The efficacy of detecting variants with small effects on the Affymetrix 6.0 platform using pooled DNA

Genome-wide genotyping of a cohort using pools rather than individual samples has long been proposed as a cost-saving alternative for performing genome-wide association (GWA) studies. However, successful disease gene mapping using pooled genotyping has thus far been limited to detecting common variants with large effect sizes, which tend not to exist for many complex common diseases or traits. Therefore, for DNA pooling to be a viable strategy for conducting GWA studies, it is important to determine whether commonly used genome-wide SNP array platforms such as the Affymetrix 6.0 array can reliably detect common variants of small effect sizes using pooled DNA. Taking obesity and age at menarche as examples of human complex traits, we assessed the feasibility of genome-wide genotyping of pooled DNA as a single-stage design for phenotype association. By individually genotyping the top associations identified by pooling, we obtained a 14- to 16-fold enrichment of SNPs nominally associated with the phenotype, but we likely missed the top true associations. In addition, we assessed whether genotyping pooled DNA can serve as an inexpensive screen as the second stage of a multi-stage design with a large number of samples by comparing the most cost-effective 3-stage designs with 80% power to detect common variants with genotypic relative risk of 1.1, with and without pooling. Given the current state of the specific technology we employed and the associated genotyping costs, we showed through simulation that a design involving pooling would be 1.07 times more expensive than a design without pooling. Thus, while a significant amount of information exists within the data from pooled DNA, our analysis does not support genotyping pooled DNA as a means to efficiently identify common variants contributing small effects to phenotypes of interest. While our conclusions were based on the specific technology and study design we employed, the approach presented here will be useful for evaluating the utility of other or future genome-wide genotyping platforms in pooled DNA studies.     

Basal lamina secreted by MDCK cells has size- and charge-selective properties

The role electrical charge plays in determining glomerular permeability to macromolecules remains unclear. If the glomerular basement membrane (GBM) has any significant role in permselectivity, physical principles would suggest a negatively charged GBM would reject similarly charged more than neutral species. However, recent in vivo studies with negative and neutral glomerular probes showed the opposite. Whether this observation is due to unique characteristics of the probes used or is a general physiological phenomenon remains to be seen. The goal of this study was to use the basement membrane deposited by Madin-Darby canine kidney epithelial cells as a simple model of a biologically derived, negatively charged filter to evaluate size- and charge-based sieving properties. Fluorescein isothiocyanate-labeled carboxymethylated Ficoll 400 (FITC-CM Ficoll 400) and amino-4-methyl-coumarin-labeled Ficoll 400 (AMC Ficoll 400) were used as negatively charged and neutral tracer molecules, respectively, during pressure-driven filtration. Streaming potential measurement indicated the presence of fixed, negative charge in the basal lamina. The sieving coefficient for neutral Ficoll 400 decreased by ～0.0013 for each 1-? increment in solute radius, compared with a decrease of 0.0023 per ? for the anionic Ficoll 400. In this system, molecular charge played a significant role in determining the sieving characteristics of the membrane, pointing to solute charge as a potential contributor to GBM permselectivity.     

Microdeletion/microduplication of proximal 15q11.2 between BP1 and BP2: a susceptibility region for neurological dysfunction including developmental and language delay

The proximal long arm of chromosome 15 has segmental duplications located at breakpoints BP1?CBP5 that mediate the generation of NAHR-related microdeletions and microduplications. The classical Prader-Willi/Angelman syndrome deletion is flanked by either of the proximal BP1 or BP2 breakpoints and the distal BP3 breakpoint. The larger Type I deletions are flanked by BP1 and BP3 in both Prader-Willi and Angelman syndrome subjects. Those with this deletion are reported to have a more severe phenotype than individuals with either Type II deletions (BP2?CBP3) or uniparental disomy 15. The BP1?CBP2 region spans approximately 500?kb and contains four evolutionarily conserved genes that are not imprinted. Reports of mutations or disturbed expression of these genes appear to impact behavioral and neurological function in affected individuals. Recently, reports of deletions and duplications flanked by BP1 and BP2 suggest an association with speech and motor delays, behavioral problems, seizures, and autism. We present a large cohort of subjects with copy number alteration of BP1 to BP2 with common phenotypic features. These include autism, developmental delay, motor and language delays, and behavioral problems, which were present in both cytogenetic groups. Parental studies demonstrated phenotypically normal carriers in several instances, and mildly affected carriers in others, complicating phenotypic association and/or causality. Possible explanations for these results include reduced penetrance, altered gene dosage on a particular genetic background, or a susceptibility region as reported for other areas of the genome implicated in autism and behavior disturbances.     

Adipose-derived stem cells for clinical applications: a review

The use of stem cells derived from adipose tissue as an autologous and self-replenishing source for a variety of differentiated cell phenotypes, provides a great deal of promise for reconstructive surgery. In this article, we review available literature encompassing methods of extraction of pluripotent adipose stem cells (ASCs) from lipoaspirate locations, their storage, options for culture, growth and differentiation, cryopreservation and its effect on stem cell survival and proliferation, and new technologies involving biomaterials and scaffolds. We will conclude by assessing potential avenues for developing this incredibly promising field.     

