In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae

Background

The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.

Results

A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.

Conclusion

In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users.     

Pharmacological characterization of INCB3344, a small molecule antagonist of human CCR2

The chemokine receptor 2 (CCR2) directs migration of monocytes and has been proposed to be a drug target for chronic inflammatory diseases. INCB3344 was first published as a small molecule nanomolar inhibitor of rodent CCR2. Here, we show that INCB3344 can also bind human CCR2 (hCCR2) with high affinity, having a dissociation constant (K_d) of approximately 5 nM. The binding of the compound to the receptor is rapid and reversible. INCB3344 potently inhibits hCCR2 binding of monocyte chemoattractant protein-1 (MCP-1) and MCP-1-induced signaling and function in hCCR2-expressing cells, including ERK phosphorylation and chemotaxis, and is competitive against MCP-1 in vitro. INCB3344 also blocks MCP-1 binding to monocytes in human whole blood, with potency consistent with in vitro studies. The whole blood binding assay described here can be used for monitoring pharmacodynamic activity of CCR2 antagonists in both preclinical models and in the clinic.     

Analyses of Current-Generating Mechanisms of Shewanella loihica PV-4 and Shewanella oneidensis MR-1 in Microbial Fuel Cells

Although members of the genus Shewanella have common features (e.g., the presence of decaheme c-type cytochromes [c-cyts]), they are widely variable in genetic and physiological features. The present study compared the current-generating ability of S. loihica PV-4 in microbial fuel cells (MFCs) with that of well-characterized S. oneidensis MR-1 and examined the roles of c-cyts in extracellular electron transfer. We found that strains PV-4 and MR-1 exhibited notable differences in current-generating mechanisms. While the MR-1 MFCs maintained a constant current density over time, the PV-4 MFCs continued to increase in current density and finally surpassed the MR-1 MFCs. Coulombic efficiencies reached 26% in the PV-4 MFC but 16% in the MR-1 MFCs. Although both organisms produced quinone-like compounds, anode exchange experiments showed that anode-attached cells of PV-4 produced sevenfold more current than planktonic cells in the same chamber, while planktonic cells of MR-1 produced twice the current of the anode-attached cells. Examination of the genome sequence indicated that PV-4 has more c-cyt genes in the metal reductase-containing locus than MR-1. Mutational analysis revealed that PV-4 relied predominantly on a homologue of the decaheme c-cyt MtrC in MR-1 for current generation, even though it also possesses two homologues of the decaheme c-cyt OmcA in MR-1. These results suggest that current generation in a PV-4 MFC is in large part accomplished by anode-attached cells, in which the MtrC homologue constitutes the main path of electrons toward the anode.Some species of dissimilatory metal-reducing bacteria (DMRB) are able to reduce solid metal oxides as terminal electron acceptors and generate currents in microbial fuel cells (MFCs) (2, 11, 14, 30, 46). Although mixed cultures are often used in MFC experiments (13), studies seeking a mechanistic understanding of electron transfer to electrode surfaces typically target pure cultures of such DMRB, due to the complexity in microbial communities. Presently, two model DMRB, Shewanella oneidensis MR-1 and Geobacter sulfurreducens PCA (2, 3, 12, 18, 31), are used in most investigations.S. oneidensis MR-1 is a metabolically diverse DMRB that has been studied extensively for its potential use in bioremediation applications. For this reason, MR-1 was the first Shewanella species to have its genome completely sequenced and annotated (10). In addition, since the first report in 1999 when this microorganism was shown to have the ability to transfer electrons to the electrode without an exogenously added mediator (14), it has also become one of the model organisms for the study of electron transfer mechanisms in MFCs.Although the molecular mechanisms for extracellular electron transfer have not yet been elucidated fully, c-type cytochromes (c-cyts) appear to be the key cellular components involved in this process (38). In S. oneidensis MR-1, OmcA and MtrC are outer membrane (OM), decaheme c-cyts that are considered to be involved in the direct (directly attached) electron transfer to solid metal oxides and anodes of MFCs (9, 20, 22, 23, 47). Several pieces of evidence suggest that OmcA and MtrC form a complex and act in a cooperative manner (33, 37, 42), and these results correlate with the fact that the genes encoding these proteins constitute an operon-like cluster in the chromosome (1). It has also been shown that MtrC and OmcA have overlapping functions as terminal reductases of metal oxides (25, 38). OmcA and MtrC are also present on the surface of nanowires and may be involved in the long-range transfer of electrons (8). In addition to direct electron transfer, MR-1 has the ability to produce water-soluble electron-shuttle compounds (quinones and flavins) that are involved in the mediated electron transfer from cells to distant solid electron acceptors (metal oxides or MFC anodes) (21, 27, 44).Recently, the genome sequences of nearly 20 Shewanella strains have been completed and annotated, opening the door to study the diversity of their extracellular electron transfer mechanisms. A comparison of their genomes has shown that although they have some consensus OM c-cyt genes, variations exist in the number and order of these genes in their metal reductase-containing loci (6). One such species is S. loihica strain PV-4, which was recently isolated from an iron-rich microbial mat near a deep-sea hydrothermal vent located on the Loihi Seamount in Hawaii (7, 32). The phenotypic and phylogenetic characteristics of PV-4 were determined, with a subsequent study focusing on the metal reduction and iron biomineralization capabilities of this bacterium (32). Initial experiments performed in our laboratory revealed that PV-4 developed a c-cyt-dependent deep red color that was much more striking than that of strain MR-1 when grown anaerobically with iron oxide as the terminal electron acceptor (26). This allowed us to assume that PV-4 could have a high extracellular electron transfer ability. Accordingly, the present study evaluated the current-producing ability of strain PV-4 in MFCs and examined the roles of some c-cyts in extracellular electron transfer. Special attention was paid to the comparison of PV-4 with MR-1 to reveal differences in mechanisms for extracellular electron transfer. We report herein differences between these strains in the roles of OM c-cyts for extracellular electron transfer, the behaviors and metabolic patterns of MFC, and the resultant MFC performances.     

