Intravesicular Solute Delivery and Surface Area Regulation in Giant Unilamellar Vesicles Driven by Cycles of Osmotic Stresses.

Phospholipid bilayers are dynamic cellular components that undergo constant changes in their topology, facilitating a broad diversity of physiological functions including endo- and exocytosis, cell division, and intracellular trafficking. These shape transformations consume energy, supplied invariably by the activity of proteins. Here, we show that cycles of oppositely directed osmotic stresses─unassisted by any protein activity─can induce well-defined remodeling of giant unilamellar vesicles, minimally recapitulating the phenomenologies of surface area homeostasis and macropinocytosis. We find that a stress cycle consisting of deflationary hypertonic stress followed by an inflationary hypotonic one prompts an elaborate sequence of membrane shape changes ultimately transporting molecular cargo from the outside into the intravesicular milieu. The initial osmotic deflation produces microscopic spherical invaginations. During the subsequent inflation, the first subpopulation contributes area to the swelling membrane, thereby providing a means for surface area regulation and tensional homeostasis. The second subpopulation vesiculates into the lumens of the mother vesicles, producing pinocytic vesicles. Remarkably, the gradients of solute concentrations between the GUV and the daughter pinocytic vesicles create cascades of water current, inducing pulsatory transient poration that enable solute exchange between the buds and the GUV interior. This results in an efficient water-flux-mediated delivery of molecular cargo across the membrane boundary. Our findings suggest a primitive physical mechanism for communication and transport across protocellular compartments driven only by osmotic stresses. They also suggest plausible physical routes for intravesicular, and possibly intracellular, delivery of ions, solutes, and molecular cargo stimulated simply by cycles of osmotic currents of water.

Hospitalized acute exacerbation in chronic obstructive pulmonary disease - impact on long-term renal outcomes.

INTRODUCTION: Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is a common and preventable event in patients with chronic obstructive pulmonary disease (COPD). Data regarding the impact of AECOPD on short- and long-term renal outcomes are lacking. METHODS: We included all COPD patients who were followed at Queen Mary Hospital (QMH) in year 2015 and reviewed their clinical/renal outcomes in subsequent five years. Relationships between AECOPD and adverse renal outcomes were evaluated. RESULTS: 371 COPD patients were included. 169 patients had hospitalized AECOPD in past one year (HAE group) while 202 patients did not (non-HAE group). 285 patients (76.8%) had renal progression/death and 102 (27.5%) patients developed acute kidney injury (AKI). HAE group showed a more rapid eGFR decline than non-HAE group (-4.64 mL/min/1.73m2/year vs. -2.40 mL/min/1.73m2/year, p = 0.025). HAE group had significantly higher risk for renal progression/death at 5 years [adjusted OR (aOR) 2.380 (95% CI = 1.144-4.954), p = 0.020]. The frequency of hospitalized AECOPD in past 3 years, any AECOPD in past 3 years, hospitalized AECOPD in past 3 years were also predictive of renal progression/death at 5 years [aOR were 1.176 (95% CI = 1.038- 1.331), 2.998 (95% CI = 1.438-6.250) and 2.887 (95% CI = 1.409-5.917) respectively; p = 0.011, 0.003 and 0.004]. HAE group also showed significantly higher risk of AKI [adjusted HR (aHR) 2.430; 95% CI = 1.306-4.519, p = 0.005]. CONCLUSIONS: AECOPD, in particular HAE, was associated with increased risk of renal progression/death and AKI. Prevention of AECOPD, especially HAE, may potentially improve short- and long-term renal outcomes in COPD patients.

An Evaluation of Gene-Diet Interaction Statistical Methods and Discovery of rs7175421-Whole Grain Interaction in Lung Cancer.

