Gene expression profiling of whole blood: Comparison of target preparation methods for accurate and reproducible microarray analysis

<p>Abstract</p> <p>Background</p> <p>Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system.</p> <p>Results</p> <p>We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples.</p> <p>Conclusion</p> <p>RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles.</p

Neuroenhancement in Military Personnel::Conceptual and Methodological Promises and Challenges

Military personnel face harsh conditions that strain their physical and mental well-being, depleting resources necessary for sustained operational performance. Future operations will impose even greater demands on soldiers in austere environments with limited support, and new training and technological approaches are essential. This report highlights the progress in cognitive neuroenhancement research, exploring techniques such as neuromodulation and neurofeedback, and emphasizes the inherent challenges and future directions in the field of cognitive neuroenhancement for selection, training, operations, and recovery

Coevolved mutations reveal distinct architectures for two core proteins in the bacterial flagellar motor

Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be the convertor element that provides mechanistic and species diversity.JK was supported by Medical Research Council grant U117581331. SK was supported by seed funds from Lahore University of Managment Sciences (LUMS) and the Molecular Biology Consortium

Toward A Brain-Based Theory of Beauty

We wanted to learn whether activity in the same area(s) of the brain correlate with the experience of beauty derived from different sources. 21 subjects took part in a brain-scanning experiment using functional magnetic resonance imaging. Prior to the experiment, they viewed pictures of paintings and listened to musical excerpts, both of which they rated on a scale of 1-9, with 9 being the most beautiful. This allowed us to select three sets of stimuli-beautiful, indifferent and ugly-which subjects viewed and heard in the scanner, and rated at the end of each presentation. The results of a conjunction analysis of brain activity showed that, of the several areas that were active with each type of stimulus, only one cortical area, located in the medial orbito-frontal cortex (mOFC), was active during the experience of musical and visual beauty, with the activity produced by the experience of beauty derived from either source overlapping almost completely within it. The strength of activation in this part of the mOFC was proportional to the strength of the declared intensity of the experience of beauty. We conclude that, as far as activity in the brain is concerned, there is a faculty of beauty that is not dependent on the modality through which it is conveyed but which can be activated by at least two sources-musical and visual-and probably by other sources as well. This has led us to formulate a brain-based theory of beauty

Genetic Editing of HBV DNA by Monodomain Human APOBEC3 Cytidine Deaminases and the Recombinant Nature of APOBEC3G

Hepatitis B virus (HBV) DNA is vulnerable to editing by human cytidine deaminases of the APOBEC3 (A3A-H) family albeit to much lower levels than HIV cDNA. We have analyzed and compared HBV editing by all seven enzymes in a quail cell line that does not produce any endogenous DNA cytidine deaminase activity. Using 3DPCR it was possible to show that all but A3DE were able to deaminate HBV DNA at levels from 10−2 to 10−5 in vitro, with A3A proving to be the most efficient editor. The amino terminal domain of A3G alone was completely devoid of deaminase activity to within the sensitivity of 3DPCR (∼10−4 to 10−5). Detailed analysis of the dinucleotide editing context showed that only A3G and A3H have strong preferences, notably CpC and TpC. A phylogenic analysis of A3 exons revealed that A3G is in fact a chimera with the first two exons being derived from the A3F gene. This might allow co-expression of the two genes that are able to restrict HIV-1Δvif efficiently

Fashionable curiosities: extreme footwear as wearable fantasies

This paper considers an aspect of the material culture fashion, focusing on shoes. Like clothes, shoes are used every day: they are essential objects that primarily allow people to perform daily and socially accepted activities, walk comfortably and adorn the body in a fashionable way. Furthermore, shoes are associated with the idea of individuality and can be highly practical or decorative, depending on their design and fashion style. With regard to their style, one phenomenon emerging in high fashion is that of the “impossible-to-wear shoes”: exhibitions and fashion shows staging designers’ bizarre shoes are becoming more frequent. During these shows, the spectators are presented with an unusual variety of footwear (e.g. heelless shoes; shoes without soles; fish-shaped shoes). These shoes are not made to fit the individual. On the contrary, their shape and forms are imposed on the individuals. This paper explores the extreme, impossible-to-wear shoes and considers the visual statements they make about contemporary society, women and femininity. I will argue that impossible-to-wear shoes are puzzling yet charming objects, epitomizing a spectacle-centered society: they are part of unexpected and personal performances, which blend the boundaries of fashion and art and allow the wearers to shift from an ordinary “self” to the extraordinary “other”