Analysis of a miR-148a Targetome in B Cell Central Tolerance.

A microRNA (miRNA) often regulates the expression of hundreds of target genes. A fundamental question in the field of miRNA research is whether a miRNA exerts its biological function through regulating a small number of key targets or through small changes in the expression of hundreds of target genes. We addressed this issue by performing functional analysis of target genes regulated by miR-148a. We previously identified miR-148a as a critical regulator of B cell central tolerance and found 119 target genes that may mediate its function. We selected 4 of them for validation and demonstrated a regulatory role for Bim, Pten, and Gadd45a in this process. In this study, we performed functional analysis of the other miR-148a target genes in in vitro and in vivo models of B cell central tolerance. Our results show that those additional target genes play a minimal role, if any, in miR-148a-mediated control of B cell central tolerance, suggesting that the function of miRNAs is mediated by a few key target genes. These findings have advanced our understanding of molecular mechanisms underlying miRNA regulation of gene expression and B cell central tolerance.

Bcl-2-Assisted Reprogramming of Mouse Astrocytes and Human Fibroblasts into Induced Neurons.

Direct neuronal reprogramming is a promising strategy to generate various types of neurons that are, otherwise, inaccessible for researchers. However, the efficiency of neuronal conversion is highly dependent on the transcription factor used, the identity of the initial cells to convert, their species' background, and the neuronal subtype to which cells will convert. Regardless of these conditioning factors, the apoptotic regulator Bcl-2 acts as a pan-neuronal reprogramming enhancer. Bcl-2 mediates its effect in reprogramming by preventing an overshot of oxidative stress during the acquisition of a neuronal oxidative metabolism, thus reducing cell death by ferroptosis and facilitating the phenotypic conversion. In this chapter, we outline two methods to obtain either mouse or human neurons derived from postnatal astrocytes and skin fibroblasts, respectively. The overall reprogramming strategy is based on the co-expression of Bcl-2 and the transcription factor Neurog2 that produces mostly excitatory neurons. However, the method can be easily adapted to achieve alternative neuronal subtypes by using additional transcription factors, such as Isl1 for motor neurons. Therefore, our approaches provide solid but flexible platforms to obtain human and mouse induced neurons in vitro that can be applied to basic or translational research.

Reversible Immobilization of Lipases on Heterofunctional Octyl-Amino Agarose Beads Prevents Enzyme Desorption.

Two different heterofunctional octyl-amino supports have been prepared using ethylenediamine and hexylendiamine (OCEDA and OCHDA) and utilized to immobilize five lipases (lipases A (CALA) and B (CALB) from Candida antarctica, lipases from Thermomyces lanuginosus (TLL), from Rhizomucor miehei (RML) and from Candida rugosa (CRL) and the phospholipase Lecitase Ultra (LU). Using pH 5 and 50 mM sodium acetate, the immobilizations proceeded via interfacial activation on the octyl layer, after some ionic bridges were established. These supports did not release enzyme when incubated at Triton X-100 concentrations that released all enzyme molecules from the octyl support. The octyl support produced significant enzyme hyperactivation, except for CALB. However, the activities of the immobilized enzymes were usually slightly higher using the new supports than the octyl ones. Thermal and solvent stabilities of LU and TLL were significantly improved compared to the OC counterparts, while in the other enzymes the stability decreased in most cases (depending on the pH value). As a general rule, OCEDA had lower negative effects on the stability of the immobilized enzymes than OCHDA and while in solvent inactivation the enzyme molecules remained attached to the support using the new supports and were released using monofunctional octyl supports, in thermal inactivations this only occurred in certain cases.