Dietary factors show different effects on genetically diverse populations. Scientific research uses gene-environment interaction models to study the effects of dietary factors on genetically diverse populations for lung cancer risk. However, previous study designs have not investigated the degree of type I error inflation and, in some instances, have not corrected for multiple testing. Using a motivating investigation of diet-gene interaction and lung cancer risk, we propose a training and testing strategy and perform real-world simulations to select the appropriate statistical methods to reduce false-positive discoveries. The simulation results show that the unconstrained maximum likelihood (UML) method controls the type I error better than the constrained maximum likelihood (CML). The empirical Bayesian (EB) method can compete with the UML method in achieving statistical power and controlling type I error. We observed a significant interaction between SNP rs7175421 with dietary whole grain in lung cancer prevention, with an effect size (standard error) of -0.312 (0.112) for EB estimate. SNP rs7175421 may interact with dietary whole grains in modulating lung cancer risk. Evaluating statistical methods for gene-diet interaction analysis can help balance the statistical power and type I error.

Surfactant-Mediated Solubilization of Myelin Figures: A Multistep Morphological Cascade.

Lamellar mesophases of insoluble lipids are readily solubilized by the micellar mesophases of soluble surfactants. This simple process underscores a broad array of biochemical methodologies, including purification, reconstitution, and crystallization of membrane proteins, as well as the isolation of detergent-resistant membrane fractions. Although much is now known about the thermodynamic driving forces of membrane solubilization, the kinetic pathways by which the surfactant alters vesicular mesophases are only beginning to be appreciated. Little is known about how these interactions affect the solubilization of more complex, multilamellar mesophases. Here, we investigate how a common zwitterionic detergent affects the solubilization of a smectic, multilamellar, cylindrical mesophase of lipids, called the myelin figure. Our results reveal that myelin solubilization occurs in a multistep manner, producing a well-defined sequence of morphologically distinct intermediates en route to complete solubilization. The kinetic processes producing these intermediates include (1) coiling, which encompasses the formation, propagation, and tightening of extended helices; (2) thinning, which reflects the unbinding of lamellae in the smectic stacks; and (3) detachment or retraction, which either dissociates the myelinic protrusion from the source lipid mass or returns the myelinic protrusion to the source lipid mass─all in transit toward complete solubilization. These occasionally overlapping steps are most pronounced in single-lipid component myelins, while compositionally graded multicomponent myelins inhibit the coiling step and detach more frequently. Taken together, the appearance of these intermediates during the solubilization of myelins suggests a complex free-energy landscape characterizing myelin solubilization populated by metastable, morphological intermediates correlated with locally minimized changes in energy dependent upon the mesophase's composition. This then predicts the accessibility of structurally distinct, kinetic intermediates─such as loose and tight coiled helices, peeled myelins, retracted tubes, and detached protrusions─before reaching the stable ground state corresponding to a dissolved suspension of mixed surfactant-lipid micelles.

Nonequilibrium Self-Organization of Lipids into Hierarchically Ordered and Compositionally Graded Cylindrical Smectics.

When a dry mass of certain amphiphiles encounters water, a spectacular interfacial instability ensues: It gives rise to the formation of ensembles of fingerlike tubular protrusions called myelin figures─tens of micrometers wide and tens to hundreds of micrometers long─representing a novel class of nonequilibrium higher-order self-organization. Here, we report that when phase-separating mixtures of unsaturated lipid, cholesterol, and sphingomyelin are hydrated, the resulting myelins break symmetry and couple their compositional degrees of freedom with the extended myelinic morphology: They produce complementary, interlamellar radial gradients of concentrations of cholesterol (and sphingomyelin) and unsaturated lipid, which stands in stark contrast to interlamellar, lateral phase separation in equilibrated morphologies. Furthermore, the corresponding gradients of molecule-specific chemistries (i.e., cholesterol extraction by methyl-ß-cyclodextrin and GM1 binding by cholera toxin) produce unusual morphologies comprising compositionally graded vesicles and buckled tubes. We propose that kinetic differences in the information processing of hydration characteristics of individual molecules while expending energy dictate this novel behavior of lipid mixtures undergoing hydration.

Peptide-Oleate Complexes Create Novel Membrane-Bound Compartments.

