Riboproteomics of the hepatitis C virus internal ribosomal entry site

Hepatitis C virus (HCV) protein translation is mediated by a cis-acting RNA, an internal ribosomal entry site (IRES), located in the 5' nontranslated region of the viral RNA. To examine proteins bound to the IRES, which could include proteins important for its function as well as potential drug targets, we used shotgun peptide sequencing to identify proteins in quadruplicate protein affinity extracts of lysed Huh7 cells, obtained using a biotinylated IRES. Twenty-six proteins bound the HCV IRES but not a reversed complementary sequence RNA or vector RNA controls. These included five ribosomal subunits, nine eukaryotic initiation factor 3 subunits, and novel interacting proteins such as the cytoskeletal-related proteins actin, FHOS (formin homologue overexpressed in spleen) and MIP-T3 (microtubule interacting protein that associates with TRAF3). Other novel HCV IRES-binding proteins included UNR (upstream of N-ras), UNR-interacting protein, and the RNA-binding proteins PAI-1 (plasminogen activator inhibitor-1) mRNA binding protein and Ewing sarcoma breakpoint 1 region protein EWS. A large set of additional proteins bound both the HCV IRES and a reversed complementary IRES sequence control, including the known HCV interactors PTB (polypyrimidine tract binding protein), the La autoantigen, and nucleolin. The discovery of these novel HCV IRES-binding proteins suggests links between IRES biology and the cytoskeleton, signal transduction, and other cellular functions.     

Crystal Structure of the Pig Pancreatic α-Amylase Complexed with ρ-Nitrophenyl-α-D-Maltoside-Flexibility in the Active Site

The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase soaked with a rho-nitrophenyl-alpha-D-maltoside (pNPG2) substrate showed a pattern of electron density corresponding to the binding of a rho-nitrophenol unit at subsite -2 of the active site. Binding of the product to subsite -2 after hydrolysis of the pNPG2 molecules, may explain the low catalytic efficiency of the hydrolysis of pNPG2 by PPA. Except a small movement of the segment from residues 304-305 the typical conformational changes of the "flexible loop" (303-309), that constitutes the surface edge of the substrate binding cleft, were not observed in the present complex structure. This result supports the hypothesis that significant movement of the loop may depend on aglycone site being filled (Payan and Qian, J. Protein Chen. 22: 275, 2003). Structural analyses have shown that pancreatic alpha-amylases undergo an induced conformational change of the catalytic residue Asp300 upon substrate binding; in the present complex the catalytic residue is observed in its unliganded orientation. The results suggest that the induced reorientation is likely due to the presence of a sugar unit at subsite -1 and not linked to the closure of the flexible surface loop. The crystal structure was refined at 2.4 A resolution to an R factor of 17.55% (Rfree factor of 23.32%).     

Noninvasive Imaging of In Vivo MuRF1 Expression during Muscle Atrophy

Numerous human diseases can lead to atrophy of skeletal muscle, and loss of this tissue has been correlated with increased mortality and morbidity rates. Clinically addressing muscle atrophy remains an unmet medical need, and the development of preclinical tools to assist drug discovery and basic research in this effort is important for advancing this goal. In this report, we describe the development of a bioluminescent gene reporter rat, based on the zinc finger nuclease-targeted insertion of a bicistronic luciferase reporter into the 3′ untranslated region of a muscle specific E3 ubiquitin ligase gene, MuRF1 (Trim63). In longitudinal studies, we noninvasively assess atrophy-related expression of this reporter in three distinct models of muscle loss (sciatic denervation, hindlimb unloading and dexamethasone-treatment) and show that these animals are capable of generating refined detail on in vivo MuRF1 expression with high temporal and anatomical resolution.     

