Nkx2.1 regulates the generation of telencephalic astrocytes during embryonic development.

The homeodomain transcription factor Nkx2.1 (NK2 homeobox 1) controls cell differentiation of telencephalic GABAergic interneurons and oligodendrocytes. Here we show that Nkx2.1 also regulates astrogliogenesis of the telencephalon from embryonic day (E) 14.5 to E16.5. Moreover we identify the different mechanisms by which Nkx2.1 controls the telencephalic astrogliogenesis. In Nkx2.1 knockout (Nkx2.1 <sup>-/-</sup> ) mice a drastic loss of astrocytes is observed that is not related to cell death. Further, in vivo analysis using BrdU incorporation reveals that Nkx2.1 affects the proliferation of the ventral neural stem cells that generate early astrocytes. Also, in vitro neurosphere assays showed reduced generation of astroglia upon loss of Nkx2.1, which could be due to decreased precursor proliferation and possibly defects in glial specification/differentiation. Chromatin immunoprecipitation analysis and in vitro co-transfection studies with an Nkx2.1-expressing plasmid indicate that Nkx2.1 binds to the promoter of glial fibrillary acidic protein (GFAP), primarily expressed in astrocytes, to regulate its expression. Hence, Nkx2.1 controls astroglial production spatiotemporally in embryos by regulating proliferation of the contributing Nkx2.1-positive precursors

Nkx2.1 regulates the proliferation and cell fate of telencephalic astrocytes during embryonic development

AbstractThe homeodomain transcription factor Nkx2.1 controls cell differentiation of telencephalic GABAergic interneurons and oligodendrocytes. Here, we show that Nkx2.1 additionally regulates astrogliogenesis of the telencephalon from embryonic day (E) 14.5 to E16.5. Our work aims to identify the different mechanisms by which Nkx2.1 controls telencephalic astrogliogenesis. InNkx2.1-/-, a drastic loss of astrocytes is observed which is not related to cell death.In vivoanalysis using BrdU incorporation reveals that Nkx2.1 affects the proliferation of ventral neural stem cells that generate early astrocytes.In vitroneurosphere assays show that Nkx2.1 additionally affects the differentiation step of Nkx2.1-derived astrocytes. Chromatin immunoprecipitation andin vitroco-transfection studies of a Nkx2.1-expressing plasmid indicate that Nkx2.1 binds to the promoter of astroglial differentiation gene GFAP, and regulates its expression. Hence, Nkx2.1 controls astroglial production spatiotemporally in embryos by regulating stem cell division and specification of the contributing Nkx2.1+precursors.</jats:p

Structure-Function Relationships Affecting the Sensing Mechanism of Monolayer-Protected Cluster Doped Xerogel Amperometric Glucose Biosensors

A systematic study of the structure–function relationships critical to understanding the sensing mechanism of 1st generation amperometric glucose biosensors with an embedded nanoparticle (NP) network is presented. Xerogel-based films featuring embedded glucose oxidase enzyme and doped with alkanethiolate-protected gold NPs, known as monolayer protected clusters (MPCs), exhibit significantly enhanced performance compared to analogous systems without NPs including higher sensitivity, faster response time, and extended linear/dynamic ranges. The proposed mechanism involves diffusion of the glucose to glucose oxidase within the xerogel, enzymatic reaction production of H2O2 with subsequent diffusion to the embedded network of MPCs where it is oxidized, an event immediately reported via fast electron transfer (ET) through the MPC system to the working electrode. Various aspects of the film construct and strategy are systematically probed using amperometry, voltammetry, and solid-state electronic conductivity measurements, including the effects of MPC peripheral chain length, MPC functionalization via place-exchange reaction, MPC core size, and the MPC density or concentration within the xerogel composite films. The collective results of these experiments support the proposed mechanism and identify interparticle spacing and the electronic communication through the MPC network is the most significant factor in the sensing scheme with the diffusional aspects of the mechanism that may be affected by film/MPC hydrophobicity and functionality (i.e., glucose and H2O2 diffusion) shown to be less substantial contributors to the overall enhanced performance. Understanding the structure–function relationships of effective sensing schemes allows for the employment of the strategy for future biosensor design toward clinically relevant targets

Cell wall degrading enzymes originating from rhizoctonia solani increase sugar beet root damage in the presence of leuconostoc mesenteroides

Sugar beet crown and root rot caused by Rhizoctonia solani is a major yield constraint. Root rot is highly increased when R. solani and Leuconostoc mesenteroides co-infect roots. We hypothesized that the absence of plant cell wall degrading enzymes in L. mesenteroides and their supply by R. solani during close contact, causes increased damage. In planta root inoculation with or without cell wall degrading enzymes showed greater rot when L. mesenteroides was combined with cellulase (49 mm rot), polygalacturonase (48 mm), and pectin lyase (35 mm) versus these enzymes (0–11 mm), R. solani (13 mm), and L. mesenteroides (22 mm) individually. Carbohydrate analysis revealed increase in simpler carbohydrates namely glucose + galactose, and fructose in the infected roots versus mock control, possibly due to the degradation of complex cell wall carbohydrates. Expression of R. solani cellulase, polygalacturonase, and pectin lyase genes during root infection corroborated well with the enzyme data. Global mRNAseq analysis identified candidate genes and highly co-expressed gene clusters (in all 3 organisms) that might be critical in host plant defense and pathogenesis. Future targeting of R. solani cell wall degrading enzymes could be an effective strategy to mitigate root damage during interaction with L. mesenteroides

Regulatory roles of small non-coding RNAs in sugar beet resistance against beet curly top virus

Beet curly top virus (BCTV) mediated yield loss in sugar beets is a major problem worldwide. The circular single-stranded DNA virus is transmitted by the beet leafhopper. Genetic sources of BCTV resistance in sugar beet are limited and commercial cultivars rely on chemical treatments versus durable genetic resistance. Phenotypic selection and double haploid production have resulted in sugar beet germplasm (KDH13-13 and KDH4-9-4) that are highly resistant to BCTV. The molecular mechanism of resistance to the virus is unknown, especially the role of small noncoding RNAs (sncRNAs) during early plant-viral interaction. Using the resistant lines along with a susceptible line (KDH19-17; 19), we demonstrate the role of sugar beet miRNAs in BCTV resistance during early infection stages when symptoms are not yet visible. The differentially expressed miRNAs altered the expression of their corresponding target genes such as pyruvate dehydrogenase (EL10Ac1g02046), carboxylesterase (EL10Ac1g01087), serine/threonine protein phosphatase (EL10Ac1g01374), and LRR receptor-like (EL10Ac7g17778), that were highly expressed in the resistant lines versus susceptible lines. Pathway enrichment analysis of the miRNA target genes showed an enrichment of genes involved in glycolysis/gluconeogenesis, galactose metabolism, starch, and sucrose metabolism to name a few. Carbohydrate analysis revealed altered glucose, galactose, fructose, and sucrose concentration in the infected leaves of resistant versus susceptible lines. We also demonstrate differential regulation of BCTV derived sncRNAs in the resistant versus susceptible lines that target sugar beet genes such as LRR (EL10Ac1g01206), 7-deoxyloganetic acid glucosyltransferase (EL10Ac5g12605), and transmembrane emp24 domain containing (EL10Ac6g14074) and altered their expression. In response to viral infection, we found that plant derived miRNAs targeted BCTV capsid protein/replication related genes and showed differences in expression among resistant and susceptible lines. The data presented here demonstrate the contribution of miRNA mediated regulation of metabolic pathways and cross-kingdom RNAi in sugar beet BCTV resistance