Non-immortalized human neural stem (NS) cells as a scalable platform for cellular assays

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells ( [Conti et al., 2005] and [Sun et al., 2008]). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133⁺CD24^−/lo cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca²⁺ imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.     

Three-way multivariate curve resolution applied to speciation of acid-base and thermal unfolding transitions of an alternating polynucleotide.

Analytical speciation of acid-base equilibria and thermal unfolding transitions of an alternating random polynucleotide containing cytosine and hypoxanthine, poly(C, I), is studied. The results are compared with those obtained previously for single-stranded polynucleotides, poly(I) and poly(C), and for the double-stranded poly(I). poly(C), to examine the influence of the secondary structure on the acid-base properties of bases. This study is based on monitoring acid-base titrations and thermal unfolding experiments by molecular absorption, CD, and molecular fluorescence spectroscopies. Experimental data were analyzed by a novel chemometric approach based on a recently developed three-way Multivariate Curve Resolution method, which allowed the simultaneous analysis of data from several spectroscopies. This procedure improves the resolution of the concentration profiles and pure spectra for the species and conformations present in folding-unfolding and acid-base equilibria. The results from acid-base studies showed the existence of only three species in the pH range 2-12 at 37 degrees C and 0.15M ionic strength. No cooperative effects were detected from the resolved concentration profiles, showing that equilibria concerning alternating polynucleotides like poly(C, I) are simpler than those involving poly(I). poly(C). Thermal unfolding experiments at neutral pH confirmed the existence of two transitions and one intermediate conformation. This intermediate conformation could only be detected and resolved without ambiguities when molecular absorption and CD spectral data were analyzed simultaneously.     

Pectin Lyase Activity in a Penicillium italicum Strain

An extracellular pectin lyase (PNL) [poly-(methoxygalacturonide)lyase; EC 4.2.2.10] produced by Penicillium italicum CECT 2294 grown on a surface bran (natural medium) or in a submerged (synthetic medium) culture was investigated. Both culture filtrates showed macerating activity at low pH on cucumber, potato, and orange tissues. The physicochemical properties of the enzyme obtained from both culture methods were identical, as well as its catalytic properties, which were assayed by different methods. The molecular mass of the PNL obtained by gel filtration chromatography was 22 kDa; the isoelectric point was 8.6, as determined by chromatofocusing; and the enzyme was able to catalyze the eliminative cleavage of pectins with low (37%) and high (from 54 to 82%) degrees of esterification. The PNL produced in liquid medium showed a K_m for pectin (degree of esterification, 70%) of 3.2 mg/ml, and the optimum pH was 6.0 to 7.0. This enzyme was stable at 50°C and at pH 8.0. The ability of this PNL to macerate plant tissues in acidic environmental conditions, its stability at low pH and temperatures up to 50°C (thus preventing mesophilic microbial growth), and the absence of pectinesterase make this preparation useful for the food industry.     

Effect of methionine sulfone on the growth of Citrobacter intermedius C3 (author's transl)]

Specific activity of glutamate dehydrogenase (GD) and glutamate synthase (GtS) has been determined in the wild strain C3 and on a non excreting pro- mutant strain. Methionine sulfone shows inhibitory effects on their growth. The addition of alpha-ketoglutarate to the medium prevents the inhibitory effect and increases the GtS specific activity in both strains. The physiological effect of methionine sulfone and its suppression by alpha-ketoglutarate is discussed.     

The ring finger protein 213 gene (Rnf213) contributes to Rift Valley fever resistance in mice

Mammalian Genome - Rift Valley fever (RVF) is an emerging viral zoonosis that primarily affects ruminants and humans. We have previously shown that wild-derived MBT/Pas mice are highly susceptible...     

Rate of Bacterial Mortality in Aquatic Environments

A method is proposed which provides a minimum estimate of the rate of bacterial mortality in growing natural populations of planktonic bacteria. This estimate is given by the rate of decrease of radioactivity from the DNA of a [³H]thymidine-labeled natural assemblage of bacteria after all added thymidine has been exhausted from the medium. Results obtained from river water, estuarine water, and seawater show overall bacterial mortality rates in the range 0.010 to 0.030 h⁻¹, in good agreement with the range of growth rates measured in the same environments. Use of selective filtration through Nuclepore filters (pore size, 2 μm) allowed us to determine the contribution of microzooplankton grazing to overall bacterial mortality. Grazing rates estimated by this method ranged from 0 to 0.02 h⁻¹.     

Pregnancy rates and metabolic profiles in cattle treated with propylene glycol prior to embryo transfer

The objective of this study was to determine the effect of a sustained propylene glycol administration to recipients of frozen/thawed in vivo derived bovine embryos. Heifers were treated with oral propylene glycol for the last 20 days before embryo transfer (n = 142), and untreated as controls (n = 133). Progesterone, insulin, insulin-like growth factor-I, glucose, urea and triglyceride were analysed in blood on Day 0 and Day 7 of the estrous cycle corresponding to embryo transfer. The heifers were selected as recipients when showing progesterone levels <2.0 ng/ml (Day 0) and >2.5 ng/ml (Day 7), according to corpus luteum quality on Day 7 by technicians unaware of animals treated. Within treated animals, significantly more recipients were selected, and increased progesterone, corpus luteum quality, pregnancy and calving rates were recorded. Day 7 progesterone concentrations were higher in heifers treated and transferred. Propylene glycol increased insulin and insulin-like-growth factor-I, but glucose, urea and triglyceride did not vary. Furthermore, insulin-like-growth factor-I, glucose and triglyceride increased at estrous time, but urea decreased and insulin remained unaltered. Together with the sustained gain in pregnancy rates throughout the experiment (2 years), other evidences suggested that the observed effects did not rely on nutritional deficiency. Thus, propylene glycol improved pregnancy rates after embryo-transfer, and progesterone, insulin and insulin-like-growth factor-I are probably involved in this effect.     

Pairwise associations between classical polymorphism in a human population from the Central Pyrenees

Analysis of pairwise associations between 17 genetic systems was performed on a sample of 160 unrelated and autochthonous individuals from the Spanish central Pyrenees. In contrast to other studies, association between ABO-Hp, ACP1-ADA or EsD-C3 systems was not detected. Significant differences between observed and expected frequencies of the joint phenotypes of Gc-P pilymorphisms are described . The significance of this association is discussed.     

Etiologic and epidemiologic study of dermatomycoses in Navarra (Spain)

We review the etiology of the dermatophytosis in Navarra (Spain) over a 5-year period and it is compared with previous studies. We have isolated 312 strains of dermatophyte fungi in 285 patients (188 men and 97 women). Trichophyton rubrum was the most frequently isolated species (58.6%) followed by Trichophyton mentagrophytes (26.2%) and Microsporum canis (10.5%). Concerning the location of the lesions, tinea pedis was the clinical pattern found in the greatest number of patients, followed by tinea corporis, tinea unguium and tinea capitis. Twenty eight percent of the isolations were accomplished in October and November. More than half of those patients questioned had had epidemiological contact with animals or practiced sports. The rise of tinea pedis in our region is emphasised. The possible causes of this increment are analyzed and some recommendations for its control are made.