Randomized Trial of BCG Vaccine to Protect against Covid-19 in Health Care Workers.

BACKGROUND: The bacille Calmette-Guérin (BCG) vaccine has immunomodulatory "off-target" effects that have been hypothesized to protect against coronavirus disease 2019 (Covid-19). METHODS: In this international, double-blind, placebo-controlled trial, we randomly assigned health care workers to receive the BCG-Denmark vaccine or saline placebo and followed them for 12 months. Symptomatic Covid-19 and severe Covid-19, the primary outcomes, were assessed at 6 months; the primary analyses involved the modified intention-to-treat population, which was restricted to participants with a negative test for severe acute respiratory syndrome coronavirus 2 at baseline. RESULTS: A total of 3988 participants underwent randomization; recruitment ceased before the planned sample size was reached owing to the availability of Covid-19 vaccines. The modified intention-to-treat population included 84.9% of the participants who underwent randomization: 1703 in the BCG group and 1683 in the placebo group. The estimated risk of symptomatic Covid-19 by 6 months was 14.7% in the BCG group and 12.3% in the placebo group (risk difference, 2.4 percentage points; 95% confidence interval [CI], -0.7 to 5.5; P = 0.13). The risk of severe Covid-19 by 6 months was 7.6% in the BCG group and 6.5% in the placebo group (risk difference, 1.1 percentage points; 95% CI, -1.2 to 3.5; P = 0.34); the majority of participants who met the trial definition of severe Covid-19 were not hospitalized but were unable to work for at least 3 consecutive days. In supplementary and sensitivity analyses that used less conservative censoring rules, the risk differences were similar but the confidence intervals were narrower. There were five hospitalizations due to Covid-19 in each group (including one death in the placebo group). The hazard ratio for any Covid-19 episode in the BCG group as compared with the placebo group was 1.23 (95% CI, 0.96 to 1.59). No safety concerns were identified. CONCLUSIONS: Vaccination with BCG-Denmark did not result in a lower risk of Covid-19 among health care workers than placebo. (Funded by the Bill and Melinda Gates Foundation and others; BRACE ClinicalTrials.gov number, NCT04327206.).

Peroxisomal ß-oxidation enzyme, DECR2, regulates lipid metabolism and promotes treatment resistance in advanced prostate cancer.

BACKGROUND: Peroxisomes are central metabolic organelles that have key roles in fatty acid homoeostasis. As prostate cancer (PCa) is particularly reliant on fatty acid metabolism, we explored the contribution of peroxisomal ß-oxidation (perFAO) to PCa viability and therapy response. METHODS: Bioinformatic analysis was performed on clinical transcriptomic datasets to identify the perFAO enzyme, 2,4-dienoyl CoA reductase 2 (DECR2) as a target gene of interest. Impact of DECR2 and perFAO inhibition via thioridazine was examined in vitro, in vivo, and in clinical prostate tumours cultured ex vivo. Transcriptomic and lipidomic profiling was used to determine the functional consequences of DECR2 inhibition in PCa. RESULTS: DECR2 is upregulated in clinical PCa, most notably in metastatic castrate-resistant PCa (CRPC). Depletion of DECR2 significantly suppressed proliferation, migration, and 3D growth of a range of CRPC and therapy-resistant PCa cell lines, and inhibited LNCaP tumour growth and proliferation in vivo. DECR2 influences cell cycle progression and lipid metabolism to support tumour cell proliferation. Further, co-targeting of perFAO and standard-of-care androgen receptor inhibition enhanced suppression of PCa cell proliferation. CONCLUSION: Our findings support a focus on perFAO, specifically DECR2, as a promising therapeutic target for CRPC and as a novel strategy to overcome lethal treatment resistance.

Pickering Emulsions of Thermotropic Liquid Crystals Stabilized by Amphiphilic Gold Nanoparticles.

