Neurotrophic Requirements of Human Motor Neurons Defined Using Amplified and Purified Stem Cell-Derived Cultures

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC₅₀ 1–2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.     

<Emphasis Type="Italic">Luteimonas aestuarii</Emphasis> sp. nov., isolated from tidal flat sediment

A novel bacterium B9^T was isolated from tidal flat sediment. Its morphology, physiology, biochemical features, and 16S rRNA gene sequence were characterized. Colonies of this strain are yellow and the cells are Gram-negative, rod-shaped, and do not require NaCl for growth. The 16S rRNA gene sequence similarity indicated that strain B9^T is associated with the genus Lysobacter (≤ 97.2%), Xanthomonas (≤ 96.8%), Pseudomonas (≤ 96.7%), and Luteimonas (≤ 96.0%). However, within the phylogenetic tree, this novel strain shares a branching point with the species Luteimonas composti CC-YY255^T (96.0%). The DNA-DNA hybridization experiments showed a DNA-DNA homology of 23.0% between strain B9^T and Luteimonas mephitis B1953/27.1^T. The G+C content of genomic DNA of the type strain is 64.7 mol% (SD, 1.1). The predominant fatty acids are iso-C_11:0, iso-C_15:0, iso-C_16:0, iso-C_17:0, iso-C_17:0 ω9c, and iso-C_11:0 3-OH. Combined analysis of the 16S rRNA gene sequences, fatty acid profile, and results from physiological and biochemical tests indicated that there is genotypic and phenotypic differentiation of the isolate from other Luteimonas species. For these reasons, strain B9^T was proposed as a novel species, named Luteimonas aestuarii. The type strain of the new species is B9^T (= KCTC 22048^T, DSM 19680^T).     

Isolation and functional analysis of a 24-residue linear alpha-helical antimicrobial peptide from Korean blackish cicada, Cryptotympana dubia (Homoptera)

A new antimicrobial peptide, cryptonin, was isolated and characterized from the adult Korean blackish cicada, Cryptotympana dubia. It consists of 24 amino acid residues and has a molecular weight of 2,704 Da on mass spectroscopy. The predicted alpha-helical structure analysis and increased helix percent in 40% trifloroethanol of cryptonin suggests that it belongs to the typical linear alpha-helix forming peptide. Binding of the biotin-labeled cryptonin at the surface of E. coli cells and increased influx of propidium iodide in E. coli after cryptonin treatment indicates that it kills microbial cells by binding bacterial cell surfaces and disrupting the cell permeability. Cryptonin showed strong antibacterial (MIC 1.56-25 microg/ml) and antifungal (MIC 3.12-50 microg/ml) activities against tested bacteria and fungi including two antibiotic-resistant bacterial strains; methicilin-resistant S. aureus and vancomycin-resistant Enterococci (MIC 25 microg/ml, each).     

Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant Chinese hamster ovary cells using a statistical design.

In order to develop a serum-free (SF) medium for the production of erythropoietin (EPO) by suspension culture of recombinant Chinese hamster ovary (rCHO) cells, a statistical optimization approach based on a Plackett-Burman design was adopted. A basal medium was prepared by supplementing Iscove's modified Dulbecco's medium (IMDM) with Fe(NO3)3.9H2O, CuCl2 and ZnSO4.7H2O which are generally contained in SF medium formulations. Insulin, transferrin and ethanolamine were also supplemented to the basal medium to determine their optimal concentrations. From this statistical analysis, glutamate, serine, methionine, phosphatidylcholine, hydrocortisone and pluronic F68 were identified as positive determinants for cell growth. The SF medium was formulated by supplementing the basal medium with components showing positive effects on cell growth in suspension culture. An EPO titer in this optimized SF medium was 79% of that in IMDM supplemented with 5% dialyzed fetal bovine serum (dFBS). Furthermore, the in vitro and in vivo biological activities of EPO produced in the SF medium were comparable to those produced in the serum-supplemented medium. Taken together, the results obtained here show that a Plackett-Burman design facilitates the development of SF media for the production of EPO by suspension culture of rCHO cells.     

Affinity biosensor for avidin using a double functionalized dendrimer monolayer on a gold electrode

We have developed an affinity biosensor system based on avidin-biotin interaction on a gold electrode. As the building block of an affinity-sensing monolayer, a fourth-generation (G4) poly(amidoamine) dendrimer having partial ferrocenyl-tethered surface groups was prepared and used. The unmodified surface amine groups from dendrimers were functionalized with biotinamidocaproate, and the biotinylated and electroactive dendritic monolayer was constructed on a gold electrode for the affinity-sensing surface interacting with avidin. An electrochemical signal from the affinity biosensor was generated by free glucose oxidase in electrolyte, depending on the degree of coverage of the sensing surface with avidin. The sensor signal decreased correlatively with increasing avidin concentration and approached a minimum level when the sensing surface was fully covered with avidin. The detection limit of avidin was about 4.5 pM, and the sensor signal was linear ranging from 1.5 pM to 10 nM under optimized conditions. From the kinetic analysis using the biotinylated glucose oxidase, an active enzyme coverage of 2.5 x 10(-12) mol/cm(2) on the avidin-pretreated surface was registered, which demonstrates the formation of a spatially ordered and compact protein layer on the derivatized electrode surface.     

Properties of L-arabinose isomerase from Escherichia coli as biocatalyst for tagatose production

L-arabinose isomerase (EC 5.3.1.4) mediates the isomerization of D-galactose into D-tagatose as well as the conversion of L-arabinose into L-ribulose. To investigate the properties of L-arabinose isomerase as a biocatalyst for the conversion of galactose to tagatose, the L-arabinose isomerase of Escherichia coli was characterized. The substrate specificity for L-arabinose was 166-fold higher than that for D-galactose. The optimal pH and temperature for the galactose isomerization reaction were 8.0 and 30 °C, respectively. The enzyme activity was stable for 1 h at temperatures below 35 °C and within a pH range of 8–10. The Michaelis constant, K _m, for galactose was 1480 mM, which is 25-fold higher than that for arabinose. The addition of Fe²⁺ and Mn²⁺ ions enhanced the conversion of galactose to tagatose, whereas the addition of Cu²⁺, Zn²⁺, Hg²⁺, and Fe³⁺ ions inhibited the reaction completely. In the presence of 1 mM Fe²⁺ ions, the K _m for galactose was found to be 300 mM.     

Innate immune responses of the airway epithelium

Barrier epithelia, especially airway epithelial cells, are persistently exposed to micro-organisms and environmental factors. To protect the host from these microbial challenges, many immune strategies have evolved. The airway epithelium participates in the critical innate immune response through the secretion of immune effectors such as mucin, antimicrobial peptides (AMP), and reactive oxygen species (ROS) to entrap or kill invading microbes. In addition, airway epithelial cells can act as mediators connecting innate and adaptive immunity by producing various cytokines and chemokines. Here, we present an overview of the role of mucosal immunity in airway epithelium, emphasizing the framework of bacterial and viral infections along with regulatory mechanisms of immune effectors in human cells and selected animal models. We also describe pathophysiological roles for immune effectors in human airway disease.     

HIV-1 Neutralizing Antibody Signatures and Application to Epitope-Targeted Vaccine Design

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