SnapShot: Microbial Hydrocarbon Bioremediation.

Hydrocarbon-degrading bacteria are phylogenetically and physiologically diverse and employ layered strategies to sense hydrocarbons, respond transcriptionally, and then move toward an oil source. They then produce biopolymers that increase hydrocarbon bioavailability. This SnapShot highlights how these bacteria respond to and then remove hydrocarbon contaminants from the environment. To view this SnapShot, open or download the PDF.

Molecular Characteristics and Biological Functions of Surface-Active and Surfactant Proteins.

Many critical biological processes take place at hydrophobic:hydrophilic interfaces, and a wide range of organisms produce surface-active proteins and peptides that reduce surface and interfacial tension and mediate growth and development at these boundaries. Microorganisms produce both small lipid-associated peptides and amphipathic proteins that allow growth across water:air boundaries, attachment to surfaces, predation, and improved bioavailability of hydrophobic substrates. Higher-order organisms produce surface-active proteins with a wide variety of functions, including the provision of protective foam environments for vulnerable reproductive stages, evaporative cooling, and gas exchange across airway membranes. In general, the biological functions supported by these diverse polypeptides require them to have an amphipathic nature, and this is achieved by a diverse range of molecular structures, with some proteins undergoing significant conformational change or intermolecular association to generate the structures that are surface active.

Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

A single parameter can predict surfactant impairment of superhydrophobic drag reduction.

Recent experimental and computational investigations have shown that trace amounts of surfactants, unavoidable in practice, can critically impair the drag reduction of superhydrophobic surfaces (SHSs), by inducing Marangoni stresses at the air-liquid interface. However, predictive models for realistic SHS geometries do not yet exist, which has limited the understanding and mitigation of these adverse surfactant effects. To address this issue, we derive a model for laminar, three-dimensional flow over SHS gratings as a function of geometry and soluble surfactant properties, which together encompass 10 dimensionless groups. We establish that the grating length g is the key geometric parameter and predict that the ratio between actual and surfactant-free slip increases with g2. Guided by our model, we perform synergistic numerical simulations and microfluidic experiments, finding good agreement with the theory as we vary surfactant type and SHS geometry. Our model also enables the estimation, based on velocity measurements, of a priori unknown properties of surfactants inherently present in microfluidic systems. For SHSs, we show that surfactant effects can be predicted by a single parameter, representing the ratio between the grating length and the interface length scale beyond which the flow mobilizes the air-water interface. This mobilization length is more sensitive to the surfactant chemistry than to its concentration, such that even trace-level contaminants may significantly increase drag if they are highly surface active. These findings advance the fundamental understanding of realistic interfacial flows and provide practical strategies to maximize superhydrophobic drag reduction.

Ionic-surfactant-mediated electro-dewetting for digital microfluidics.

The ability to manipulate droplets on a substrate using electric signals1-known as digital microfluidics-is used in optical2,3, biomedical4,5, thermal6 and electronic7 applications and has led to commercially available liquid lenses8 and diagnostics kits9,10. Such electrical actuation is mainly achieved by electrowetting, with droplets attracted towards and spreading on a conductive substrate in response to an applied voltage. To ensure strong and practical actuation, the substrate is covered with a dielectric layer and a hydrophobic topcoat for electrowetting-on-dielectric (EWOD)11-13; this increases the actuation voltage (to about 100 volts) and can compromise reliability owing to dielectric breakdown14, electric charging15 and biofouling16. Here we demonstrate droplet manipulation that uses electrical signals to induce the liquid to dewet, rather than wet, a hydrophilic conductive substrate without the need for added layers. In this electrodewetting mechanism, which is phenomenologically opposite to electrowetting, the liquid-substrate interaction is not controlled directly by electric field but instead by field-induced attachment and detachment of ionic surfactants to the substrate. We show that this actuation mechanism can perform all the basic fluidic operations of digital microfluidics using water on doped silicon wafers in air, with only ±2.5 volts of driving voltage, a few microamperes of current and about 0.015 times the critical micelle concentration of an ionic surfactant. The system can also handle common buffers and organic solvents, promising a simple and reliable microfluidic platform for a broad range of applications.

Controlling heme redox properties in peptide amphiphile fibers with sequence and heme loading ratio.