A challenging question in evolutionary theory is the origin of cell division and plausible molecular mechanisms involved. Here, we made the surprising observation that complexes formed by short alpha-helical peptides and oleic acid can create multiple membrane-enclosed spaces from a single lipid vesicle. The findings suggest that such complexes may contain the molecular information necessary to initiate and sustain this process. Based on these observations, we propose a new molecular model to understand protocell division.

Minimal Reconstitution of Membranous Web Induced by a Vesicle-Peptide Sol-Gel Transition.

Positive strand RNA viruses replicate in specialized niches called membranous web within the cytoplasm of host cells. These virus replication organelles sequester viral proteins, RNA, and a variety of host factors within a fluid, amorphous matrix of clusters of endoplasmic reticulum (ER) derived vesicles. They are thought to form by the actions of a nonstructural viral protein NS4B, which remodels the ER and produces dense lipid-protein condensates. Here, we used in vitro reconstitution to identify the minimal components and elucidate physical mechanisms driving the web formation. We found that the N-terminal amphipathic domain of NS4B (peptide 4BAH2) and phospholipid vesicles (∼100-200 nm in diameter) were sufficient to produce a gel-like, viscoelastic condensate. This condensate coexists with the surrounding aqueous phase and affords rapid exchange of molecules. Together, it recapitulates the essential properties of the virus-induced membranous web. Our data support a novel phase separation mechanism in which phospholipid vesicles provide a supramolecular template spatially organizing multiple self-associating peptides thereby generating programmable multivalency de novo and inducing macroscopic phase separation.

Response of microbial membranes to butanol: interdigitation vs. disorder.

Biobutanol production by fermentation is potentially a sustainable alternative to butanol production from fossil fuels. However, the toxicity of butanol to fermentative bacteria, resulting largely from cell membrane fluidization, limits production titers and is a major factor limiting the uptake of the technology. Here, studies were undertaken, in vitro and in silico, on the butanol effects on a representative bacterial (i.e. Escherichia coli) inner cell membrane. A critical butanol : lipid ratio for stability of 2 : 1 was observed, computationally, consistent with complete interdigitation. However, at this ratio the bilayer was ∼20% thicker than for full interdigitation. Furthermore, butanol intercalation induced acyl chain bending and increased disorder, measured as a 27% lateral diffusivity increase experimentally in a supported lipid bilayer. There was also a monophasic Tm reduction in butanol-treated large unilamellar vesicles. Both behaviours are inconsistent with an interdigitated gel. Butanol thus causes only partial interdigitation at physiological temperatures, due to butanol accumulating at the phospholipid headgroups. Acyl tail disordering (i.e. splaying and bending) fills the subsequent voids. Finally, butanol short-circuits the bilayer and creates a coupled system where interdigitated and splayed phospholipids coexist. These findings will inform the design of strategies targeting bilayer stability for increasing biobutanol production titers.

Informed Molecular Design of Conjugated Oligoelectrolytes To Increase Cell Affinity and Antimicrobial Activity.

Membrane-intercalating conjugated oligoelectrolytes (COEs) are emerging as potential alternatives to conventional, yet increasingly ineffective, antibiotics. Three readily accessible COEs, belonging to an unreported series containing a stilbene core, namely D4, D6, and D8, were designed and synthesized so that the hydrophobicity increases with increasing side-chain length. Decreased aqueous solubility correlates with increased uptake by E. coli. The minimum inhibitory concentration (MIC) of D8 is 4 µg mL-1 against both E. coli and E. faecalis, with an effective uptake of 72 %. In contrast, the MIC value of the shortest COE, D4, is 128 µg mL-1 owing to the low cellular uptake of 3 %. These findings demonstrate the application of rational design to generate efficacious antimicrobial COEs that have potential as low-cost antimicrobial agents.

Pulsatile Lipid Vesicles under Osmotic Stress.

