Establishment and Partial Characterization of a Continuous Human Malignant Glioma Cell Line: NG97

1. A human glioma cell line, NG97, was established from tissue obtained from a patient diagnosed with a grade III astrocytoma.2. The NG97 cell line has been subcultured for more than 100 passages in standard culture media without feeder layer or collagen coatings.3. NG97 cells grow in vitro as two subpopulations with distinct morphological appearance: stellate cells with pleomorphic nuclei, and small round cells with few processes. The cells have a doubling time of about 72 h and a plating efficiency of 1%. The injection of NG97 cells into congenitally athymic mice induced the formation of solid tumor masses that could be retransplanted every 4 weeks. The cells obtained from tumor mass when cultivated in vitro had a morphology comparable to those of the initial culture.4. This cell line may prove useful for cellular and molecular studies as well as in studies of gliomas treatment.     

Prevalence of chronic obstructive pulmonary disease and variation in risk factors across four geographically diverse resource-limited settings in Peru

Background

It is unclear how geographic and social diversity affects the prevalence of chronic obstructive pulmonary disease (COPD). We sought to characterize the prevalence of COPD and identify risk factors across four settings in Peru with varying degrees of urbanization, altitude, and biomass fuel use.

Methods

We collected sociodemographics, clinical history, and post-bronchodilator spirometry in a randomly selected, age-, sex- and site-stratified, population-based sample of 2,957 adults aged ≥35 years (median age was 54.8 years and 49.3% were men) from four resource-poor settings: Lima, Tumbes, urban and rural Puno. We defined COPD as a post-bronchodilator FEV₁/FVC < 70%.

Results

Overall prevalence of COPD was 6.0% (95% CI 5.1%–6.8%) but with marked variation across sites: 3.6% in semi-urban Tumbes, 6.1% in urban Puno, 6.2% in Lima, and 9.9% in rural Puno (p < 0.001). Population attributable risks (PARs) of COPD due to smoking ≥10 pack-years were less than 10% for all sites, consistent with a low prevalence of daily smoking (3.3%). Rather, we found that PARs of COPD varied by setting. In Lima, for example, the highest PARs were attributed to post-treatment tuberculosis (16% and 22% for men and women, respectively). In rural Puno, daily biomass fuel for cooking among women was associated with COPD (prevalence ratio 2.22, 95% CI 1.02–4.81) and the PAR of COPD due to daily exposure to biomass fuel smoke was 55%.

Conclusions

The burden of COPD in Peru was not uniform and, unlike other settings, was not predominantly explained by tobacco smoking. This study emphasizes the role of biomass fuel use, and highlights pulmonary tuberculosis as an often neglected risk factor in endemic areas.     

Effect of intraluminal pillars on particle motion in bifurcated microchannels

A central feature of intussusceptive angiogenesis is the development of an intravascular pillar that bridges the opposing sides of the microvessel lumen. In this report, we created polydimethyl siloxane (PDMS) microchannels with geometric proportions based on corrosion casts of the colon microcirculation. The structure of the PDMS microchannels was a bifurcated channel with an intraluminal pillar in the geometric center of the bifurcation. The effect of the intraluminal pillar on particle flow paths was investigated using an in vitro perfusion system. The microchannels were perfused with fluorescent particles, and the particle movements were recorded using fluorescence videomicroscopy. We found that the presence of an intravascular pillar significantly decreased particle velocity in the bifurcation system (p < 0.05). In addition, the pillar altered the trajectory of particles in the center line of the flow stream. The particle trajectory resulted in prolonged pillar contact as well as increased residence time within the bifurcation system (p < 0.001). Our results suggest that the intravascular pillar not only provides a mechanism of increasing resistance to blood flow but may also participate in spatial redistribution of cells within the flow stream. Supported in part by NIH Grants HL47078, HL75426, HL054885, HL070542 and HLO74022.     

Signs of selective pressure on genetic variants affecting human height

Many decades of scientific investigation have proved the role of selective pressure in Homo Sapiens at least at the level of individual genes or loci. Nevertheless, there are examples of polygenic traits that are bound to be under selection, but studies devoted to apply population genetics methods to unveil such occurrence are still lacking. Stature provides a relevant example of well-studied polygenic trait for which is now available a genome-wide association study which has identified the genes involved in this trait, and which is known to be under selection. We studied the behavior of F_ST in a simulated toy model to detect population differentiation on a generic polygenic phenotype under selection. The simulations showed that the set of alleles involved in the trait has a higher mean F_ST value than those undergoing genetic drift only. In view of this we looked for an increase in the mean F_ST value of the 180 variants associated to human height. For this set of alleles we found F_ST to be significantly higher than the genomic background (p = 0.0356). On the basis of a statistical analysis we excluded that the increase was just due to the presence of outliers. We also proved as marginal the role played by local adaptation phenomena, even on different phenotypes in linkage disequilibrium with genetic variants involved in height. The increase of F_ST for the set of alleles involved in a polygenic trait seems to provide an example of symmetry breaking phenomenon concerning the population differentiation. The splitting in the allele frequencies would be driven by the initial conditions in the population dynamics which are stochastically modified by events like drift, bottlenecks, etc, and other stochastic events like the born of new mutations.     