We report emulsions of thermotropic liquid crystals (LCs) in water that are stabilized using amphiphilic gold nanoparticles (AuNPs) and retain their ability to respond to aqueous analytes for extended periods (e.g., up to 1 year after preparation). These LC emulsions exhibit exceptional colloidal stability that results from the adsorption of AuNPs that are functionalized with thiol-terminated poly(ethylene glycol) (PEG-thiol) and hexadecanethiol (C16-thiol) to LC droplet interfaces. These stabilized LC emulsions respond to the presence of model anionic (SDS), cationic (C12TAB), and nonionic (C12E4) surfactants in the surrounding aqueous media, as evidenced by ordering transitions in the LC droplets that can be readily observed using polarized light microscopy. Our results reveal significant differences in the sensitivity of the stabilized LC droplets toward each of these analytes. In particular, these stabilized droplets can detect the cationic C12TAB at concentrations that are lower than those required for bare LC droplets under similar experimental conditions (0.5 and 2 mM, respectively). These results demonstrate an enhanced sensitivity of the LC toward C12TAB when the PEG/C16-thiol-coated AuNPs are adsorbed at LC droplet interfaces. In contrast, the concentrations of SDS required to observe optical transformations in the stabilized LC droplets are higher than those required for the bare LC droplets, suggesting that the presence of the PEG/C16-thiol AuNPs reduces the sensitivity of the LC toward this analyte. When combined, our results show that this Pickering stabilization approach using amphiphilic AuNPs as stabilizing agents for LC-in-water emulsions provides a promising platform for developing LC droplet-based optical sensors with long-term colloidal stability as well as opportunities to tune the sensitivity and selectivity of the response to target aqueous analytes.

Engineering Synthetic Electron Transfer Chains from Metallopeptide Membranes.

The energetic and geometric features enabling redox chemistry across the copper cupredoxin fold contain key components of electron transfer chains (ETC), which have been extended here by templating the cross-ß bilayer assembly of a synthetic nonapeptide, HHQALVFFA-NH2 (K16A), with copper ions. Similar to ETC cupredoxin plastocyanin, these assemblies contain copper sites with blue-shifted (λmax 573 nm) electronic transitions and strongly oxidizing reduction potentials. Electron spin echo envelope modulation and X-ray absorption spectroscopies define square planar Cu(II) sites containing a single His ligand. Restrained molecular dynamics of the cross-ß peptide bilayer architecture support metal ion coordination stabilizing the leaflet interface and indicate that the relatively high reduction potential is not simply the result of distorted coordination geometry (entasis). Cyclic voltammetry (CV) supports a charge-hopping mechanism across multiple copper centers placed 10-12 Å apart within the assembled peptide leaflet interface. This metal-templated scaffold accordingly captures the electron shuttle and cupredoxin functionality in a peptide membrane-localized electron transport chain.

Spatial MS multiomics on clinical prostate cancer tissues.

Mass spectrometry (MS) and MS imaging (MSI) are used extensively for both the spatial and bulk characterization of samples in lipidomics and proteomics workflows. These datasets are typically generated independently due to different requirements for sample preparation. However, modern omics technologies now provide higher sample throughput and deeper molecular coverage, which, in combination with more sophisticated bioinformatic and statistical pipelines, make generating multiomics data from a single sample a reality. In this workflow, we use spatial lipidomics data generated by matrix-assisted laser desorption/ionization MSI (MALDI-MSI) on prostate cancer (PCa) radical prostatectomy cores to guide the definition of tumor and benign tissue regions for laser capture microdissection (LCM) and bottom-up proteomics all on the same sample and using the same mass spectrometer. Accurate region of interest (ROI) mapping was facilitated by the SCiLS region mapper software and dissected regions were analyzed using a dia-PASEF workflow. A total of 5525 unique protein groups were identified from all dissected regions. Lysophosphatidylcholine acyltransferase 1 (LPCAT1), a lipid remodelling enzyme, was significantly enriched in the dissected regions of cancerous epithelium (CE) compared to benign epithelium (BE). The increased abundance of this protein was reflected in the lipidomics data with an increased ion intensity ratio for pairs of phosphatidylcholines (PC) and lysophosphatidylcholines (LPC) in CE compared to BE.

Targeting hyaluronan-mediated motility receptor (HMMR) enhances response to androgen receptor signalling inhibitors in prostate cancer.

