SUMMARY Recent advances in three dimensional (3D) culture systems have led to the generation of brain organoids that resemble different human brain regions; however, a 3D organoid model of the midbrain containing functional midbrain dopaminergic (mDA) neurons has not been reported. We developed a method to differentiate human pluripotent stem cells into a large multicellular organoid-like structure that contains distinct layers of neuronal cells expressing characteristic markers of human midbrain. Importantly, we detected electrically active and functionally mature mDA neurons and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using 2D methods or MLOs generated from mouse embryonic stem cells, our human MLOs produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a tractable in vitro system to study the human midbrain and its related diseases.

Chronic myeloid leukemia responds well to therapy targeting the oncogenic fusion protein BCR-ABL1 in chronic phase, but is resistant to treatment after it progresses to blast crisis (BC). BC is characterized by elevated β-catenin signaling in granulocyte macrophage progenitors (GMPs), which enables this population to function as leukemia stem cells (LSCs) and act as a reservoir for resistance. Because normal hematopoietic stem cells (HSCs) and LSCs depend on β-catenin signaling for self-renewal, strategies to specifically target BC will require identification of drugable factors capable of distinguishing between self-renewal in BC LSCs and normal HSCs. Here, we show that the MAP kinase interacting serine/threonine kinase (MNK)-eukaryotic translation initiation factor 4E (eIF4E) axis is overexpressed in BC GMPs but not normal HSCs, and that MNK kinase-dependent eIF4E phosphorylation at serine 209 activates β-catenin signaling in BC GMPs. Mechanistically, eIF4E overexpression and phosphorylation leads to increased β-catenin protein synthesis, whereas MNK-dependent eIF4E phosphorylation is required for nuclear translocation and activation of β-catenin. Accordingly, we found that a panel of small molecule MNK kinase inhibitors prevented eIF4E phosphorylation, β-catenin activation, and BC LSC function in vitro and in vivo. Our findings identify the MNK-eIF4E axis as a specific and critical regulator of BC self-renewal, and suggest that pharmacologic inhibition of the MNK kinases may be therapeutically useful in BC chronic myeloid leukemia.cancer stem cell | Wnt pathway | xenograft | biomarker

Because the Sprouty (Spry) proteins were shown to be inhibitors of the mainstream Ras/ERK pathway, there has been considerable interest in ascertaining their mechanism of action especially since a possible role as tumor suppressors for these inhibitory proteins has been suggested. We compared the ability of the mammalian Spry isoforms to inhibit the Ras/ERK pathway in the context of fibroblast growth factor receptor (FGFR) signaling. Spry2 is considerably more inhibitory than Spry1 or Spry4, and this correlates with the binding to Grb2 via a C-terminal proline-rich sequence that is found exclusively on Spry2. This PXXPXR motif binds directly to the N-terminal Src homology domain 3 of Grb2, and when added onto the C terminus of Spry4 the resultant chimera inhibits the Ras/ERK pathway. The ability to inhibit neurite outgrowth in PC-12 cells correlates with the propensity of Spry isoforms and engineered constructs to inhibit the phosphorylation of ERK1/2. The PXXPXR motif is cryptic in unstimulated cells, and it is postulated that Spry2 undergoes a conformational change following FGFR stimulation, enabling the subsequent interaction with Grb2. We present evidence that Spry2 can compete with the RasGEF (guanine nucleotide exchange factor) SOS1 for binding to Grb2, resulting in the inhibition of phosphorylation of ERK1/2.

In the context of fibroblast growth factor (FGF) signaling, Sprouty2 (Spry2) is the most profound inhibitor of the Ras/ERK pathway as compared with other Spry isoforms. An exclusive, necessary, but cryptic PXXPXR motif in the C terminus of Spry2 is revealed upon stimulation. The activation of Spry2 appears to be linked to sequences in the N-terminal half of the protein and correlated with a bandshifting seen on SDS-PAGE. The bandshifting is likely caused by changes in the phosphorylation status of key Ser and Thr residues following receptor stimulation. Dephosphorylation of at least two conserved Ser residues (Ser-112 and Ser-115) within a conserved Ser/Thr sequence is accomplished upon stimulation by a phosphatase that binds to Spry2 around residues 50 -60. We show that human Spry2 coimmunoprecipitates with both the catalytic and the regulatory subunits of protein phosphatase 2A (PP2A-C and PP2A-A, respectively) in cells upon FGF receptor (FGFR) activation. PP2A-A binds directly to Spry2, but not to Spry2⌬50 -60 (⌬50 -60), and the activity of PP2A increases with both FGF treatment and FGFR1 overexpression. c-Cbl and PP2A-A compete for binding centered around Tyr-55 on Spry2. We show that there are at least two distinct pools of Spry2, one that binds PP2A and another that binds c-Cbl. c-Cbl binding likely targets Spry2 for ubiquitin-linked destruction, whereas the phosphatase binding and activity are necessary to dephosphorylate specific Ser/Thr residues. The resulting change in tertiary structure enables the Pro-rich motif to be revealed with subsequent binding of Grb2, a necessary step for Spry2 to act as a Ras/ERK pathway inhibitor in FGF signaling.

This study compared the use of different test models to assess the cytotoxicity of a dental composite. The cytotoxicity of a composite polymerized using two halogen-based light-curing units (LCUs) (Max LC and Astralis) and two light-emitting diode LCUs (E-light and Freelight) served as the basis of comparison. Disk-shaped specimens (7 mm diameter, 2 mm high) were fabricated using the four different light sources. The specimens were used in several cytotoxicity test models: direct and indirect contact tests as well as an extract test with an established cell line L-929. The cells were stained with neutral red after cell-material contact for 48 h. Neutral red-stained areas (in mm2, for direct and indirect tests) and absorbance readings (for extract tests) were analysed statistically using ANOVA and the Tukey post hoc test, with P < 0.05 considered to be significantly different. Good correlation between direct and indirect contact tests (r = 0.903) was found. The extract test was the least correlated among the three tests. It was found that the E-light + Freelight-cured composite elicited cytotoxicity from the correlated studies. Uncured specimens were most detrimental to the cells in all tests. Our data demonstrated that composite cured with light-emitting diode LCUs were cytotoxic to L-929 cells. Different test models were found to give rise to different findings. Thus, a good cell-material contact method would replicate more closely the physiological situation in vivo. This in turn would give more clinically relevant results.