Invadopodia are actin-rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen-binding domains of Camelid heavy-chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA-MB-231 breast and PC-3 prostate cancer cells. A nanobody (K(d)~35 nM, 1:1 stoichiometry) that disrupts fascin F-actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion. Cortactin-SH3 dependent WIP recruitment toward the plasma membrane was specifically inhibited by a cortactin nanobody (K(d)~75 nM, 1:1 stoichiometry). This functional domain is shown to be important for formation of properly organized invadopodia, MMP-9 secretion, matrix degradation, and cancer cell invasion. Notably, using a subcellular delocalization strategy to trigger protein loss of function, we uncovered a fascin-bundling-independent role in MMP-9 secretion. Hence, we demonstrate that nanobodies enable high resolution protein function mapping in cells.

Invadopodia and filopodia are dynamic, actin-based protrusions contributing to cancer cell migration, invasion, and metastasis. The force of actin bundles is essential for their protrusive activity. The bundling protein fascin is known to play a role in both invadopodia and filopodia. As it is more and more acknowledged that functionally related proteins cooperate, it is unlikely that only fascin bundles actin in these protrusions. Another interesting candidate is L-plastin, normally expressed in hematopoietic cells, but considered a common marker of many cancer types. We identified L-plastin as a new component of invadopodia, where it contributes to degradation and invasiveness. By means of specific, high-affinity nanobodies inhibiting bundling of fascin or L-plastin, we further unraveled their cooperative mode of action. We show that the bundlers cannot compensate for each other due to strikingly different bundling characteristics: L-plastin bundles are much thinner and less tightly packed. Composite bundles adopt an intermediate phenotype, with fascin delivering the rigidity and strength for protrusive force and structural stability, whereas L-plastin accounts for the flexibility needed for elongation. Consistent with this, elevated L-plastin expression promotes elongation and reduces protrusion density in cells with relatively lower L-plastin than fascin levels.Filopodia are finger-like protrusions coordinating movement at the cell front by "sensing" extracellular stimuli (1). Invadopodia on the other hand are formed at the ventral cell side, enabling matrix degradation by localized proteolysis (2). As both actin-based protrusions contribute to migration and invasion, they are associated with cancer metastasis (3, 4), which has also been shown in vivo (5, 6).The architecture and dynamics of filopodia and invadopodia are controlled by a broad variety of actin-binding proteins (7,8). Moreover, these regulating proteins show highly similar spatial and temporal distributions in filopodia and invadopodia (9).

No abstract

Diffusion magnetic resonance imaging biomarkers can provide quantifiable information of the brain tissue after a mild traumatic brain injury (mTBI). However, the commonly applied diffusion tensor imaging (DTI) model is not very specific to changes in the underlying cellular structures. To overcome these limitations, other diffusion models have recently emerged to provide a more complete view on the damage profile following TBI. In this study, we investigated longitudinal changes in advanced diffusion metrics following experimental mTBI, utilising three different diffusion models in a rat model of mTBI, including DTI, diffusion kurtosis imaging and a white matter model. Moreover, we investigated the association between the diffusion metrics with histological markers, including glial fibrillary acidic protein (GFAP), neurofilaments and synaptophysin in order to investigate specificity. Our results revealed significant decreases in mean diffusivity in the hippocampus and radial diffusivity and radial extra axonal diffusivity (RadEAD) in the cingulum one week post injury. Furthermore, correlation analysis showed that increased values of fractional anisotropy one day post injury in the hippocampus was highly correlated with GFAP reactivity three months post injury. Additionally, we observed a positive correlation between GFAP on one hand and the kurtosis parameters in the hippocampus on the other hand three months post injury. This result indicated that prolonged glial activation three months post injury is related to higher kurtosis values at later time points. In conclusion, our findings point out to the possible role of kurtosis metrics as well as metrics from the white matter model as prognostic biomarker to monitor prolonged glial reactivity and inflammatory responses after a mTBI not only at early timepoints but also several months after injury.

