Fragment-based modelling of single stranded RNA bound to RNA recognition motif containing proteins

Protein-RNA complexes are important for many biological processes. However, structural modeling of such complexes is hampered by the high flexibility of RNA. Particularly challenging is the docking of single-stranded RNA (ssRNA). We have developed a fragment-based approach to model the structure of ssRNA bound to a protein, based on only the protein structure, the RNA sequence and conserved contacts. The conformational diversity of each RNA fragment is sampled by an exhaustive library of trinucleotides extracted from all known experimental protein–RNA complexes. The method was applied to ssRNA with up to 12 nucleotides which bind to dimers of the RNA recognition motifs (RRMs), a highly abundant eukaryotic RNA-binding domain. The fragment based docking allows a precise de novo atomic modeling of protein-bound ssRNA chains. On a benchmark of seven experimental ssRNA–RRM complexes, near-native models (with a mean heavy-atom deviation of <3 Å from experiment) were generated for six out of seven bound RNA chains, and even more precise models (deviation < 2 Å) were obtained for five out of seven cases, a significant improvement compared to the state of the art. The method is not restricted to RRMs but was also successfully applied to Pumilio RNA binding proteins.     

First evidence of polychaete intermediate hosts for Neospirorchis spp. marine turtle blood flukes (Trematoda: Spirorchiidae)

Life cycles of spirorchiids that infect the vascular system of turtles are poorly understood. Few life cycles of these blood flukes have been elucidated and all intermediate hosts reported are gastropods (Mollusca), regardless of whether the definitive host is a freshwater or a marine turtle. During a recent survey of blood fluke larvae in polychaetes on the coast of South Carolina, USA, spirorchiid-like cercariae were found to infect the polychaetes Amphitrite ornata (Terebellidae) and Enoplobranchus sanguineus (Polycirridae). Cercariae were large, furcate, with a ventral acetabulum, but no eyespots were observed. Partial sequences of D1–D2 domains of the large ribosomal subunit, the internal transcribed spacer 2, and the mitochondrial cytochrome oxidase 1 genes allowed the identification of sporocysts and cercariae as belonging to two unidentified Neospirorchis species reported from the green turtle, Chelonia mydas, in Florida: Neospirorchis sp. (Neogen 13) in A. ornata and Neospirorchis sp. (Neogen 14) in E. sanguineus. Phylogenetic analysis suggests that infection of annelids by blood flukes evolved separately in aporocotylids and spirorchiids. Our results support the contention that the Spirorchiidae is not a valid family and suggest that Neospirorchis is a monophyletic clade within the paraphyletic Spirorchiidae. Since specificity of spirorchiids for their intermediate hosts is broader than it was thus far assumed, surveys of annelids in turtle habitats are necessary to further our understanding of the life history of these pathogenic parasites.     

Micro-chemical imaging of cesium distribution in Arabidopsis thaliana plant and its interaction with potassium and essential trace elements

Cesium as an alkali element exhibits a chemical reactivity similar to that of potassium, an essential element for plants. It has been suggested that Cs phytotoxicity might be due either to its competition with potassium to enter the plant, resulting in K starvation, or to its intracellular competition with K binding sites in cells. Such elemental interactions can be evidenced by chemical imaging, which determines the elemental distributions. In this study, the model plant Arabidopsis thaliana was exposed to 1 mM cesium in the presence (20 mM) or not of potassium. The quantitative imaging of Cs and endogenous elements (P, S, Cl, K, Ca, Mn, Fe, and Zn) was carried out using ion beam micro-chemical imaging with 5 microm spatial resolution. Chemical imaging was also evidenced by microfocused synchrotron-based X-ray fluorescence (microXRF) which presents a better lateral resolution (<1 microm) but is not quantitative. Cesium distribution was similar to potassium which suggests that Cs can compete with K binding sites in cells. Cesium and potassium were mainly concentrated in the vascular system of stems and leaves. Cs was also found in lower concentration in leaves mesophyll/epidermis. This late representing the larger proportion in mass, mesophyll/epidermis can be considered as the major storage site for cesium in A. thaliana. Trichomes were not found to accumulate cesium. Interestingly, increased Mn, Fe, and Zn concentrations were observed in leaves at high chlorosis. Mn and Fe increased more in the mesophyll than in veins, whereas zinc increased more in veins than in the mesophyll suggesting a tissue specific interaction of Cs with these trace elements homeostasis. This study illustrates the sensitivity of ion beam microprobe and microfocused synchrotron-based X-ray fluorescence to investigate concentrations and distributions of major and trace elements in plants. It also shows the suitability of these analytical imaging techniques to complement biochemical investigations of metallic stress in plants.     

Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga

Mixotrophic microorganisms are able to use organic carbon as well as inorganic carbon sources and thus, play an essential role in the biogeochemical carbon cycle. In aquatic ecosystems, the alteration of carbon dioxide (CO₂) fixation by toxic metals such as cadmium – classified as a priority pollutant – could contribute to the unbalance of the carbon cycle. In consequence, the investigation of cadmium impact on carbon assimilation in mixotrophic microorganisms is of high interest. We exposed the mixotrophic microalga Chlamydomonas reinhardtii to cadmium in a growth medium containing both CO₂ and labelled ¹³C-[1,2] acetate as carbon sources. We showed that the accumulation of cadmium in the pyrenoid, where it was predominantly bound to sulphur ligands, impaired CO₂ fixation to the benefit of acetate assimilation. Transmission electron microscopy (TEM)/X-ray energy dispersive spectroscopy (X-EDS) and micro X-ray fluorescence (μXRF)/micro X-ray absorption near-edge structure (μXANES) at Cd L_III-edge indicated the localization and the speciation of cadmium in the cellular structure. In addition, nanoscale secondary ion mass spectrometry (NanoSIMS) analysis of the ¹³C/¹²C ratio in pyrenoid and starch granules revealed the origin of carbon sources. The fraction of carbon in starch originating from CO₂ decreased from 73 to 39% during cadmium stress. For the first time, the complementary use of high-resolution elemental and isotopic imaging techniques allowed relating the impact of cadmium at the subcellular level with carbon assimilation in a mixotrophic microalga.     

Plasma levels of osteopontin identify patients at risk for organ damage in systemic lupus erythematosus

Introduction

Osteopontin (OPN) has been implicated as a mediator of Th17 regulation via type I interferon (IFN) receptor signaling and in macrophage activity at sites of tissue repair. This study assessed whether increased circulating plasma OPN (cOPN) precedes development of organ damage in pediatric systemic lupus erythematosus (pSLE) and compared it to circulating plasma neutrophil gelatinase-associated lipocalin (cNGAL), a predictor of increased SLE disease activity.

Methods

cOPN and cNGAL were measured in prospectively followed pSLE (n = 42) and adult SLE (aSLE; n = 23) patients and age-matched controls. Time-adjusted cumulative disease activity and disease damage were respectively assessed using adjusted-mean SLE disease activity index (SLEDAI) (AMS) and SLICC/ACR damage index (SDI).

Results

Compared to controls, elevated cOPN and cNGAL were observed in pSLE and aSLE. cNGAL preceded worsening SLEDAI by 3-6 months (P = 0.04), but was not associated with increased 6-month AMS. High baseline cOPN, which was associated with high IFNalpha activity and expression of autoantibodies to nucleic acids, positively correlated with 6-month AMS (r = 0.51 and 0.52, P = 0.001 and 0.01 in pSLE and aSLE, respectively) and was associated with SDI increase at 12 months in pSLE (P = 0.001). Risk factors for change in SDI in pSLE were cOPN (OR 7.5, 95% CI [2.9-20], P = 0.03), but not cNGAL, cumulative prednisone, disease duration, immunosuppression use, gender or ancestry using univariate and multivariate logistic regression. The area under the curve (AUC) when generating the receiver-operating characteristic (ROC) of baseline cOPN sensitivity and specificity for the indication of SLE patients with an increase of SDI over a 12 month period is 0.543 (95% CI 0.347-0.738; positive predictive value 95% and negative predictive value 38%).

Conclusion

High circulating OPN levels preceded increased cumulative disease activity and organ damage in SLE patients, especially in pSLE, and its value as a predictor of poor outcome should be further validated in large longitudinal cohorts.     

The phenotype and genotype of rheumatoid arthritis in the Democratic Republic of Congo

Introduction

Little is known about rheumatoid arthritis in the black, particularly in Congolese, populations. Our objective was to describe the phenotype and genotype of rheumatoid arthritis (RA) in Congolese.

Methods

All consecutive rheumatoid arthritis (RA) patients attending Kinshasa University Hospital in a three-year time period were included. Demographics, clinical features and tobacco consumption were noted. Disease Activity Score (DAS)-28 based on the erythrocyte sedimentation rate (ESR), Health Assessment Questionnaire (HAQ), anti-citrullinated peptide antibodies (CCP) antibodies and rheumatoid factor (RF) were determined. Radiographs were scored according to Sharp-van der Heijde. On a subset of patients and controls HLA-DRB1 typing was performed.

