CSIG-09. ASSESSMENT OF TUMOR TREATING FIELDS (TTFields) COMBINED WITH TRICHOSTATIN A (TSA) IN PATIENT-DERIVED GLIOBLASTOMA (GBM) CELLS

BACKGROUND TTFields therapy is a non-invasive biophysical approach for GBM patient treatment. TTFields produce an anti-mitotic effect by destabilizing microtubule polymerization and interrupting cell division. However, epigenetic modifications that may be induced by TTFields remain unknown. To address this, TTFields were applied to patient-derived GBM cells ± the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and cellular proliferation was assessed. HDAC activity was also measured after TTFields application. METHODS GBM cells were isolated from a newly-diagnosed patient tumor (IDH-WT, unmethylated MGMT promoter). Cells were grown in DMEM/F12 media with 10% FBS and gentamicin. Cells were plated on plastic coverslips (1×104cells/coverslip) and incubated overnight. TTFields were applied at 200 kHz (field intensity of ~1.6 V/cm) for 6 days. For the last 2 days of TTFields, cells were incubated with DMSO or 250 nM TSA. Cells were harvested and counted to assess proliferation (n=4-5/group). To determine if TTFields have a direct effect on HDAC activity, TTFields were applied as described and nuclei were extracted. Nuclear HDAC activity was measured using a commercial kit (n=2-3/group). Cell counts were compared with one-way ANOVA and Tukey multiple comparisons with p< 0.05 considered significant. RESULTS Cell counts for the DMSO control group, TSA only, TTFields only, and the combination were: 643,400 ± 111,384, 233,800 ± 144,200, 90,775 ± 45,209, and 41,520 ± 36,168, respectively (mean ± SD; ANOVA p< 0.0001). There was no statistical difference between the TTFields only and TSA only or TTFields + TSA groups, with all other comparisons statistically significant. For the HDAC activity measurements, control nuclei were 1068 ± 135 pmol/min/mg and TTFields-treated nuclei were 1301 ± 313.6 pmol/min/mg and were not statistically different. CONCLUSIONS The addition of TSA to TTFields did not alter cellular proliferation more than TTFields alone and TTFields did not alter HDAC activity in these GBM cells.

FABP7 Binds to Fatty Acid Micelles: Implications for Lipid Transport

The transport of hydrophobic molecules, including long-chain fatty acids, within cells is highly dynamic. Hydrophobic molecules are unable to freely diffuse through the aqueous cytoplasm without a transporter. Fatty acid binding proteins (FABP) transport these molecules to different cellular compartments. As part of their transport, FABPs often associate with cell membranes to acquire and deliver their bound cargo. Understanding the nature of this transport is becoming increasingly important because lipid signaling functions are associated with metabolic pathways impacting disease pathologies such as carcinomas, autism and schizophrenia. Herein, we focus on Brain fatty acid binding protein (FABP7), which demonstrates localization to the cytoplasm and nucleus, influencing transcription and fatty acid metabolism. We use a combined biophysical approach to elucidate the interaction between FABP7 and model membranes. Specifically, we use microscale thermophoresis to show that FABP7 can bind oleic acid (OA) and docosahexaenoic acid (DHA) micelles, while differential scanning fluorimetry experiments show binding lowers the melting temperature of FABP7. Structural data from NMR and multiscale molecular dynamics simulations reveals that the interaction between FABP7 and micelles is through FABP7 portal region residues. Our simulations also capture binding events where fatty acids dissociate from the model membrane and bind to FABP7. Overall, our data reveals a novel interaction between FABP7 and OA or DHA micelles and provides key structural insight into the transport of hydrophobic molecules.

The Natural Capital Value of the Seagrass Posidonia oceanica in the North-Western Mediterranean

Posidonia oceanica is an endemic Mediterranean seagrass used as a ‘biological quality element’ in monitoring programmes of the EU Water Framework Directive, providing information about coastal ecosystems status. The regression of P. oceanica meadows caused a growing interest among policy makers to assess the value of seagrasses and to increase their protection. An evaluation of P. oceanica meadows located in the Ligurian-Provençal basin (NW Mediterranean) through a biophysical approach is here developed. Six meadows located in Liguria (Italy) and Corsica (France) were investigated by applying the emergy analysis to assess the natural capital (NC) stocked by leaves and rhizomes components. Results highlighted the importance of carrying out an analysis of the variations in the NC value in both components: rhizomes defined the growth stage and the capacity to store NC over time; leaves provided information on the variability due to disturbances in the water column. Emergy analysis allows defining the NC, in terms of resources needed to maintain the meadows and to provide services to coastal communities. This research is inserted into the effort of incorporating the NC evaluation into marine planning and decision making to achieve nature conservation goals, while ensuring the sustainable exploitation of marine resources.