PurposeEven with an optimal treatment protocol, the median survival of glioblastoma (GB) patients is only 12–15 months. Hence, there is need for novel effective therapies that improve survival outcomes. Recent evidence suggests an important role for connexin (Cx) proteins (especially Cx43) in the microenvironment of malignant glioma. Cx43-mediated gap junctional communication has been observed between tumor cells, between astrocytes and between tumor cells and astrocytes. Therefore, gap junction directed therapy using a pharmacological suppressor or modulator, such as tonabersat, could be a promising target in the treatment of GB. In this preclinical study, we evaluated the possible therapeutic potential of tonabersat in the F98 model.ProceduresFemale Fischer rats were inoculated with ± 25.000 F98 tumor cells in the right frontal lobe. Eight days post-inoculation contrast-enhanced T1-weighted (CE-T1w) magnetic resonance (MR) images were acquired to confirm tumor growth in the brain. After tumor confirmation, rats were randomized into a Control Group, a Connexin Modulation Group (CM), a Standard Medical Treatment Group (ST), and a Standard Medical Treatment with adjuvant Connexin Modulation Group (STCM). To evaluate therapy response, T2-weighted (T2w) and CE-T1w sequences were acquired at several time points. Tumor volume analysis was performed on CE-T1w images and statistical analysis was performed using a linear mixed model.ResultsSignificant differences in estimated geometric mean tumor volumes were found between the ST Group and the Control Group and also between the STCM Group and the Control Group. In addition, significant differences in estimated geometric mean tumor volumes between the ST Group and the STCM Group were demonstrated. No significant differences in estimated geometric mean tumor volumes were found between the Control Group and the CM Group.ConclusionOur results demonstrate a therapeutic potential of tonabersat for the treatment of GB when used in combination with radiotherapy and temozolomide chemotherapy.

Recently, a 18F-labeled derivative of the widely used 68Ga-PSMA-11 was developed for PET imaging of prostate cancer. Although 18F-PSMA-11 has already been evaluated in a Phase I and Phase II clinical trial, preclinical evaluation of this radiotracer is important for further understanding its dynamic behavior. Saturation binding experiments were conducted by incubation of LNCaP cells with 18F-PSMA-11 or 68Ga-PSMA-11 for 1 h, followed by determination of the specific and aspecific binding. Mice bearing LNCaP or PC-3 xenografts each received ± 3.7 MBq 18F-PSMA-11 and 68Ga-PSMA-11 followed by dynamic acquisition of 2.5 h as well as ± 15 MBq 18F-FDG followed by static acquisition at 1 h post injection (p.i.). Uptake was evaluated by comparison of uptake parameters (SUVmean, SUVmax, TBRmean and TBRmax). Mice underwent ex vivo biodistribution where 18F-PSMA-11 activity was measures in excretory organs (kidneys, bladder and liver) as well as bone fragments (femur, humerus, sternum and skull) to evaluate bone uptake. The dissociation constant (Kd) of 18F-PSMA-11 and 68Ga-PSMA-11 was 2.95 ± 0.87 nM and 0.49 ± 0.20 nM, respectively. Uptake parameters were significantly higher in LNCaP compared to PC-3 xenografts for both 18F-PSMA-11 and 68Ga-PSMA-11, while no difference was found for 18F-FDG uptake (except for SUVmax). Tumor uptake of 18F-PSMA-11 showed a similar trend over time as 68Ga-PSMA-11, although all uptake parameter curves of the latter were considerably lower. When comparing early (60 min p.i.) to delayed (150 min p.i.) imaging for both radiotracers individually, TBRmean and TBRmax were significantly higher at the later timepoint, as well as the SUVmax of 68Ga-PSMA-11. The highest %ID/g was determined in the kidneys (94.0 ± 13.6%ID/g 1 h p.i.) and the bladder (6.48 ± 2.18%ID/g 1 h p.i.). No significant increase in bone uptake was seen between 1 and 2 h p.i. Both radiotracers showed high affinity for the PSMA receptor. Over time, all uptake parameters were higher for 18F-PSMA-11 compared to 68Ga-PSMA-11. Delayed imaging with the latter may improve tumor visualization, while no additional benefits could be found for late 18F-PSMA-11 imaging. Ex vivo biodistribution demonstrated fast renal clearance of 18F-PSMA-11 as well as no significant increase in bone uptake.

