Phenazine Cations as Anticancer Theranostics†.

The biological properties of two water-soluble organic cations based on polypyridyl structures commonly used as ligands for photoactive transition metal complexes designed to interact with biomolecules are investigated. A cytotoxicity screen employing a small panel of cell lines reveals that both cations show cytotoxicity toward cancer cells but show reduced cytotoxicity to noncancerous HEK293 cells with the more extended system being notably more active. Although it is not a singlet oxygen sensitizer, the more active cation also displayed enhanced potency on irradiation with visible light, making it active at nanomolar concentrations. Using the intrinsic luminescence of the cations, their cellular uptake was investigated in more detail, revealing that the active compound is more readily internalized than its less lipophilic analogue. Colocalization studies with established cell probes reveal that the active cation predominantly localizes within lysosomes and that irradiation leads to the disruption of mitochondrial structure and function. Stimulated emission depletion (STED) nanoscopy and transmission electron microscopy (TEM) imaging reveal that treatment results in distinct lysosomal swelling and extensive cellular vacuolization. Further imaging-based studies confirm that treatment with the active cation induces lysosomal membrane permeabilization, which triggers lysosome-dependent cell-death due to both necrosis and caspase-dependent apoptosis. A preliminary toxicity screen in the Galleria melonella animal model was carried out on both cations and revealed no detectable toxicity up to concentrations of 80 mg/kg. Taken together, these studies indicate that this class of synthetically easy-to-access photoactive compounds offers potential as novel therapeutic leads.

Exposure to Aldehyde Cherry e-Liquid Flavoring and Its Vaping Byproduct Disrupt Pulmonary Surfactant Biophysical Function.

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.

The potential impacts of micro-and-nano plastics on various organ systems in humans.

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.

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.

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.

The impact of e-cigarette exposure on different organ systems: A review of recent evidence and future perspectives.

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.

From Chemotherapy to Phototherapy - Changing the Therapeutic Action of a Metallo-Intercalating RuII -ReI Luminescent System by Switching its Sub-Cellular Location.

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.

Surfactant Proteins SP-B and SP-C in Pulmonary Surfactant Monolayers: Physical Properties Controlled by Specific Protein-Lipid Interactions.

The lining of the alveoli is covered by pulmonary surfactant, a complex mixture of surface-active lipids and proteins that enables efficient gas exchange between inhaled air and the circulation. Despite decades of advancements in the study of the pulmonary surfactant, the molecular scale behavior of the surfactant and the inherent role of the number of different lipids and proteins in surfactant behavior are not fully understood. The most important proteins in this complex system are the surfactant proteins SP-B and SP-C. Given this, in this work we performed nonequilibrium all-atom molecular dynamics simulations to study the interplay of SP-B and SP-C with multicomponent lipid monolayers mimicking the pulmonary surfactant in composition. The simulations were complemented by z-scan fluorescence correlation spectroscopy and atomic force microscopy measurements. Our state-of-the-art simulation model reproduces experimental pressure-area isotherms and lateral diffusion coefficients. In agreement with previous research, the inclusion of either SP-B and SP-C increases surface pressure, and our simulations provide a molecular scale explanation for this effect: The proteins display preferential lipid interactions with phosphatidylglycerol, they reside predominantly in the lipid acyl chain region, and they partition into the liquid expanded phase or even induce it in an otherwise packed monolayer. The latter effect is also visible in our atomic force microscopy images. The research done contributes to a better understanding of the roles of specific lipids and proteins in surfactant function, thus helping to develop better synthetic products for surfactant replacement therapy used in the treatment of many fatal lung-related injuries and diseases.

Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells.

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 vs 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.

Sphingomyelin Depletion Inhibits CXCR4 Dynamics and CXCL12-Mediated Directed Cell Migration in Human T Cells.

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.

Identification of a New Cholesterol-Binding Site within the IFN-γ Receptor that is Required for Signal Transduction.

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.

Adaptive Lipid Immiscibility and Membrane Remodeling Are Active Functional Determinants of Primary Ciliogenesis.

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.

Super-Resolution Microscopy Using a Bioorthogonal-Based Cholesterol Probe Provides Unprecedented Capabilities for Imaging Nanoscale Lipid Heterogeneity in Living Cells.

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.

A Dinuclear Osmium(II) Complex Near-Infrared Nanoscopy Probe for Nuclear DNA.

