of lipid heterogeneity in pulmonary surfactant monolayers at the atomistic level, Frontiers in Cell and Developmental Biology, Vol: 8, Pages: 1-16, ISSN: 2296-634X
Abstract Pulmonary surfactant is a complex mixture of lipids and proteins lining the interior of the alveoli, and constitutes the first barrier to both oxygen and pathogens as they progress toward blood circulation. Despite decades of study, the behavior of the pulmonary surfactant is poorly understood on the molecular scale, which hinders the development of effective surfactant replacement therapies, useful in the treatment of several lung-related diseases. In this work, we combined all-atom molecular dynamics simulations, Langmuir trough measurements, and AFM imaging to study synthetic four-component lipid monolayers designed to model protein-free pulmonary surfactant. We characterized the structural and dynamic properties of the monolayers with a special focus on lateral heterogeneity. Remarkably, simulations reproduce almost quantitatively the experimental data on pressure–area isotherms and the presence of lateral heterogeneities highlighted by AFM. Quite surprisingly, the pressure–area isotherms do not show a plateau region, despite the presence of liquid-condensed nanometer–sized domains at surface pressures larger than 20 mN/m. In the simulations, the domains were small and transient, but they did not coalesce to yield a separate phase. The liquid–condensed domains were only slightly enriched in DPPC and cholesterol, and their chemical composition remained very similar to the overall composition of the monolayer membrane. Instead, they differed from liquid-expanded regions in terms of membrane thickness (in agreement with AFM data), diffusion rates, acyl chain packing, and orientation. We hypothesize that such lateral heterogeneities are crucial for lung surfactant function, as they allow both efficient packing, to achieve low surface tension, and sufficient fluidity, critical for rapid adsorption to the air–liquid interface during the breathing cycle.
Liekkinen J, de Santos Moreno B, Paananen RO, et al., 2020, Understanding the Functional Properties of Lipid Heterogeneity in Pulmonary Surfactant Monolayers at the Atomistic Level, Frontiers in Cell and Developmental Biology, Vol: 8
Gutowska-Owsiak D, Podobas EI, Eggeling C, et al., 2020, Addressing Differentiation in Live Human Keratinocytes by Assessment of Membrane Packing Order, Frontiers in Cell and Developmental Biology, Vol: 8
Gutowska-Owsiak D, Podobas EI, Eggeling C, et al., 2020, Addressing differentiation in live human keratinocytes by assessment of membrane packing order, Frontiers in Cell and Developmental Biology, Vol: 8, Pages: 1-11, ISSN: 2296-634X
Differentiation of keratinocytes is critical for epidermal stratification and formation of a protective stratum corneum. It involves a series of complex processes leading through gradual changes in characteristics and functions of keratinocytes up to their programmed cell death via cornification. The stratum corneum is an impermeable barrier, comprised of dead cell remnants (corneocytes) embedded within lipid matrix. Corneocyte membranes are comprised of specialized lipids linked to late differentiation proteins, contributing to the formation of a highly stiff and mechanically strengthen layer. To date, the assessment of the progression of keratinocyte differentiation is only possible by determination of specific differentiation markers, e.g. by using proteomics-based approaches. Unfortunately, this requires fixation or cell lysis, and currently there is no robust methodology available to study differentiation in living cells, neither at a single cell, nor in high throughput. Here, we explore a new live-cell based approaches for screening differentiation advancement in keratinocytes, in a “calcium switch” model. We employ a polarity-sensitive dye, Laurdan, and Laurdan general polarization function (GP) as a reporter of the degree of membrane lateral packing order or condensation, as an adequate marker of differentiation. We show that the assay is straightforward and can be conducted either on a single cell level using confocal spectral imaging or on the ensemble level using a fluorescence plate reader. Such systematic quantification may become useful for understanding mechanisms of keratinocyte differentiation, such as the role of membrane inhomogeneities in stiffness, and for future therapeutic development.
Smitten KL, Thick EJ, Southam HM, et al., 2020, Mononuclear ruthenium(ii) theranostic complexes that function as broad-spectrum antimicrobials in therapeutically resistant pathogens through interaction with DNA, Chemical Science, Vol: 11, Pages: 8828-8838, ISSN: 2041-6520
Six luminescent, mononuclear ruthenium(II) complexes based on the tetrapyridophenazine (tpphz) and dipyridophenazine (dppz) ligands are reported. The therapeutic activities of the complexes against Gram-negative bacteria (E. coli, A. baumannii, P. aeruginosa) and Gram-positive bacteria (E. faecalis and S. aureus) including pathogenic multi- and pan-drug resistant strains were assessed. Estimated minimum inhibitory and bactericidal concentrations show the activity of the lead compound is comparable to ampicillin and oxacillin in therapeutically sensitive strains and this activity was retained in resistant strains. Unlike related dinuclear analogues the lead compound does not damage bacterial membranes but is still rapidly taken up by both Gram-positive and Gram-negative bacteria in a glucose independent manner. Direct imaging of the complexes through super-resolution nanoscopy and transmission electron microscopy reveals that once internalized the complexes' intracellular target for both Gram-negative and Gram-positive strains is bacterial DNA. Model toxicity screens showed the compound is non-toxic to Galleria mellonella even at exposure concentrations that are orders of magnitude higher than the bacterial MIC.