Glucosinolate polymorphism in wild cabbage (Brassica oleracea) influences the structure of herbivore communities

Natural plant populations often show substantial heritable variation in chemical structure of secondary metabolites. Despite a great deal of evidence from laboratory studies that these chemicals influence herbivore behaviour and life history, there exists little evidence for the structuring of natural herbivore communities according to plant chemical profiles. Brassica oleracea (Brassicaceae) produces aliphatic glucosinolates, which break down into toxins when leaf tissue is damaged. Structural diversity in these glucosinolates is heritable, and varies considerably at two ecological scales in the UK: both within and between populations. We surveyed herbivore attack on plants producing different glucosinolates, using 12 natural B. oleracea populations. In contrast to the results of previous studies in this system, which suffered low statistical power, we found significant differential responses of herbivore species to heritable glucosinolates, both within and between plant populations. We found significant correlations between herbivore infestation rates and the presence or absence of two heritable glucosinolates: sinigrin and progoitrin. There was variation between herbivore species in the direction of response, the ecological scale at which responses were identified, and the correlations for some herbivore species changed at different times of the year. We conclude that variation in plant secondary metabolites can structure the community of herbivores that attack them, and propose that herbivore-mediated differential selection deserves further investigation as a mechanism maintaining the observed diversity of glucosinolates in wild Brassica.     

Occurrence and diversity of naphthalene dioxygenase genes in soil microbial communities from the Maritime Antarctic

The diversity of naphthalene dioxygenase genes ( ndo ) in soil environments from the Maritime Antarctic was assessed, dissecting as well the influence of the two vascular plants that grow in the Antarctic : Deschampsia antarctica and Colobanthus quitensis . Total community DNA was extracted from bulk and rhizosphere soil samples from Jubany station and Potter Peninsula, South Shetland Islands. ndo genes were amplified by a nested PCR and analysed by denaturant gradient gel electrophoresis approach (PCR-DGGE) and cloning and sequencing. The ndo- DGGE fingerprints of oil-contaminated soil samples showed even and reproducible patterns, composed of four dominant bands. The presence of vascular plants did not change the relative abundance of ndo genotypes compared with bulk soil. For non-contaminated sites, amplicons were not obtained for all replicates and the variability among the fingerprints was comparatively higher, likely reflecting a lower abundance of ndo genes. The phylogenetic analyses showed that all sequences were affiliated to the nahAc genes closely related to those described for Pseudomonas species and related mobile genetic elements. This study revealed that a microdiversity of nahAc -like genes exists in microbial communities of Antarctic soils and quantitative PCR indicated that their relative abundance was increased in response to anthropogenic sources of pollution.     