Novel integrated microdialysis-amperometric system for in vitro detection of dopamine secreted from PC12 cells: design, construction, and validation

A novel dual channel in vitro apparatus, derived from a previously described design, has been coupled with dopamine (DA) microsensors for the flow-through detection of DA secreted from PC12 cells. The device, including two independent microdialysis capillaries, was loaded with a solution containing PC12 cells while a constant phosphate-buffered saline (PBS) medium perfusion was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular calcium was removed from extracellular fluid of the second capillary. After a first period of stabilization and DA baseline recording, KCl (75 mM) was added to the perfusion fluid of both capillaries. In this manner, a simultaneous “treatment–control” experimental design was performed to detect K⁺-evoked calcium-dependent DA secretion. For this purpose, self-referencing DA microsensors were developed, and procedures for making, testing, and calibrating them are described in detail. The electronic circuitry was derived from previously published schematics and optimized for dual sensor constant potential amperometry applications. The microdialysis system was tested and validated in vitro under different experimental conditions, and DA secretion was confirmed by high-performance liquid chromatography with electrochemical detection (HPLC–EC). PC12 cell viability was quantified before and after each experiment. The proposed apparatus serves as a reliable model for studying the effects of different drugs on DA secretion through the direct comparison of extracellular DA increase in treatment–control experiments performed on the same initial PC12 cell population.     

HPLC enantioseparation and absolute configuration of novel anti-inflammatory pyrrole derivatives

The assignment of the absolute configuration of novel anti-inflammatory pyrrole derivatives has been accomplished by a combined strategy based on independent physical methods. The key step of our stereochemical characterization approach is the production at mg-scale of enantiomerically pure forms by HPLC on Chiralpak IA stationary phase.     

Targeting Notch signaling cross-talk with estrogen receptor and ErbB-2 in breast cancer

The Notch signaling pathway is receiving considerable interest because of its pervasive importance in developmental biology and more recently, in the post-natal functions of the immune system and in cancer biology.Our observations, together with those of other laboratories, support a context-dependent role for Notch signaling in breast cancer.Targeting Notch signaling paves the way to new therapeutic strategy.     

Identification of an Iron-Sulfur Cluster That Modulates the Enzymatic Activity in NarE, a Neisseria meningitidis ADP-ribosyltransferase

In prokaryotes, mono-ADP-ribose transfer enzymes represent a family of exotoxins that display activity in a variety of bacterial pathogens responsible for causing disease in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report here that NarE, a putative ADP-ribosylating toxin previously identified from Neisseria meningitidis, which shares structural homologies with Escherichia coli heat labile enterotoxin and toxin from Vibrio cholerae, possesses an iron-sulfur center. The recombinant protein was expressed in E. coli, and when purified at high concentration, NarE is a distinctive golden brown in color. Evidence from UV-visible spectrophotometry and EPR spectroscopy revealed characteristics consistent of an iron-binding protein. The presence of iron was determined by colorimetric method and by an atomic absorption spectrophotometer. To identify the amino acids involved in binding iron, a combination of site-directed mutagenesis and UV-visible and enzymatic assays were performed. All four cysteine residues were individually replaced by serine. Substitution of Cys⁶⁷ and Cys¹²⁸ into serine caused a drastic reduction in the E₄₂₀/E₂₈₀ ratio, suggesting that these two residues are essential for the formation of a stable coordination. This modification led to a consistent loss in ADP-ribosyltransferase activity, while decrease in NAD-glycohydrolase activity was less dramatic in these mutants, indicating that the correct assembly of the iron-binding site is essential for transferase but not hydrolase activity. This is the first observation suggesting that a member of the ADP-ribosyltransferase family contains an Fe-S cluster implicated in catalysis. This observation may unravel novel functions exerted by this class of enzymes.     

Gene expression in mycorrhizal orchid protocorms suggests a friendly plant–fungus relationship

Main conclusion

Orchid mycorrhiza has been often interpreted as an antagonistic relationship. Our data on mycorrhizal protocorms do not support this view as plant defence genes were not induced, whereas some nodulin-like genes were significantly up-regulated. Orchids fully depend on symbiotic interactions with specific soil fungi for seed germination and early development. Germinated seeds give rise to a protocorm, a heterotrophic organ that acquires nutrients, including organic carbon, from the mycorrhizal partner. It has long been debated if this interaction is mutualistic or antagonistic. To investigate the molecular bases of the orchid response to mycorrhizal invasion, we developed a symbiotic in vitro system between Serapias vomeracea, a Mediterranean green meadow orchid, and the rhizoctonia-like fungus Tulasnella calospora. 454 pyrosequencing was used to generate an inventory of plant and fungal genes expressed in mycorrhizal protocorms, and plant genes could be reliably identified with a customized bioinformatic pipeline. A small panel of plant genes was selected and expression was assessed by real-time quantitative PCR in mycorrhizal and non-mycorrhizal protocorm tissues. Among these genes were some markers of mutualistic (e.g. nodulins) as well as antagonistic (e.g. pathogenesis-related and wound/stress-induced) genes. None of the pathogenesis or wound/stress-related genes were significantly up-regulated in mycorrhizal tissues, suggesting that fungal colonization does not trigger strong plant defence responses. In addition, the highest expression fold change in mycorrhizal tissues was found for a nodulin-like gene similar to the plastocyanin domain-containing ENOD55. Another nodulin-like gene significantly more expressed in the symbiotic tissues of mycorrhizal protocorms was similar to a sugar transporter of the SWEET family. Two genes coding for mannose-binding lectins were significantly up-regulated in the presence of the mycorrhizal fungus, but their role in the symbiosis is unclear.