Imperial College London

DrJorgeBernardino de la Serna

Faculty of MedicineNational Heart & Lung Institute

Senior Lecturer in Inhalation Toxicology and Pharmacology
 
 
 
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Contact

 

+44 (0)20 7594 3277j.bernardino-de-la-serna

 
 
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Location

 

102Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
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76 results found

Morana O, Nieto-Garai JA, Björkholm P, Bernardino de la Serna J, Terrones O, Arboleya A, Ciceri D, Rojo-Bartolomé I, Blouin CM, Lamaze C, Lorizate M, Contreras F-Xet 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.

Journal article

Droge F, Noakes FF, Archer SA, Sreedharan S, Raza A, Robertson CC, MacNeil S, Haycock JW, Carson H, Meijer AJHM, Smythe CGW, de la Serna JB, Dietzek-Ivansic B, Thomas JAet 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

Journal article

Lorizate M, Terrones O, NietoGarai JA, RojoBartolomé I, Ciceri D, Morana O, OlazarIntxausti J, Arboleya A, Martin A, Szynkiewicz M, CallejaFelipe M, Bernardino de la Serna J, Contreras Fet 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.

Journal article

Bernabé-Rubio M, Bosch-Fortea M, Alonso MA, Bernardino de la Serna Jet 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.

Journal article

Bernabe-Rubio M, Bosch-Fortea M, Garcia E, Bernardino de la Serna J, Alonso MAet 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.

Journal article

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.

Journal article

Dushianthan A, Clark H, Madsen J, Mogg R, Matthews L, Berry L, Bernardino de la Serna J, Batchelor J, Brealey D, Hussell T, Porter J, Djukanovic R, Feelisch M, Postle A, Grocott MPWet 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

Journal article

Liekkinen J, de Santos Moreno B, Paananen R, Vattulainen I, Monticelli L, Bernardino de la Serna J, Javanainen Met 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.

Journal article

Gutowska-Owsiak D, Podobas EI, Eggeling C, Ogg G, Bernardino de la Serna Jet 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.

Journal article

Smitten KL, Thick EJ, Southam HM, Bernardino de la Serna J, Foster SJ, Thomas JAet 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.

Journal article

Pickford P, Lucey M, Fang Z, Bitsi S, Bernardino de la Serna J, Broichhagen J, Hodson DJ, Minnion J, Rutter GA, Bloom SR, Tomas A, Jones Bet 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.

Journal article

Bello-Gamboa A, Velasco M, Moreno S, Herranz G, Ilie R, Huetos S, Dávila S, Sánchez A, Bernardino De La Serna J, Calvo V, Izquierdo Met 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.

Journal article

Bernardino de la Serna J, Mellado M, Dustin ML, Garcia-Parajo MF, Morikis Det al., 2020, Editorial: ImmunoPhysics and ImmunoEngineering, Frontiers of Physics, Vol: 8, Pages: 1-4, ISSN: 2095-0462

Journal article

Saeed HK, Sreedharan S, Jarman PJ, Archer SA, Fairbanks SD, Foxon SP, Auty AJ, Chekulaev D, Keane T, Meijer AJHM, Weinstein JA, Smythe CGW, Bernardino de la Serna J, Thomas JAet 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

Journal article

Smitten KL, Fairbanks SD, Robertson CC, Bernardino de la Serna J, Foster SJ, Thomas JAet 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.

Journal article

Bernabé-Rubio M, Bosch-Fortea M, García E, de la Serna JB, Alonso MAet 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>

Journal article

Smitten KL, Southam HM, Bernardino de la Serna J, Gill MR, Jarman PJ, Smythe CGW, Poole RK, Thomas JAet 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.

Journal article

Owen J, Kamila S, Shrivastava S, Carugo D, Bernardino de la Serna J, Mannaris C, Pereno V, Browning R, Beguin E, McHale AP, Callan JF, Stride Eet al., 2018, The Role of PEG-40-stearate in the Production, Morphology, and Stability of Microbubbles., Langmuir

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.

Journal article

Compte M, Lykke Harwood S, Munoz IG, Navarro R, Zonca M, Perez-Chacon G, Erce-Llamazares A, Merino N, Tapia-Galisteo A, Cuesta AM, Mikkelsen K, Caleiras E, Nunez-Prado N, Aznar MA, Lykkemark S, Martinez-Torrecuadrada J, Melero I, Blanco FJ, Bernardino de la Serna J, Zapata JM, Sanz L, Alvarez-Vallina Let al., 2018, A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity, Nature Communications, Vol: 9, ISSN: 2041-1723

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.

