Asymmetric Genome Organization in an RNA Virus Revealed via Graph-Theoretical Analysis of Tomographic Data

Cryo-electron microscopy permits 3-D structures of viral pathogens to be determined in remarkable detail. In particular, the protein containers encapsulating viral genomes have been determined to high resolution using symmetry averaging techniques that exploit the icosahedral architecture seen in many viruses. By contrast, structure determination of asymmetric components remains a challenge, and novel analysis methods are required to reveal such features and characterize their functional roles during infection. Motivated by the important, cooperative roles of viral genomes in the assembly of single-stranded RNA viruses, we have developed a new analysis method that reveals the asymmetric structural organization of viral genomes in proximity to the capsid in such viruses. The method uses geometric constraints on genome organization, formulated based on knowledge of icosahedrally-averaged reconstructions and the roles of the RNA-capsid protein contacts, to analyse cryo-electron tomographic data. We apply this method to the low-resolution tomographic data of a model virus and infer the unique asymmetric organization of its genome in contact with the protein shell of the capsid. This opens unprecedented opportunities to analyse viral genomes, revealing conserved structural features and mechanisms that can be targeted in antiviral drug design.     

Multimodal Imaging of Orthotopic Mouse Model of Endometrial Carcinoma

Background

Orthotopic endometrial cancer models provide a unique tool for studies of tumour growth and metastatic spread. Novel preclinical imaging methods also have the potential to quantify functional tumour characteristics in vivo, with potential relevance for monitoring response to therapy.

Methods

After orthotopic injection with luc-expressing endometrial cancer cells, eleven mice developed disease detected by weekly bioluminescence imaging (BLI). In parallel the same mice underwent positron emission tomography–computed tomography (PET-CT) and magnetic resonance imaging (MRI) employing ¹⁸F-fluorodeoxyglocose (¹⁸F-FDG) or ¹⁸F- fluorothymidine (¹⁸F-FLT) and contrast reagent, respectively. The mice were sacrificed when moribund, and post-mortem examination included macroscopic and microscopic examination for validation of growth of primary uterine tumours and metastases. PET-CT was also performed on a patient derived model (PDX) generated from a patient with grade 3 endometrioid endometrial cancer.

Results

Increased BLI signal during tumour growth was accompanied by increasing metabolic tumour volume (MTV) and increasing MTV x mean standard uptake value of the tumour (SUV_mean) in ¹⁸F-FDG and ¹⁸F-FLT PET-CT, and MRI conspicuously depicted the uterine tumour. At necropsy 82% (9/11) of the mice developed metastases detected by the applied imaging methods. ¹⁸F-FDG PET proved to be a good imaging method for detection of patient derived tumour tissue.

Conclusions

We demonstrate that all imaging modalities enable monitoring of tumour growth and metastatic spread in an orthotopic mouse model of endometrial carcinoma. Both PET tracers, ¹⁸F-FDG and ¹⁸F-FLT, appear to be equally feasible for detecting tumour development and represent, together with MRI, promising imaging tools for monitoring of patient-derived xenograft (PDX) cancer models.     

Kinetics of polyglucose breakdown in Chlorobium

Washed cells of Chlorobium limicola f. thiosulfatophilum photoassimilate CO₂ and acetate into polyglucose, which is laid down within the cells as rosette-like granules. When the cells are incubated in the dark, the polyglucose is broken down. Experiments using electron microscopy and labelling with ¹⁴C-acetate have revealed that degradation of polyglucose occurs in such a way that all the granules are subject to degradation simultaneously and the polyglucose which has been formed most recently in the light, becomes metabolized in the dark first.     