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.

Relationships, Decisions, and Physical Effort in the Marro Traditional Sporting Game: A Multimodal Approach.

The purpose of this study was to examine the players' decisions-making in Marro (a Traditional Sporting Game) through a multimodal approach. Each player's decision-making assumes specific accelerations and decelerations associated with different effort. The research objectives were: (i) to study the decision-making associated with the roles of Hunter and Hare; (ii) to know the physical effort by the roles (Hunters and Hares); (iii) to reveal T-Patterns in the multimodal strategic approach (integrated with decisions and different physical effort) with a direct incidence on the scoring by roles. The study was performed with 22 male and 2 female players aged 18 to 25 (M = 19.4; SD = 1.3). The Marro game was played by two groups for eight minutes. An observational methodology was used, through a type III design. The observational design was nomothetic, one-time, and multidimensional. An 'ad hoc' tool was built to ensure the data quality. Univariate analyses were performed using Crosstabs Command, with adjusted residuals (AR), Classification Trees (Chaid model) and T-Pattern Analysis (TPA). Significant differences were found between matches using the scoring (p < 0.001; ES = 0.26), role (p < 0.001; ES = 0.31), or the organic variables of the study, the speed (p < 0.001; ES = 0.73), the metabolic power and the acceleration/deceleration the speed (p = 0.023; ES = 0.43), while the predictive model pointed to the variable role (p < 0.001) as the main factor responsible for the model growth. TPA (p < 0.005) revealed differences attributable to internal logic in the yellow (first match) and orange (second match) teams, while organic variables were more changeable in the violet (first match) and green (second match) teams. This study advances the individualization of the decision-making process. These results may be useful to better understand the internal of functioning of the Marro game 360° since the use of various methodologies and variables (multimodal approach) provided original findings.

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.

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.

Effect of Ball Screen and One-on-One on the Level of Opposition and Effectiveness of Shots in the ACB.

In light of the importance of shots on the final result in basketball matches, this research aims to reveal the predictive ability of variables associated with different game actions (ball screen, one-on-one near the basket and one-on-one far away from the basket), shot zone (near to or far from the basket) and players' position (outside or inside) of the success of shots and of the type of shot, defended or not defended, in three seasons (2014 to 2016) of the Copa del Rey in the ACB (Asociación de Clubes de Baloncesto) League in Spain. Observational methodology was used, and an ad hoc instrument was created using Lince software. The study was conducted on a total of 7695 game actions, of which an analysis of 877 shots was made using CHAID (Chi-squared Automatic Interaction Detection) classification trees. The results obtained in this research have led us to the following conclusions: (a) the most important variable in the success of shots is to find "open shots", regardless of the game action, (b) one-on-one actions outside and inside the zone have a clear connection to the finishes of the plays and end up in defended shots, (c) shots by ball screen lead to more open shots than in one-on-one and (d) play with passes may facilitate to find "open shots". These results can be of interest for coaches to prepare their teams in the best possible way, but they must be interpreted with caution as the sample was very specific (Copa del Rey in the ACB League) and, therefore, it is necessary to continue investigating this topic in other competition contexts.

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.