TO (mg!dl) Cholest. (mg/dl) SN 9 0.22 0.02 56.9 5.0 48.9 2.6 Yoshida 9 0.43 0.07' 123.3 8.1" 203.3 14.8" Yosh.+Exfract 5 0.52 0.07 60.1 11.3' 111.9 17.4'The dried hydro-alcoholic extract of V myrtillus leaves was given per os, as an aqueous solution, at the dose of 3 g/kg, twice daily for four consecutive days. Treated rats were compared to untreated controls for statistically significant differences, using Student't t-test. The results are shown in Table 1.Oral administration of the extract significantly decreased plasma TG and cholesterol levels but did not affect FFA concentration. We were also interested in determining the effects of the extract on basal lipidemia in normolipidemic rats. In this case, no significant effect on plasma TG and FFA levels could be detected. These results indicate that V myrtillus leaves, which are known as a hypoglycemic agent, may be active also in lipid metabolism alterations.Little is known about the active constituents present in the leaves of Vaccinium myrtillus; in early studies the presence of "neomyrtilline" was reported (3), probably a glucoside of trihydroxybenzoic acid, but also the recent literature is not rich on this topic (4). Our investigation is to be extended in order to obtain active fractions of the extract and to further elucidate its pharmacological activity on different models of hyperlipidemia and experimental diabetes.

Objective: Intestinal inflammation triggers postoperative ileus (POI), commonly seen after abdominal surgery and characterized by impaired gastrointestinal transit; when prolonged, this leads to increased morbidity. Hydrogen sulfide (H 2 S) is recognized as an important mediator of many (patho)physiological processes, including inflammation, and is now investigated for anti-inflammatory application. Therefore, the aim of this study was to investigate the effect of the H 2 S-releasing naproxen derivative ATB-346, developed to reduce gastrointestinal injury by naproxen, and the slow-release H 2 S donor GYY4137 on intestinal inflammation and delayed gastrointestinal transit in murine POI. Methods: C57Bl6J mice were fasted for 6 h, anesthetized and after laparotomy, POI was induced by compressing the small intestine with two cotton applicators for 5 min (intestinal manipulation; IM). GYY4137 (50 mg/kg, intraperitoneally), ATB-346 (16 mg/kg, intragastrically) or naproxen (10 mg/kg, intragastrically) were administered 1 h before IM. At 24 h postoperatively, gastrointestinal transit was assessed via fluorescent imaging, and mucosa-free muscularis segments were prepared for later analysis. Inflammatory parameters and activity of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 were measured. Histological examination of whole tissue sections was done on hematoxylin-eosin stained slides. Results: Pre-treatment with GYY4137 (geometric center; GC: 7.6 ± 0.5) and ATB-346 (GC: 8.4 ± 0.3) prevented the delayed transit induced by IM (GC: 3.6 ± 0.5 vs. 9.0 ± 0.4 in non-operated controls) while naproxen only partially did (GC: 5.9 ± 0.5; n = 8 for all groups). GYY4137 and ATB-346 significantly reduced the IM-induced increase in muscular myeloperoxidase (MPO) activity and protein levels of interleukin (IL)-6, IL-1β and monocyte chemotactic protein 1; the reduction by naproxen was less pronounced and only reached significance for MPO activity and IL-6 levels. All treatments significantly reduced the increase in COX-2 activity caused by IM, whereas only GYY4137 significantly reduced the increase in iNOS activity. Naproxen treatment caused significant histological damage of intestinal villi. Conclusion: The study shows that naproxen partially prevents POI, probably through its inhibitory effect on COX-2 activity. Both ATB-346 and GYY4137 were more effective, the result with GYY4137 showing that H 2 S per se can prevent POI.