Photoassimilation of acetate and metabolism of carbohydrate in Chlorobium thiosulfatophilum

1. Washed cell suspensions of Chlorobium thiosulfatophilum form large amounts of a polyglucose in the light. Addition of acetate to the cells increases the formation of polysaccharide considerably. During incubation in the dark, polysaccharide decreases with time, and organic acids such as succinic and propionic acid are excreted into the medium. 2. Glucose isolated from cells which had photoassimilated 1-, 2-, and U-¹⁴C-acetate had a specific activity which lay between 1 and 2 times that of the acetate substrates. 3. To analyse the distribution of radioactivity in the glucose units formed during photoassimilation of ¹⁴C-acetate, 2 microbial degradations, with bakers' yeast and Zymomonas mobilis respectively, were used. The results show that acetate gives rise to carbon atoms 1+2 and 5+6 of glucose, whereas carbon atomes 3+4 are not labelled. Further, the results indicate that glucose is not formed via the reductive pentose phosphate cycle when acetate is present.     

Regulation of gene expression is associated with tolerance of the Arctic copepod Calanus glacialis to CO2‐acidified sea water

Ocean acidification is the increase in seawater pCO₂ due to the uptake of atmospheric anthropogenic CO₂, with the largest changes predicted to occur in the Arctic seas. For some marine organisms, this change in pCO₂, and associated decrease in pH, represents a climate change‐related stressor. In this study, we investigated the gene expression patterns of nauplii of the Arctic copepod Calanus glacialis cultured at low pH levels. We have previously shown that organismal‐level performance (development, growth, respiration) of C. glacialis nauplii is unaffected by low pH. Here, we investigated the molecular‐level response to lowered pH in order to elucidate the physiological processes involved in this tolerance. Nauplii from wild‐caught C. glacialis were cultured at four pH levels (8.05, 7.9, 7.7, 7.5). At stage N6, mRNA was extracted and sequenced using RNA‐seq. The physiological functionality of the proteins identified was categorized using Gene Ontology and KEGG pathways. We found that the expression of 151 contigs varied significantly with pH on a continuous scale (93% downregulated with decreasing pH). Gene set enrichment analysis revealed that, of the processes downregulated, many were components of the universal cellular stress response, including DNA repair, redox regulation, protein folding, and proteolysis. Sodium:proton antiporters were among the processes significantly upregulated, indicating that these ion pumps were involved in maintaining cellular pH homeostasis. C. glacialis significantly alters its gene expression at low pH, although they maintain normal larval development. Understanding what confers tolerance to some species will support our ability to predict the effects of future ocean acidification on marine organisms.     

Assembly models for Papovaviridae based on tiling theory

A vital constituent of a virus is its protein shell, called the viral capsid, that encapsulates and hence provides protection for the viral genome. Assembly models are developed for viral capsids built from protein building blocks that can assume different local bonding structures in the capsid. This situation occurs, for example, for viruses in the family of Papovaviridae, which are linked to cancer and are hence of particular interest for the health sector. More specifically, the viral capsids of the (pseudo-) T = 7 particles in this family consist of pentamers that exhibit two different types of bonding structures. While this scenario cannot be described mathematically in terms of Caspar-Klug theory (Caspar D L D and Klug A 1962 Cold Spring Harbor Symp. Quant. Biol. 27 1), it can be modelled via tiling theory (Twarock R 2004 J. Theor. Biol. 226 477). The latter is used to encode the local bonding environment of the building blocks in a combinatorial structure, called the assembly tree, which is a basic ingredient in the derivation of assembly models for Papovaviridae along the lines of the equilibrium approach of Zlotnick (Zlotnick A 1994 J. Mol. Biol. 241 59). A phase space formalism is introduced to characterize the changes in the assembly pathways and intermediates triggered by the variations in the association energies characterizing the bonds between the building blocks in the capsid. Furthermore, the assembly pathways and concentrations of the statistically dominant assembly intermediates are determined. The example of Simian virus 40 is discussed in detail.     

