Hypothesis generation in signaling networks.

Biological signaling networks comprise the chemical processes by which cells detect and respond to changes in their environment. Such networks have been implicated in the regulation of important cellular activities, including cellular reproduction, mobility, and death. Though technological and scientific advances have facilitated the rapid accumulation of information about signaling networks, utilizing these massive information resources has become infeasible except through computational methods and computer-based tools. To date, visualization and simulation tools have received significant emphasis. In this paper, we present a graph-theoretic formalization of biological signaling network models that are in wide but informal use, and formulate two problems on the graph: the Constrained Downstream and Minimum Knockout Problems. Solutions to these problems yield qualitative tools for generating hypotheses about the networks, which can then be experimentally tested in a laboratory setting. Using established graph algorithms, we provide a solution to the Constrained Downstream Problem. We also show that the Minimum Knockout Problem is NP-Hard, propose a heuristic, and assess its performance. In tests on the Epidermal Growth Factor Receptor (EGFR) network, we find that our heuristic reports the correct solution to the problem in seconds. Source code for the implementations of both solutions is available from the authors upon request.     

Seroprevalence of Toxoplasma gondii in wild kangaroos using an ELISA

Infection with Toxoplasma gondii is a significant problem in Australian marsupials, and can lead to devastating disease and predispose animals to predation. T. gondii infection in kangaroos is also of public health significance due to the kangaroo meat trade. A moderate seroprevalence of T. gondii was observed in a study of western grey kangaroos located in the Perth metropolitan area in Western Australia. Of 219 kangaroos tested, 15.5% (95%CI: 10.7–20.3) were positive for T. gondii antibodies using an ELISA developed to detect T. gondii IgG in macropod marsupials. When compared with the commercially available MAT (modified agglutination test), the ELISA developed was in absolute agreement and yielded a κ coefficient of 1.00. Of 18 kangaroos tested for the presence of T. gondii DNA by PCR, the 9 ELISA positive kangaroos tested PCR positive and the 9 ELISA negative kangaroos tested PCR negative indicating the ELISA protocol was both highly specific and sensitive and correlated 100% with the more labour intensive PCR assay.     

Nanomicellar Topical Aqueous Drop Formulation of Rapamycin for Back-of-the-Eye Delivery

The objective of this study was to develop a clear, aqueous rapamycin-loaded mixed nanomicellar formulations (MNFs) for the back-of-the-eye delivery. MNF of rapamycin (0.2%) was prepared with vitamin E tocopherol polyethylene glycol succinate (TPGS) (Vit E TPGS) and octoxynol-40 (Oc-40) as polymeric matrix. MNF was characterized by various parameters such as size, charge, shape, and viscosity. Proton nuclear magnetic resonance (¹H NMR) was used to identify unentrapped rapamycin in MNF. Cytotoxicity was evaluated in human retinal pigment epithelial (D407) and rabbit primary corneal epithelial cells (rPCECs). In vivo posterior ocular rapamycin distribution studies were conducted in male New Zealand white rabbits. The optimized MNF has excellent rapamycin entrapment and loading efficiency. The average size of MNF was 10.98 ± 0.089 and 10.84 ± 0.11 nm for blank and rapamycin-loaded MNF, respectively. TEM analysis revealed that nanomicelles are spherical in shape. Absence of free rapamycin in the MNF was confirmed by ¹H NMR studies. Neither placebo nor rapamycin-loaded MNF produced cytotoxicity on D407 and rPCECs indicating formulations are tolerable. In vivo studies demonstrated a very high rapamycin concentration in retina-choroid (362.35 ± 56.17 ng/g tissue). No drug was identified in the vitreous humor indicating the sequestration of rapamycin in lipoidal retinal tissues. In summary, a clear, aqueous MNF comprising of Vit E TPGS and Oc-40 loaded with rapamycin was successfully developed. Back-of-the-eye tissue distribution studies demonstrated a very high rapamycin levels in retina-choroid (place of drug action) with a negligible drug partitioning into vitreous humor.KEY WORDS: back-of-the-eye, drug delivery, formulation, mixed nanomicelles, posterior, rabbits, rapamycin/sirolimus, retina/choroid, sclera, topical eye drops     

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PP_i-dependent phosphofructokinase (TvPP_i-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPP_i-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to “short” bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.     