Results

A total of 114 females and 14 males aged 51.2 ± 14.9 were included. Mean duration of symptoms was four years. Moderate tobacco consumption was reported in a minority of patients. DAS-28 at first visit was >5.1 and HAQ ≥0.5 in all patients. X-rays showed joint erosions and/or joint space narrowing, mostly of a moderate grade in 55.8% of patients. Anti-CCP and/or RF were present in 48.6% of patients with available data (n = 72) and in 3.0% of controls (n = 67). Radiographic changes and nodules were more frequent in RF or anti-CCP positive patients. One copy of the shared epitope was found in 13 patients (35.1%) and 3 controls (12.5%). Two copies were found in one patient (2.7%) and in one control (4.2%).

Conclusion

Congolese patients with RA consult long after disease onset. Despite this delay, the majority presents without major damage and is RF, anti-CCP and SE negative. We put forward the hypothesis that besides different environmental factors there is probably also a particular genetic risk profile in Congolese patients, different from the HLA-DRB1 shared epitope.     

Raltegravir flexibility and its impact on recognition by the HIV‐1 IN targets

HIV‐1 IN is a pertinent target for the development of AIDS chemotherapy. The first IN‐specific inhibitor approved for the treatment of HIV/AIDS, RAL, was designed to block the ST reaction. We characterized the structural and conformational features of RAL and its recognition by putative HIV‐1 targets – the unbound IN, the vDNA, and the IN?vDNA complex – mimicking the IN states over the integration process. RAL binding to the targets was studied by performing an extensive sampling of the inhibitor conformational landscape and by using four different docking algorithms: Glide, Autodock, VINA, and SurFlex. The obtained data evidenced that: (i) a large binding pocket delineated by the active site and an extended loop in the unbound IN accommodates RAL in distinct conformational states all lacking specific interactions with the target; (ii) a well‐defined cavity formed by the active site, the vDNA, and the shortened loop in the IN?vDNA complex provide a more optimized inhibitor binding site in which RAL chelates Mg²⁺ cations; (iii) a specific recognition between RAL and the unpaired cytosine of the processed DNA is governed by a pair of strong H‐bonds similar to those observed in DNA base pair G‐C. The identified RAL pose at the cleaved vDNA shed light on a putative step of RAL inhibition mechanism. This modeling study indicates that the inhibition process may include as a first step RAL recognition by the processed vDNA bound to a transient intermediate IN state, and thus provides a potentially promising route to the design of IN inhibitors with improved affinity and selectivity. Copyright © 2013 John Wiley & Sons, Ltd.     

A novel harmony search-K means hybrid algorithm for clustering gene expression data

Recent progress in bioinformatics research has led to the accumulation of huge quantities of biological data at various data sources. The DNA microarray technology makes it possible to simultaneously analyze large number of genes across different samples. Clustering of microarray data can reveal the hidden gene expression patterns from large quantities of expression data that in turn offers tremendous possibilities in functional genomics, comparative genomics, disease diagnosis and drug development. The k- ¬means clustering algorithm is widely used for many practical applications. But the original k-¬means algorithm has several drawbacks. It is computationally expensive and generates locally optimal solutions based on the random choice of the initial centroids. Several methods have been proposed in the literature for improving the performance of the k-¬means algorithm. A meta-heuristic optimization algorithm named harmony search helps find out near-global optimal solutions by searching the entire solution space. Low clustering accuracy of the existing algorithms limits their use in many crucial applications of life sciences. In this paper we propose a novel Harmony Search-K means Hybrid (HSKH) algorithm for clustering the gene expression data. Experimental results show that the proposed algorithm produces clusters with better accuracy in comparison with the existing algorithms.     

Mutation D816V alters the internal structure and dynamics of c-KIT receptor cytoplasmic region: implications for dimerization and activation mechanisms

The type III receptor tyrosine kinase (RTK) KIT plays a crucial role in the transmission of cellular signals through phosphorylation events that are associated with a switching of the protein conformation between inactive and active states. D816V KIT mutation is associated with various pathologies including mastocytosis and cancers. D816V-mutated KIT is constitutively active, and resistant to treatment with the anti-cancer drug Imatinib. To elucidate the activating molecular mechanism of this mutation, we applied a multi-approach procedure combining molecular dynamics (MD) simulations, normal modes analysis (NMA) and binding site prediction. Multiple 50-ns MD simulations of wild-type KIT and its mutant D816V were recorded using the inactive auto-inhibited structure of the protein, characteristic of type III RTKs. Computed free energy differences enabled us to quantify the impact of D816V on protein stability in the inactive state. We evidenced a local structural alteration of the activation loop (A-loop) upon mutation, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. A thorough normal mode analysis of several MD conformations led to a plausible molecular rationale to propose that JMR is able to depart its auto-inhibitory position more easily in the mutant than in wild-type KIT and is thus able to promote kinase mutant dimerization without the need for extra-cellular ligand binding. Pocket detection at the surface of NMA-displaced conformations finally revealed that detachment of JMR from the kinase domain in the mutant was sufficient to open an access to the catalytic and substrate binding sites.