Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.     

BAC libraries construction from the ancestral diploid genomes of the allotetraploid cultivated peanut

Background

Cultivated peanut, Arachis hypogaea is an allotetraploid of recent origin, with an AABB genome. In common with many other polyploids, it seems that a severe genetic bottle-neck was imposed at the species origin, via hybridisation of two wild species and spontaneous chromosome duplication. Therefore, the study of the genome of peanut is hampered both by the crop's low genetic diversity and its polyploidy. In contrast to cultivated peanut, most wild Arachis species are diploid with high genetic diversity. The study of diploid Arachis genomes is therefore attractive, both to simplify the construction of genetic and physical maps, and for the isolation and characterization of wild alleles. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaënsis with genome types AA and BB respectively.

Results

We constructed and characterized two large-insert libraries in Bacterial Artificial Chromosome (BAC) vector, one for each of the diploid ancestral species. The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb. Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues.

Conclusion

These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map.

     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Guidepost neurons for the lateral olfactory tract: Expression of metabotropic glutamate receptor 1 and innervation by glutamatergic olfactory bulb axons

The guidepost neurons for the lateral olfactory tract, which are called lot cells, are the earliest‐generated neurons in the neocortex. They migrate tangentially and ventrally further down this tract, and provide scaffolding for the olfactory bulb axons projecting into this pathway. The molecular profiles of the lot cells are largely uncharacterized. We found that lot cells specifically express metabotropic glutamate receptor subtype‐1 at a very early stage of development. This receptor is functionally competent and responds to a metabotropic glutamate receptor agonist with a transient increase in the intracellular calcium ion concentration. When the glutamatergic olfactory bulb axons were electrically stimulated, lot cells responded to the stimulation with a calcium increase mainly via ionotropic glutamate receptors, suggesting potential neurotransmission between the axons and lot cells during early development. Together with the finding that lot cells themselves are glutamatergic excitatory neurons, our results provide another notable example of precocious interactions between the projecting axons and their intermediate targets. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Involvement of liver aldehyde oxidase in the reduction of nicotinamide N-oxide

The present paper describes that mammalian liver aldehyde oxidase is involved in the reduction of nicotinamide N-oxide to nicotinamide. Rabbit liver aldehyde oxidase supplemented with its electron donor exhibited a significant nicotinamide N-oxide reductase activity under anaerobic conditions. Liver cytosols from rabbits, hogs, guinea pigs, hamsters, rats and mice, all of them, similarly exhibited the N-oxide reductase activity in the presence of an electron donor of aldehyde oxidase, but not xanthine oxidase. The cytosolic N-oxide reductase activity was almost completely inhibited by menadione, an inhibitor of aldehyde oxidase.     

Deer herbivory affects the functional diversity of forest floor plants via changes in competition-mediated assembly rules

Distorted plant diversity patterns due to ungulate herbivory could be explained by changes in community assembly processes, but the effects of ungulate herbivory on plant community assembly remain unclear. Here, we examined the role of deer herbivory in the regulation of the assembly processes of a forest floor plant community by assessing species and functional diversity in over- and no-grazing plots (control and exclosure plots, respectively) in Shiretoko National Park in Japan. Compared with the exclosure plot, vegetation coverage was considerably lower, and species richness and diversity were higher in the control plot. Functional traits associated with competitive ability (leaf area and chlorophyll content) were significantly higher in the exclosure plot. The pattern of functional diversity changed from overdispersion to clustering with an increase in local crowdedness. This trait clustering indicates that the local communities that were free from ungulate disturbance gradually became dominated by some competitively superior plant species, which led to low species diversity and biotic homogenization. In contrast, the reduction in vegetation due to overgrazing by deer resulted in an increase in the relative importance of stochastic assembly processes, which enabled the coexistence of various species, including less competitive ones. Our results emphasize that although deer overabundance is of concern, their complete exclusion has a negative consequence from an ecological perspective. Because deer herbivory is an inherent process that affects the biodiversity of plants on the forest floor, the establishment of fences requires careful consideration to ensure the conservation of ecological processes and their associated biodiversity.     

Purification of NADPH-linked alpha,beta-ketoalkene double bond reductase from rat liver

NADPH-linked alpha,beta-ketoalkene double bond reductase was purified from rat liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose. AF-Blue Toyopearl and hydroxyapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 39,500 by the electrophoresis and by HPLC gel filtration on a TSK gel G3000 SWXL column. The double bond of 2-alkenals was also reduced by the enzyme, but to a lesser extent. The enzyme activity was inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuribenzoic acid, N-ethylmaleimide, iodoacetamide, dicumarol, quercitrin, and disulfirum. However, the enzyme was insensitive to oxygen.     

Apurinic/apyrimidinic endonuclease sensitive sites as intermediates in the in vitro degradation of deoxyribonucleic acid by neocarzinostatin

Neocarzinostatin (NCS) induces alkali-labile sites in DNA which are stabilized by NaBH4 reduction. The stabilized sites are sensitive to an AP endonuclease from human lymphoma cells. NCS-induced degradation of supercoiled Col E1 DNA proceeds in stepwise fashion with apurinic/apyrimidinic (AP) sites as intermediates. Degradation is increased when reaction occurs in the presence of AP endonuclease, and DNA reacted with NCS can be shown to have numerous AP endonuclease sensitive sites.