BACKGROUND: Resistance to androgen receptor signalling inhibitors (ARSIs) represents a major clinical challenge in prostate cancer. We previously demonstrated that the ARSI enzalutamide inhibits only a subset of all AR-regulated genes, and hypothesise that the unaffected gene networks represent potential targets for therapeutic intervention. This study identified the hyaluronan-mediated motility receptor (HMMR) as a survival factor in prostate cancer and investigated its potential as a co-target for overcoming resistance to ARSIs. METHODS: RNA-seq, RT-qPCR and Western Blot were used to evaluate the regulation of HMMR by AR and ARSIs. HMMR inhibition was achieved via siRNA knockdown or pharmacological inhibition using 4-methylumbelliferone (4-MU) in prostate cancer cell lines, a mouse xenograft model and patient-derived explants (PDEs). RESULTS: HMMR was an AR-regulated factor that was unaffected by ARSIs. Genetic (siRNA) or pharmacological (4-MU) inhibition of HMMR significantly suppressed growth and induced apoptosis in hormone-sensitive and enzalutamide-resistant models of prostate cancer. Mechanistically, 4-MU inhibited AR nuclear translocation, AR protein expression and subsequent downstream AR signalling. 4-MU enhanced the growth-suppressive effects of 3 different ARSIs in vitro and, in combination with enzalutamide, restricted proliferation of prostate cancer cells in vivo and in PDEs. CONCLUSION: Co-targeting HMMR and AR represents an effective strategy for improving response to ARSIs.

Interactions of Bacterial Quorum Sensing Signals with Model Lipid Membranes: Influence of Membrane Composition on Membrane Remodeling.

We report the influence of membrane composition on the multiscale remodeling of multicomponent lipid bilayers initiated by contact with the amphiphilic bacterial quorum sensing signal N-(3-oxo)-dodecanoyl-l-homoserine lactone (3-oxo-C12-AHL) and its anionic headgroup hydrolysis product, 3-oxo-C12-HS. We used fluorescence microscopy and quartz crystal microbalance with dissipation (QCM-D) to characterize membrane reformation that occurs when these amphiphiles are placed in contact with supported lipid bilayers (SLBs) composed of (i) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) containing varying amounts of cholesterol or (ii) mixtures of DOPC and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, a conical zwitterionic lipid) or 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS, a model anionic lipid). In general, we observe these mixed-lipid membranes to undergo remodeling events, including the formation and subsequent collapse of long tubules and the formation of hemispherical caps, upon introduction to biologically relevant concentrations of 3-oxo-C12-AHL and 3-oxo-C12-HS in ways that differ substantially from those observed in single-component DOPC membranes. These differences in bilayer reformation and their associated dynamics can be understood in terms of the influence of membrane composition on the time scales of molecular flip-flop, lipid packing defects, and lipid phase segregation in these materials. The lipid components investigated here are representative of classes of lipids that comprise both naturally occurring cell membranes and many useful synthetic soft materials. These studies thus represent a first step toward understanding the ways in which membrane composition can impact interactions with this important class of bacterial signaling molecules.

Dynamic exchange controls the assembly structure of nucleic-acid-peptide chimeras.

Recent attempts to develop the next generation of functional biomaterials focus on systems chemistry approaches exploiting dynamic networks of hybrid molecules. This task is often found challenging, but we herein present ways for profiting from the multiple interaction interfaces forming Nucleic-acid-Peptide assemblies and tuning their formation. We demonstrate that the formation of well-defined structures by double-stranded DNA-peptide conjugates (dsCon) is restricted to a specific range of environmental conditions and that precise DNA hybridization, satisfying the interaction interfaces, is a crucial factor in this process. We further reveal the impact of external stimuli, such as competing free DNA elements or salt additives, which initiate dynamic interconversions, resulting in hybrid structures exhibiting spherical and fibrillar domains or a mixture of spherical and fibrillar particles. This extensive analysis of the co-assembly systems chemistry offers new insights into prebiotic hybrid assemblies that may now facilitate the design of new functional materials. We discuss the implications of these findings for the emergence of function in synthetic materials and during early chemical evolution.

Cohort profile: GRACE - a residential aged care cohort examining factors influencing antimicrobial resistance carriage.

