Modularity analysis based on predicted protein-protein interactions provides new insights into pathogenicity and cellular process of Escherichia coli O157:H7

We review grounding issues that influence the scientific usefulness of any biomedical multiscale model (MSM). Groundings are the collection of units, dimensions, and/or objects to which a variable or model constituent refers. To date, models that primarily use continuous mathematics rely heavily on absolute grounding, whereas those that primarily use discrete software paradigms (e.g., object-oriented, agent-based, actor) typically employ relational grounding. We review grounding issues and identify strategies to address them. We maintain that grounding issues should be addressed at the start of any MSM project and should be reevaluated throughout the model development process. We make the following points. Grounding decisions influence model flexibility, adaptability, and thus reusability. Grounding choices should be influenced by measures, uncertainty, system information, and the nature of available validation data. Absolute grounding complicates the process of combining models to form larger models unless all are grounded absolutely. Relational grounding facilitates referent knowledge embodiment within computational mechanisms but requires separate model-to-referent mappings. Absolute grounding can simplify integration by forcing common units and, hence, a common integration target, but context change may require model reengineering. Relational grounding enables synthesis of large, composite (multi-module) models that can be robust to context changes. Because biological components have varying degrees of autonomy, corresponding components in MSMs need to do the same. Relational grounding facilitates achieving such autonomy. Biomimetic analogues designed to facilitate translational research and development must have long lifecycles. Exploring mechanisms of normal-to-disease transition requires model components that are grounded relationally. Multi-paradigm modeling requires both hyperspatial and relational grounding.     

The chromosome-scale assembly of endive (Cichorium endivia) genome provides insights into the sesquiterpenoid biosynthesis

Endive (Cichorium endivia L.) is a leafy vegetable in the Asteraceae family. Sesquiterpene lactones (STLs) in endive leaves bring a bitter taste that varies between varieties. Despite their importance in breeding varieties with unique flavours, sesquiterpenoid biosynthesis pathways in endive are poorly understood. We assembled a chromosome-scale endive genome of 641 Mb with a contig N50 of 5.16 Mb and annotated 46,711 protein-coding genes. Several gene families, especially terpene synthases (TPS) genes, expanded significantly in the C. endivia genome. STLs biosynthesis-related genes and TPS genes in more bitter varieties have shown a higher level of expression, which could be attributed to genomic variations. Our results penetrate the origin and diversity of bitter taste and facilitate the molecular breeding of endive varieties with unique bitter tastes. The high-quality endive assembly would provide a reference genome for studying the evolution and diversity of Asteraceae.     

Structure-function relationships in apolipoprotein(a): insights into lipoprotein(a) assembly and pathogenicity

PURPOSE OF REVIEW: Lipoprotein(a) is a structurally and functionally unique lipoprotein consisting of the glycoprotein apolipoprotein(a) covalently linked to LDL. Lipoprotein(a) is assembled extracellularly by a two-step mechanism, still incompletely understood, in which initial non-covalent interactions between apolipoprotein(a) and apolipoprotein B precede specific disulfide bond formation. Elevated concentrations of plasma lipoprotein(a) are a risk factor for a variety of vascular diseases, including coronary heart disease, ischaemic stroke and venous thrombosis. Whereas many pathogenic mechanisms have been proposed for lipoprotein(a), it remains to be conclusively demonstrated which mechanisms are relevant to human disease. RECENT FINDINGS: Structural and functional studies have verified that apolipoprotein(a) kringle 4 types 6-8 contain lysine binding sites of a weaker affinity for lysine analogues than kringle 4 type 10. Recent evidence has conclusively shown a role for kringle 4 types 7 and 8 in lipoprotein(a) assembly. Moreover, apolipoprotein(a) has been shown to undergo a conformational change, from a closed to an open form, which accelerates the rate of covalent lipoprotein(a) assembly. Functional studies in vitro have identified the domains in apolipoprotein(a) that mediate its inhibitory effects on fibrin clot lysis, binding to fibrin and other biological substrates, and pro-inflammatory and anti-angiogenic properties. SUMMARY: Extensive structure-function studies of apolipoprotein(a) have begun to yield important insights into the domains in apolipoprotein(a) that mediate lipoprotein(a) assembly and the pathogenic effects of this lipoprotein. Continued investigations of these relationships will contribute critically to unravelling the many outstanding questions about lipoprotein(a) metabolism and pathophysiology.     

Calculating the electrostatic properties of RNA provides new insights into molecular interactions and function.

Solutions to the nonlinear Poisson-Boltzmann equation were used to obtain the electrostatic potentials of RNA molecules that have known three-dimensional structures. The results are described in terms of isopotential contours and surface electrostatic potential maps. Both representations have unexpected features: 'cavities' within isopotential contours and areas of enhanced negative potential on molecular surfaces. Intriguingly, the sites of unusual electrostatic features correspond to functionally important regions, suggesting that electrostatic properties play a key role in RNA recognition and stabilization. These calculations reveal that the electrostatic potentials generated by RNA molecules have a variety of functionally important characteristics that cannot be discerned by simple visual inspection of the molecular structure.     

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.     

Bioinformatics analysis of the locus for enterocyte effacement provides novel insights into type-III secretion

Background  

Like many other pathogens, enterohaemorrhagic and enteropathogenic strains of Escherichia coli employ a type-III secretion system to translocate bacterial effector proteins into host cells, where they then disrupt a range of cellular functions. This system is encoded by the locus for enterocyte effacement. Many of the genes within this locus have been assigned names and functions through homology with the better characterised Ysc-Yop system from Yersinia spp. However, the functions and homologies of many LEE genes remain obscure.     