Calibrating the Human Mutation Rate via Ancestral Recombination Density in Diploid Genomes

The human mutation rate is an essential parameter for studying the evolution of our species, interpreting present-day genetic variation, and understanding the incidence of genetic disease. Nevertheless, our current estimates of the rate are uncertain. Most notably, recent approaches based on counting de novo mutations in family pedigrees have yielded significantly smaller values than classical methods based on sequence divergence. Here, we propose a new method that uses the fine-scale human recombination map to calibrate the rate of accumulation of mutations. By comparing local heterozygosity levels in diploid genomes to the genetic distance scale over which these levels change, we are able to estimate a long-term mutation rate averaged over hundreds or thousands of generations. We infer a rate of 1.61 ± 0.13 × 10⁻⁸ mutations per base per generation, which falls in between phylogenetic and pedigree-based estimates, and we suggest possible mechanisms to reconcile our estimate with previous studies. Our results support intermediate-age divergences among human populations and between humans and other great apes.     

Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.     

Mechanical forces regulate elastase activity and binding site availability in lung elastin

Many fundamental cellular and extracellular processes in the body are mediated by enzymes. At the single molecule level, enzyme activity is influenced by mechanical forces. However, the effects of mechanical forces on the kinetics of enzymatic reactions in complex tissues with intact extracellular matrix (ECM) have not been identified. Here we report that physiologically relevant macroscopic mechanical forces modify enzyme activity at the molecular level in the ECM of the lung parenchyma. Porcine pancreatic elastase (PPE), which binds to and digests elastin, was fluorescently conjugated (f-PPE) and fluorescent recovery after photobleach was used to evaluate the binding kinetics of f-PPE in the alveolar walls of normal mouse lungs. Fluorescent recovery after photobleach indicated that the dissociation rate constant (k_off) for f-PPE was significantly larger in stretched than in relaxed alveolar walls with a linear relation between k_off and macroscopic strain. Using a network model of the parenchyma, a linear relation was also found between k_off and microscopic strain on elastin fibers. Further, the binding pattern of f-PPE suggested that binding sites on elastin unfold with strain. The increased overall reaction rate also resulted in stronger structural breakdown at the level of alveolar walls, as well as accelerated decay of stiffness and decreased failure stress of the ECM at the macroscopic scale. These results suggest an important role for the coupling between mechanical forces and enzyme activity in ECM breakdown and remodeling in development, and during diseases such as pulmonary emphysema or vascular aneurysm. Our findings may also have broader implications because in vivo, enzyme activity in nearly all cellular and extracellular processes takes place in the presence of mechanical forces.     

Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.     

Cryoelectron tomographic analysis of an HIV-neutralizing protein and its complex with native viral gp120

Identifying structural determinants of human immunodeficiency virus (HIV) neutralization is an important component of rational drug and vaccine design. We used cryoelectron tomography and atomic force microscopy to characterize the structure of an extremely potent HIV-neutralizing protein, D1D2-Ig alpha tp (abbreviated as D1D2-IgP), a polyvalent antibody construct that presents dodecameric CD4 in place of the Fab regions. We show that D1D2-IgP has a novel structure, displaying greater flexibility of its antibody arms than the closely related IgM. Using simian immunodeficiency virus in complex with D1D2-IgP, we present unequivocal evidence that D1D2-IgP can cross-link surface spikes on the same virus and on neighboring viruses. The observed binding to the viral envelope spikes is the result of specific CD4-gp120 interaction, because binding was not observed with MICA-IgP, a construct that is identical to D1D2-IgP except that major histocompatibility complex Class I-related Chain A (MICA) replaces the CD4 moiety. CD4-mediated binding was also associated with a significantly elevated proportion of ruptured viruses. The ratio of inactivated to CD4-liganded gp120-gp41 spikes can be much greater than 1:1, because all gp120-gp41 spikes on the closely apposed surfaces of cross-linked viruses should be incapable of accessing the target cell surface and mediating entry, as a result of inter-virus spike cross-linking. These results implicate flexibility rather than steric bulk or polyvalence per se as a structural explanation for the extreme potency of D1D2-IgP and thus suggest polyvalence presented on a flexible scaffold as a key design criterion for small molecule HIV entry inhibitors.