BACKGROUND: The emergence of antimicrobial-resistant bacteria represents a considerable threat to human health, particularly for vulnerable populations such as those living in residential aged care. However, antimicrobial resistance carriage and modes of transmission remain incompletely understood. The Generating evidence on antimicrobial Resistance in the Aged Care Environment (GRACE) study was established to determine principal risk factors of antimicrobial resistance carriage and transmission in residential aged care facilities (RACFs). This article describes the cohort characteristics, national representation, and planned analyses for this study. METHODS: Between March 2019 and March 2020, 279 participants were recruited from five South Australian RACFs. The median age was 88.6 years, the median period in residence was 681 days, and 71.7% were female. A dementia diagnosis was recorded in 54.5% and more than two thirds had moderate to severe cognitive impairment (68.8%). 61% had received at least one course of antibiotics in the 12 months prior to enrolment. RESULTS: To investigate the representation of the GRACE cohort to Australians in residential aged care, its characteristics were compared to a subset of the historical cohort of the Registry of Senior Australians (ROSA). This included 142,923 individuals who were permanent residents of RACFs on June 30th, 2017. GRACE and ROSA cohorts were similar in age, sex, and duration of residential care, prevalence of health conditions, and recorded dementia diagnoses. Differences were observed in care requirements and antibiotic exposure (both higher for GRACE participants). GRACE participants had fewer hospital visits compared to the ROSA cohort, and a smaller proportion were prescribed psycholeptic medications. CONCLUSIONS: We have assembled a cohort of aged care residents that is representative of the Australian aged care population, and which provides a basis for future analyses. Metagenomic data isolated from participants and built environments will be used to determine microbiome and resistome characteristics of an individual and the facility. Individual and facility risk exposures will be aligned with metagenomic data to identify principal determinants for antimicrobial resistance carriage. Ultimately, this analysis will inform measures aimed at reducing the emergence and spread of antimicrobial resistant pathogens in this high-risk population.

Selective Transcription Factor Blockade Reduces Human Retinal Endothelial Cell Expression of Intercellular Adhesion Molecule-1 and Leukocyte Binding.

The interaction between leukocytes and cytokine-activated retinal endothelium is an initiating step in non-infectious uveitis involving the posterior eye, mediated by cell adhesion molecules. However, because cell adhesion molecules are required for immune surveillance, therapeutic interventions would ideally be employed indirectly. Using 28 primary human retinal endothelial cell isolates, this study sought to identify transcription factor targets for reducing levels of the key retinal endothelial cell adhesion molecule, intercellular adhesion molecule (ICAM)-1, and limiting leukocyte binding to the retinal endothelium. Five candidate transcription factors-C2CD4B, EGR3, FOSB, IRF1, and JUNB-were identified by differential expression analysis of a transcriptome generated from IL-1ß- or TNF-α-stimulated human retinal endothelial cells, interpreted in the context of the published literature. Further filtering involved molecular studies: of the five candidates, C2CD4B and IRF1 consistently demonstrated extended induction in IL-1ß- or TNF-α-activated retinal endothelial cells and demonstrated a significant decrease in both ICAM-1 transcript and ICAM-1 membrane-bound protein expression by cytokine-activated retinal endothelial cells following treatment with small interfering RNA. RNA interference of C2CD4B or IRF1 significantly reduced leukocyte binding in a majority of human retinal endothelial cell isolates stimulated by IL-1ß or TNF-α. Our observations suggest that the transcription factors C2CD4B and IRF1 may be potential drug targets for limiting leukocyte-retinal endothelial cell interactions in non-infectious uveitis involving the posterior eye.

Long-term perturbation of the peripheral immune system months after SARS-CoV-2 infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious respiratory virus which is responsible for the coronavirus disease 2019 (COVID-19) pandemic. It is increasingly clear that recovered individuals, even those who had mild COVID-19, can suffer from persistent symptoms for many months after infection, a condition referred to as "long COVID", post-acute sequelae of COVID-19 (PASC), post-acute COVID-19 syndrome, or post COVID-19 condition. However, despite the plethora of research on COVID-19, relatively little is known about the molecular underpinnings of these long-term effects. METHODS: We have undertaken an integrated analysis of immune responses in blood at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 patients recovering from mild, moderate, severe, or critical COVID-19 in comparison to healthy uninfected controls. Twenty-one of these patients were referred to a long COVID clinic and > 50% reported ongoing symptoms more than 6 months post-infection. RESULTS: Anti-Spike and anti-RBD IgG responses were largely stable up to 24 wpi and correlated with disease severity. Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3+ monocytes) and adaptive immune populations (T helper, T follicular helper, and regulatory T cells) in convalescent individuals compared to healthy controls, which were most strongly evident at 12 and 16 wpi. RNA sequencing revealed significant perturbations to gene expression in COVID-19 convalescents until at least 6 months post-infection. We also uncovered significant differences in the transcriptome at 24 wpi of convalescents who were referred to a long COVID clinic compared to those who were not. CONCLUSIONS: Variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals.