Pickford P, Lucey M, Fang Z, et al., 2020, Signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide., British Journal of Pharmacology, Vol: 177, Pages: 3905-3923, ISSN: 0007-1188
BACKGROUND AND PURPOSE: Amino acid substitutions at the N-termini of glucagon-like peptide-1 receptor agonist (GLP-1RA) peptides result in distinct patterns of intracellular signalling, sub-cellular trafficking and efficacy in vivo. Here we aimed to determine whether sequence differences at the ligand C-termini of clinically approved GLP-1RAs exendin-4 and lixisenatide lead to similar phenomena. EXPERIMENTAL APPROACH: Exendin-4, lixisenatide, and N-terminally substituted analogues with biased signalling characteristics were compared across a range of in vitro trafficking and signalling assays in different cell types. Fluorescent ligands and new time-resolved FRET approaches were developed to study agonist behaviours at the cellular and sub-cellular level. Anti-hyperglycaemic and anorectic effects of each parent ligand, and their biased derivatives, were assessed in mice. KEY RESULTS: Lixisenatide and exendin-4 showed equal binding affinity, but lixisenatide was 5-fold less potent for cAMP signalling. Both peptides induced extensive GLP-1R clustering in the plasma membrane and were rapidly endocytosed, but the GLP-1R recycled more slowly to the cell surface after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti-hyperglycaemic and anorectic effects in mice with lixisenatide. N-terminal substitution of His1 by Phe1 to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose. CONCLUSION AND IMPLICATIONS: Changes to the C-terminus of exendin-4 affect signalling potency and GLP-1R trafficking via mechanisms unrelated to GLP-1R occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.
Bello-Gamboa A, Velasco M, Moreno S, et al., 2020, Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes, Journal of Extracellular Vesicles, Vol: 9, Pages: 1-21, ISSN: 2001-3078
T-cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubule-organizing centre (MTOC) and the polarization of the MTOC to the immune synapse (IS). These events lead to exosome secretion at the IS. We describe here that upon IS formation centrosomal area F-actin decreased concomitantly with MTOC polarization to the IS. PKCδ-interfered T cell clones showed a sustained level of centrosomal area F-actin associated with defective MTOC polarization. We analysed the contribution of two actin cytoskeleton-regulatory proteins, FMNL1 and paxillin, to the regulation of cortical and centrosomal F-actin networks. FMNL1β phosphorylation and F-actin reorganization at the IS were inhibited in PKCδ-interfered clones. F-actin depletion at the central region of the IS, a requirement for MTOC polarization, was associated with FMNL1β phosphorylation at its C-terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization; nonetheless, FMNL1β re-expression restored MTOC polarization in a centrosomal area F-actin reorganization-independent manner. Moreover, PKCδ-interfered clones exhibited decreased paxillin phosphorylation at the MTOC, which suggests an alternative actin cytoskeleton regulatory pathway. Our results infer that PKCδ regulates MTOC polarization and secretory traffic leading to exosome secretion in a coordinated manner by means of two distinct pathways, one involving FMNL1β regulation and controlling F-actin reorganization at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F-actin reorganization.
Bernardino de la Serna J, Mellado M, Dustin ML, et al., 2020, Editorial: ImmunoPhysics and ImmunoEngineering, Frontiers of Physics, Vol: 8, Pages: 1-4, ISSN: 2095-0462
Saeed HK, Sreedharan S, Jarman PJ, et al., 2020, Making the Right Link to Theranostics: The Photophysical and Biological Properties of Dinuclear Ru-II-Re-I dppz Complexes Depend on Their Tether, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 142, Pages: 1101-1111, ISSN: 0002-7863
Smitten KL, Fairbanks SD, Robertson CC, et al., 2019, Ruthenium based antimicrobial theranostics – using nanoscopy to identify therapeutic targets and resistance mechanisms in Staphylococcus aureus, Chemical Science, Vol: 11, Pages: 70-79, ISSN: 2041-6520
In previous studies we reported that specific dinuclear RuII complexes are particularly active against pathogenic Gram-negative bacteria and, unusually for this class of compounds, appeared to display lowered activity against Gram-positive bacteria. With the aim of identifying resistance mechanisms specific to Gram-positive bacteria, the uptake and antimicrobial activity of the lead complex against Staphylococcus aureus SH1000 and other isolates, including MRSA was investigated. This revealed differential, strain specific, sensitivity to the complex. Exploiting the inherent luminescent properties of the RuII complex, super-resolution STED nanoscopy was used to image its initial interaction with S. aureus and confirm its cellular internalization. Membrane damage assays and transmission electron microscopy confirm that the complex disrupts the bacterial membrane structure before internalization, which ultimately results in a small amount of DNA damage. A known resistance mechanism against cationic antimicrobials in Gram-positive bacteria involves increased expression of the mprF gene as this results in an accumulation of positively charged lysyl-phosphatidylglycerol on the outer leaflet of the cytoplasmic membrane that electrostatically repel cationic species. Consistent with this model, it was found that an mprF deficient strain was particularly susceptible to treatment with the lead complex. More detailed co-staining studies also revealed that the complex was more active in S. aureus strains missing, or with altered, wall teichoic acids.
