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We show, via molecular simulations, that not only does cholesterol induce a lipid order, but the lipid order also enhances cholesterol localization within the lipid leaflets. Therefore, there is a strong interdependence between these two phenomena. In the ordered phase, cholesterol molecules are predominantly present in the bilayer leaflets and orient themselves parallel to the bilayer normal. In the disordered phase, cholesterol molecules are mainly present near the center of the bilayer at the midplane region and are oriented orthogonal to the bilayer normal. At the melting temperature of the lipid bilayers, cholesterol concentration in the leaflets and the bilayer midplane is equal. This result suggests that the localization of cholesterol in the lipid bilayers is mainly dictated by the degree of ordering of the lipid bilayer. We validate our findings on 18 different lipid bilayer systems, obtained from three different phospholipid bilayers with varying concentrations of cholesterol. To cover a large temperature range in simulations, we employ the Dry Martini force field. We demonstrate that the Dry and the Wet Martini (with polarizable water) force fields produce comparable results.

Understanding the principles that guide the uptake of engineered nanomaterials (ENMs) by cells is of interest in biomedical and occupational health research. While evidence has started to accumulate on the role of membrane proteins in ENM uptake, the role of membrane lipid chemistry in regulating ENM endocytosis has remained largely unexplored. Here, we have addressed this issue by altering the plasma membrane lipid composition directly in live cells using a methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange method. Our observations, in an alveolar epithelial cell line and using silica nanoparticles, reveal that the lipid composition of the plasma membrane outer leaflet plays a significant role in ENM endocytosis and the intracellular fate of ENMs, by affecting nonspecific ENM diffusion into the cell, changing membrane fluidity, and altering the activity of scavenger receptors (SRs) involved in active endocytosis. These results have implications for understanding ENM uptake in different subsets of cells, depending on cell membrane lipid composition.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores, whereby macrophages either aid in clearance or serve as a niche for its dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high order plasma membrane domains enriched in cholesterol and sphingomyelin) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages’ ability to phagocytose C. neoformans. We found that cholesterol or sphingomyelin depletion resulted in significantly deficient IgG-mediated phagocytosis of the fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency while a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable sphingomyelin (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis whereas raft-inhibiting conformation (cis-AzoSM) significantly blunted phagocytosis in a reversible manner. We observed that the effect on phagocytosis may be mediated by facilitating Fcγ receptor (FcγR) function, whereby IgG immune complexes cross-link to FcγRIII, resulting in tyrosine phosphorylation of FcR γ-subunit (FcRγ), an important accessory protein in the FcγR signaling cascade. Correspondingly, cholesterol or sphingomyelin depletion resulted in decreased FcRγ phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcRγ phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating FcγRIII-mediated phagocytosis of C. neoformans.

The increasing industrial and biomedical applications of nanomaterials have enhanced the need to educate a well-trained nanotechnology workforce. This need has led to efforts to introduce hands-on, nanotechnology-based, experimental modules into high school or college-level courses in science or engineering. However, the majority of such efforts have focused on nanoparticle synthesis techniques, and an equally important aspect of working with nanomaterials, i.e. nanoparticle characterization, has received less attention. Herein, we report a series of nanoparticle characterization experiments, as part of a newly developed "Nano and Biointerfaces" course, to familiarize upper undergraduate students as well as graduate students in chemical engineering with nanoparticle characterization techniques. An inquiry-based approach was used in that the composition and properties of nanoparticles were not revealed to the students beforehand and students were asked to perform experiments to characterize nanoparticle composition, size, morphology, and surface area. The results of these experiments were compared with certificates of analysis for particles, provided by the vendor, and the differences in measured properties were discussed. Assessment was performed through evaluation of laboratory memos and presentations, a question in the end of semester final exam, and a student survey. The modular nature of these experiments allows for them to be implemented, with modifications as needed, in other higher education institutions, or in high schools, to familiarize students with nanoparticle characterization.

