Aging refers to the physical and functional decline of the tissues over time that often leads to age-related degenerative diseases. Accumulating evidence implicates that the senescence of neural stem cells (NSCs) is of paramount importance to the aging of central neural system (CNS). However, exploration of the underlying molecular mechanisms has been hindered by the lack of proper aging models to allow the mechanistic examination within a reasonable time window. In the present study, we have utilized a hydroxyurea (HU) treatment protocol and effectively induced postnatal subventricle NSCs to undergo cellular senescence as determined by augmented senescence-associated-β-galactosidase (SA-β-gal) staining, decreased proliferation and differentiation capacity, increased G0/G1 cell cycle arrest, elevated reactive oxygen species (ROS) level and diminished apoptosis. These phenotypic changes were accompanied by a significant increase in p16, p21 and p53 expression, as well as a decreased expression of key proteins in various DNA repair pathways such as xrcc2, xrcc3 and ku70. Further proteomic analysis suggests that multiple pathways are involved in the HU-induced NSC senescence, including genes related to DNA damage and repair, mitochondrial dysfunction and the increase of ROS level. Intriguingly, compensatory mechanisms may have also been initiated to interfere with apoptotic signaling pathways and to minimize the cell death by downregulating Bcl2-associated X protein (BAX) expression. Taken together, we have successfully established a cellular model that will be of broad utilities to the molecular exploration of NSC senescence and aging.

Excitatory transmission in the brain is commonly mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In amyotrophic lateral sclerosis (ALS), AMPA receptors allow cytotoxic levels of calcium into neurons, contributing to motor neuron injury. We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents compared with vulnerable spinal motor neurons. We have also shown that PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown via siRNA promotes motor neuron survival in models of spinal muscular atrophy (SMA) and ALS. It has been reported that inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. In addition, leptin can regulate AMPA receptor trafficking via PTEN inhibition. Thus, we speculate that manipulation of AMPA receptors by PTEN may represent a potential therapeutic strategy for neuroprotective intervention in ALS and other neurodegenerative disorders. To this end, the first step is to establish a fibroblast-iPS-motor neuron in vitro cell model to study AMPA receptor manipulation. Here we report that iPS-derived motor neurons from human fibroblasts express AMPA receptors. PTEN depletion decreases AMPA receptor expression and AMPA-mediated whole-cell currents, resulting in inhibition of AMPA-induced neuronal death in primary cultured and iPS-derived motor neurons. Taken together, our results imply that PTEN depletion may protect motor neurons by inhibition of excitatory transmission that represents a therapeutic strategy of potential benefit for the amelioration of excitotoxicity in ALS and other neurodegenerative disorders.

Free ADP-ribose is a turnover product of NAD+, protein-bound polymeric and monomeric ADP-ribose, and cyclic ADP-ribose. But little is known about the specific cellular roles or metabolism of free ADP-ribose. ADP-ribose pyrophosphatase (EC 3.6.1.13), which hydrolyzes ADP-ribose into AMP and ribose-5'-phosphate, was purified from human erythrocytes. Purification was achieved to homogeneity by successive chromatographic steps, resulting in a final purification of 75,790-fold from the hemolysate. The purified enzyme showed a single band with the molecular weight of 34 kDa on SDS-PAGE both in the presence and absence of 2-mercaptoethanol. The molecular weight of the native enzyme calculated by gel filtration was 68 kDa, indicating that the active enzyme is a dimer of identical subunits. The enzyme requiring Mg2+ showed highest activity toward ADP-ribose, and about 40-70% activities with IDP-ribose, ADP-mannose and GDP-mannose. The enzyme showed a Km of 169 +/- 11 microM for ADP-ribose, broad pH optimum around pH 9.5, and pI of 5.1. ADP was a potent noncompetitive inhibitor with a Ki of 16 +/- 1.2 microM. These results suggest that our enzyme is unique, and different from the other ADP-ribose pyrophosphatases reported. ADP-ribose pyrophosphatase may play an important role in the regulation of intracellular steady-state of free ADP-ribose.

Rubus coreanus fruits have been employed as a traditional medicine for centuries in the Asia-Pacific region. Its pharmacological action differs according to the different extraction methods utilized and the degree of fruit ripening. In this study, we determined the cellular effect of different ethanol extracts of mature and immature Rubus coreanus fruits in human hepatic cell line, HepG2 cells. The antioxidant activity, effect on superoxide dismutase activity and cholesterol biosynthesis efficiency was also evaluated. Immature Rubus coreanus extract showed higher antioxidant capability, compared with that of its mature fractions. Cellular antioxidant proteins including HO-1, Cu/Zn-superoxide dismutase and catalase were highly expressed in the presence of Rubus coreanus. Cholesterol levels in HepG2 cells treated with the water fraction of immature Rubus coreanus were significantly reduced. This antihyperlipidaemic action of Rubus coreanus is a consequence of cholesterol biosynthesis and extracellular secretion in HepG2 cells. These results indicate that among different ethanol fraction of mature and immature Rubus coreanus fruit extracts, water extract of immature fruit extract shows higher antioxidant as well as higher antihyperlipidaemic action.

