Ontogenetic oligodendrocyte maturation through gestational iron deprivation: The road not taken.

Developmental iron deficiency (dID) models facilitate the study of specific oligodendrocyte (OL) requirements for their progression to a mature state and subsequent contribution to myelination. In the current work, we used the dID model in transgenic mice expressing green fluorescence protein under the CNPase promoter allowing the identification of cells belonging to the oligodendroglial lineage, and the visualization of the entire myelin structure and single OL morphology. The present work evaluates dID effects on OL complexity in different brain areas. Control animals showed an increase in OL complexity both during development and along the anterior-posterior axis. In contrast, dID animals exhibited an initial increase in CNPase+ cells with prevalence of immature-OL (i-OL), an effect later compensated during development by selective death of those i-OL. As a consequence, developmental behavior was impaired in terms of body balance, muscle response, and sensorimotor functions. To explore why i-OL fail to mature in dID, expression levels of transcriptional factors involved in the maturation of the OL lineage were studied. In nuclear fractions, dID animals showed an increase in Hes5, which prevents the maturation of i-OL, and a decrease in Sox10, a positive regulator of OL maturation. The cytoplasmic fractions showed a decrease in Olig1, which is critical for precursor cell differentiation into premyelinating OL. Overall, the expression levels of Hes5, Sox10, and Olig1 in dID conditions correlated with an unfavorable OL maturation profile. In sum, the current results provide further evidence of dID impact on myelination, keeping OL away from the maturational path.

The Divalent Metal Transporter 1 (DMT1) Is Required for Iron Uptake and Normal Development of Oligodendrocyte Progenitor Cells.

The divalent metal transporter 1 (DMT1) is a multimetal transporter with a primary role in iron transport. Although DMT1 has been described previously in the CNS, nothing was known about the role of this metal transporter in oligodendrocyte maturation and myelination. To determine whether DMT1 is required for oligodendrocyte progenitor cell (OPC) maturation, we used siRNAs and the Cre-lox system to knock down/knock out DMT1 expression in vitro as well as in vivo Blocking DMT1 synthesis in primary cultures of OPCs reduced oligodendrocyte iron uptake and significantly delayed OPC development. In vivo, a significant hypomyelination was found in DMT1 conditional knock-out mice in which DMT1 was postnatally deleted in NG2- or Sox10-positive OPCs. The brain of DMT1 knock-out animals presented a decrease in the expression levels of myelin proteins and a substantial reduction in the percentage of myelinated axons. This reduced postnatal myelination was accompanied by a decrease in the number of myelinating oligodendrocytes and a rise in proliferating OPCs. Furthermore, using the cuprizone model of demyelination, we established that DMT1 deletion in NG2-positive OPCs lead to less efficient remyelination of the adult brain. These results indicate that DMT1 is vital for OPC maturation and for the normal myelination of the mouse brain.SIGNIFICANCE STATEMENT To determine whether divalent metal transporter 1 (DMT1), a multimetal transporter with a primary role in iron transport, is essential for oligodendrocyte development, we created two conditional knock-out mice in which DMT1 was postnatally deleted in NG2- or Sox10-positive oligodendrocyte progenitor cells (OPCs). We have established that DMT1 is necessary for normal OPC maturation and is required for an efficient remyelination of the adult brain. Since iron accumulation by OPCs is indispensable for myelination, understanding the iron incorporation mechanism as well as the molecules involved is critical to design new therapeutic approaches to intervene in diseases in which the myelin sheath is damaged or lost.

Characterization, optimization, and scale-up of cellulases production by trichoderma reesei cbs 836.91 in solid-state fermentation using agro-industrial products.

The application of cellulases in saccharification processes is restricted by its production cost. Consequently, new fungal strains able to elaborate higher cellulases titers and with special activity profiles are required to make the process economical. The aim of this investigation was to find a promising wild-type Trichoderma strain for cellulases production. The Trichoderma reesei strain 938 (CBS 836.91) was selected among twenty strains on the basis of cellulase-agar-plate screening. Evaluation of the selected strain on six solid substrates indicated the highest activities to be obtained from wheat bran. Statistical analyses of the experimental design indicated a significant effect of pH and moisture on the generation of endoglucanase (EGA) and filter-paper (FPA) activity. Furthermore, a central-composite design-based optimization revealed that pH values between 6.4 and 6.6 and moisture from 74 to 94% were optimal for cellulases production. Under these conditions, 8-10 IU gds(-1) of FPA and 15.6-17.8 IU gds(-1) of EGA were obtained. In addition, cultivation in a rotating-drum reactor under optimal conditions gave 8.2 IU gds(-1) FPA and 13.5 IU gds(-1) EGA. Biochemical characterization of T. reesei 938 cellulases indicated a substantially higher resistance to 4 mM Fe(+2) and a slightly greater tolerance to alkaline pH in comparison to Celluclast(®). These results suggest that T. reesei 938 could be a promising candidate for improved cellulases production through direct-evolution strategies.

Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method.

Endo-polygalacturonase (endo-PGase) activity determinations generally rely on viscosity changes or reducing sugar ends produced by this activity over polygalacturonic acid. Torres and coworkers [Enzyme Microb. Technol. 48 (2011) 123-128] showed that ruthenium red (RR) is useful for endo-PGase determination. In this article, we present a high-throughput liquid-based endo-PGase assay based on the RR method and compare it with the viscosity determination method. The reduced assay uses a small volume of enzyme solution, 40 µg of polygalacturonic acid, and 45 µg of RR for each sample determination. Furthermore, we obtained an interconversion factor for RR and viscosity activities.