The plasma membrane of eukaryotic cells is asymmetric with respect to its phospholipid composition. Analysis of the lipid composition of the outer leaflet is important for understanding cell membrane biology in health and disease. Here, a method based on cyclodextrin-mediated lipid exchange to characterize the phospholipids in the outer leaflet of red blood cells (RBCs) is reported. Methyl-α-cyclodextrin, loaded with exogenous lipids, was used to extract phospholipids from the membrane outer leaflet, while delivering lipids to the cell to maintain cell membrane integrity. Thin layer chromatography and lipidomics demonstrated that the extracted lipids were from the membrane outer leaflet. Phosphatidylcholines (PC) and sphingomyelins (SM) were the most abundant phospholipids in the RBCs outer leaflet with PC 34:1 and SM 34:1 being the most abundant species. Fluorescence quenching confirmed the delivery of exogenous lipids to the cell outer leaflet. The developed lipid exchange method was then used to remove phosphatidylserine, a phagocyte recognition marker, from the outer leaflet of senescent RBCs. Senescent RBCs with reconstituted membranes were phagocytosed in significantly lower amounts compared to control cells, demonstrating the efficiency of the lipid exchange process and its application in modifying cell–cell interactions.

Fluorescence-based techniques have been an integral factor in the study of cellular and model membranes. Fluorescence studies carried out on model membranes have provided valuable structural information and have helped reveal mechanistic detail regarding the formation and properties of ordered lipid domains, commonly known as lipid rafts. This chapter focuses on four techniques, based on fluorescence spectroscopy or microscopy, which are commonly used to analyze lipid rafts. The techniques described in this chapter may be used in a variety of ways and in combination with other techniques to provide valuable information regarding lipid order and domain formation, especially in model membranes.

The plasma membrane of eukaryotic cells is commonly believed to contain ordered lipid domains. The interest in understanding the origin of such domains has led to extensive studies on the phase behavior of mixed lipid systems. Three-component phase diagrams, composed of a high melting temperature (Tm) lipid, cholesterol, and a low Tm lipid have been valuable in studying lipid phase behavior. However, developing phase diagrams over the entire composition space and with precise tie-lines requires significant experimental effort. In this study, a machine learning approach was used to predict the Tm of lipids and generate phase diagrams from lipid mixtures. First, artificial neural network (ANN) was used for the prediction of Tm. The network was trained using available Tm data and was able to generate Tm values that closely matched literature results for its testing dataset. This model was then used to predict the Tm for lipids that have not yet been experimentally tested. Then, random forests (RF) and support vector machines (SVM) were trained and tested for their ability to predict a test three-component phase diagram. The model from the RF algorithm was able to generate a diagram that closely matched published results. This model was then used to generate phase diagrams for lipid mixtures at various temperatures and various degrees of unsaturation. This machine learning approach to the generation of lipid phase diagrams has the potential to save significant time and resources in studies of lipid phase behavior.

With the increasing industrial and biomedical applications of engineered nanomaterials (ENMs), concerns have been raised regarding the increased risk of exposure. Exposure to ENMs can potentially lead to adverse health effects including cell toxicity. The plasma membrane, a lipid bilayer surrounding all cells, is the first cellular entity that comes into contact with foreign particles, and membrane damage by ENMs is one of the potential mechanisms through which ENMs induce cytotoxicity. In recent years, significant effort has been focused on elucidating the complex interactions at the particle–plasma membrane interface. Such studies have primarily relied on membrane models to tease out what particle physicochemical properties might perturb the structure and function of the cell plasma membrane. However, the diversity of membrane models has made it difficult to translate the results obtained from studies with one model to another and ultimately to live cells. This review summarizes the current knowledge on ENM–plasma membrane interactions based on studies in membrane models including lipid monolayers, supported lipid bilayers, and lipid vesicles. The mechanisms of membrane disruption by the ENMs in each model have been discussed in detail and the role of the membrane model itself in modulating the results has been described. In addition, results in membrane models have been compared with the current knowledge about cells. Finally, some of the challenges toward improving the current membrane models and enhancing the environmental relevance of studies are discussed.

Plasma membrane damage is one of the primary mechanisms through which engineered nanoparticles induce cell toxicity. Over the past decade, a number of mechanistic studies have used membrane models to examine how nanoparticles alter membrane structure and integrity. However, the role of membrane lipid asymmetry, considering that the plasma membrane has a different lipid composition in the exofacial leaflet compared to the cytofacial leaflet, in regulating nanoparticle–membrane interactions has remained obscure. In the current study, the role of individual membrane leaflets in regulating the interactions of membrane models and erythrocytes with engineered silica nanoparticles (50 and 100 nm) was examined. It was found that silica nanoparticles bind to and disrupt synthetic vesicles mimicking the exofacial leaflet but not those mimicking the cytofacial leaflet of the erythrocyte plasma membrane. Forster resonance energy transfer (FRET) studies revealed that nanoparticles disrupted vesicle integrity by inducing pore formation in vesicles. Imaging giant unilamellar vesicles and further FRET experiments revealed that nanoparticles that disrupt membrane integrity show significant localization at the vesicle surface. Nanoparticles that disrupted vesicles mimicking the exofacial leaflet also induced hemolysis in erythrocytes, suggesting that the exofacial leaflet is the primary regulator of nanoparticle-induced membrane damage. This was confirmed by demonstrating that nanoparticles caused a similar disruptive behavior in symmetric and asymmetric vesicles, which had similar outer leaflet, but different inner leaflet lipid compositions. Together, these studies reveal that membrane lipid asymmetry plays a minor role in nanoparticle-induced membrane disruption with the exofacial leaflet being the primary regulator of interactions.

