Advances in Immunotherapy for the Treatment of Adult Glioblastoma: Overcoming Chemical and Physical Barriers.

Glioblastoma, or glioblastoma multiforme (GBM, WHO Grade IV), is a highly aggressive adult glioma. Despite extensive efforts to improve treatment, the current standard-of-care (SOC) regimen, which consists of maximal resection, radiotherapy, and temozolomide (TMZ), achieves only a 12-15 month survival. The clinical improvements achieved through immunotherapy in several extracranial solid tumors, including non-small-cell lung cancer, melanoma, and non-Hodgkin lymphoma, inspired investigations to pursue various immunotherapeutic interventions in adult glioblastoma patients. Despite some encouraging reports from preclinical and early-stage clinical trials, none of the tested agents have been convincing in Phase III clinical trials. One, but not the only, factor that is accountable for the slow progress is the blood-brain barrier, which prevents most antitumor drugs from reaching the target in appreciable amounts. Herein, we review the current state of immunotherapy in glioblastoma and discuss the significant challenges that prevent advancement. We also provide thoughts on steps that may be taken to remediate these challenges, including the application of ultrasound technologies.

Interaction of Two Amphipathic α-Helix Bundle Proteins, ApoLp-III and ApoE 3, with the Oil-Aqueous Interface.

Protein-lipid interactions govern the structure and function of lipoprotein particles, which transport neutral lipids and other hydrophobic cargo through the blood stream. Apolipoproteins cover the surface of lipoprotein particles, including low-density (LDL) and high-density (HDL) lipoproteins, and determine their function. Previous work has focused on small peptides derived from these apolipoproteins or used such artificial lipid systems as Langmuir monolayers or the lipid disc assay to determine how apolipoproteins interact with the neutral lipid interface. Here, we focus on a recurring protein domain found in many neutral lipid-binding proteins, the amphipathic α-helix bundle. We use liquid droplet tensiometry to investigate protein-lipid interactions on an oil droplet, which mimics the real lipoprotein interface. The N-terminus of apoE 3 and full-length apoLp-III serve as model proteins. We find that each protein interacts with lipid monolayers at the oil-aqueous interface in unique ways. For the first time, we show that helix bundle unfolding is critical for proper protein insertion into the lipid monolayer at the oil-aqueous interface and that specific membrane lipids promote the rebinding of protein upon fluctuation in droplet size. These results shed new light on how amphipathic apolipoprotein α-helix bundles interact with neutral lipid particles.

The Spo7 sequence LLI is required for Nem1-Spo7/Pah1 phosphatase cascade function in yeast lipid metabolism.

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase that catalyzes the dephosphory-lation of Pah1 phosphatidate phosphatase, required for its translocation to the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7/Pah1 phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of the Nem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54-56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7Δ mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo Consistent with the role of the Nem1-Spo7 complex in activating the function of Pah1, we found that the mutational effects of the Spo7 LLI sequence were on the Nem1-Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1-Spo7 complex formation and its role in the phosphatase cascade that regulates the function of Pah1 phosphatidate phosphatase.

Protein kinase C mediates the phosphorylation of the Nem1-Spo7 protein phosphatase complex in yeast.

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase required for the nuclear/endoplasmic reticulum membrane localization of Pah1, a phosphatidate phosphatase that produces diacylglycerol for triacylglycerol synthesis at the expense of phospholipid synthesis. In a previous study, we showed that the protein phosphatase is subject to phosphorylation by protein kinase A (PKA). Here, we demonstrate that Nem1-Spo7 is regulated through its phosphorylation by protein kinase C (PKC), which plays multiple roles, including the regulation of lipid synthesis and cell wall integrity. Phosphorylation analyses of Nem1-Spo7 and its synthetic peptides indicate that both subunits of the complex are bona fide PKC substrates. Site-directed mutagenesis of NEM1 and SPO7, coupled with phosphopeptide mapping and immunoblotting with a phosphoserine-specific PKC substrate antibody, revealed that Ser-201 in Nem1 and Ser-22/Ser-28 in Spo7 are major PKC target sites of phosphorylation. Activity analysis of mutant Nem1-Spo7 complexes indicates that the PKC phosphorylation of Nem1 exerts a stimulatory effect, but the phosphorylation of Spo7 has no effect. Lipid-labeling analysis of cells expressing the phosphorylation-deficient alleles of NEM1 and SPO7 indicates that the stimulation of the Nem1-Spo7 activity has the effect of increasing triacylglycerol synthesis. Prephosphorylation of Nem1-Spo7 by PKC inhibits the PKA phosphorylation of Nem1, whereas prephosphorylation of the phosphatase complex by PKA inhibits the PKC phosphorylation of Spo7. Collectively, this work advances the understanding of the Nem1-Spo7 regulation by phosphorylation and its impact on lipid synthesis.