Journal article

Schneider F, Waithe D, Galiani S, Bernardino de la Serna J, Sezgin E, Eggeling Cet al., 2018, Nanoscale spatiotemporal diffusion modes measured by simultaneous confocal and stimulated emission depletion nanoscopy imaging, Nano Letters, Vol: 18, Pages: 4233-4240, ISSN: 1530-6984

The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.

Journal article

Schneider F, Waithe D, Galiani S, de la Serna JB, Sezgin E, Eggeling Cet al., 2018, Nanoscale spatio-temporal diffusion modes measured by simultaneous confocal and STED imaging, Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 18, Pages: 4233-4240, ISSN: 1530-6984

The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.

Journal article

Waithe D, Schneider F, Chojnacki J, Clausen MP, Shrestha D, Bernardino de la Serna J, Eggeling Cet al., 2018, Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data, Methods, Vol: 140-141, Pages: 62-73, ISSN: 1046-2023

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

Journal article

Gutowska-Owsiak D, Bernardino de la Serna J, Fritzsche M, Naeem A, Podobas EI, Leeming M, Colin-York H, O'Shaughnessy R, Eggeling C, Ogg GSet al., 2018, Orchestrated control of filaggrin-actin scaffolds underpins cornification, Cell Death and Disease, Vol: 9, ISSN: 2041-4889

Epidermal stratification critically depends on keratinocyte differentiation and programmed death by cornification, leading to formation of a protective skin barrier. Cornification is dynamically controlled by the protein filaggrin, rapidly released from keratohyalin granules (KHGs). However, the mechanisms of cornification largely remain elusive, partly due to limitations of the observation techniques employed to study filaggrin organization in keratinocytes. Moreover, while the abundance of keratins within KHGs has been well described, it is not clear whether actin also contributes to their formation or fate. We employed advanced (super-resolution) microscopy to examine filaggrin organization and dynamics in skin and human keratinocytes during differentiation. We found that filaggrin organization depends on the cytoplasmic actin cytoskeleton, including the role for α- and β-actin scaffolds. Filaggrin-containing KHGs displayed high mobility and migrated toward the nucleus during differentiation. Pharmacological disruption targeting actin networks resulted in granule disintegration and accelerated cornification. We identified the role of AKT serine/threonine kinase 1 (AKT1), which controls binding preference and function of heat shock protein B1 (HspB1), facilitating the switch from actin stabilization to filaggrin processing. Our results suggest an extended model of cornification in which filaggrin utilizes actins to effectively control keratinocyte differentiation and death, promoting epidermal stratification and formation of a fully functional skin barrier.

Journal article

Santos AM, Ponjavic A, Fritzsche M, Fernandes RA, Bernardino de la Serna J, Wilcock MJ, Schneider F, Urbancic I, McColl J, Anzilotti C, Ganzinger KA, Assmann M, Depoil D, Cornal RJ, Dustin ML, Klenerman D, Davis SJ, Eggeling C, Lee SFet al., 2018, Capturing resting T cells: the perils of PLL, NATURE IMMUNOLOGY, Vol: 19, Pages: 203-205, ISSN: 1529-2908

Journal article

Garcia E, Bernardino de la Serna J, 2017, Dissecting single–cell molecular spatiotemporal mobility and clustering at Focal Adhesions in polarised cells by fluorescence fluctuation spectroscopy methods

<jats:title>Abstract</jats:title><jats:p>Quantitative fluorescence fluctuation spectroscopy from optical microscopy datasets is a very powerful tool to resolve multiple spatiotemporal cellular and subcellular processes at the molecular level. In particular, raster image correlation spectroscopy (RICS) and number and brightness analyses (N&amp;B) yield molecular mobility and clustering dynamic information extracted from real-time cellular processes. This quantitative information can be inferred in a highly flexibly and detailed manner, i.e. 1) at the localisation level: from full-frame datasets and multiple regions of interest within; and 2) at the temporal level: not only from full-frame and multiple regions, but also intermediate temporal events. Here we build on previous research in deciphering the molecular dynamics of paxillin, a main component of focal adhesions. Cells use focal adhesions to attach to the extracellular matrix and interact with their local environment. Through focal adhesions and other adhesion structures, cells sense their local environment and respond accordingly; due to this continuous communication, these structures can be highly dynamic depending on the extracellular characteristics. By using a previously well-characterised model like paxillin, we examine powerful sensitivity characteristics and some limitations of RICS and N&amp;B analyses. We show that cells upon contact to different surfaces show differential self-assembly dynamics in terms of molecular diffusion and oligomerisation. In addition, single-cell studies show that these dynamics change gradually following an antero-posterior gradient.</jats:p>