The novel binding mode of N-alkyl-N'-hydroxyguanidine to neuronal nitric oxide synthase provides mechanistic insights into NO biosynthesis

A series of N-alkyl-N'-hydroxyguanidine compounds have recently been characterized as non-amino acid substrates for all three nitric oxide synthase (NOS) isoforms which mimic NO formation from N(omega)-hydroxy-L-arginine. Crystal structures of the nNOS heme domain complexed with either N-isopropyl-N'-hydroxyguanidine or N-butyl-N'-hydroxyguanidine reveal two different binding modes in the substrate binding pocket. The binding mode of the latter is consistent with that observed for the substrate N(omega)-hydroxy-L-arginine bound in the nNOS active site. However, the former binds to nNOS in an unexpected fashion, thus providing new insights into the mechanism on how the hydroxyguanidine moiety leads to NO formation. Structural features of substrate binding support the view that the OH-substituted guanidine nitrogen, instead of the hydroxyl oxygen, is the source of hydrogen supplied to the active ferric-superoxy species for the second step of the NOS catalytic reaction.     

Cryo-electron tomography provides novel insights into nuclear pore architecture: implications for nucleocytoplasmic transport

To go beyond the current structural consensus model of the nuclear pore complex (NPC), we performed cryo-electron tomography of fully native NPCs from Xenopus oocyte nuclear envelopes (NEs). The cytoplasmic face of the NPC revealed distinct anchoring sites for the cytoplasmic filaments, whereas the nuclear face was topped with a massive distal ring positioned above the central pore with indications of the anchoring sites for the nuclear basket filaments and putative intranuclear filaments. The rather "spongy" central framework of the NPC was perforated by an elaborate channel and void system, and at the membrane pore interface it exhibited distinct "handles" protruding into the lumen of the NE. The most variable structural moiety of the NPC was a rather tenuous central plug partially obstructing the central pore. Its mobile character was documented by time-lapse atomic force microscopy. Taken together, the new insights we gained into NPC structure support the notion that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargoes.     

Spatiotemporal dynamics of glycolytic waves provides new insights into the interactions between immobilized yeast cells and gels

The immobilization of cells or enzymes is a promising tool for the development of biosensors, yet the interactions between the fixative materials and the cells are not fully understood, especially with respect to their impact on both cell metabolism and cell-to-cell signaling. We show that the spatiotemporal dynamics of waves of metabolic synchronization of yeast cells provides a new criterion to distinguish the effect of different gels on the cellular metabolism, which otherwise could not be detected. Cells from the yeast Saccharomyces carlsbergensis were immobilized into agarose gel, silica gel (TMOS), or a mixture of TMOS and alginate. We compared these immobilized cells with respect to their ability to generate temporal, intracellular oscillations in glycolysis as well as propagating, extracellular synchronization waves. While the temporal dynamics, as measured by the period and the number of oscillatory cycles, was similar for all three immobilized cell populations, significant differences have been observed with respect to the shape of the waves, wave propagation direction and velocity in the three gel matrices used.     

The pathogenicity of splicing defects: mechanistic insights into pre‐mRNA processing inform novel therapeutic approaches

Removal of introns from pre‐mRNA precursors (pre‐mRNA splicing) is a necessary step for the expression of most genes in multicellular organisms, and alternative patterns of intron removal diversify and regulate the output of genomic information. Mutation or natural variation in pre‐mRNA sequences, as well as in spliceosomal components and regulatory factors, has been implicated in the etiology and progression of numerous pathologies. These range from monogenic to multifactorial genetic diseases, including metabolic syndromes, muscular dystrophies, neurodegenerative and cardiovascular diseases, and cancer. Understanding the molecular mechanisms associated with splicing‐related pathologies can provide key insights into the normal function and physiological context of the complex splicing machinery and establish sound basis for novel therapeutic approaches.     

Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly)₁₀, suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2′ positions, which may be attributed to the larger space available for substrate binding at the S2 and S2′ sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase.     

Genome sequence of foxtail millet (Setaria italica) provides insights into grass evolution and biofuel potential

Foxtail millet (Setaria italica), a member of the Poaceae grass family, is an important food and fodder crop in arid regions and has potential for use as a C(4) biofuel. It is a model system for other biofuel grasses, including switchgrass and pearl millet. We produced a draft genome (～423 Mb) anchored onto nine chromosomes and annotated 38,801 genes. Key chromosome reshuffling events were detected through collinearity identification between foxtail millet, rice and sorghum including two reshuffling events fusing rice chromosomes 7 and 9, 3 and 10 to foxtail millet chromosomes 2 and 9, respectively, that occurred after the divergence of foxtail millet and rice, and a single reshuffling event fusing rice chromosome 5 and 12 to foxtail millet chromosome 3 that occurred after the divergence of millet and sorghum. Rearrangements in the C(4) photosynthesis pathway were also identified.     

Structure of the extracellular portion of CD46 provides insights into its interactions with complement proteins and pathogens

The human membrane cofactor protein (MCP, CD46) is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6), Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4) that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.     

Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei

Burkholderia pseudomallei is a potential bioterror agent and the causative agent of melioidosis, a severe disease that is endemic in areas of Southeast Asia and Northern Australia. Infection is often associated with bacterial dissemination to distant sites, and there are many possible disease manifestations, with melioidosis septic shock being the most severe. Eradication of the organism following infection is difficult, with a slow fever-clearance time, the need for prolonged antibiotic therapy and a high rate of relapse if therapy is not completed. Mortality from melioidosis septic shock remains high despite appropriate antimicrobial therapy. Prevention of disease and a reduction in mortality and the rate of relapse are priority areas for future research efforts. Studying how the disease is acquired and the host-pathogen interactions involved will underpin these efforts; this review presents an overview of current knowledge in these areas, highlighting key topics for evaluation.