Analysis of induced error by susceptibility effect in low-density gel dosimeters.

PURPOSE: In low-density (LD) gel dosimeter, diffusive spin-spin relaxation rate (R2)-dispersion caused by susceptibility-induced internal gradient leads to a significant deviation in the measured R2 from the real value. In this study, the effect of induced internal gradient on R2 was visualized and quantified algebraically as an important cause of inaccuracy in LD gel dosimeters. MATERIALS AND METHODS: In this method, two sets of LD and unit-density (UD) gel dosimeters were prepared. The LD gel was made by mixing the UD gel with expanded polystyrene spheres. The R2 was used to determine the spatially resolved decay rates due to diffusion in internal magnetic field. The internal gradient was calculated for a multiple spin-echo sequence. RESULTS: It is shown that in a LD gel, the internal gradient leads to overestimation of mean R2 value (R2mean). Pixel-by-pixel R2 measurements inside a LD gel showed significant deviation from R2 mapping in UD gel. CONCLUSION: It appears that significant differences between R2mean in a selected region of interest and pixel-by-pixel R2 values are the main source of inaccuracy in dose mapping of a LD gel.

A novel quantification method for low-density gel dosimeter.

AIM: Low signal-to-noise ratio (SNR) images of lung-like (low-density [LD]) gel dosimeters, compared to unit-density (UD) gels, necessitate the use of different quantification methods. SETTING AND DESIGN: In this study, a new method is introduced based on noise correction and exponential (NCEXP) fitting. The feasibility of NCEXP method for quantifying dose absorption in LD gels is evaluated. MATERIALS AND METHODS: Sensitivity, dose resolution, detectable dynamic range, and correlation of the calibration curve for both UD and LD gel dosimeters are the parameters, which we analyze to investigate the consequences of new method. Results of NCEXP method are compared to maximum likelihood estimation of rician distribution (MLE-R) and variable echo number (VAREC) quantification methods. RESULTS: Dose response of LD gel dosimeter shows wider detectable dynamic range as compared to UD gel. Using NCEXP method for both LD and UD dosimeter gels, a more sensitive calibration curve with a superior dose resolution is obtained. The advantage of new quantification method is more significant for LD dosimeter gel analysis, where SNR decreases as a result of higher absorbed doses (≥10 Gy). Despite the inverse effect of the VAREC method on detectable dose range of UD gel, no specific changes are observed in dynamic dose range of LD gel dosimeter with different quantification methods. The correlations obtained with different methods were approximately of the same order for UD and LD gels. CONCLUSION: NCEXP method seems to be more effective than the MLE-R and VAREC methods for quantification of LD dosimeter gel, especially where high-dose absorption and steep-dose gradients exist such as those in intensity-modulated radiation therapy and stereotactic radiosurgery.

Interaction of a model apolipoprotein, apoLp-III, with an oil-phospholipid interface.

Lipid droplets are "small" organelles that play an important role in de novo synthesis of new membrane, and steroid hormones, as well as in energy storage. The way proteins interact specifically with the oil-(phospho-)lipid monolayer interface of lipid droplets is a relatively unexplored but crucial question. Here, we use our home built liquid droplet tensiometer to mimic intracellular lipid droplets and study protein-lipid interactions at this interface. As model neutral lipid binding protein, we use apoLp-III, an amphipathic α-helix bundle protein. This domain is also found in proteins from the perilipin family and in apoE. Protein binding to the monolayer is studied by the decrease in the oil/water surface tension. Previous work used POPC (one of the major lipids found on lipid droplets) to form the phospholipid monolayer on the triolein surface. Here we expand this work by incorporating other lipids with different physico-chemical properties to study the effect of charge and lipid head-group size. This study sheds light on the affinity of this important protein domain to interact with lipids.

Chiral nematic liquid crystal microlenses.

Nematic liquid crystals (NLCs) of achiral molecules and racemic mixtures of chiral ones form flat films and show uniform textures between circular polarizers when suspended in sub-millimeter size grids and immersed in water. On addition of chiral dopants to the liquid crystal, the films exhibit optical textures with concentric ring patterns and radial variation of the birefringence color. Both are related to a biconvex shape of the chiral liquid crystal film; the rings are due to interference. The curvature radii of the biconvex lens array are in the range of a few millimeters. This curvature leads to a radial variation of the optical axis along the plane of the film. Such a Pancharatnam-type phase lens dominates the imaging and explains the measured focal length of about one millimeter. To our knowledge, these are the first spontaneously formed Pancharatnam devices. The unwinding of the helical structure at the grid walls drives the lens shape. The relation between the lens curvature and material properties such as helical pitch, the twist elastic constant, and the interfacial tensions, is derived. This simple, novel method for spontaneously forming microlens arrays can also be used for various sensors.

