4-Methylumbelliferone enhances the effects of chemotherapy on both temozolomide-sensitive and resistant glioblastoma cells.

Glioblastoma (GBM) is the most frequent malignant primary tumor of the CNS in adults, with a median survival of 14.6 months after diagnosis. The effectiveness of GBM therapies remains poor, highlighting the need for new therapeutic alternatives. In this work, we evaluated the effect of 4-methylumbelliferone (4MU), a coumarin derivative without adverse effects reported, in combination with temozolomide (TMZ) or vincristine (VCR) on U251, LN229, U251-TMZ resistant (U251-R) and LN229-TMZ resistant (LN229-R) human GBM cells. We determined cell proliferation by BrdU incorporation, migration through wound healing assay, metabolic and MMP activity by XTT and zymography assays, respectively, and cell death by PI staining and flow cytometry. 4MU sensitizes GBM cell lines to the effect of TMZ and VCR and inhibits metabolic activity and cell proliferation on U251-R cells. Interestingly, the lowest doses of TMZ enhance U251-R and LN229-R cell proliferation, while 4MU reverts this and even sensitizes both cell lines to TMZ and VCR effects. We showed a marked antitumor effect of 4MU on GBM cells alone and in combination with chemotherapy and proved, for the first time, the effect of 4MU on TMZ-resistant models, demonstrating that 4MU would be a potential therapeutic alternative for improving GBM therapy even on TMZ-refractory patients.

Paving the way for adequate myelination: The contribution of galectin-3, transferrin and iron.

Considering the worldwide incidence of well characterized demyelinating disorders such as Multiple Sclerosis (MS) and the increasing number of pathologies recently found to involve hypomyelinating factors such as micronutrient deficits, elucidating the molecular basis of central nervous system (CNS) demyelination, remyelination and hypomyelination becomes essential to the development of future neuroregenerative therapies. In this context, this review discusses novel findings on the contribution of galectin-3 (Gal-3), transferrin (Tf) and iron to the processes of myelination and remyelination and their potentially positive regulation of oligodendroglial precursor cell (OPC) differentiation. Studies were conducted in cuprizone (CPZ)-induced demyelination and iron deficiency (ID)-induced hypomyelination, and the participation of glial and neural stem cells (NSC) in the remyelination process was evaluated by means of both in vivo and in vitro assays on primary cell cultures.

Optimizing culture medium composition to improve oligodendrocyte progenitor cell yields in vitro from subventricular zone-derived neural progenitor cell neurospheres.

Neural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC.

Improving arylsulfatase activity determination in dried blood spots: Screening and diagnostic approaches for Maroteaux-Lamy syndrome (MPS VI).

BACKGROUND: Mucopolysaccharidosis type VI can be screened by measuring the lysosomal arylsulfatase B (ARSB) residual enzyme activity in dried blood spots (DBS) using synthetic substrates. However, we have found experimental obstacles when determining ARSB activity with the fluorescent method due to the significant quenching effect rendered by DBS components. METHODS: We adapted the methods originally described by Chamoles et al. [1] and Civallero et al. [2] and put forward 2 distinct approaches for ARSB activity quantification from DBS samples by measuring the 4-methylumbelliferone (ß-MU) fluorescence generated from the ARSB 4-methylumbelliferone sulfate (ß-MUS) substrate. RESULTS: We demonstrate the high throughput feasibility of a novel approach for measuring ARSB activities by incorporating tailor-made calibration curves according to each patient's DBS sample quenching properties. The second method is used to calculate ARSB activities by measuring the fluorescence and absorbance parameters in each reaction sample with a single DBS-free calibration curve. CONCLUSIONS: The quantitative correlation between the DBS sample absorbance and its quenching effect can be used to calculate predictive ARSB activities and would serve as an affordable first tier screening test. The method described herein demonstrates the critical importance of adapting the ß-MU calibration curves to each patient's unique DBS sample matrix and its positive impact on the accuracy and reliability of ARSB activity measurements.

The Notch-target gene hairy2a impedes the involution of notochordal cells by promoting floor plate fates in Xenopus embryos.

We have previously shown that the early Xenopus organiser contains cells equally potent to give rise to notochord or floor plate, and that Notch signalling triggers a binary decision, favouring the floor plate fate at the expense of the notochord. Now, we present evidence that Delta1 is the ligand that triggers the binary switch, which is executed through the Notch-mediated activation of hairy2a in the surrounding cells within the organiser, impeding their involution through the blastopore and promoting their incorporation into the hairy2a+ notoplate precursors (future floor-plate cells) in the dorsal non-involuting marginal zone.

Notch activates sonic hedgehog and both are involved in the specification of dorsal midline cell-fates in Xenopus.

We analysed the role of Notch signalling during the specification of the dorsal midline in Xenopus embryos. By activating or blocking the pathway we found that Notch expands the floor plate domain of sonic hedgehog and pintallavis and represses the notochordal markers chordin and brachyury, with a concomitant reduction of the notochord size. We propose that within a population of the early organiser with equivalent potential to develop either as notochord or floor plate, Notch activation favours floor plate development at the expense of the notochord, preferentially before mid gastrula. We present evidence that sonic hedgehog down-regulates chordin, suggesting that secreted Sonic hedgehog may be involved or reinforcing the cell-fate switch executed by Notch. We also show that Notch signalling requires Presenilin to modulate this switch.