Owen J, Kamila S, Shrivastava S, et al., 2019, The role of PEG-40-stearate in the production, morphology, and stability of microbubbles, Langmuir: the ACS journal of surfaces and colloids, Vol: 35, Pages: 10014-10024, ISSN: 0743-7463
Phospholipid coated microbubbles are currently in widespread clinical use as ultrasound contrast agents and under investigation for therapeutic applications. Previous studies have demonstrated the importance of the coating nanostructure in determining microbubble stability and its dependence upon both composition and processing method. While the influence of different phospholipids has been widely investigated, the role of other constituents such as emulsifiers has received comparatively little attention. Herein, we present an examination of the impact of polyethylene glycol (PEG) derivatives upon microbubble structure and properties. We present data using both pegylated phospholipids and a fluorescent PEG-40-stearate analogue synthesized in-house to directly observe its distribution in the microbubble coating. We examined microbubbles of clinically relevant sizes, investigating both their surface properties and population size distribution and stability. Domain formation was observed only on the surface of larger microbubbles, which were found to contain a higher concentration of PEG-40-stearate. Lipid analogue dyes were also found to influence domain formation compared with PEG-40-stearate alone. "Squeezing out" of PEG-40-stearate was not observed from any of the microbubble sizes investigated. At ambient temperature, microbubbles formulated with DSPE-PEG(2000) were found to be more stable than those containing PEG-40-stearate. At 37 °C, however, the stability in serum was found to be the same for both formulations, and no difference in acoustic backscatter was detected. This could potentially reduce the cost of PEGylated microbubbles and facilitate simpler attachment of targeting or therapeutic species. However, whether PEG-40-stearate sufficiently shields microbubbles to inhibit physiological clearance mechanisms still requires investigation.
Bernabé-Rubio M, Bosch-Fortea M, García E, et al., 2019, The ciliary membrane of polarized epithelial cells stems from a midbody remnant-associated membrane patch with condensed nanodomains
<jats:title>Abstract</jats:title><jats:p>The primary cilium is a specialized plasma membrane protrusion that harbors receptors involved in important signaling pathways. Despite its central role in regulating cellular behavior, the biogenesis of the primary cilium is not fully understood. In fact, the source of the ciliary membrane remains a mystery in cell types that assemble their primary cilium entirely at the cell surface, such as polarized renal epithelial cells. After cytokinesis, the remnant of the midbody of these cells moves to the center of the apical surface, where it licenses the centrosome for ciliogenesis through an unidentified mechanism. Here, to investigate the origin of the ciliary membrane and the role of the midbody remnant, we analyzed membrane compaction and lipid dynamics at the microscale and nanoscale in living renal epithelial MDCK cells. We found that a specialized patch made of condensed membranes with restricted lipid lateral mobility surrounds the midbody remnant. This patch accompanies the remnant on its journey towards the centrosome and, once the two structures have met, the remnant delivers part of membranes of the patch to build the ciliary membrane. In this way, we have determined the origin of the ciliary membrane and the contribution of the midbody remnant to primary cilium formation in cells whose primary cilium is assembled at the plasma membrane.</jats:p>
Smitten KL, Southam HM, Bernardino de la Serna J, et al., 2019, Using nanoscopy to probe the biological activity of antimicrobial leads that display potent activity against pathogenic, multidrug resistant, gram-negative bacteria., ACS Nano, Vol: 13, Pages: 5133-5146, ISSN: 1936-0851
Medicinal leads that are also compatible with imaging technologies are attractive, as they facilitate the development of therapeutics through direct mechanistic observations at the molecular level. In this context, the uptake and antimicrobial activities of several luminescent dinuclear RuII complexes against E. coli were assessed and compared to results obtained for another ESKAPE pathogen, the Gram-positive major opportunistic pathogen Enterococcus faecalis, V583. The most promising lead displays potent activity, particularly against the Gram-negative bacteria, and potency is retained in the uropathogenic multidrug resistant EC958 ST131 strain. Exploiting the inherent luminescent properties of this complex, super-resolution STED nanoscopy was used to image its initial localization at/in cellular membranes and its subsequent transfer to the cell poles. Membrane damage assays confirm that the complex disrupts the bacterial membrane structure before internalization. Mammalian cell culture and animal model studies indicate that the complex is not toxic to eukaryotes, even at concentrations that are several orders of magnitude higher than its minimum inhibitory concentration (MIC). Taken together, these results have identified a lead molecular architecture for hard-to-treat, multiresistant, Gram-negative bacteria, which displays activities that are already comparable to optimized natural product-based leads.
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