Understanding the mechanisms by which engineered nanomaterials disrupt the cell plasma membrane is crucial in advancing the industrial and biomedical applications of nanotechnology. While the role of nanoparticle properties in inducing membrane damage has received significant attention, the role of the lipid chemical structure in regulating such interactions is less explored. Here, we investigated the role of the lipid chemical structure in the disruption of lipid vesicles by unmodified silica, carboxyl-modified silica, and unmodified polystyrene nanoparticles (50 nm). The role of the lipid headgroup was examined by comparing nanoparticle effects on vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vs an inverse phosphocholine (PC) with the same acyl chain structure. The role of acyl chain saturation was examined by comparing nanoparticle effects on saturated vs unsaturated PCs and sphingomyelins. Nanoparticle effects on PCs (glycerol backbone) vs sphingomyelins (sphingosine backbone) were also examined. Results showed that the lipid headgroup, backbone, and acyl chain saturation affect nanoparticle binding to and disruption of the membranes. A low headgroup tilt angle and the presence of a trimethylammonium moiety at the vesicle surface are required for unmodified nanoparticles to induce membrane disruption. Lipid backbone structure significantly affects nanoparticle-membrane interactions, with carboxyl-modified particles only disrupting lipids containing cis unsaturation and a sphingosine backbone. Acyl chain saturation makes vesicles more resistant to particles by increasing lipid packing in vesicles, impeding molecular interactions. Finally, nanoparticles were capable of changing the lipid packing, resulting in pore formation in the process. These observations are important in interpreting nanoparticle toxicity to biological membranes.

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic cells is the loss of compositional asymmetry of the cell membrane, leading to the translocation of phosphatidylserine to the membrane outer leaflet. This process is triggered by increased intracellular concentration of Ca(2+), which activates scramblase, an enzyme that facilitates bidirectional movement of phospholipids between membrane leaflets. Given the importance of eryptosis in various diseased conditions, there have been efforts to induce eryptosis in vitro. Such efforts have generally relied on the calcium ionophore, ionomycin, to enhance intracellular Ca(2+) concentration and induce eryptosis. However, many discrepancies have been reported in the literature regarding the procedure for inducing eryptosis using ionomycin. Herein, we report a step-by-step protocol for ionomycin-induced eryptosis in human erythrocytes. We focus on important steps in the procedure including the ionophore concentration, incubation time, and glucose depletion, and provide representative result. This protocol can be used to reproducibly induce eryptosis in the laboratory.

Despite a number of fundamental studies, the interactions of engineered nanoparticles with the cell plasma membrane are still not well understood. Membrane models, such as lipid monolayers and bilayers, have been used to provide a mechanistic understanding of nanoparticle effects on the structure and integrity of the cell membrane. However, the role of the membrane model itself in regulating nanoparticle–lipid interactions has generally been overlooked. In an effort to elucidate how changing the membrane model affects the resulting interactions with nanoparticles, the current study investigates the interaction of silica nanoparticles (104 ± 5 nm) with different surface-functional groups: plain (silanol), amine, and poly(ethylene glycol) (PEG), with various molecular weights of 2k, 5k, and 20k Daltons, with lipid monolayers and bilayers of the same composition. Studies with lipid monolayers demonstrated that PEGylated particles, regardless of the PEG molecular weight, and unlike plain and amine-modified particles, were able to penetrate the monolayer and disrupt the structure of lipids as evidenced by tensiometry and atomic force microscopy. In contrast, when interacting with lipid bilayers in suspension (i.e. vesicles), plain, amine-modified, and PEG 20k-modified particles disrupted vesicle structure, causing significant leakage, while PEG 2k- and 5k-modified particles did not affect the integrity of the vesicles. In summary, while the interactions of silica nanoparticles with membrane models is dependent on particle surface properties, a clear difference is observed in particle interactions with lipid monolayers compared to bilayers.