Contrasting diversity patterns of soil mites and nematodes in secondary succession

Soil biodiversity has been recognized as a key feature of ecosystem functioning and stability. However, soil biodiversity is strongly impaired by agriculture and relatively little is known on how and at what spatial and temporal scales soil biodiversity is restored after the human disturbances have come to an end. Here, a multi-scale approach was used to compare diversity patterns of soil mites and nematodes at four stages (early, mid, late, reference site) along a secondary succession chronosequence from abandoned arable land to heath land. In each field four soil samples were taken during four successive seasons. We determined soil diversity within samples (α-diversity), between samples (β-diversity) and within field sites (γ-diversity). The patterns of α- and γ-diversity developed similarly along the chronosequence for oribatid mites, but not for nematodes. Nematode α-diversity was highest in mid- and late-successional sites, while γ-diversity was constant along the chronosequence. Oribatid mite β-diversity was initially high, but decreased thereafter, whereas nematode β-diversity increased when succession proceeded; indicating that patterns of within-site heterogeneity diverged for oribatid mites and nematodes. The spatio-temporal diversity patterns after land abandonment suggest that oribatid mite community development depends predominantly on colonization of new taxa, whereas nematode community development depends on shifts in dominance patterns. This would imply that at old fields diversity patterns of oribatid mites are mainly controlled by dispersal, whereas diversity patterns of nematodes are mainly controlled by changing abiotic or biotic soil conditions. Our study shows that the restoration of soil biodiversity along secondary successional gradients can be both scale- and phylum-dependent.     

Legionella pneumophila Strain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins

Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.Many bacterial pathogens use specialized protein secretion systems to deliver into host cells virulence effector proteins that interfere with the antimicrobial responses of the host and facilitate the survival of the pathogen (5, 10, 22, 76). The components of these secretion systems are highly conserved. Comparative bioinformatic analysis of pathogen genomes revealed an ever-increasing number of proteins that are likely to be translocated virulence effectors. Only a few effectors have been characterized, and their biochemical functions are unknown, yet the identification of translocated effector proteins and their mechanism of action is fundamental to understanding the pathogenesis of many bacterial infections.Legionella pneumophila is the etiological agent of Legionnaires’ disease, which is an acute form of pneumonia (34, 66). L. pneumophila serogroup 1 accounts for more than 90% of all cases worldwide. Although L. pneumophila is an environmental organism, its ability to survive and replicate in amoebae, such as Acanthamoeba castellanii, has equipped the organism with the capacity to replicate in human cells (45, 58, 68, 80). Following the inhalation of aerosols containing L. pneumophila into the human lung, the bacteria promote their uptake by alveolar macrophages and epithelial cells (21, 44, 71), where they replicate within an intracellular vacuole that avoids fusion with the endocytic pathway (46, 47). L. pneumophila evades endosome fusion by establishing a replicative vacuole that shares many characteristics with the endoplasmic reticulum (ER) (48, 53, 89). The formation of the unique Legionella-containing vacuole (LCV) requires the Dot (defective in organelle trafficking)/Icm (intracellular multiplication) type IV secretion system (T4SS) (85, 91).Type IV secretion systems are versatile multiprotein complexes that can transport DNA and proteins to recipient bacteria or host cells (19, 36). Based on structural and organizational similarity, three main T4SS classes have been distinguished: T4SSA, T4SSB, and genomic island-associated T4SS (GI-T4SS) (3, 51). The genetic organization and components of T4SSA have high similarity to the classical VirB4/VirD4 transfer DNA (T-DNA) transfer system of Agrobacterium tumefaciens (3). In the sequenced L. pneumophila strains, three distinct T4SSAs with different prevalences among strains have been described: Lvh, Trb-1, and Trb-2 (37, 83, 86). The Lvh (Legionella vir homologues) T4SSA is not required for intracellular bacterial replication in macrophages and amoebae but seems to contribute to infection at lower temperatures and inclusion in Acanthamoeba castellanii cysts (6, 78, 86).The Dot/Icm T4SSB secretes and translocates multiple bacterial effector proteins into the vacuolar membrane and cytosol of the host cell (31, 70). The functions of the great majority of these proteins are unknown. Several effectors have similarity to eukaryotic proteins or carry eukaryotic motifs (7, 16, 25). They are predicted to allow L. pneumophila to manipulate host cell processes by functional mimicry (31, 70). Many of the effectors have paralogues or belong to related protein families that are likely to have overlapping functions.Comparative analysis of the recent L. pneumophila genome sequences has revealed their diversity and plasticity (16, 18, 88). This plasticity enables the bacterium to acquire new genetic factors, including new effector proteins that enhance bacterial replication and survival in eukaryotic cells. This has resulted in a diverse species in which 7 to 11% of the genes in each L. pneumophila isolate are strain specific (38). Some of the diversity occurs among genes encoding Dot/Icm effectors, including those within the same family. For example some ankyrin repeat and F-box effector genes are highly conserved, while others vary considerably between L. pneumophila isolates (16, 41, 62, 73, 75). Even though it is not experimentally proven, the subsequent selection of Dot/Icm effectors among different L. pneumophila isolates might reflect their usefulness in host-pathogen interactions, whereby different effector repertoires are maintained during adaptation to different environmental niches or hosts. This may then translate into differences in virulence during opportunistic infection.In this study, we sequenced the genome of L. pneumophila serogroup 1 strain 130b (ATCC BAA-74, also known as Wadsworth or AA100) (29, 30) and analyzed the sequence for T4SSs and novel Dot/Icm effectors. This analysis established that the strain encodes a unique combination of T4SSs and a set of Dot/Icm effectors that had not been described previously but that are present in a range of clinical and environmental L. pneumophila isolates. The new effectors represent the latest members of an ever-growing list of T4SS substrates and presumably reflect the great capacity of L. pneumophila for adaptation to a variety of hosts.     