Insertion of perilipin 3 into a glycero(phospho)lipid monolayer depends on lipid headgroup and acyl chain species.

Lipid droplets (LDs) are organelles that contribute to various cellular functions that are vital for life. Aside from acting as a neutral lipid storage depot, they are also involved in building new membranes, synthesis of steroid hormones, and cell signaling. Many aspects of LD structure and function are not yet well-understood. Here we investigate the interaction of perilipin 3, a member of the perilipin family of LD binding proteins, and three N-terminal truncation mutants with lipid monolayers. The interaction is studied as a function of surface pressure for a series of systematically chosen lipids. We find that the C terminus of perilipin 3 has different insertion behavior from that of the longer truncation mutants and the full-length protein. Inclusion of N-terminal sequences with the C terminus decreases the ability of the protein construct to insert in lipid monolayers. Coupling of anionic lipids to negative spontaneous curvature facilitates protein interaction and insertion. The C terminus shows strong preference for lipids with more saturated fatty acids. This work sheds light on the LD binding properties and function of the different domains of perilipin 3.

Insertion of apoLp-III into a lipid monolayer is more favorable for saturated, more ordered, acyl-chains.

Neutral lipid transport in mammals is complicated involving many types of apolipoprotein. The exchangeable apolipoproteins mediate the transfer of hydrophobic lipids between tissues and particles, and bind to cell surface receptors. Amphipathic α-helices form a common structural motif that facilitates their lipid binding and exchangeability. ApoLp-III, the only exchangeable apolipoprotein found in insects, is a model amphipathic α-helix bundle protein and its three dimensional structure and function mimics that of the mammalian proteins apoE and apoAI. Even the intracellular exchangeable lipid droplet protein TIP47/perilipin 3 contains an α-helix bundle domain with high structural similarity to that of apoE and apoLp-III. Here, we investigated the interaction of apoLp-III from Locusta migratoria with lipid monolayers. Consistent with earlier work we find that insertion of apoLp-III into fluid lipid monolayers is highest for diacylglycerol. We observe a preference for saturated and more highly ordered lipids, suggesting a new mode of interaction for amphipathic α-helix bundles. X-ray reflectivity shows that apoLp-III unfolds at a hydrophobic interface and flexible loops connecting the amphipathic α-helices stay in solution. X-ray diffraction indicates that apoLp-III insertion into diacylglycerol monolayers induces additional ordering of saturated acyl-chains. These results thus shed important new insight into the protein-lipid interactions of a model exchangeable apolipoprotein with significant implications for its mammalian counterparts.

Effects of fluid viscosity on a moving sonoluminescing bubble.

Based on the quasi-adiabatic model, the parameters of the bubble interior for a moving single bubble sonoluminescence in water, adiponitrile, and N-methylformamide are calculated for various fluid viscosities. By using a complete form of the hydrodynamic force, the bubble trajectory is calculated for a moving single bubble sonoluminescence (m-SBSL). It is found that as the fluid viscosity increases, the unique circular path changes to an ellipsoidal and then linear form and along this incrementally increase of viscosity the light intensity increases. By using the Bremsstrahlung model to describe the bubble radiation, gradual increase of the viscosity results in brighter emissions. It is found that in fluids with higher viscosity the light intensity decreases as time passes.

Interaction of two oscillating sonoluminescence bubbles in sulfuric acid.

The mutual interaction of two oscillating gas bubbles in different concentrations of sulfuric acid is numerically investigated. A nonlinear oscillation for spherical symmetric bubbles with equilibrium radii smaller than 10 µm at a frequency of 37 kHz in a strong driving acoustical field P(a)=1.8 bar is assumed. The calculations are based on the investigation of the secondary Bjerknes force with regard to adiabatic model for the bubble interior which appears as repulsion or attraction interaction force. In this work the influence of the various concentrations of sulfuric acid in uncoupled and coupled distances between bubbles has been investigated. It is found that the sign and value of the secondary Bjerknes force depend on the sulfuric acid viscosity and its amount would be decreased by liquid viscosity enhancement. The results show that big change in the parameters of produced bubbles occurs in the sulfuric acid with concentrations from 65% to 85%.