Viral population estimation using pyrosequencing

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an EM algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.Comment: 23 pages, 13 figure

Indeterminacy of Reverse Engineering of Gene Regulatory Networks: The Curse of Gene Elasticity

Gene Regulatory Networks (GRNs) have become a major focus of interest in recent years. A number of reverse engineering approaches have been developed to help uncover the regulatory networks giving rise to the observed gene expression profiles. However, this is an overspecified problem due to the fact that more than one genotype (network wiring) can give rise to the same phenotype. We refer to this phenomenon as “gene elasticity.” In this work, we study the effect of this particular problem on the pure, data-driven inference of gene regulatory networks.We simulated a four-gene network in order to produce “data” (protein levels) that we use in lieu of real experimental data. We then optimized the network connections between the four genes with a view to obtain the original network that gave rise to the data. We did this for two different cases: one in which only the network connections were optimized and the other in which both the network connections as well as the kinetic parameters (given as reaction probabilities in our case) were estimated. We observed that multiple genotypes gave rise to very similar protein levels. Statistical experimentation indicates that it is impossible to differentiate between the different networks on the basis of both equilibrium as well as dynamic data.We show explicitly that reverse engineering of GRNs from pure expression data is an indeterminate problem. Our results suggest the unsuitability of an inferential, purely data-driven approach for the reverse engineering transcriptional networks in the case of gene regulatory networks displaying a certain level of complexity

Spatial Extent of Charge Repulsion Regulates Assembly Pathways for Lysozyme Amyloid Fibrils

Formation of large protein fibrils with a characteristic cross β-sheet architecture is the key indicator for a wide variety of systemic and neurodegenerative amyloid diseases. Recent experiments have strongly implicated oligomeric intermediates, transiently formed during fibril assembly, as critical contributors to cellular toxicity in amyloid diseases. At the same time, amyloid fibril assembly can proceed along different assembly pathways that might or might not involve such oligomeric intermediates. Elucidating the mechanisms that determine whether fibril formation proceeds along non-oligomeric or oligomeric pathways, therefore, is important not just for understanding amyloid fibril assembly at the molecular level but also for developing new targets for intervening with fibril formation. We have investigated fibril formation by hen egg white lysozyme, an enzyme for which human variants underlie non-neuropathic amyloidosis. Using a combination of static and dynamic light scattering, atomic force microscopy and circular dichroism, we find that amyloidogenic lysozyme monomers switch between three different assembly pathways: from monomeric to oligomeric fibril assembly and, eventually, disordered precipitation as the ionic strength of the solution increases. Fibril assembly only occurred under conditions of net repulsion among the amyloidogenic monomers while net attraction caused precipitation. The transition from monomeric to oligomeric fibril assembly, in turn, occurred as salt-mediated charge screening reduced repulsion among individual charged residues on the same monomer. We suggest a model of amyloid fibril formation in which repulsive charge interactions are a prerequisite for ordered fibril assembly. Furthermore, the spatial extent of non-specific charge screening selects between monomeric and oligomeric assembly pathways by affecting which subset of denatured states can form suitable intermolecular bonds and by altering the energetic and entropic requirements for the initial intermediates emerging along the monomeric vs. oligomeric assembly path

Distinctive Gut Microbiota of Honey Bees Assessed Using Deep Sampling from Individual Worker Bees

Surveys of 16S rDNA sequences from the honey bee, Apis mellifera, have revealed the presence of eight distinctive bacterial phylotypes in intestinal tracts of adult worker bees. Because previous studies have been limited to relatively few sequences from samples pooled from multiple hosts, the extent of variation in this microbiota among individuals within and between colonies and locations has been unclear. We surveyed the gut microbiota of 40 individual workers from two sites, Arizona and Maryland USA, sampling four colonies per site. Universal primers were used to amplify regions of 16S ribosomal RNA genes, and amplicons were sequenced using 454 pyrotag methods, enabling analysis of about 330,000 bacterial reads. Over 99% of these sequences belonged to clusters for which the first blastn hits in GenBank were members of the known bee phylotypes. Four phylotypes, one within Gammaproteobacteria (corresponding to “Candidatus Gilliamella apicola”) one within Betaproteobacteria (“Candidatus Snodgrassella alvi”), and two within Lactobacillus, were present in every bee, though their frequencies varied. The same typical bacterial phylotypes were present in all colonies and at both sites. Community profiles differed significantly among colonies and between sites, mostly due to the presence in some Arizona colonies of two species of Enterobacteriaceae not retrieved previously from bees. Analysis of Sanger sequences of rRNA of the Snodgrassella and Gilliamella phylotypes revealed that single bees contain numerous distinct strains of each phylotype. Strains showed some differentiation between localities, especially for the Snodgrassella phylotype

LEDGF gene silencing impairs the tumorigenicity of prostate cancer DU145 cells by abating the expression of Hsp27 and activation of the Akt/ERK signaling pathway