Journal article

Sreedharan S, Gill MR, Garcia E, Saeed HK, Robinson D, Byrne A, Cadby A, Keyes TE, Smythe C, Pellett P, Bernardino de la Serna J, Thomas JAet al., 2017, Multimodal Super-resolution Optical Microscopy Using a Transition-Metal-Based Probe Provides Unprecedented Capabilities for Imaging Both Nuclear Chromatin and Mitochondria, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 139, Pages: 15907-15913, ISSN: 0002-7863

Journal article

Waithe D, Schneider F, Chojnacki J, Clausen M, Shrestha D, Bernardino de la Serna J, Eggeling Cet al., 2017, Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data, Publisher: bioRxiv

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

Working paper

Mao B, Calatayud DG, Mirabello V, Kuganathan N, Ge H, Jacobs RMJ, Shepherd AM, Ribeiro Martins JA, Bernardino de la Serna J, Hodges BJ, Botchway SW, Pascu SIet al., 2017, Fluorescence-lifetime imaging and super-resolution microscopies shed light on the directed- and self-assembly of functional porphyrins onto carbon nanotubes and flat surfaces, Chemistry - A European Journal, Vol: 23, Pages: 9772-9789, ISSN: 0947-6539

Functional porphyrins have attracted intense attention due to their remarkably high extinction coefficients in the visible region and potential for optical and energy‐related applications. Two new routes to functionalised SWNTs have been established using a bulky ZnII‐porphyrin featuring thiolate groups at the periphery. We probed the optical properties of this zinc(II)‐substituted, bulky aryl porphyrin and those of the corresponding new nano‐composites with single walled carbon nanotube (SWNTs) and coronene, as a model for graphene. We report hereby on: i) the supramolecular interactions between the pristine SWNTs and ZnII‐porphyrin by virtue of π–π stacking, and ii) a novel covalent binding strategy based on the Bingel reaction. The functional porphyrins used acted as dispersing agent for the SWNTs and the resulting nanohybrids showed improved dispersibility in common organic solvents. The synthesized hybrid materials were probed by various characterisation techniques, leading to the prediction that supramolecular polymerisation and host–guest functionalities control the fluorescence emission intensity and fluorescence lifetime properties. For the first time, XPS studies highlighted the differences in covalent versus non‐covalent attachments of functional metalloporphyrins to SWNTs. Gas‐phase DFT calculations indicated that the ZnII‐porphyrin interacts non‐covalently with SWNTs to form a donor–acceptor complex. The covalent attachment of the porphyrin chromophore to the surface of SWNTs affects the absorption and emission properties of the hybrid system to a greater extent than in the case of the supramolecular functionalisation of the SWNTs. This represents a synthetic challenge as well as an opportunity in the design of functional nanohybrids for future sensing and optoelectronic applications.

Journal article

Fritzsche M, Fernandes RA, Chang VT, Colin-York H, Clausen MP, Felce JH, Galiani S, Erlenkamper C, Santos AM, Heddleston JM, Pedroza-Pacheco I, Waithe D, Bernardino de la Serna J, Lagerholm BC, Liu T-L, Chew T-L, Betzig E, Davis SJ, Eggeling Cet al., 2017, Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation, Science Advances, Vol: 3, Pages: 1-18, ISSN: 2375-2548

T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse. This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament lengths of two differently sized filamentous actin populations, wherein formin-mediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact with activating surfaces and no cytoskeletal rearrangements. Our work suggests that incipient signaling in T cells initiates global cytoskeletal rearrangements across the whole cell, including a stiffening process for possibly mechanically supporting contact formation at the immunological synapse interface as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions.

Journal article

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