Controlling the reduction midpoint potential of heme B is a key factor in many bioelectrochemical reactions, including long-range electron transport. Currently, there are a number of globular model protein systems to study this biophysical parameter; however, there are none for large polymeric protein model systems (e.g., the OmcS protein from G. sulfurreducens). Peptide amphiphiles, short peptides with a lipid tail that polymerize into fibrous structures, fill this gap. Here, we show a peptide amphiphile model system where one can tune the electrochemical potential of heme B by changing the loading ratio and peptide sequence. Changing the loading ratio resulted in the most significant increase, with values as high as -22 mV down to -224 mV. Circular dichroism spectra of certain sequences show Cotton effects at lower loading ratios that disappear as more heme B is added, indicating an ordered environment that becomes disrupted if heme B is overpacked. These findings can contribute to the design of functional self-assembling biomaterials.

High-Throughput Extracellular Matrix Proteomics of Human Lungs Enabled by Photocleavable Surfactant and diaPASEF.

The extracellular matrix (ECM) is a complex assembly of proteins that provide interstitial scaffolding and elastic recoil for human lungs. The pulmonary extracellular matrix is increasingly recognized as an independent bioactive entity, by creating biochemical and mechanical signals that influence disease pathogenesis, making it an attractive therapeutic target. However, the pulmonary ECM proteome ("matrisome") remains challenging to analyze by mass spectrometry due to its inherent biophysical properties and relatively low abundance. Here, we introduce a strategy designed for rapid and efficient characterization of the human pulmonary ECM using the photocleavable surfactant Azo. We coupled this approach with trapped ion mobility MS with diaPASEF to maximize the depth of matrisome coverage. Using this strategy, we identify nearly 400 unique matrisome proteins with excellent reproducibility that are known to be important in lung biology, including key core matrisome proteins.

Impact of Surfactants on Cumulative Trypsin Activity in Bottom-Up Proteome Analysis.

Trypsin digestion plays a pivotal role in successful bottom-up peptide characterization and quantitation. While denaturants are often incorporated to enhance protein solubility, surfactants are recognized to inhibit enzyme activity. However, several reports have suggested that incorporating surfactants or other solvent additives may enhance digestion and MS detection. Here, we assess the impacts of ionic surfactants on cumulative trypsin activity and subsequently evaluate the total digestion efficiency of a proteome mixture by quantitative MS. Although low surfactant concentrations, such as 0.01% SDS or 0.2% SDC, significantly enhanced the initial trypsin activity (by 14 or 42%, respectively), time course assays revealed accelerated enzyme deactivation, evident by 10- or 40-fold reductions in trypsin activity half-life at these respective surfactant concentrations. Despite enhanced initial tryptic activity, quantitative MS analysis of a common liver proteome extract, digested with various surfactants (0.01 or 0.1% SDS, 0.5% SDC), consistently revealed decreased peptide counts and signal intensity, indicative of a lower digestion efficiency compared to a nonsurfactant control. Furthermore, including detergents for digestion did not improve the detection of membrane proteins, nor hydrophobic peptides. These results stress the importance of assessing cumulative enzyme activity when optimizing the digestion of a proteome mixture, particularly in the presence of denaturants.

Comparative biophysical study of clinical surfactants using constrained drop surfactometry.

Surfactant replacement therapy is crucial in managing neonatal respiratory distress syndrome (RDS). Currently licensed clinical surfactants in the United States and Europe, including Survanta, Infasurf, Curosurf, and Alveofact, are all derived from bovine or porcine sources. We conducted a comprehensive examination of the biophysical properties of these four clinical surfactant preparations under physiologically relevant conditions, using constrained drop surfactometry (CDS). The assessed biophysical properties included the adsorption rate, quasi-static and dynamic surface activity, resistance to surfactant inhibition by meconium, and the morphology of the adsorbed surfactant films. This comparative study unveiled distinct in vitro biophysical properties of these clinical surfactants and revealed correlations between their chemical composition, lateral film structure, and biophysical functionality. Notably, at 1 mg/mL, Survanta exhibited a significantly lower adsorption rate compared with the other preparations at the same surfactant concentration. At 10 mg/mL, Infasurf, Curosurf, and Survanta all demonstrated excellent dynamic surface activity, whereas Alveofact exhibited the poorest quasi-static and dynamic surface activity. The suboptimal surface activity of Alveofact is found to be correlated with its unique monolayer-predominant morphology, in contrast to other surfactants forming multilayers. Curosurf, in particular, showcased superior resistance to biophysical inhibition by meconium compared with other preparations. Understanding the diverse biophysical behaviors of clinical surfactants provides crucial insights for precision and personalized design in treating RDS and other respiratory conditions. The findings from this study contribute valuable perspectives for the development of more efficacious and fully synthetic surfactant preparations.NEW & NOTEWORTHY A thorough investigation into the biophysical properties of four animal-derived clinical surfactant preparations was conducted through constrained drop surfactometry under physiologically relevant conditions. This comparative study unveiled unique in vitro biophysical characteristics among these clinical surfactants, establishing correlations between their chemical composition, lateral film structure, and biophysical functionality. The acquired knowledge offers essential insights for the precise and personalized design of clinical surfactant for the treatment of respiratory distress syndrome and other respiratory conditions.