Aberrant fibroblast growth factor receptor (FGFR) activation/expression is a common feature in lung cancer (LC). In this study, we evaluated the antitumor activity of and the mechanisms underlying acquired resistance to two potent selective FGFR inhibitors, AZD4547 and BAY116387, in LC cell lines. The antitumor activity of AZD4547 and BAY1163877 was screened in 24 LC cell lines, including 5 with FGFR1 amplification. Two cell lines containing FGFR1 amplifications, H1581 and DMS114, were sensitive to FGFR inhibitors (IC50<250 nm). Clones of FGFR1-amplified H1581 cells resistant to AZD4547 or BAY116387 (H1581AR and H1581BR cells, respectively) were established. Receptor tyrosine kinase (RTK) array and immunoblotting analyses showed strong overexpression and activation of Met in H1581AR/BR cells, compared with that in the parental cells. Gene set enrichment analysis against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that cytokine-cytokine receptor interaction pathways were significantly enriched in H1581AR/BR cells, with Met contributing significantly to the core enrichment. Genomic DNA quantitative PCR and fluorescent in situ hybridization analyses showed MET amplification in H1581AR, but not in H1581BR, cells. Met amplification drives acquired resistance to AZD4547 in H1581AR cells by activating ErbB3. Combination treatment with FGFR inhibitors and an anaplastic lymphoma kinase (ALK)/Met inhibitor, crizotinib, or Met-specific short interfering RNA (siRNA) synergistically inhibited cell proliferation in both H1581AR and H1581BR cells. Conversely, ectopic expression of Met in H1581 cells conferred resistance to AZD4547 and BAY1163877. Acquired resistance to FGFR inhibitors not only altered cellular morphology, but also promoted migration and invasion of resistant clones, in part by inducing epithelial-to-mesenchymal transition. Taken together, our data suggest that Met activation is sufficient to bypass dependency on FGFR signaling. Concurrent inhibition of the Met and FGFR pathways may have synergistic clinical benefits when targeting FGFR-dependent LC.

GLE1 mutations cause lethal congenital contracture syndrome 1 (LCCS1), a severe autosomal recessive fetal motor neuron disease, and more recently have been associated with amyotrophic lateral sclerosis (ALS). The gene encodes a highly conserved protein with an essential role in mRNA export. The mechanism linking Gle1 function to motor neuron degeneration in humans has not been elucidated, but increasing evidence implicates abnormal RNA processing as a key event in the pathogenesis of several motor neuron diseases. Homozygous gle1(-/-) mutant zebrafish display various aspects of LCCS, showing severe developmental abnormalities including motor neuron arborization defects and embryonic lethality. A previous gene expression study on spinal cord from LCCS fetuses indicated that oligodendrocyte dysfunction may be an important factor in LCCS. We therefore set out to investigate the development of myelinating glia in gle1(-/-) mutant zebrafish embryos. While expression of myelin basic protein (mbp) in hindbrain oligodendrocytes appeared relatively normal, our studies revealed a prominent defect in Schwann cell precursor proliferation and differentiation in the posterior lateral line nerve. Other genes mutated in LCCS have important roles in Schwann cell development, thereby suggesting that Schwann cell deficits may be a common factor in LCCS pathogenesis. These findings illustrate the potential importance of glial cells such as myelinating Schwann cells in motor neuron diseases linked to RNA processing defects.

Identifying the extent of environmental contamination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for infection control and prevention. The extent of environmental contamination has not been fully investigated in the context of severe coronavirus disease (COVID-19) patients.

Searches of zebrafish EST and whole genome shotgun sequence databases for sequences encoding the sterol-sensing domain (SSD) protein motif identified two sets of DNA sequences with significant homology to the Drosophila dispatched gene required for release of secreted Hedgehog protein. Using morpholino antisense oligonucleotides, we found that inhibition of one of these genes, designated Disp1, results in a phenotype similar to that of the "you-type" mutants, previously implicated in signalling by Hedgehog proteins in the zebrafish embryo. Injection of disp1 mRNA into embryos homozygous for one such mutation, chameleon (con) results in rescue of the mutant phenotype. Radiation hybrid mapping localised disp1 to the same region of LG20 to which the con mutation was mapped by meiotic recombination analysis. Sequence analysis of disp1 cDNA derived from homozygous con mutant embryos revealed that both mutant alleles are associated with premature termination codons in the disp1 coding sequence. By analysing the expression of markers of specific cell types in the neural tube, pancreas and myotome of con mutant and Disp1 morphant embryos, we conclude that Disp1 activity is essential for the secretion of lipid-modified Hh proteins from midline structures.