cAMP effector mechanisms. Novel twists for an 'old' signaling system

Cyclic AMP (cAMP) has traditionally been thought to act exclusively through cAMP-dependent protein kinase (cAPK, PKA), but a growing number of cAMP effects are not attributable to general activation of cAPK. At present, cAMP is known also to directly regulate ion channels and the ubiquitous Rap guanine exchange factors Epac 1 and 2. Adding to the sophistication of cAMP signaling is the fact that (1) the cAPK holoenzyme is incompletely dissociated even at saturating cAMP, the level of free R subunit of cAPK being able to regulate the maximal activity of cAPK, (2) cAPK activity can be modulated by oxidative glutathionylation, and (3) cAPK is anchored close to relevant substrates, other signaling enzymes, and local compartments of cAMP. Finally, we will demonstrate an example of fine-tuning of cAMP signaling through synergistic induction of neurite extensions by cAPK and Epac.     

Rational engineering of enzyme stability

During the past 15 years there has been a continuous flow of reports describing proteins stabilized by the introduction of mutations. These reports span a period from pioneering rational design work on small enzymes such as T4 lysozyme and barnase to protein design, and directed evolution. Concomitantly, the purification and characterization of naturally occurring hyperstable proteins has added to our understanding of protein stability. Along the way, many strategies for rational protein stabilization have been proposed, some of which (e.g. entropic stabilization by introduction of prolines or disulfide bridges) have reasonable success rates. On the other hand, comparative studies and efforts in directed evolution have revealed that there are many mutational strategies that lead to high stability, some of which are not easy to define and rationalize. Recent developments in the field include increasing awareness of the importance of the protein surface for stability, as well as the notion that normally a very limited number of mutations can yield a large increase in stability. Another development concerns the notion that there is a fundamental difference between the "laboratory stability" of small pure proteins that unfold reversibly and completely at high temperatures and "industrial stability", which is usually governed by partial unfolding processes followed by some kind of irreversible inactivation process (e.g. aggregation). Provided that one has sufficient knowledge of the mechanism of thermal inactivation, successful and efficient rational stabilization of enzymes can be achieved.     

LPS from Neisseria meningitidis is crucial for inducing monocyte- and microparticle-associated tissue factor activity but not for tissue factor expression

Neisseria meningitidis causes sepsis with coagulopathy. The present study evaluated the tissue factor (TF)-inducing capacity of bacterial LPS in different presentation forms, i.e. membrane-bound LPS versus purified LPS, and of non-LPS components of N. meningitidis. By using a wild-type N. meningitidis, a mutant N. meningitidis lacking LPS (LPS-deficient N. meningitidis), purified LPS from N. meningitidis and Escherichia coli, we measured TF-expression and TF-activity on human monocytes and microparticles (MPs). The effect of TF-modulators, such as phosphatidylserine (PS), tissue factor pathway inhibitor (TFPI) and recombinant IL-10 (rhIL-10) was investigated. In plasmas from meningococcal patients, fibrinopeptide A (FPA), LPS and IL-10 were quantified. Monocytes and MPs exposed to purified LPS or wild-type N. meningitidis had much higher TF-activity than monocytes and MPs exposed to LPS-deficient N. meningitidis (clot formation assay). Incubation with wild-type N. meningitidis, but also LPS-deficient N. meningitidis, resulted in TF-expression on monocytes (flow cytometry, qRT-PCR). Increased cellular TF-activity is associated with coincident surface-exposure of PS and the number of monocytes positive for both PS and TF was significantly higher for monocytes exposed to wild-type N. meningitidis (7.6%) compared with monocytes exposed to LPS-deficient N. meningitidis (1.8%). Treatment with rhIL-10 reduced monocyte- and MP-associated TF-activity, the number of monocytes positive for both TF and PS, and microvesiculation. Patients with meningococcal septicemia had significantly higher levels of LPS, FPA and IL-10 than patients with distinct meningitis. Our results indicate that LPS from N. meningitidis is crucial for inducing TF-activity, but not for monocyte- and MP-associated TF-expression. TF-activity seems to require coincident expression of TF and PS on monocytes, and LPS induces such double-positive monocytes.