Poly(vinylpyrrolidone)-Enhanced CRISPR-Cas System for Robust Nucleic Acid Diagnostics.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has opened a new path for molecular diagnostics based on RNA programmed trans-cleavage activity. However, their accessibility for highly sensitive clinical diagnostics remains insufficient. In this study, we systematically investigated the impact of various surfactants on the CRISPR-Cas12a system and found that poly(vinylpyrrolidone) (PVP), a nonionic surfactant, showed the highest enhancement effect among these tested surfactants. Additionally, the enhancement effects of PVP are compatible and versatile to CRISPR-Cas12b and Cas13a systems, improving the sensitivity of these CRISPR-Cas systems toward synthetic targets by 1-2 orders of magnitude. By integrating the PVP-enhanced CRISPR system with isothermal nucleic acid amplification, both the two- and one-step assays exhibited comparable sensitivity and specificity to gold-standard quantitative polymerase chain reaction (qPCR) in the assay of clinical human papillomavirus (HPV) samples, thereby holding significant promise for advancing clinical diagnostics and biomedical research.

Amphiphilic Rhodamine Fluorescent Probes Combined with Basal Imaging for Fine Structures of the Cell Membrane.

Confocal fluorescence imaging of fine structures of the cell membrane is important for understanding their biofunctions but is often neglected due to the lack of an effective method. Herein, we develop new amphiphilic rhodamine fluorescent probe RMGs in combination with basal imaging for this purpose. The probes show high signal-to-noise ratio and brightness and low internalization rate, making them suitable for imaging the fine substructures of the cell membrane. Using the representative probe RMG3, we not only observed the cell pseudopodia and intercellular nanotubes but also monitored the formation of migrasomes in real time. More importantly, in-depth imaging studies on more cell lines revealed for the first time that hepatocellular carcinoma cells secreted much more adherent extracellular vesicles than other cell lines, which might serve as a potential indicator of liver cells. We believe that RMGs may be useful for investigating the fine structures of the cell membrane.

Quantification of Biopharmaceutically Relevant Nonionic Surfactant Excipients Using Benchtop qNMR.

Nonionic surfactant excipients (NISEs) are commonly added to biologics formulations to mitigate the effects of stress incurred by the active biotherapeutic during manufacturing, transport, and storage. During manufacturing, NISEs are added by dilution of a stock solution directly into a protein formulation, and their accurate addition is critical in maintaining the quality and integrity of the drug product and thus ensuring patient safety. This is especially true for the common NISEs, polysorbates 20 and 80 (PS20 and PS80, respectively) and poloxamer 188 (P188). With the increasing diversity of biologic modalities within modern pharmaceutical pipelines, there is thus a critical need to develop and deploy convenient and user-accessible analytical techniques that can rapidly and reliably quantify these NISEs under biopharmaceutically relevant conditions. We thus pursued 60 MHz benchtop quantitative NMR (qNMR) as a nondestructive and user-friendly analytical technique for the quantification of PS20, PS80, and P188 under such conditions. We demonstrated the ability of benchtop qNMR (1) to quantify simulated PS20, PS80, and P188 stock solutions representative of those used during the drug substance (DS) formulation step in biomanufacturing and (2) to quantify these NISEs at and below their target concentrations (≤0.025% w/v) directly in biologics formulations containing histidine, sucrose, and one of three biotherapeutic modalities (monoclonal antibody, antibody-drug conjugate, and Fc-fusion protein). Our results demonstrate that benchtop qNMR offers a fit-for-purpose, reliable, user-friendly, and green analytical route by which NISE of interest to the biopharmaceutical industry may be readily and reliably quantified. We conclude that benchtop qNMR has the potential to be applied to other excipient formulation components in the presence of various biological modalities as well as the potential for routine integration within analytical and QC laboratories across pharmaceutical development and manufacturing sites.

Thermoascus aurantiacus harbors an esterase/lipase that is highly activated by anionic surfactant.