Osh6 Revisited: Control of PS Transport by the Concerted Actions of PI4P and Sac1 Phosphatase

Osh6, a member of the oxysterol-binding protein–related protein (ORP) family, is a lipid transport protein that is involved in the transport of phosphatidylserine (PS) between the endoplasmic reticulum (ER) and the plasma membrane (PM). We used a biophysical approach to characterize its transport mechanism in detail. We examined the transport of all potential ligands of Osh6. PI4P and PS are the best described lipid cargo molecules; in addition, we showed that PIP2 can be transported by Osh6 as well. So far, it was the exchange between the two cargo molecules, PS and PI4P, in the lipid-binding pocket of Osh6 that was considered an essential driving force for the PS transport. However, we showed that Osh6 can efficiently transport PS along the gradient without the help of PI4P and that PI4P inhibits the PS transport along its gradient. This observation highlights that the exchange between PS and PI4P is indeed crucial, but PI4P bound to the protein rather than intensifying the PS transport suppresses it. We considered this to be important for the transport directionality as it prevents PS from returning back from the PM where its concentration is high to the ER where it is synthesized. Our results also highlighted the importance of the ER resident Sac1 phosphatase that enables the PS transport and ensures its directionality by PI4P consumption. Furthermore, we showed that the Sac1 activity is regulated by the negative charge of the membrane that can be provided by PS or PI anions in the case of the ER membrane.

Active site characterization and activity of the human aspartyl (asparaginyl) β-hydroxylase

Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the β-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.

Spectroscopic Fourier Transform Infrared Study of Poly (N-Isopropylacrylamide)-Human Recombinant Platelet-Derived Growth Factor- BB Protein as a Strategic Biobetter Regenerative Material

Biobetter or Biosuperior are recombinant protein drugs with one or more characteristics that are better than the original formulation. Platelet-Derived Growth Factor (PDGF) is a potent mitogen that can induce pulp stem cell proliferation and promote angiogenesis in vitro. PDGF-BB is required for wound healing, and they are chemoattractants that can activate macrophages and fibroblast activation in vitro. Poly(n-isopropylacrylamide) (PNIPAM) polymer is a thermoresponsive polymer widely used for drug delivery. A lyophilized combination of PNIPAM and 5µg of Recombinant Human PDGF-BB(rhPDGF-BB)(sigma Aldrich-P3201) protein was prepared in the present study. This combination was subjected to Spectroscopic Fourier-Transform Infrared(FTIR) study. The results suggested that a biophysical approach like FTIR enables assessing protein stability and aggregation tendency of recombinant proteins, contributing as a useful method to identify samples with prospective high therapeutic values.

The Amyloid Region of Hfq Riboregulator Promotes DsrA:rpoS RNAs Annealing

Hfq is a bacterial RNA chaperone which promotes the pairing of small noncoding RNAs to target mRNAs, allowing post-transcriptional regulation. This RNA annealing activity has been attributed for years to the N-terminal region of the protein that forms a toroidal structure with a typical Sm-fold. Nevertheless, many Hfqs, including that of Escherichia coli, have a C-terminal region with unclear functions. Here we use a biophysical approach, Synchrotron Radiation Circular Dichroism (SRCD), to probe the interaction of the E. coli Hfq C-terminal amyloid region with RNA and its effect on RNA annealing. This C-terminal region of Hfq, which has been described to be dispensable for sRNA:mRNA annealing, has an unexpected and significant effect on this activity. The functional consequences of this novel property of the amyloid region of Hfq in relation to physiological stress are discussed.

A biophysical approach to assess weather impacts on honey bee colony winter mortality

The western honey bee ( Apis mellifera ) is one of the most important insects kept by humans, but high colony losses are reported around the world. While the effects of general climatic conditions on colony winter mortality were already demonstrated, no study has investigated specific weather conditions linked to biophysical processes governing colony vitality. Here, we quantify the comparative relevance of four such processes that co-determine the colonies' fitness for wintering during the annual hive management cycle, using a 10-year dataset of winter colony mortality in Austria that includes 266 378 bee colonies. We formulate four process-based hypotheses for wintering success and operationalize them with weather indicators. The empirical data is used to fit simple and multiple linear regression models on different geographical scales. The results show that approximately 20% of winter mortality variability can be explained by the analysed weather conditions, and that it is most sensitive to the duration of extreme cold spells in mid and late winter. Our approach shows the potential of developing weather indicators based on biophysical processes and discusses the way forward for applying them in climate change studies.