Background Despite their growing popularity, the potential respiratory toxicity of electronic cigarettes (e-cigarettes) remains largely unknown. One potential aspect of e-cigarette toxicity is the effect of e-cigarette vapor on lung surfactant function. Lung surfactant is a mixture of lipids and proteins that lines the alveolar region. The surfactant layer reduces the surface tension of the alveolar fluid, thereby playing a crucial role in lung stability. Due to their small size, particulates in e-cigarette vapor can penetrate the deep lungs and come into contact with the lung surfactant. The current study sought to examine the potential adverse effects of e-cigarette vapor and conventional cigarette smoke on lung surfactant interfacial properties. Methods Infasurf®, a clinically used and commercially available calf lung surfactant extract, was used as lung surfactant model. Infasurf® films were spread on top of an aqueous subphase in a Langmuir trough with smoke particulates from conventional cigarettes or vapor from different flavors of e-cigarettes dispersed in the subphase. Surfactant interfacial properties were measured in real-time upon surface compression while surfactant lateral structure after exposure to smoke or vapor was examined using atomic force microscopy (AFM). Results E-cigarette vapor regardless of the dose and flavoring of the e-liquid did not affect surfactant interfacial properties. In contrast, smoke from conventional cigarettes had a drastic, dose-dependent effect on Infasurf® interfacial properties reducing the maximum surface pressure from 65.1 ± 0.2 mN/m to 46.1 ± 1.3 mN/m at the highest dose. Cigarette smoke and e-cigarette vapor both altered surfactant microstructure resulting in an increase in the area of lipid multilayers. Studies with individual smoke components revealed that tar was the smoke component most disruptive to surfactant function. Conclusions While both e-cigarette vapor and conventional cigarette smoke affect surfactant lateral structure, only cigarette smoke disrupts surfactant interfacial properties. The surfactant inhibitory compound in conventional cigarettes is tar, which is a product of burning and is thus absent in e-cigarette vapor.

Fungal glucosylceramide (GlcCer) is a plasma membrane sphingolipid in which the sphingosine backbone is unsaturated in carbon position 8 (C8) and methylated in carbon position 9 (C9). Studies in the fungal pathogen, Cryptococcus neoformans, have shown that loss of GlcCer synthase activity results in complete loss of virulence in the mouse model. However, whether the loss of virulence is due to the lack of the enzyme or to the loss of the sphingolipid is not known. In this study, we used genetic engineering to alter the chemical structure of fungal GlcCer and studied its effect on fungal growth and pathogenicity. Here we show that unsaturation in C8 and methylation in C9 is required for virulence in the mouse model without affecting fungal growth in vitro or common virulence factors. However, changes in GlcCer structure led to a dramatic susceptibility to membrane stressors resulting in increased cell membrane permeability and rendering the fungal mutant unable to grow within host macrophages. Biophysical studies using synthetic vesicles containing GlcCer revealed that the saturated and unmethylated sphingolipid formed vesicles with higher lipid order that were more likely to phase separate into ordered domains. Taken together, these studies show for the first time that a specific structure of GlcCer is a major regulator of membrane permeability required for fungal pathogenicity.

INTRODUCTION: Invasion of other tissues during bloodborne metastasis in part requires adhesion of cancer cells to vascular endothelium by specific fluid shear-dependent receptor–ligand interactions. This study investigates the hypothesis that the adhesion is mediated by ligands shared between endothelial E-selectin and Galectin-1 (Gal-1), both of which are upregulated during inflammation and cancer. METHODS: Flow chamber adhesion and dynamic biochemical tissue analysis (DBTA) assays were used to evaluate whether Gal-1 modulates E-selectin adhesive interactions of breast cancer cells and tissues under dynamic flow conditions, while immunocytochemistry, immunohistochemistry, western blotting, and fluorescence anisotropy were used to study molecular interactions under static conditions. RESULTS: Dynamic adhesion assays revealed a shear-dependent binding interaction between Gal-1hFc treated breast cancer cells and tissues and E-selectin-coated beads, causing ~ 300% binding increase of the beads compared to negative controls. Immunocyto- and immunohistochemical analyses showed that Gal-1 and E-selectin fluorescent signals colocalized on cells and tissues at ~ 75% for each assay. Immunoprecipitation and Western blotting of Mac-2BP from breast cancer cell lysates revealed that Gal-1 and E-selectin share Mac-2BP as a ligand, while fluorescence anisotropy and circulating tumor cell model systems exhibited competitive or antagonistic binding between Gal-1 and E-selectin for shared ligands, including Mac-2BP. Furthermore, Mac-2BP functional blockade inhibited the effects of Gal-1 on E-selectin binding. CONCLUSIONS: In summary, this investigation reveals a shear-dependent interaction between E-selectin and Gal-1 that may be due to intermediation by a similar or shared ligand(s), including Mac-2BP, which may provide a rational basis for development of novel diagnostics or therapeutics for breast cancer.