Chaste: An Open Source C++ Library for Computational Physiology and Biology

Chaste — Cancer, Heart And Soft Tissue Environment — is an open source C++ library for the computational simulation of mathematical models developed for physiology and biology. Code development has been driven by two initial applications: cardiac electrophysiology and cancer development. A large number of cardiac electrophysiology studies have been enabled and performed, including high-performance computational investigations of defibrillation on realistic human cardiac geometries. New models for the initiation and growth of tumours have been developed. In particular, cell-based simulations have provided novel insight into the role of stem cells in the colorectal crypt. Chaste is constantly evolving and is now being applied to a far wider range of problems. The code provides modules for handling common scientific computing components, such as meshes and solvers for ordinary and partial differential equations (ODEs/PDEs). Re-use of these components avoids the need for researchers to ‘re-invent the wheel’ with each new project, accelerating the rate of progress in new applications. Chaste is developed using industrially-derived techniques, in particular test-driven development, to ensure code quality, re-use and reliability. In this article we provide examples that illustrate the types of problems Chaste can be used to solve, which can be run on a desktop computer. We highlight some scientific studies that have used or are using Chaste, and the insights they have provided. The source code, both for specific releases and the development version, is available to download under an open source Berkeley Software Distribution (BSD) licence at http://www.cs.ox.ac.uk/chaste, together with details of a mailing list and links to documentation and tutorials.

This is a PLOS Computational Biology Software Article

     

Positional Bias of MHC Class I Restricted T-Cell Epitopes in Viral Antigens Is Likely due to a Bias in Conservation

The immune system rapidly responds to intracellular infections by detecting MHC class I restricted T-cell epitopes presented on infected cells. It was originally thought that viral peptides are liberated during constitutive protein turnover, but this conflicts with the observation that viral epitopes are detected within minutes of their synthesis even when their source proteins exhibit half-lives of days. The DRiPs hypothesis proposes that epitopes derive from Defective Ribosomal Products (DRiPs), rather than degradation of mature protein products. One potential source of DRiPs is premature translation termination. If this is a major source of DRiPs, this should be reflected in positional bias towards the N-terminus. By contrast, if downstream initiation is a major source of DRiPs, there should be positional bias towards the C-terminus. Here, we systematically assessed positional bias of epitopes in viral antigens, exploiting the large set of data available in the Immune Epitope Database and Analysis Resource. We show a statistically significant degree of positional skewing among epitopes; epitopes from both ends of antigens tend to be under-represented. Centric-skewing correlates with a bias towards class I binding peptides being over-represented in the middle, in parallel with a higher degree of evolutionary conservation.     

Hepcidin Bound to α2-Macroglobulin Reduces Ferroportin-1 Expression and Enhances Its Activity at Reducing Serum Iron Levels

Hepcidin regulates iron metabolism by down-regulating ferroportin-1 (Fpn1). We demonstrated that hepcidin is complexed to the blood transport protein, α₂-macroglobulin (α₂M) (Peslova, G., Petrak, J., Kuzelova, K., Hrdy, I., Halada, P., Kuchel, P. W., Soe-Lin, S., Ponka, P., Sutak, R., Becker, E., Huang, M. L., Suryo Rahmanto, Y., Richardson, D. R., and Vyoral, D. (2009) Blood 113, 6225–6236). However, nothing is known about the mechanism of hepcidin binding to α₂M or the effects of the α₂M·hepcidin complex in vivo. We show that decreased Fpn1 expression can be mediated by hepcidin bound to native α₂M and also, for the first time, hepcidin bound to methylamine-activated α₂M (α₂M-MA). Passage of high molecular weight α₂M·hepcidin or α₂M-MA·hepcidin complexes (≈725 kDa) through a Sephadex G-25 size exclusion column retained their ability to decrease Fpn1 expression. Further studies using ultrafiltration indicated that hepcidin binding to α₂M and α₂M-MA was labile, resulting in some release from the protein, and this may explain its urinary excretion. To determine whether α₂M-MA·hepcidin is delivered to cells via the α₂M receptor (Lrp1), we assessed α₂M uptake and Fpn1 expression in Lrp1^−/− and Lrp1^+/+ cells. Interestingly, α₂M·hepcidin or α₂M-MA·hepcidin demonstrated similar activities at decreasing Fpn1 expression in Lrp1^−/− and Lrp1^+/+ cells, indicating that Lrp1 is not essential for Fpn1 regulation. In vivo, hepcidin bound to α₂M or α₂M-MA did not affect plasma clearance of α₂M/α₂M-MA. However, serum iron levels were reduced to a significantly greater extent in mice treated with α₂M·hepcidin or α₂M-MA·hepcidin relative to unbound hepcidin. This effect could be mediated by the ability of α₂M or α₂M-MA to retard kidney filtration of bound hepcidin, increasing its half-life. A model is proposed that suggests that unlike proteases, which are irreversibly bound to activated α₂M, hepcidin remains labile and available to down-regulate Fpn1.     