Fungal lipolytic enzymes play crucial roles in various lipid bio-industry processes. Here, we elucidated the biochemical and structural characteristics of an unexplored fungal lipolytic enzyme (TaLip) from Thermoascus aurantiacus var. levisporus, a strain renowned for its significant industrial relevance in carbohydrate-active enzyme production. TaLip belongs to a poorly understood phylogenetic branch within the class 3 lipase family and prefers to hydrolyze mainly short-chain esters. Nonetheless, it also displays activity against natural long-chain triacylglycerols. Furthermore, our analyses revealed that the surfactant sodium dodecyl sulfate (SDS) enhances the hydrolytic activity of TaLip on pNP butyrate by up to 5.0-fold. Biophysical studies suggest that interactions with SDS may prevent TaLip aggregation, thereby preserving the integrity and stability of its monomeric form and improving its performance. These findings highlight the resilience of TaLip as a lipolytic enzyme capable of functioning in tandem with surfactants, offering an intriguing enzymatic model for further exploration of surfactant tolerance and activation in biotechnological applications.

Ultrathin Flexible Silica Nanosheets with Surface Chemistry-Modulated Affinity to Mammalian Cells.

Flexibility of nanomaterials is challenging but worthy to tune for biomedical applications. Biocompatible silica nanomaterials are under extensive exploration but are rarely observed to exhibit flexibility despite the polymeric nature. Herein, a facile one-step route is reported to ultrathin flexible silica nanosheets (NSs), whose low thickness and high diameter-to-thickness ratio enables folding. Thickness and diameter can be readily tuned to enable controlled flexibility. Mechanism study reveals that beyond the commonly used surfactant, the "uncommon" one bearing two hydrophobic tails play a guiding role in producing sheeted/layered/shelled structures, while addition of ethanol appropriately relieved the strong interfacial tension of the assembled surfactants, which will otherwise produce large curled sheeted structures. With these ultrathin NSs, it is further shown that the cellular preference for particle shape and rigidity is highly dependent on surface chemistry of nanoparticles: under high particle-cell affinity, NSs, and especially the flexible ones will be preferred by mammalian cells for internalization or attachment, while this preference is basically invalid when the affinity is low. Therefore, properties of the ultrathin silica NSs can be effectively expanded and empowered by surface chemistry to realize improved bio-sensing or drug delivery.

Biosensing with Oleosin-Stabilized Liquid Crystal Droplets.

Liquid crystals (LCs) are emerging as novel platforms for chemical, physical, and biological sensing. They can be used to detect biological amphiphiles such as lipids, fatty acids, digestive surfactants, and bacterial endotoxins. However, designing LC-based sensors in a manner that preserves their sensitivity and responsiveness to these stimuli, and possibly improves biocompatibility, remains challenging. In this work, the stabilization of LC droplets by oleosins, plant-sourced and highly surface active proteins due to their extended amphipathic helix, is investigated. Purified oleosins, at sub-micromolar concentrations, are shown to readily stabilize nematic LC droplets without switching their alignment, allowing them to detect surfactants at micromolar concentrations. Direct evidence of localization of oleosins at the LC-water interface is provided with fluorescent labeling, and the stabilized droplets remain stable over months. Interestingly, chiral LC droplets readily switch in the presence of nanomolar oleosin concentrations, an unexpected behavior that is explained by accounting for the energy barriers required for switching the alignment between the two cases. This leads thus to a twofold conclusion: oleosin-stabilized nematic LC droplets present a biocompatible alternative for bioanalyte detection, while chiral LCs can be further investigated for use as highly sensitive sensors for detecting amphipathic helices in biological systems.

Comparative Analysis of PEG-Free and PEG-Based Self-Emulsifying Drug Delivery Systems for Enhanced Oral Bioavailability of Therapeutic (Poly) Peptides.