Environmentally Responsive Emulsions of Thermotropic Liquid Crystals with Exceptional Long-Term Stability and Enhanced Sensitivity to Aqueous Amphiphiles.

We report colloidally stable emulsions of thermotropic liquid crystals (LCs) that can detect the presence of amphiphilic analytes in aqueous environments. Our approach makes use of a Pickering stabilization strategy consisting of surfactant-nanoparticle complexes (SiO2/CnTAB, n = 8, 12, 16) that adsorb to aqueous/LC droplet interfaces. This strategy can stabilize LC emulsions against coalescence for at least 3 months. These stabilized LC emulsions also retain the ability to respond to the presence of model anionic, cationic, and nonionic amphiphiles (e.g., SDS, C12TAB, C12E4) in aqueous solutions by undergoing "bipolar-to-radial" changes in LC droplet configurations that can be readily observed and quantified using polarized light microscopy. Our results reveal these ordering transitions to depend upon the length of the hydrocarbon tail of the CnTAB surfactant used to form the stabilizing complexes. In general, increasing CnTAB surfactant tail length leads to droplets that respond at lower analyte concentrations, demonstrating that this Pickering stabilization strategy can be used to tune the sensitivities of the stabilized LC droplets. Finally, we demonstrate that these colloidally stable LC droplets can report the presence of rhamnolipid, a biosurfactant produced by the bacterial pathogen Pseudomonas aeruginosa. Overall, our results demonstrate that this Pickering stabilization strategy provides a useful tool for the design of LC droplet-based sensors with substantially improved colloidal stability and new strategies to tune their sensitivities. These advances could increase the potential practical utility of these responsive soft materials as platforms for the detection and reporting of chemical and biological analytes.

Antibody-Targeted Liposomes for Enhanced Targeting of the Blood-Brain Barrier.

The blood-brain barrier (BBB) hinders therapeutic delivery to the central nervous system (CNS), thereby impeding the development of therapies for brain injury and disease. Receptor-mediated transcytosis (RMT) systems are a promising way to shuttle a targeted therapeutic into the brain. Here, we developed and evaluated an RMT antibody-targeted liposomal system. A previously identified antibody, scFv46.1, that binds to the human and murine BBB and can pass through the murine BBB by transcytosis after intravenous injection was used to decorate the surface of liposomes. Using an in vitro BBB model, we demonstrated the cellular uptake of scFv46.1-modified liposomes (46.1-Lipo). Next, the biodistribution and brain uptake capacity of 46.1-targeted liposomes were assessed after intravenous administration. Our results showed that 46.1-Lipo can lead to increased brain accumulation through targeting of the brain vasculature. Initial rate pharmacokinetic experiments and biodistribution analyses indicated that 46.1-Lipo loaded with pralidoxime exhibited a 10-fold increase in brain accumulation compared with a mock-targeted liposomal group, and this increased accumulation was brain-specific. These studies indicate the potential of this 46.1-Lipo system as a synthetic vehicle for the targeted transport of therapeutic molecules into the CNS.

National Trends in Antibiotic Use in Australian Residential Aged Care Facilities, 2005-2016.

BACKGROUND: Understanding current patterns of antibiotic use in residential aged care facilities (RACFs) is essential to inform stewardship activities, but limited utilization data exist. This study examined changes in prevalence and consumption of antibiotics in Australian RACFs between 2005-2006 and 2015-2016. METHODS: This population-based, repeated cross-sectional analysis included all long-term permanent residents of Australian RACFs between July 2005 and June 2016 who were aged ≥ 65 years. The yearly prevalence rate of antibiotic use and number of defined daily doses (DDDs) of systemic antibiotics per 1000 resident-days were determined annually from linked pharmaceutical claims data. Trends were assessed using ordinary least squares regression. RESULTS: This study included 502 752 residents from 3218 RACFs, with 424.9 million resident-days analyzed. Antibiotics were dispensed on 5 608 126 occasions during the study period, of which 88% were for oral use. Cefalexin, amoxicillin-clavulanic acid, and trimethoprim were the most commonly dispensed antibiotics. The annual prevalence of antibiotic use increased from 63.8% (95% confidence interval [CI], 63.3%-64.4%) to 70.3% (95% CI, 69.9%-70.7%) between 2005-2006 and 2015-2016 (0.8% average annual increase, P < .001). There was a 39% relative increase in total consumption of systemic antibiotics, with utilization increasing from 67.6 to 93.8 DDDs/1000 resident-days during the study period (average annual increase of 2.8 DDDs/1000 resident-days, P < .001). CONCLUSIONS: This nationwide study showed substantial increases in both prevalence of use and total consumption of antibiotics in Australian RACFs between 2005 and 2016. The increasingly widespread use of antibiotics in Australian RACFs is concerning and points to a need for enhanced efforts to optimize antibiotic use in this setting.