The response of lipid bilayers to osmotic stress is an important part of cellular function. Recent experimental studies showed that when cell-sized giant unilamellar vesicles (GUVs) are exposed to hypotonic media, they respond to the osmotic assault by undergoing a cyclical sequence of swelling and bursting events, coupled to the membrane's compositional degrees of freedom. Here, we establish a fundamental and quantitative understanding of the essential pulsatile behavior of GUVs under hypotonic conditions by advancing a comprehensive theoretical model of vesicle dynamics. The model quantitatively captures the experimentally measured swell-burst parameters for single-component GUVs, and reveals that thermal fluctuations enable rate-dependent pore nucleation, driving the dynamics of the swell-burst cycles. We further extract constitutional scaling relationships between the pulsatile dynamics and GUV properties over multiple timescales. Our findings provide a fundamental framework that has the potential to guide future investigations on the nonequilibrium dynamics of vesicles under osmotic stress.

HAMLET - A protein-lipid complex with broad tumoricidal activity.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a tumoricidal protein-lipid complex with broad effects against cancer cells of different origin. The therapeutic potential is emphasized by a high degree of specificity for tumor tissue. Here we review early studies of HAMLET, in collaboration with the Orrenius laboratory, and some key features of the subsequent development of the HAMLET project. The early studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. In subsequent studies, we have identified a sequence of interactions that starts with the membrane integration of HAMLET and the activation of ion fluxes followed by HAMLET internalization, progressive inhibition of MAPK kinases and GTPases and sorting of HAMLET to different cellular compartments, including the nuclei. Therapeutic efficacy of HAMLET has been demonstrated in animal models of glioblastoma, bladder cancer and intestinal cancer. In clinical studies, HAMLET has been shown to target skin papillomas and bladder cancers. The findings identify HAMLET as a new drug candidate with promising selectivity for cancer cells and a strong therapeutic potential.

The novel small leucine-rich protein chondroadherin-like (CHADL) is expressed in cartilage and modulates chondrocyte differentiation.

The constitution and biophysical properties of extracellular matrices can dramatically influence cellular phenotype during development, homeostasis, or pathogenesis. These effects can be signaled through a differentially regulated assembly of collagen fibrils, orchestrated by a family of collagen-associated small leucine-rich proteins (SLRPs). In this report, we describe the tissue-specific expression and function of a previously uncharacterized SLRP, chondroadherin-like (CHADL). We developed antibodies against CHADL and, by immunohistochemistry, detected CHADL expression mainly in skeletal tissues, particularly in fetal cartilage and in the pericellular space of adult chondrocytes. In situ hybridizations and immunoblots on tissue lysates confirmed this tissue-specific expression pattern. Recombinant CHADL bound collagen in cell culture and inhibited in vitro collagen fibrillogenesis. After Chadl shRNA knockdown, chondrogenic ATDC5 cells increased their differentiation, indicated by increased transcript levels of Sox9, Ihh, Col2a1, and Col10a1. The knockdown increased collagen II and aggrecan deposition in the cell layers. Microarray analysis of the knockdown samples suggested collagen receptor-related changes, although other upstream effects could not be excluded. Together, our data indicate that the novel SLRP CHADL is expressed in cartilaginous tissues, influences collagen fibrillogenesis, and modulates chondrocyte differentiation. CHADL appears to have a negative regulatory role, possibly ensuring the formation of a stable extracellular matrix.

Mixing Water, Transducing Energy, and Shaping Membranes: Autonomously Self-Regulating Giant Vesicles.

Giant lipid vesicles are topologically closed compartments bounded by semipermeable flexible shells, which isolate femto- to picoliter quantities of the aqueous core from the surrounding bulk. Although water equilibrates readily across vesicular walls (10(-2)-10(-3) cm(3) cm(-2) s(-1)), the passive permeation of solutes is strongly hindered. Furthermore, because of their large volume compressibility (∼10(9)-10(10) N m(-2)) and area expansion (10(2)-10(3) mN m(-1)) moduli, coupled with low bending rigidities (10(-19) N m), vesicular shells bend readily but resist volume compression and tolerate only a limited area expansion (∼5%). Consequently, vesicles experiencing solute concentration gradients dissipate the available chemical energy through the osmotic movement of water, producing dramatic shape transformations driven by surface-area-volume changes and sustained by the incompressibility of water and the flexible membrane interface. Upon immersion in a hypertonic bath, an increased surface-area-volume ratio promotes large-scale morphological remodeling, reducing symmetry and stabilizing unusual shapes determined, at equilibrium, by the minimal bending-energy configurations. By contrast, when subjected to a hypotonic bath, walls of giant vesicles lose their thermal undulation, accumulate mechanical tension, and, beyond a threshold swelling, exhibit remarkable oscillatory swell-burst cycles, with the latter characterized by damped, periodic oscillations in vesicle size, membrane tension, and phase behavior. This cyclical pattern of the osmotic influx of water, pressure, membrane tension, pore formation, and solute efflux suggests quasi-homeostatic self-regulatory behavior allowing vesicular compartments produced from simple molecular components, namely, water, osmolytes, and lipids, to sense and regulate their microenvironment in a negative feedback loop.