The number of deaths from cryptococcal meningitis is around 180,000 per year. The disease is the second leading cause of mortality among individuals with AIDS. Antifungal treatment is costly and associated with adverse effects and resistance, evidencing the urgency of development of both therapeutic and prophylactic tools. Here we demonstrate the key roles of polysaccharide- and glycolipid-containing structures in a vaccination model to prevent cryptococcosis., Cryptococcus neoformans is an encapsulated fungal pathogen that causes meningoencephalitis. There are no prophylactic tools for cryptococcosis. Previously, our group showed that a C. neoformans mutant lacking the gene encoding sterylglucosidase (Δsgl1) induced protection in both immunocompetent and immunocompromised murine models of cryptococcosis. Since sterylglucosidase catalyzes degradation of sterylglucosides (SGs), accumulation of this glycolipid could be responsible for protective immunity. In this study, we analyzed whether the activity of SGs is sufficient for the protective effect induced by the Δsgl1 strain. We observed that the accumulation of SGs impacted several properties of the main polysaccharide that composes the fungal capsule, glucuronoxylomannan (GXM). We therefore used genetic manipulation to delete the SGL1 gene in the acapsular mutant Δcap59 to generate a double mutant (strain Δcap59/Δsgl1) that was shown to be nonpathogenic and cleared from the lung of mice within 7 days post-intranasal infection. The inflammatory immune response triggered by the Δcap59/Δsgl1 mutant in the lung differed from the response seen with the other strains. The double mutant did not induce protection in a vaccination model, suggesting that SG-related protection requires the main capsular polysaccharide. Finally, GXM-containing extracellular vesicles (EVs) enriched in SGs delayed the acute lethality of Galleria mellonella against C. neoformans infection. These studies highlighted a key role for GXM and SGs in inducing protection against a secondary cryptococcal infection, and, since EVs notoriously contain GXM, these results suggest the potential use of Δsgl1 EVs as a vaccination strategy for cryptococcosis.

Antarctic notothenioids are noted for extreme stenothermy, yet underpinnings of their thermal limits are not fully understood. We hypothesized that properties of ventricular membranes could explain previously observed differences among notothenioids in temperature onset of cardiac arrhythmias and persistent asystole. Microsomes were prepared using ventricles from six species of notothenioids, including four species from the hemoglobin-less (Hb−) family Channichthyidae (icefishes), which also differentially express cardiac myoglobin (Mb), and two species from the (Hb+) Nototheniidae. We determined membrane fluidity and structural integrity by quantifying fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and leakage of 5(6)-carboxyfluorescein, respectively, over a temperature range from ambient (0 °C) to 20 °C. Compositions of membrane phospholipids and cholesterol contents were also quantified. Membranes from all four species of icefishes exhibited greater fluidity than membranes from the red-blooded species N. coriiceps. Thermal sensitivity of fluidity did not vary among species. The greatest thermal sensitivity to leakage occurred between 0 and 5 °C for all species, while membranes from the icefish, Chaenocephalus aceratus (Hb−/Mb−) displayed leakage that was nearly 1.5-fold greater than leakage in N. coriiceps (Hb+/Mb+). Contents of phosphatidylethanolamine (PE) were approximately 1.5-fold greater in icefishes than in red-blooded fishes, and phospholipids had a higher degree of unsaturation in icefishes than in Hb + notothenioids. Cholesterol contents were lowest in Champsocephalus gunnari (Hb−/Mb−) and highest in the two Hb+/Mb + species, G. gibberifrons and N. coriiceps. Our results reveal marked differences in membrane properties and indicate a breach in membrane fluidity and structural integrity at a lower temperature in icefishes than in red-blooded notothenioids.