OBJECTIVES: SARS-Cov-2 virus preferentially binds to the Angiotensin Converting Enzyme 2 (ACE2) on alveolar epithelial type II cells, initiating an inflammatory response and tissue damage which may impair surfactant synthesis contributing to alveolar collapse, worsening hypoxia and leading to respiratory failure. The objective of this study is to evaluate the feasibility, safety and efficacy of nebulised surfactant in COVID-19 adult patients requiring mechanical ventilation for respiratory failure. TRIAL DESIGN: This study is a dose-escalating randomized open-label clinical trial of 20 COVID-19 patients. PARTICIPANTS: This study is conducted in two centres: University Hospital Southampton and University College London Hospitals. Eligible participants are aged ≥18, hospitalised with COVID-19 (confirmed by PCR), who require endotracheal intubation and are enrolled within 24 hours of mechanical ventilation. For patients unable to consent, assent is obtained from a personal legal representative (PerLR) or professional legal representative (ProfLR) prior to enrolment. The following are exclusion criteria: imminent expected death within 24 hours; specific contraindications to surfactant administration (e.g. known allergy, pneumothorax, pulmonary hemorrhage); known or suspected pregnancy; stage 4 chronic kidney disease or requiring dialysis (i.e., eGFR < 30); liver failure (Child-Pugh Class C); anticipated transfer to another hospital, which is not a study site, within 72 hours; current or recent (within 1 month) participation in another study that, in the opinion of the investigator, would prevent enrollment for safety reasons; and declined consent or assent. INTERVENTION AND COMPARATOR: Intervention: The study is based on an investigational drug/device combination product. The surfactant product is Bovactant (Alveofact®), a natural animal derived (bovine) lung surfactant formulated as a lyophilized powder in 108 mg vials and reconstituted to 45 mg/mL in buffer supplied in a prefilled syringe. 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, lyso-phosphatidylcholine, water and 0.3% calcium. The Drug Delivery Device is the AeroFact-COVID™ nebulizer, an investigational 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 and 24 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, and 30 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 (3240 mg) 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 and 24 hours. 2 controls. 2 controls with no placebo intervention. The trial steering committee, advised by the data monitoring committee, will review trial progression and dose escalation/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 discontinued on the basis of lack of efficacy. The trial may be stopped early on the basis of safety or feasibility concerns. Comparator: No placebo intervention. All participants will receive usual standard of care in accordance with the local policies for mechanically ventilated patients and all other treatments will be left to the discretion of the attending physician. MAIN OUTCOMES: The co-primary outcome is the improvement in oxygenation (PaO2/FiO2 ratio) and pulmonary ventilation (Ventilation Index (VI), where VI = [RR x (PIP - PEEP) × PaCO2]/1000) at 48 hours after study initiation. The secondary outcomes include frequency and severity of adverse events (AEs), Adverse Device Effects (ADEs), Serious Adverse Events (SAEs) and Serious Adverse Device Events (SADEs), change in pulmonary compliance, change in positive end-expiratory pressure (PEEP) requirement of ventilatory support at 24 and 48 hours after study initiation, clinical improvement defined by time to one improvement point on the ordinal scale described in the WHO master protocol (2020) recorded while hospitalised, days of mechanical ventilation, mechanical ventilator free days (VFD) at day 21, length of intensive care unit stay, number of days hospitalised and mortality at day 28. Exploratory end points will include quantification of SARS-CoV-2 viral load from tracheal aspirates using PCR, surfactant dynamics (synthesis and turnover) and function (surface tension reduction) from deep tracheal aspirate samples (DTAS), surfactant phospholipid concentrations in plasma and DTAS, inflammatory markers (cellular and cytokine) in plasma and DTAS, and blood oxidative stress markers. RANDOMISATION: After informed assent, patients fulfilling inclusion criteria will be randomised to 3:2 for the treatment and control arms using an internet-based block randomization service (ALEA tool for clinical trials, FormsVision BV) in combination with electronic data collection. Randomisation will be done by the recruiting centre with a unique subject identifier specific to that centre. BLINDING (MASKING): This is an open-labelled unblinded study. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): The total sample size is 20 COVID-19 mechanically ventilated patients (12 intervention; 8 control). TRIAL STATUS: Current protocol version is V2 dated 5th of June 2020. The recruitment is currently ongoing and started on the 14th of October 2020. The anticipated study completion date is November 2021. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04362059 (Registered 24 April 2020), EUDAMED number: CIV-GB-20-06-033328, EudraCT number: 2020-001886-35 (Registered 11 May 2020) FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).

Biotechnology, nanotechnology and medicine.

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.

Understanding the Functional Properties of Lipid Heterogeneity in Pulmonary Surfactant Monolayers at the Atomistic Level.

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 at the molecular scale is poorly understood, 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 liquid-condensed domains were small and transient, but they did not coalesce to yield a separate phase. They 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, as well as 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.

Addressing Differentiation in Live Human Keratinocytes by Assessment of Membrane Packing Order.

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 a relatively impermeable barrier, comprised of dead cell remnants (corneocytes) embedded in lipid matrix. Corneocyte membranes are comprised of specialized lipids linked to late differentiation proteins, contributing to the formation of a stiff and mechanically strengthened layer. To date, the assessment of the progression of keratinocyte differentiation is only possible through 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 keratinocyte differentiation in living cells in real-time. Here, we explore 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 in homogeneities in stiffness, and for future therapeutic development.