Brownian Dynamics of Electrostatically Adhering Small Vesicles to a Membrane Surface Induces Domains and Probes Viscosity.

Using single-particle tracking, we investigate the interaction of small unilamellar vesicles (SUVs) that are electrostatically tethered to the freestanding membrane of a giant unilamellar vesicle (GUV). We find that the surface mobility of the GUV-riding SUVs is Brownian, insensitive to the bulk viscosity, vesicle size, and vesicle fluidity but strongly altered by the viscosity of the underlying membrane. Analyzing the diffusional behavior of SUVs within the Saffman-Delbrück model for the dynamics of membrane inclusions supports the notion that the mobility of the small vesicles is coupled to that of dynamically induced lipid clusters within the target GUV membrane. The reversible binding also offers a nonperturbative means for measuring the viscosity of biomembranes, which is an important parameter in cell physiology and function.

Lipids as tumoricidal components of human α-lactalbumin made lethal to tumor cells (HAMLET): unique and shared effects on signaling and death.

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 µm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 µm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.

The impact of lowering the study design significance threshold to 0.005 on sample size in randomized cancer clinical trials.

The proposal of improving reproducibility by lowering the significance threshold to 0.005 has been discussed, but the impact on conducting clinical trials has yet to be examined from a study design perspective. The impact on sample size and study duration was investigated using design setups from 125 phase II studies published between 2015 and 2022. The impact was assessed using percent increase in sample size and additional years of accrual with the medians being 110.97% higher and 2.65 years longer respectively. The results indicated that this proposal causes additional financial burdens that reduce the efficiency of conducting clinical trials.

Kinetic control of shape deformations and membrane phase separation inside giant vesicles.

A variety of cellular processes use liquid-liquid phase separation (LLPS) to create functional levels of organization, but the kinetic pathways by which it proceeds remain incompletely understood. Here in real time, we monitor the dynamics of LLPS of mixtures of segregatively phase-separating polymers inside all-synthetic, giant unilamellar vesicles. After dynamically triggering phase separation, we find that the ensuing relaxation-en route to the new equilibrium-is non-trivially modulated by a dynamic interplay between the coarsening of the evolving droplet phase and the interactive membrane boundary. The membrane boundary is preferentially wetted by one of the incipient phases, dynamically arresting the progression of coarsening and deforming the membrane. When the vesicles are composed of phase-separating mixtures of common lipids, LLPS within the vesicular interior becomes coupled to the membrane's compositional degrees of freedom, producing microphase-separated membrane textures. This coupling of bulk and surface phase-separation processes suggests a physical principle by which LLPS inside living cells might be dynamically regulated and communicated to the cellular boundaries.

Information-Distilled Generative Label-Free Morphological Profiling Encodes Cellular Heterogeneity.