Phylogeography and niche modelling of the relict plant Amborella trichopoda (Amborellaceae) reveal multiple Pleistocene refugia in New Caledonia

Amborella trichopoda Baill. (Amborellaceae, Amborellales), the sole living member of the sister group to all other extant angiosperms, is endemic to New Caledonia. We addressed the intraspecific phylogeography of Amborella by investigating whether its present population genetic structure could be related to its current and past habitats. We found moderate range‐wide genetic diversity based on nuclear microsatellite data and detected four well‐differentiated, geographically distinct genetic groups using Bayesian clustering analyses. We modelled the ecological niche of Amborella based on the current climatic and environmental conditions. The predictive ability of the model was very good throughout the Central East mainland zone, but Amborella was predicted in the northern part of the island where this plant has not been reported. Furthermore, no significant barrier was detected based on habitat suitability that could explain the genetic differentiation across the area. Conversely, we found that the main genetic clusters could be related to the distribution of the suitable habitat at the last glacial maximum (LGM, c. 21 000 years BP), when Amborella experienced a dramatic 96.5% reduction in suitable area. At least two lineages survived in distinct putative refugia located in the Massif des Lèvres and in the vicinity of Mount Aoupinié. Our findings finally confirmed the importance of LGM rainforest refugia in shaping the current intra‐ and interspecific diversity in New Caledonian plants and revealed the possibility of an as yet unreported refugium. The combination of niche modelling and population genetics thereby offered novel insight into the biogeographical history of an emblematic taxon.     

The impact of cytomegalovirus infection on clinical severity and outcomes in kidney transplant recipients with Pneumocystis jirovecii pneumonia

Cytomegalovirus (CMV) infection is associated with Pneumocystis jirovecii pneumonia (PJP) in kidney transplant recipients (KTRs), but its impact on clinical severity and outcomes in KTRs with PJP is unknown. We reviewed 1994 medical records of KTRs from January 1997 to March 2019. PJP or CMV infection was diagnosed by polymerase chain reaction or culturing using blood or respiratory specimens. We divided patients into PJP and PJP+CMV groups, and evaluated the clinical severity and outcomes. Fifty two patients had PJP (2.6%) in the whole study cohort. Among patients with PJP, 38 (73.1%) had PJP alone and 14 (26.9%) had combined PJP and CMV co-infection. The PJP+CMV group showed worse laboratory findings (serum albumin and C-reactive protein, P = 0.010 for both) and higher requirement of continuous renal replacement therapy than the PJP group (P = 0.050). The pneumonia severity was worse in the PJP+CMV group than in the PJP group (P < 0.05), and CMV infection was a high risk factor of pneumonia severity (odds ratio 16.0; P = 0.002). The graft function was worse in the PJP+CMV group (P < 0.001), and the incidence of graft failure was higher in the PJP+CMV group than in the PJP group (85.7% vs 36.8%; P < 0.001). Mortality was double in the PJP+CMV group than in the PJP group, but not statistically significant (21.4% vs 10.5%; P = 0.370). Our results show that approximately one in four patients with PJP after kidney transplantation develops CMV with increased clinical severity and risk of graft failure. The possibility of increased clinical severity and worse clinical outcomes by CMV co-infection should be considered in KTRs with PJP.     

Mechanisms underlying acquired platinum resistance in high grade serous ovarian cancer - a mini review

Background

Advanced epithelial ovarian cancer is one of the hardest human malignancies to treat. Standard treatment involves cytoreductive surgery and platinum-based chemotherapy, however, median progression-free survival for patients diagnosed with advanced stage disease (FIGO stages III and IV) is approximately 18?months. There has been little improvement in overall survival over the past decade and less than half of women with advanced stage disease will be living 5?years after diagnosis. A majority of patients initially have a favourable response to platinum-based chemotherapy, but most will eventually relapse and their disease will become platinum resistant.