The Effect of Freeze-Thaw Cycles on Gene Expression Levels in Lymphoblastoid Cell Lines

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene expression. We report a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene expression levels in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. We found that previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that while insight drawn from gene regulatory studies in mature LCLs may generally not be affected by the artificial nature of the LCL model system, many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures.     

Enhancing protein crystallization through precipitant synergy

Suitable conditions for protein crystallization are commonly identified by screening combinations of independent factors that affect crystal formation. Because precipitating agents are prime determinants of crystallization, we investigated whether a systematic exploration of combinations of mechanistically distinct precipitants would enhance crystallization. A crystallization screen containing 64 precipitant mixtures was devised. Tests with ten HIV envelope-related proteins demonstrated that use of precipitant mixtures significantly enhanced both the probability of crystallization as well as the quality of optimized crystals. Tests with hen egg white lysozyme generated a novel C2 crystal from a salt/organic solvent mixture; structure solution at 2 A resolution revealed a lattice held together by both hydrophobic and electrostatic dyad interactions. The results indicate that mechanistically distinct precipitants can synergize, with precipitant combinations adding unique dimensions to protein crystallization.     

Evaluating automated parameter constraining procedures of neuron models by experimental and surrogate data

Neuron models, in particular conductance-based compartmental models, often have numerous parameters that cannot be directly determined experimentally and must be constrained by an optimization procedure. A common practice in evaluating the utility of such procedures is using a previously developed model to generate surrogate data (e.g., traces of spikes following step current pulses) and then challenging the algorithm to recover the original parameters (e.g., the value of maximal ion channel conductances) that were used to generate the data. In this fashion, the success or failure of the model fitting procedure to find the original parameters can be easily determined. Here we show that some model fitting procedures that provide an excellent fit in the case of such model-to-model comparisons provide ill-balanced results when applied to experimental data. The main reason is that surrogate and experimental data test different aspects of the algorithm’s function. When considering model-generated surrogate data, the algorithm is required to locate a perfect solution that is known to exist. In contrast, when considering experimental target data, there is no guarantee that a perfect solution is part of the search space. In this case, the optimization procedure must rank all imperfect approximations and ultimately select the best approximation. This aspect is not tested at all when considering surrogate data since at least one perfect solution is known to exist (the original parameters) making all approximations unnecessary. Furthermore, we demonstrate that distance functions based on extracting a set of features from the target data (such as time-to-first-spike, spike width, spike frequency, etc.)—rather than using the original data (e.g., the whole spike trace) as the target for fitting—are capable of finding imperfect solutions that are good approximations of the experimental data.     

Niche evolution in Australian terrestrial mammals? Clarifying scale-dependencies in phylogenetic and functional drivers of co-occurrence

Interactive forces between competition and habitat filtering drive many biogeographic patterns over evolutionary time scales. However, the responsiveness of assemblages to these two forces is highly influenced by spatial scale, forming complex patterns of niche separation. We explored these spatial dependencies by quantifying the influence of phylogeny and functional traits in shaping present day native terrestrial mammal assemblages at multiple scales, principally by identifying the spatial scales at which niche evolution operates. We modelled the distribution of 53 native terrestrial mammal species across New South Wales, Australia. Using predicted distributions, we estimated the range overlap between each pair of species at increasing grain sizes (~0.8, 5.1, 20, 81, 506, 2,025, 8,100 km²). We employed a decision tree to identify how interactions among functional traits and phylogenetic relatedness translated to levels of sympatry at increasing spatial scales. We found that Australian terrestrial mammals displayed phylogenetic over-dispersion that was inversely related to spatial scale, suggesting that ecological processes were more influential than biogeographic sympatry patterns in defining assemblages of species. While the contribution of phylogenetic relatedness to patterns of co-occurrence decreased as spatial scale increased, the reverse was true for habitat preferences. At the same time, functional traits also operated at different scales, as dietary preferences dominated at local spatial scales (<10 km²) while body mass has a stronger effect at larger spatial scales. Our findings show that ecological and evolutionary processes operate at different scales and that Australian terrestrial mammals diverged slower along their micro-scale niche compared to their macro-scale niche. By combining phylogenetic and niche methods through the modelling of species distributions, we assessed whether specific traits were related to a particular niche. More importantly, conducting multi-scale spatial analysis avoids categorical assignment of traits-to-niches, providing a clearer relationship between traits and a species ecological niche and a more precise scaling for the axes of niche evolution.     