Image-based cytometry faces challenges due to technical variations arising from different experimental batches and conditions, such as differences in instrument configurations or image acquisition protocols, impeding genuine biological interpretation of cell morphology. Existing solutions, often necessitating extensive pre-existing data knowledge or control samples across batches, have proved limited, especially with complex cell image data. To overcome this, "Cyto-Morphology Adversarial Distillation" (CytoMAD), a self-supervised multi-task learning strategy that distills biologically relevant cellular morphological information from batch variations, is introduced to enable integrated analysis across multiple data batches without complex data assumptions or extensive manual annotation. Unique to CytoMAD is its "morphology distillation", symbiotically paired with deep-learning image-contrast translation-offering additional interpretable insights into label-free cell morphology. The versatile efficacy of CytoMAD is demonstrated in augmenting the power of biophysical imaging cytometry. It allows integrated label-free classification of human lung cancer cell types and accurately recapitulates their progressive drug responses, even when trained without the drug concentration information. CytoMAD  also allows joint analysis of tumor biophysical cellular heterogeneity, linked to epithelial-mesenchymal plasticity, that standard fluorescence markers overlook. CytoMAD can substantiate the wide adoption of biophysical cytometry for cost-effective diagnosis and screening.

Endobronchial ultrasound-guided transbronchial needle aspiration in lung cancer: the first experience in Hong Kong.

OBJECTIVE: To investigate the diagnostic performance and safety of endobronchial ultrasound-guided transbronchial needle aspiration in patients presenting with radiological features of lung cancer. DESIGN: Prospective case series. SETTING: University teaching hospital, Hong Kong. PATIENTS: Consecutive patients with mediastinal or hilar abnormalities suspected of or confirmed as having lung cancer underwent endobronchial ultrasound-guided transbronchial needle aspiration and presented between August 2006 and December 2010. MAIN OUTCOME MEASURES: Diagnostic performance (including sensitivity, specificity, negative predictive value and accuracy), procedural complications, and tissue adequacy for molecular profiling. RESULTS: A total of 269 procedures were performed in 259 patients, with malignancy confirmed in 210 (81%) of them. In the whole cohort with confirmed or suspected lung cancer, the overall sensitivity, specificity, negative predictive value, and accuracy of endobronchial ultrasound-guided transbronchial needle aspiration were 87%, 100%, 74%, and 91%, respectively. Among 42 patients with tumour samples sent for mutation tests (epidermal growth factor receptor and/or anaplastic lymphoma kinase), 40 (95%) were found to be adequate. No complication or mortality ensued from these procedures. CONCLUSION: Endobronchial ultrasound-guided transbronchial needle aspiration is highly effective in determining the diagnosis and lymph node staging in patients with lung cancer. In combination with its excellent safety profile, it should be considered a frontline diagnostic test for patients presenting with mediastinal abnormalities suspicious of lung cancer.

Assessing surrogacy using restricted mean survival time ratio for overall survival in liver cancer: a narrative review.

BACKGROUND AND OBJECTIVE: The application of immunotherapy in cancers, including liver cancer, has been increasing. However, non-proportional hazard (NPH) is often observed in cancer immunotherapy trials. In presence of violation of proportional hazard (PH) assumption, restricted mean survival time (RMST) ratio was proposed as an alternative to hazard ratio (HR) for evaluating the treatment effects of such trials. To shorten the total study duration, an intermediate endpoint with shorter follow-up such as progression-free survival (PFS) is used as the primary endpoint. Our aim is to evaluate the applicability of RMST ratio in addition to the HR in assessing the level of PFS serving as a surrogacy of overall survival (OS). METHODS: Phase II or phase III hepatocellular carcinoma (HCC) immunotherapy studies that were published between January 2013 and August 2022 were identified via the search in PubMed. Weighted least-square regression (WLSR) was applied to analyze the trial level data with the sample size of study being set as the weight. The evaluation was conducted twice with RMST ratio and HR being applied in respective evaluation to examine the level of PFS as a surrogacy for OS. KEY CONTENT AND FINDINGS: Based on the results of eight included trials, the R-square values of WLSR with either HR or RMST ratio being applied were 0.31 and 0.16 separately, indicating a moderate and low correlation between PFS and OS respectively. CONCLUSIONS: In this study, our results demonstrated the potential of RMST ratio in addition to HR for evaluating the level of surrogacy in immunotherapy trials. Furthermore, including more large scale and homogeneous studies into the research may help better understand the level of surrogacy in liver cancer.