Contribution of facility level factors to variation in antibiotic use in long-term care facilities: a national cohort study.

OBJECTIVES: To examine national variation in systemic antibiotic use in long-term care facilities (LTCFs) and identify facility characteristics associated with antibiotic utilization. METHODS: This retrospective cohort study included 312 375 residents of 2536 Australian LTCFs between 2011 and 2016. LTCFs were categorized as low, medium or high antibiotic use facilities according to tertiles of DDDs of systemic antibiotics dispensed per 1000 resident-days. Multivariable logistic regression estimated the associations between facility characteristics (ownership, size, location, medication quality indicator performance, prevalence of after-hours medical practitioner services) and antibiotic use (low versus high). RESULTS: LTCFs in the lowest and highest antibiotic use categories received a median of 54.3 (IQR 46.5-60.5) and 106.1 (IQR 95.9-122.3) DDDs/1000 resident-days, respectively. Compared with not-for-profit LTCFs in major cities, government-owned non-metropolitan LTCFs were less likely to experience high antibiotic use [adjusted OR (aOR) 0.47, 95% CI 0.24-0.91]. LTCFs with 69-99 residents were less likely to experience high antibiotic use (aOR 0.69, 95% CI 0.49-0.97) than those with 25-47 residents annually. Greater prevalence of medical practitioner services accessed after-hours was associated with high antibiotic use [aOR 1.10 (per 10% increase in after-hours services), 95% CI 1.01-1.21]. South Australian LTCFs (aOR 2.17, 95% CI 1.38-3.39) were more likely, while Queensland (0.43, 95% CI 0.30-0.62) and Western Australian (aOR 0.34, 95% CI 0.21-0.57) LTCFs were less likely to experience high antibiotic use than New South Wales LTCFs. CONCLUSIONS: Considerable facility level variation in systemic antibiotic use was observed across Australian LTCFs. Identification of facility characteristics associated with antibiotic use provides a basis for targeted stewardship initiatives.

Interactions of Bacterial Quorum Sensing Signals with Model Lipid Membranes: Influence of Acyl Tail Structure on Multiscale Response.

Many common bacteria use amphiphilic N-acyl-L-homoserine lactones (AHLs) as signaling molecules to coordinate group behaviors at high cell densities. Past studies demonstrate that AHLs can adsorb to and promote the remodeling of lipid membranes in ways that could underpin cell-cell or host-cell interactions. Here, we report that changes in AHL acyl tail group length and oxidation state (e.g., the presence or absence of a 3-oxo group) can lead to differences in the interactions of eight naturally occurring AHLs in solution and in contact with model lipid membranes. Our results reveal that the presence of a 3-oxo group impacts remodeling when AHLs are placed in contact with supported lipid bilayers (SLBs) of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Whereas AHLs that have 3-oxo groups generally promote the formation of microtubules, AHLs that lack 3-oxo groups generally form hemispherical caps on the surfaces of SLBs. These results are interpreted in terms of the time scales on which AHLs translocate across bilayers to relieve asymmetrical bilayer stress. Quartz crystal microbalance with dissipation measurements also reveal that 3-oxo AHLs associate with DOPC bilayers to a greater extent than their non-3-oxo analogues. In contrast, we observed no monotonic relationship between AHL tail length and bilayer reformation. Finally, we observed that 3-oxo AHLs facilitate greater transport or leakage of molecular cargo across the membranes of DOPC vesicles relative to AHLs without 3-oxo groups, also suggesting increased bilayer disruption and destabilization. These fundamental studies hint at interactions and associated multiscale phenomena that may inform current interpretations of the behaviors of AHLs in biological contexts. These results could also provide guidance useful for the design of new classes of synthetic materials (e.g., sensor elements or drug delivery vehicles) that interact with or respond selectively to communities of bacteria that use 3-oxo AHLs for cell-cell communication.