Concerted release of substrate domains from GroEL by ATP is demonstrated with FRET

The chaperonin GroEL assists protein folding by undergoing ATP-induced conformational changes that are concerted within each of its two back-to-back stacked rings. Here we examined whether concerted allosteric switching gives rise to all-or-none release and folding of domains in a chimeric fluorescent protein substrate, CyPet-YPet. Using this substrate, it was possible to determine the folding yield of each domain from its intrinsic fluorescence and that of the entire chimera by measuring Förster resonance energy transfer between the two domains. Hence, it was possible to determine whether release of one domain is accompanied by release of the other domain (concerted mechanism), or whether their release is not coupled. Our results show that the chimera's release tends to be concerted when folding is assisted by a wild-type GroEL variant, but not when assisted by the F44W/D155A mutant that undergoes a sequential allosteric switch. A connection between the allosteric mechanism of this molecular machine and its biological function in assisting folding is thus established.     

DNA methylation patterns associate with genetic and gene expression variation in HapMap cell lines

Background  

DNA methylation is an essential epigenetic mechanism involved in gene regulation and disease, but little is known about the mechanisms underlying inter-individual variation in methylation profiles. Here we measured methylation levels at 22,290 CpG dinucleotides in lymphoblastoid cell lines from 77 HapMap Yoruba individuals, for which genome-wide gene expression and genotype data were also available.     

Heterologous epitope-scaffold prime:boosting immuno-focuses B cell responses to the HIV-1 gp41 2F5 neutralization determinant

The HIV-1 envelope glycoproteins (Env) gp120 and gp41 mediate entry and are the targets for neutralizing antibodies. Within gp41, a continuous epitope defined by the broadly neutralizing antibody 2F5, is one of the few conserved sites accessible to antibodies on the functional HIV Env spike. Recently, as an initial attempt at structure-guided design, we transplanted the 2F5 epitope onto several non-HIV acceptor scaffold proteins that we termed epitope scaffolds (ES). As immunogens, these ES proteins elicited antibodies with exquisite binding specificity matching that of the 2F5 antibody. These novel 2F5 epitope scaffolds presented us with the opportunity to test heterologous prime:boost immunization strategies to selectively boost antibody responses against the engrafted gp41 2F5 epitope. Such strategies might be employed to target conserved but poorly immunogenic sites on the HIV-1 Env, and, more generally, other structurally defined pathogen targets. Here, we assessed ES prime:boosting by measuring epitope specific serum antibody titers by ELISA and B cell responses by ELISpot analysis using both free 2F5 peptide and an unrelated ES protein as probes. We found that the heterologous ES prime:boosting immunization regimen elicits cross-reactive humoral responses to the structurally constrained 2F5 epitope target, and that incorporating a promiscuous T cell helper epitope in the immunogens resulted in higher antibody titers against the 2F5 graft, but did not result in virus neutralization. Interestingly, two epitope scaffolds (ES1 and ES2), which did not elicit a detectable 2F5 epitope-specific response on their own, boosted such responses when primed with the ES5. Together, these results indicate that heterologous ES prime:boost immunization regimens effectively focus the humoral immune response on the structurally defined and immunogen-conserved HIV-1 2F5 epitope.     

Maximally efficient prediction in the early fly visual system may support evasive flight maneuvers

The visual system must make predictions to compensate for inherent delays in its processing. Yet little is known, mechanistically, about how prediction aids natural behaviors. Here, we show that despite a 20-30ms intrinsic processing delay, the vertical motion sensitive (VS) network of the blowfly achieves maximally efficient prediction. This prediction enables the fly to fine-tune its complex, yet brief, evasive flight maneuvers according to its initial ego-rotation at the time of detection of the visual threat. Combining a rich database of behavioral recordings with detailed compartmental modeling of the VS network, we further show that the VS network has axonal gap junctions that are critical for optimal prediction. During evasive maneuvers, a VS subpopulation that directly innervates the neck motor center can convey predictive information about the fly’s future ego-rotation, potentially crucial for ongoing flight control. These results suggest a novel sensory-motor pathway that links sensory prediction to behavior.