This study aims to compare the potential of Polyethylene glycol (PEG-free and PEG-based self-emulsifying drug delivery systems (SEDDS) for the oral administration of insulin glargine (IG). Hydrophobic ion pairs (HIPs) of IG are formed using various counterions. HIPs are assessed for log P octanol/water and dissociation behavior. They are incorporated into SEDDS based on polyglycerol (PG) and zwitterionic surfactant (ZW) using response surface methodology and compared to conventional PEG-SEDDS in size, stability, and log D SEDDS/release medium. Oral IG bioavailability in PG/ZW-SEDDS and PEG-SEDDS is evaluated in rats. Among the various counterions studied, IG-BIS (bis(isotridecyl)sulfosuccinate) HIPs demonstrated the highest log P and an improved dissociation profile. PG/ZW-SEDDS and PEG-SEDDS have similar ≈40 nm sizes and are stable over 24 h. Both formulations have log D > 4 in water and >2 in 50 mM phosphate buffer pH 6.8. PG/ZW-SEDDS yielded an oral bioavailability of 2.13 ± 0.66% for IG, while the employment of PEG-SEDDS resulted in an oral bioavailability of 1.15 ± 0.35%. This study highlights the prospective utilization of PEG-free SEDDS involving the concurrent application of PG and ZW surfactants, an alternative to conventional PEG surfactants, for improved oral therapeutic (poly) peptide delivery.

An Assay for Immunogenic Detection of Anti-PEG Antibody.

With the increasing use of polyethylene glycol (PEG) based proteins and drug delivery systems, anti-PEG antibodies have commonly been detected among the population, causing the accelerated blood clearance and hypersensitivity reactions, poses potential risks to the clinical efficacy and safety of PEGylated drugs. Therefore, vigilant monitoring of anti-PEG antibodies is crucial for both research and clinical guidance regarding PEGylated drugs. The enzyme-linked immunosorbent assay (ELISA) is a common method for detecting anti-PEG antibodies. However, diverse coating methods, blocking solutions and washing solutions have been employed across different studies, and unsuitable use of Tween 20 as the surfactant even caused biased results. In this study, we established the optimal substrate coating conditions, and investigated the influence of various surfactants and blocking solutions on the detection accuracy. The findings revealed that incorporating 1 % bovine serum albumin into the serum dilution in the absence of surfactants will result the credible outcomes of anti-PEG antibody detection.

Electrokinetic transport of CTAB induces multiphasic behavior during capillary adsorption and desorption.

Cationic surfactant coatings (e.g., CTAB) are commonly used in CE to control EOF and thereby improve separation efficiencies. However, our understanding of surfactant adsorption and desorption dynamics under EOF conditions is limited. Here, we apply automated zeta potential analysis to study the adsorption and desorption kinetics of CTAB in a capillary under different transport conditions: diameter, length, voltage alternation pattern and frequency, and applied pressure. In contrast to other studies, we observe slower kinetics at distinct capillary wall zeta potential ranges. Within these ranges, which we call "stagnant regimes," the EOF mobility significantly counteracts the electrophoretic (EP) mobility of CTA+ and hinders the net transport. By constructing a numerical model to compare with our experiments and recasting our experimental data in terms of the net CTA+ transport volume normalized by surface area, we reveal that the EP mobility of CTA+ and the capillary surface-area-to-volume ratio dictate the zeta potential range and the duration of the stagnant regime and thereby govern the overall reaction kinetics. Our results indicate that further transport-oriented studies can significantly aid in the understanding and design of electrokinetic systems utilizing CTAB and other charged surfactants.

Advances in high-throughput screening approaches for biosurfactants: current trends, bottlenecks and perspectives.

The market size of biosurfactants (BSs) has been expanding at an extremely fast pace due to their broad application scope. Therefore, the re-construction of cell factories with modified genomic and metabolic profiles for desired industrial performance has been an intriguing aspect. Typical mutagenesis approaches generate huge mutant libraries, whereas a battery of specific, robust, and cost-effective high-throughput screening (HTS) methods is requisite to screen target strains for desired phenotypes. So far, only a few specialized HTS assays have been developed for BSs that were successfully applied to obtain anticipated mutants. The most important milestones to reach, however, continue to be: specificity, sensitivity, throughput, and the potential for automation. Here, we discuss important colorimetric and fluorometric HTS approaches for possible intervention on automated HTS platforms. Moreover, we explain current bottlenecks in developing specialized HTS platforms for screening high-yielding producers and discuss possible perspectives for addressing such challenges.

Cellular Uptake of Cell-Penetrating Peptides Activated by Amphiphilic p-Sulfonatocalix[4]arenes.

We report the synthesis of a series of amphiphilic p-sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4-Cn) and their application as efficient counterion activators for membrane transport of cell-penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO-K1, HCT 116, and KTC-1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4-Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure-activity relationships for calixarene-based counterion activators and show that CX4-Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4-C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP-counterion complexes into the cytosol and highlights the potential benefits of CX4-Cn for efficient and direct translocation of CPPs and CPP-conjugated cargo molecules into the cytosol of live cells.