An Open-Label,Randomised Study of Dihydroartemisinin-Piperaquine Versus Artesunate-Mefloquine for Falciparum Malaria in Asia

Background

The artemisinin-based combination treatment (ACT) of dihydroartemisinin (DHA) and piperaquine (PQP) is a promising novel anti-malarial drug effective against multi-drug resistant falciparum malaria. The aim of this study was to show non-inferiority of DHA/PQP vs. artesunate-mefloquine (AS+MQ) in Asia.

Methods and Findings

This was an open-label, randomised, non-inferiority, 63-day follow-up study conducted in Thailand, Laos and India. Patients aged 3 months to 65 years with Plasmodium falciparum mono-infection or mixed infection were randomised with an allocation ratio of 2∶1 to a fixed-dose DHA/PQP combination tablet (adults: 40 mg/160 mg; children: 20 mg/320 mg; n = 769) or loose combination of AS+MQ (AS: 50 mg, MQ: 250 mg; n = 381). The cumulative doses of study treatment over the 3 days were of about 6.75 mg/kg of DHA and 54 mg/kg of PQP and about 12 mg/kg of AS and 25 mg/kg of MQ. Doses were rounded up to the nearest half tablet. The primary endpoint was day-63 polymerase chain reaction (PCR) genotype-corrected cure rate. Results were 87.9% for DHA/PQP and 86.6% for AS+MQ in the intention-to-treat (ITT; 97.5% one-sided confidence interval, CI: >−2.87%), and 98.7% and 97.0%, respectively, in the per protocol population (97.5% CI: >−0.39%). No country effect was observed. Kaplan-Meier estimates of proportions of patients with new infections on day 63 (secondary endpoint) were significantly lower for DHA/PQP than AS+MQ: 22.7% versus 30.3% (p = 0.0042; ITT). Overall gametocyte prevalence (days 7 to 63; secondary endpoint), measured as person-gametocyte-weeks, was significantly higher for DHA/PQP than AS+MQ (10.15% versus 4.88%; p = 0.003; ITT). Fifteen serious adverse events were reported, 12 (1.6%) in DHA/PQP and three (0.8%) in AS+MQ, among which six (0.8%) were considered related to DHA/PQP and three (0.8%) to AS+MQ.

Conclusions

DHA/PQP was a highly efficacious drug for P. falciparum malaria in areas where multidrug parasites are prevalent. The DHA/PQP combination can play an important role in the first-line treatment of uncomplicated falciparum malaria.

Trial Registration

Controlled-Trials.com ISRCTN81306618     

Regulation of IFN response gene activity during infliximab treatment in rheumatoid arthritis is associated with clinical response to treatment

Introduction  

Cross-regulation between TNF and type I IFN has been postulated to play an important role in autoimmune diseases. Therefore, we determined the effect of TNF blockade in rheumatoid arthritis (RA) on the type I IFN response gene activity in relation to clinical response.