Sedum cools soil and can improve neighboring plant performance during water deficit on a green roof

Green roofs have the potential to function as islands of biodiversity within urban and suburban environments. However, plant diversity is constrained by the harsh environment of a green roof, especially summertime water deficit and heat stress. We hypothesized that Sedum species, which are highly tolerant of the roof-top environment, would reduce peak soil temperature and increase performance of neighboring plants during summer water deficit. To test these hypotheses, we grew focal plant species with and without Sedum on a green roof. We then monitored growth during wet periods and drought tolerance during dry periods. During a three-year experiment, S. album reduced maximum growth of neighbor plants, Agastache rupestris and Asclepias verticillata, during favorable growth conditions, but increased performance of neighbors during summer water deficit. In a second experiment, four species of Sedum were each found to decrease peak soil temperature by 5-7 °C. All species decreased total growth of neighboring Agastache ‘Black Adder’ during favorable growth conditions, but again increased performance during summer water deficit. These results suggest that the palette of green roof plants can be expanded by using Sedum species as nurse plants.     

Preparation and antibacterial evaluation of decarboxylated fluoroquinolones

Decarboxylated ciprofloxacin (3) has been reported in the literature to have antibacterial activities against Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Bacillus subtilis, Enterobacter cloacae, Serratia marcescens and especially potent activity against Escherichia coli. Herein, we report our syntheses of 3 and five additional decarboxylated fluoroquinolones (FQs). We have re-evaluated the antibacterial activity of these FQs. In contrast to previously reported data, none of these decarboxylated fluoroquinolones showed significant antibacterial activity in our assays using both the broth dilution and agar methods. Our study confirmed that the presence of a carboxylic acid group at the 3-position of the fluoroquinolone scaffold is essential for antibacterial activity.     

7-Alkyl-N(2)-substituted-3-deazaguanines. Synthesis, DNA polymerase III inhibition and antibacterial activity

Several 2-anilino- and 2-benzylamino-3-deaza-6-oxopurines [3-deazaguanines] and selected 8-methyl and 8-aza analogs have been synthesized. 7-Substituted N²-(3-ethyl-4-methylphenyl)-3-deazaguanines were potent and selective inhibitors of Gram+ bacterial DNA polymerase (pol) IIIC, and 7-substituted N²-(3,4-dichlorobenzyl)-3-deazaguanines were potent inhibitors of both pol IIIC and pol IIIE from Gram+ bacteria, but weakly inhibited pol IIIE from Gram− bacteria. Potent enzyme inhibitors in both classes inhibited the growth of Gram+ bacteria (MICs 2.5-10 μg/ml), and were inactive against the Gram− organism Escherichia coli. Several derivatives had moderate protective activity in Staphylococcus aureus-infected mice.     

Functional gene expression profiling in yeast implicates translational dysfunction in mutant huntingtin toxicity

Huntington disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (htt) protein. To uncover candidate therapeutic targets and networks involved in pathogenesis, we integrated gene expression profiling and functional genetic screening to identify genes critical for mutant htt toxicity in yeast. Using mRNA profiling, we have identified genes differentially expressed in wild-type yeast in response to mutant htt toxicity as well as in three toxicity suppressor strains: bna4Δ, mbf1Δ, and ume1Δ. BNA4 encodes the yeast homolog of kynurenine 3-monooxygenase, a promising drug target for HD. Intriguingly, despite playing diverse cellular roles, these three suppressors share common differentially expressed genes involved in stress response, translation elongation, and mitochondrial transport. We then systematically tested the ability of the differentially expressed genes to suppress mutant htt toxicity when overexpressed and have thereby identified 12 novel suppressors, including genes that play a role in stress response, Golgi to endosome transport, and rRNA processing. Integrating the mRNA profiling data and the genetic screening data, we have generated a robust network that shows enrichment in genes involved in rRNA processing and ribosome biogenesis. Strikingly, these observations implicate dysfunction of translation in the pathology of HD. Recent work has shown that regulation of translation is critical for life span extension in Drosophila and that manipulation of this process is protective in Parkinson disease models. In total, these observations suggest that pharmacological manipulation of translation may have therapeutic value in HD.     

Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture

We investigated the migration of human leukocytes through endothelial cells (EC), and particularly their underlying basement membrane (BM). EC were cultured for 20 days on 3 μm-pore filters or collagen gels to form a distinct BM, and then treated with tumour necrosis factor-α, interleukin-1β or interferon-γ. Neutrophil migration through the cytokine-treated EC and BM was delayed for 20-day compared to 4-day cultures. The BM alone obstructed chemotaxis of neutrophils, and if fresh EC were briefly cultured on stripped BM, there was again a hold-up in migration. In studies with lymphocytes and monocytes, we could detect little hold-up of migration for 20-day versus 4-day cultures, in either the filter- or gel-based models. Direct microscopic observations showed that BM also held-up neutrophil migration under conditions of flow. Treatment of upper and/or lower compartments of filters with antibodies against integrins, showed that neutrophil migration through the endothelial monolayer was dependent on β₂-integrins, but not β1- or β₃-integrins. Migration from the subendothelial compartment was supported by β1- and β₂-integrins for all cultures, but blockade of β₃-integrin only inhibited migration effectively for 20-day cultures. Flow cytometry indicated that there was no net increase in expression of β1- or β3-integrins during neutrophil migration, and that their specific subendothelial function was likely dependent on turnover of integrins during migration. These studies show that BM is a distinct barrier to migration of human neutrophils, and that β₃-integrins are particularly important in crossing this barrier. The lesser effect of BM on lymphocytes and monocytes supports the concept that crossing the BM is a separate, leukocyte-specific, regulated step in migration.