Bacterial Quorum Sensing Signals Promote Large-Scale Remodeling of Lipid Membranes.

We report that N-acyl-l-homoserine lactones (AHLs), a class of nonionic amphiphiles that common bacteria use as signals to coordinate group behaviors, can promote large-scale remodeling in model lipid membranes. Characterization of supported lipid bilayers (SLBs) of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) by fluorescence microscopy and quartz crystal microbalance with dissipation (QCM-D) reveals the well-studied AHL signal 3-oxo-C12-AHL and its anionic head group hydrolysis product (3-oxo-C12-HS) to promote the formation of long microtubules that can retract into hemispherical caps on the surface of the bilayer. These transformations are dynamic, reversible, and dependent upon the head group structure. Additional experiments demonstrate that 3-oxo-C12-AHL can promote remodeling to form microtubules in lipid vesicles and promote molecular transport across bilayers. Molecular dynamics (MD) simulations predict differences in thermodynamic barriers to translocation of these amphiphiles across a bilayer that are reflected in both the type and extent of reformation and associated dynamics. Our experimental observations can thus be interpreted in terms of accumulation and relief of asymmetric stresses in the inner and outer leaflets of a bilayer upon intercalation and translocation of these amphiphiles. Finally, experiments on Pseudomonas aeruginosa, a pathogen that uses 3-oxo-C12-AHL for cell-to-cell signaling, demonstrate that 3-oxo-C12-AHL and 3-oxo-C12-HS can promote membrane remodeling at biologically relevant concentrations and in the absence of other biosurfactants, such as rhamnolipids, that are produced at high population densities. Overall, these results have implications for the roles that 3-oxo-C12-AHL and its hydrolysis product may play in not only mediating intraspecies bacterial communication but also processes such as interspecies signaling and bacterial control of host-cell response. Our findings also provide guidance that could prove useful for the design of synthetic self-assembled materials that respond to bacteria in ways that are useful in the context of sensing, drug delivery, and in other fundamental and applied areas.

Protonation-Driven Aqueous Lyotropic Self-Assembly of Synthetic Six-Tail Lipidoids.

We report the aqueous lyotropic mesophase behaviors of protonated amine-based "lipidoids," a class of synthetic lipid-like molecules that mirrors essential structural features of the multitail bacterial amphiphile lipid A. Small-angle X-ray scattering (SAXS) studies demonstrate that the protonation of the tetra(amine) headgroups of six-tail lipidoids in aqueous HCl, HNO3, H2SO4, and H3PO4 solutions variably drives their self-assembly into lamellar (Lα) and inverse micellar (III) lyotropic liquid crystals (LLCs), depending on acid identity and concentration, amphiphile tail length, and temperature. Lipidoid assemblies formed in H2SO4(aq) exhibit rare inverse body-centered cubic (BCC) and inverse face-centered cubic (FCC) micellar morphologies, the latter of which unexpectedly coexists with zero mean curvature Lα phases. Complementary atomistic molecular dynamics (MD) simulations furnish detailed insights into this unusual self-assembly behavior. The unique aqueous lyotropic mesophase behaviors of ammonium lipidoids originate in their dichotomous ability to adopt both inverse conical and chain-extended molecular conformations depending on the number of counterions and their identity, which lead to coexisting supramolecular assemblies with remarkably different mean interfacial curvatures.

Biomolecular Assemblies: Moving from Observation to Predictive Design.

Biomolecular assembly is a key driving force in nearly all life processes, providing structure, information storage, and communication within cells and at the whole organism level. These assembly processes rely on precise interactions between functional groups on nucleic acids, proteins, carbohydrates, and small molecules, and can be fine-tuned to span a range of time, length, and complexity scales. Recognizing the power of these motifs, researchers have sought to emulate and engineer biomolecular assemblies in the laboratory, with goals ranging from modulating cellular function to the creation of new polymeric materials. In most cases, engineering efforts are inspired or informed by understanding the structure and properties of naturally occurring assemblies, which has in turn fueled the development of predictive models that enable computational design of novel assemblies. This Review will focus on selected examples of protein assemblies, highlighting the story arc from initial discovery of an assembly, through initial engineering attempts, toward the ultimate goal of predictive design. The aim of this Review is to highlight areas where significant progress has been made, as well as to outline remaining challenges, as solving these challenges will be the key that unlocks the full power of biomolecules for advances in technology and medicine.