Disruption of plasma membrane integrity is a primary mechanism of nanoparticle toxicity in cells. Mechanistic studies on nanoparticle-induced membrane damage have been commonly performed using model membranes with a focus on symmetric bilayers, overlooking the fact that the membrane has an asymmetric phospholipid composition. In this study, erythrocytes with normal and scrambled membrane asymmetry were utilized to examine how the loss of membrane asymmetry and the resulting alterations in the outer leaflet lipid composition affect nanoparticle-membrane interactions. Unmodified, amine-modified, and carboxyl-modified silica (30 nm) were used as nanoparticle models. Loss of membrane asymmetry was achieved by induction of eryptosis, using a calcium ionophore. Erythrocyte membrane disruption (hemolysis) by unmodified silica nanoparticles was significantly reduced in eryptotic compared to healthy cells. Amine- and carboxyl-modified particles did not cause hemolysis in either cell. In agreement, a significant reduction in the binding of unmodified silica nanoparticles to the membrane was observed upon loss of membrane asymmetry. Unmodified silica particles also caused significant cell deformation, changing healthy erythrocytes into a spheroid shape. In agreement with findings in the cells, unmodified particles disrupted vesicles mimicking the erythrocyte outer leaflet lipid composition. The degree of disruption and nanoparticle binding to the membrane was reduced in vesicles mimicking the composition of scrambled membranes. Cryo-electron microscopy revealed the presence of lipid layers on particle surfaces, pointing to lipid adsorption as the mechanism for vesicle damage. Together, findings indicate an important role for the lipid composition of the membrane outer leaflet in nanoparticle-induced membrane damage in both vesicles and erythrocytes.

We asked young scientists this question: How do broad interests benefit your science? Scientists with a variety of hobbies responded that their extracurricular activities have enhanced a wide range of skills, from creativity to communication to resilience. Many also mentioned the value of clearing their minds and relaxing. Follow NextGen and share your own hobbies on Twitter with #NextGenSci. As a rock climber, you have to risk falling in order to become better ; the same principle applies in science. Realizing this has made me look at research failures in a more positive light—less as a demonstration of incompetence, and more of an opportunity to learn something and build resilience. I've learned that reminding yourself of your own competence (on the wall or in the lab) is a useful way to build up the courage to take more risks.

Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM., IMPORTANCE Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

There has been considerable evidence in recent years suggesting that plasma membrane lipids are important regulators of fungal pathogenicity. Various glycolipids have been shown to impart virulent properties in several fungal species, while others have been shown to play a role in host defense. In addition to their role as virulence factors, lipids also contribute to other virulence mechanisms such as drug resistance, biofilm formation, and release of extracellular vesicles. In addition, lipids also affect the mechanical properties of the plasma membrane through the formation of packed microdomains composed mainly of sphingolipids and sterols. Changes in the composition of lipid microdomains have been shown to disrupt the localization of virulence factors and affect fungal pathogenicity. This review gathers evidence on the various roles of plasma membrane lipids in fungal virulence and how lipids might contribute to the different processes that occur during infection and treatment. Insight into the role of lipids in fungal virulence can lead to an improved understanding of the process of fungal pathogenesis and the development of new lipid-mediated therapeutic strategies.

Cryptococcus neoformans is a fungal pathogen that causes pulmonary infections, which may progress into life-threatening meningitis. In commonly used mouse models of C. neoformans infections, fungal cells are not contained in the lungs, resulting in dissemination to the brain. We have previously reported the generation of an engineered C. neoformans strain ( C. neoformans Δ gcs1 ) which can be contained in lung granulomas in the mouse model and have shown that granuloma formation is dependent upon the enzyme sphingosine kinase 1 (SK1) and its product, sphingosine 1-phosphate (S1P). In this study, we have used four mouse models, CBA/J and C57BL6/J (both immunocompetent), Tgε26 (an isogenic strain of strain CBA/J lacking T and NK cells), and SK −/− (an isogenic strain of strain C57BL6/J lacking SK1), to investigate how the granulomatous response and SK1-S1P pathway are interrelated during C. neoformans infections. S1P and monocyte chemotactic protein-1 (MCP-1) levels were significantly elevated in the bronchoalveolar lavage fluid of all mice infected with C. neoformans Δ gcs1 but not in mice infected with the C. neoformans wild type. SK1 −/− mice did not show elevated levels of S1P or MCP-1. Primary neutrophils isolated from SK1 −/− mice showed impaired antifungal activity that could be restored by the addition of extracellular S1P. In addition, high levels of tumor necrosis factor alpha were found in the mice infected with C. neoformans Δ gcs1 in comparison to the levels found in mice infected with the C. neoformans wild type, and their levels were also dependent on the SK1-S1P pathway. Taken together, these results suggest that the SK1-S1P pathway promotes host defense against C. neoformans infections by regulating cytokine levels, promoting extracellular killing by phagocytes, and generating a granulomatous response.

Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans.

The function of dipalmitoyl phosphatidylcholine (DPPC) Langmuir monolayers was studied after exposure to various concentrations of 200 nm carboxyl-modified polystyrene particles by a combination of surface pressure and surfactant microstructure studies. The presence of particles in the subphase at the lowest concentrations tested (10 −5 to 10 −4 g/L) did not influence the π – A isotherms. However, at the highest concentration (10 −3 g/L), changes in the hysteresis areas of the isotherms were observed. The formation of LC domains during compression was significantly altered by the presence of the particles, resulting in the formation of smaller but more numerous domains. Fluorescence and atomic force microscopy images suggested that particles remained in the subphase but were closely associated with the condensed domains.

A novel amino acid supplementation strategy was developed for enhancing the production of IL-2 (interleukin-2; as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2) in fed-batch highcell-density cultures. The amino acids most needed and their amounts were determined using a stoichiometric model, and full factorial design experiments were conducted to determine the effects of single amino acids and amino acid mixtures on production. One of the most effective amino acid mixtures was found to be leucine, aspartic acid and glycine. This amino acid mixture was utilized for the production of IL-2 in batch and fed-batch fermentations. The amount of IL-2 produced increased from 403 to 722 mg/l and from 5.15 5 10 3 to 8.08 x 10 3 mg/l in batch and fed-batch cultures respectively. The results also revealed that the above amino acid mixture specifically increases IL-2 concentration in the cells.

Despite numerous advances in cancer research, lung cancer has remained the major cause of cancer-related mortality worldwide, mainly because of metastasis and the lack of curative systemic therapy. It has been well established that lipid rafts, the membrane microdomains containing cholesterol and glycosphingolipids, play an important role in cell adhesion and spreading. Notably, there are elevated levels of cholesterol-rich lipid rafts observed in cancer cells relative to normal ones. Thus, alterations in lipid rafts, such as through cholesterol depletion, have been considered as a strategy for cancer metastasis prevention. In this study three different treatments - methyl-beta-cyclodextrin (mβCD), sphingomyelinase (smase), and simvastatin (simva) - were used to investigate the effects of cholesterol depletion on the mechanics of adhesion and rolling velocities of multiple non-small cell lung cancer (NSCLC) cell lines (H1299, H23, H460, and A569) and a small cell lung cancer (SCLC) cell line (SHP-77) on E-selectin, a vascular endothelial molecule hypothesized to initiate recruitment of circulating tumor cells at metastatic sites. Under physiological conditions in a parallel plate chamber, it was observed that the number of adherent NSCLC cells significantly decreased through mβCD and simva treatments, while smase treatment did not show significant effect on those cells. In addition, only for NSCLC cells, significant increases in rolling velocities were detected after treatments with mβCD and simva, but not via smase. In contrast, cholesterol depletion did not affect the number of attached cells and rolling velocity of the SCLC cells. Our results show that cholesterol might regulate E-selectin-mediated adhesion of NSCLC cells to endothelium, most likely by disturbing functional E-selectin ligands expressed on the surface of the cancer cells. We have previously reported that CD44 expressed on colon cancer and breast cancer cells are functional E-selectin ligands that mediate cell adhesion and rolling on endothelium. Protein structure analysis by flow cytometry and western blot showed that all NSCLC cell lines strongly expressed CD44. After the cholesterol depletion through the various treatments, the molecular surface expression of CD44 was down regulated for all these cells. In contrast, the SCLC line did not show any expression of CD44. Although further studies must be performed to verify CD44 function as an E-selectin ligand, the data in total imply protein loss or absence due to the defection of the cell membrane integrity could impact cell adhesion and rolling, and consequently metastasis. Therefore, cholesterol depletion could have the potential to help prevent cancer metastasis through down regulation of relevant adhesion molecules. Citation Format: Amina Mohammadalipour, Christian Showalter, Harrison T. Muturi, Vishva Sharma, Amir M. Farnoud, Vishwajeet Puri, Sonia M. Najjar, Monica M. Burdick. Cell membrane cholesterol modulates lung cancer cell adhesion and rolling on E-selectin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 98.