Lens epithelium-derived growth factor (LEDGF) maintains survival pathways by augmenting the transcription of stress-response genes such as small heat-shock protein 27. Recently, aberrant expression of LEDGF was found in prostate cancer (PC). Herein, we showed that LEDGF overexpression upregulated Hsp27 in PC cells, DU145, PC-3 and LNCaP and promoted antiapoptotic pathways in PCs. We found that these cells had higher abundance of Hsp27, which was correlated with the levels of LEDGF expression. Transactivation assay in DU145 cells revealed that transactivation of Hsp27 was related to the magnitude of LEDGF expression. Silencing of LEDGF in DU145 cells abrogated Hsp27 expression and inhibited stimulated cell proliferation, invasiveness and migration. These cells were arrested in S and G2 phase, and failed to accumulate cyclin B1, and showed increased apoptosis. Furthermore, LEDGF-depleted DU145 cells displayed elevated Bax and cleaved caspase 9 expression and reduced levels of Bcl2, Bcl-XL. The activated survival pathway(s), ERK1/2 and Akt, were selectively decreased in these cells, which characteristically have lower tumorigenicity. Conversely, the depleted cells, when re-overexpressed with LEDGF or Hsp27, regained tumorigenic properties. Collectively, results reveal the involvement of LEDGF-mediated elevated expression of Hsp27-dependent survival pathway(s) in PC. Our findings suggest new lines of investigation aimed at developing therapies by targeting LEDGF or its aberrant expression-associated stimulated antiapoptotic pathway(s)

Goal Setting with ICF (International Classification of Functioning, Disability and Health) and Multidisciplinary Team Approach in Stroke Rehabilitation

Stroke-associated impairments display a wide variety of clinical signs and symptoms. Therefore, a multidisciplinary team with different experts working closely together is necessary for effective stroke rehabilitation

Towards a Synthetic Chloroplast

The evolution of eukaryotic cells is widely agreed to have proceeded through a series of endosymbiotic events between larger cells and proteobacteria or cyanobacteria, leading to the formation of mitochondria or chloroplasts, respectively. Engineered endosymbiotic relationships between different species of cells are a valuable tool for synthetic biology, where engineered pathways based on two species could take advantage of the unique abilities of each mutualistic partner.We explored the possibility of using the photosynthetic bacterium Synechococcus elongatus PCC 7942 as a platform for studying evolutionary dynamics and for designing two-species synthetic biological systems. We observed that the cyanobacteria were relatively harmless to eukaryotic host cells compared to Escherichia coli when injected into the embryos of zebrafish, Danio rerio, or taken up by mammalian macrophages. In addition, when engineered with invasin from Yersinia pestis and listeriolysin O from Listeria monocytogenes, S. elongatus was able to invade cultured mammalian cells and divide inside macrophages.Our results show that it is possible to engineer photosynthetic bacteria to invade the cytoplasm of mammalian cells for further engineering and applications in synthetic biology. Engineered invasive but non-pathogenic or immunogenic photosynthetic bacteria have great potential as synthetic biological devices

Monotone and near-monotone biochemical networks

Monotone subsystems have appealing properties as components of larger networks, since they exhibit robust dynamical stability and predictability of responses to perturbations. This suggests that natural biological systems may have evolved to be, if not monotone, at least close to monotone in the sense of being decomposable into a “small” number of monotone components, In addition, recent research has shown that much insight can be attained from decomposing networks into monotone subsystems and the analysis of the resulting interconnections using tools from control theory. This paper provides an expository introduction to monotone systems and their interconnections, describing the basic concepts and some of the main mathematical results in a largely informal fashion

Coordinating Environmental Genomics and Geochemistry Reveals Metabolic Transitions in a Hot Spring Ecosystem

We have constructed a conceptual model of biogeochemical cycles and metabolic and microbial community shifts within a hot spring ecosystem via coordinated analysis of the “Bison Pool” (BP) Environmental Genome and a complementary contextual geochemical dataset of ∼75 geochemical parameters. 2,321 16S rRNA clones and 470 megabases of environmental sequence data were produced from biofilms at five sites along the outflow of BP, an alkaline hot spring in Sentinel Meadow (Lower Geyser Basin) of Yellowstone National Park. This channel acts as a >22 m gradient of decreasing temperature, increasing dissolved oxygen, and changing availability of biologically important chemical species, such as those containing nitrogen and sulfur. Microbial life at BP transitions from a 92°C chemotrophic streamer biofilm community in the BP source pool to a 56°C phototrophic mat community. We improved automated annotation of the BP environmental genomes using BLAST-based Markov clustering. We have also assigned environmental genome sequences to individual microbial community members by complementing traditional homology-based assignment with nucleotide word-usage algorithms, allowing more than 70% of all reads to be assigned to source organisms. This assignment yields high genome coverage in dominant community members, facilitating reconstruction of nearly complete metabolic profiles and in-depth analysis of the relation between geochemical and metabolic changes along the outflow. We show that changes in environmental conditions and energy availability are associated with dramatic shifts in microbial communities and metabolic function. We have also identified an organism constituting a novel phylum in a metabolic “transition” community, located physically between the chemotroph- and phototroph-dominated sites. The complementary analysis of biogeochemical and environmental genomic data from BP has allowed us to build ecosystem-based conceptual models for this hot spring, reconstructing whole metabolic networks in order to illuminate community roles in shaping and responding to geochemical variability