Development of inexpensive and simple culture media and appropriate induction conditions are always favorable for industry. In this research, chemical composition and stoichiometric data for gamma-interferon production and recombinant Escherichia coli growth were used in order to achieve a simple medium and favorable induction conditions. To achieve this goal, the effects of medium composition and induction conditions on the production of gamma-interferon were investigated in batch culture of E. coli BL21 (DE3) [pET3a-ifngamma]. These conditions were considered as suitable conditions for the production of gamma-interferon: 2.5x M9 medium, supplemented with a mixture of amino acids (milligram per liter), including glutamic acid 215, aspartic acid 250, lysine 160, and phenylalanine 90, and induction at late-log phase (OD(600) = 4.5). Under these conditions, dry cell weight of 6 +/- 0.2 g/l and gamma-interferon concentration of 2.15 +/- 0.1 g/l were obtained. Later, without changing the concentration ratio of amino acids and glucose, the effect of increase in the primary glucose concentration on productivity of gamma-interferon was investigated. It was found that 25 g/l glucose will result in maximum attainable biomass and recombinant human gamma-interferon. At improved conditions, a dry cell weight of 14 +/- 0.2 g/l, concentration and overall productivity of gamma-interferon 4.2 +/- 0.1 g/l and 420 +/- 10 mg/l h, respectively, were obtained.

In order to increase the productivity of human IL-2 (interleukin-2), a stoichiometric model has been used to determine the most essential amino acids and precise values of their amounts to be added to the culture during expression of human IL-2 (as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2). Experiments were performed to investigate the effect of chosen amino acids and their interactions on expression of human IL-2. Glutamine, a mixture of leucine, aspartic acid and glycine, and a mixture of leucine, glutamine and aspartic acid, were the most effective for the expression of IL-2. The most promising amino acids were then chosen for further experiments at three different levels to determine whether altering their stoichiometry can lead to better expression levels. The optimized value of glutamine in the flask was 0.316 g/l; a mixture of leucine, glutamine and aspartic acid at concentrations of 0.124, 0.316 and 0.212 g/l respectively and of leucine, aspartic acid and glycine in concentrations of 0.124, 0.212, 0.111 g/l respectively were chosen to be added to the flask. The effect of glutamine, as one of the amino acids most influencing the expression of IL-2 in batch and fed-batch high-cell-density cultures, was studied. The results revealed that the amount of expressed IL-2 compared with the control culture increased from 81 to 195 mg/l in the shake flask, 403 to 594 mg/l in the fermentor and 5.15 to 10.01 g/l in the fermentor under fed-batch cultivation.

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.

Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [ N ′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo- N ′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5 , COS111 , MKK1 , and STE2 , which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. IMPORTANCE Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

The lipid bilayer of the plasma membrane is thought to be compartmentalized by the presence of lipid-protein microdomains. In eukaryotic cells, microdomains composed of sterols and sphingolipids packed in a liquid-ordered state, commonly known as lipid rafts, are believed to exist. While less studied in bacterial cells, reports on the presence of sterol or protein-mediated microdomains in bacterial cell membranes are also appearing with increasing frequency. Recent efforts have been focused on addressing the biophysical and biochemical properties of lipid rafts. However, most studies have been focused on synthetic membranes, mammalian cells, and/or model, non-pathogenic microorganisms. Much less is known about microdomains in the plasma membrane of pathogenic microorganisms. This review attempts to provide an overview of the current state of knowledge of lipid rafts in pathogenic fungi and the developing field of microdomains in pathogenic bacteria. The current literature on the structure and function and of microdomains is reviewed and the potential role of microdomains in growth, pathogenesis, and drug resistance of pathogens are discussed. Better insight into the structure and function of membrane microdomains in pathogenic microorganisms might lead to a better understanding of the process of pathogenesis and development of raft-mediated approaches for new methods of therapy.

Background: Recent studies have shown that colloidal particles can disrupt the interfacial properties of lung surfactant and thus key functional abilities of lung surfactant. However, the mechanisms underlying the interactions between aerosols and surfactant films remain poorly understood, as our ability to expose films to particles via the aerosol route has been limited. The aim of this study was to develop a method to reproducibly apply aerosols with a quantifiable particle dose on lung surfactant films and investigate particle-induced changes to the interfacial properties of the surfactant under conditions that more closely mimic those in vivo. Methods: Films of DPPC and Infasurf® were exposed to aerosols containing polystyrene particles generated using a Dry Powder Insufflator™. The dose of particles deposited on surfactant films was determined via light absorbance. The interfacial properties of the surfactant were studied using a Langmuir-Wilhelmy balance during surfactant compression to fil...