Patients with rheumatoid arthritis (RA) have altered levels of exhaled nitric oxide (NO) compared with healthy controls. Here, we investigated whether the clinical features of and immunological factors in RA pathogenesis could be linked to the NO lung dynamics in early disease. A total of 44 patients with early RA and anti-citrullinated peptide antibodies (ACPAs), specified as cyclic citrullinated peptide 2 (CCP2), were included. Their exhaled NO levels were measured, and the alveolar concentration, the airway compartment diffusing capacity and the airway wall concentration of NO were estimated using the Högman–Meriläinen algorithm. The disease activity was measured using the Disease Activity Score for 28 joints. Serum samples were analysed for anti-CCP2, rheumatoid factor, free secretory component, secretory component containing ACPAs, antibodies against Porphyromonas gingivalis (Rgp) and total levels of IgA, IgA1 and IgA2. Significant negative correlations were found between the airway wall concentration of NO and the number of swollen joints (Rho −0.48, p = 0.004), between the airway wall concentration of NO and IgA rheumatoid factor (Rho −0.41, p = 0.017), between the alveolar concentration and free secretory component (Rho −0.35, p = 0.023) and between the alveolar concentration and C-reactive protein (Rho −0.36, p = 0.016), but none were found for anti-CCP2, IgM rheumatoid factor or the anti-Rgp levels. In conclusion, altered NO levels, particularly its production in the airway walls, may have a role in the pathogenesis of ACPA-positive RA.

Background/Aim: Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors. Patient and Methods: Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed. Results: Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3' end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient. Conclusion: We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse.

Patients with established rheumatoid arthritis (RA) and disease modifying treatments have lower nitric oxide (NO) levels in the alveolar compartment (C A NO) and in the airway wall (C aw NO), but also higher diffusion capacities for NO in the airways (D aw NO) compared to matched controls. The aim of the present study was to investigate the NO lung dynamics in patients with recent onset RA before and after immune suppression with methotrexate therapy. Patients with early RA and antibodies against anticitrullinated peptides (ACPA) were recruited. Measurement of exhaled NO and inflammatory markers in serum were performed. Clinical disease activity was evaluated with Disease Activity Score for 28 joints. Healthy individuals were used as matched controls. Data are presented as median (lower quartile, upper quartile) values. RA patients (n = 44) had lower exhaled NO (F E NO 50 ) 16 (10–24) ppb compared to controls 21 (15, 29) ppb, p = 0.013. In NO-dynamics, C A NO was lower in RA patients 1.6 (1.0, 2.2) ppb compared to the control subjects 2.3 (1.3, 3.1) ppb, p = 0.007. C aw NO was also lower in the RA patients 55 (24, 106) ppb compared to control subjects 124 (110, 170) ppb, p < 0.001, but D aw NO was higher 17 (8, 30) mL/s and 9 (5, 11) mL/s respectively, p < 0.001. Methotrexate treatment for three months reduced disease activity, but did not change the NO dynamics. In conclusion, the altered NO dynamics of the lung in ACPA-positive RA patients are already present in the early stages of the disease before any treatments and do not change after methotrexate therapy suggesting a role in the pathogenesis.

BACKGROUND/AIM:Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors.PATIENT AND METHODS:Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed.RESULTS:Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3' end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient.CONCLUSION:We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse.

Patients with established rheumatoid arthritis (RA) and recent onset RA have lower nitric oxide (NO) levels in the alveolar compartment (CANO) and in the airway wall (CawNO), but also a higher diffusion capacity for NO in the airways (DawNO) compared with matched controls. The aim was to study recent onset RA subjects, before treatment and 3 months of immunosuppressive treatment with methotrexate (MTX). RA subjects, ACPA positive, were recruited. Disease activity was measured using Disease Activity Score for 28 joints (DAS28), and disability according to Health Assessment Questionnaire (HAQ). The NO dynamics (median (IQR)) were estimated by the Högman-Meriläinen algorithm. When the non-smoking RA subjects (n=22) were compared with smoking subjects (n=8) there were differences in FENO50 of 19 (12, 25) ppb and 9 (5, 14) ppb resp. (p=0.004) and in NO dynamics CANO 2.1 (1.1, 2.3) ppb and 0.9 (0.3, 1.2) ppb resp. (p=0.007), while no differences were found in CawNO 65 (38, 119) ppb and 21 (17, 61) ppb resp. (p=0.12), and DawNO 16 (8, 31) mL/s and 17 (8, 27) mL/s resp. (p=0.68). The weekly MTX dose was 20 (15, 20) mg. There were reductions of DAS28, HAQ and s-CRP on MTX treatment with no differences between non-smokers and current smokers. Neither FENO50 nor any of the NO parameters changed after MTX. There were no correlations between FENO50 and NO parameters with DAS28, HAQ and s-CRP before or after treatment. The altered NO dynamics of the lung in ACPA positive RA patients are present in the early stage of the disease, before any treatments are initiated and do not change after MTX therapy. The HMA can be an additional tool to study the early pulmonary events in the development of the RA pathology.

Rheumatoid arthritis (RA) is an autoimmune disorder that primarily affects the joints. The aetiology of RA is not known, but appears to be multifactorial resulting in a pathologic immune reaction with antibody formation. The lungs might be the initial site for the immune reaction and involved in the development of RA. Exhaled NO analysis has to our knowledge never been applied in RA patients. The aim of the study was to apply the extended NO analysis in patients with rheumatoid arthritis. RA patients were recruited at their regular visit at the clinic. NO parameters were estimated by the Högman & Meriläinen algorithm (HMA); alveolar NO (CANO), airway compartment diffusing capacity (DawNO) and tissue concentration of the airway wall (CawNO). Healthy subjects, matched by age, gender and height were used as controls. Data is given in mean and geometric mean1 (25, 75 percentile). RA patients (n=36) had significantly altered values in the NO parameters compared to the control group (n=36). DawNO1 was higher 22 (15,36) vs. 8 (5,11) mL/s, CANO 1.4 (0.5,1.8) vs. 2.5 (2.0,3.0) ppb and CawNO1 were lower 48 (22,87) vs. 116 (76,160) ppb in the RA patients compared to the controls (all p<0.001), while there was no difference in JawNO1 (p=0.36). There is a lung involvement in RA patients demonstrated by the use of extended NO analysis. DawNO is in line with severe atopic asthma while CawNO is in line with current smokers. Because of the relatively long duration of RA among the subjects, it can be argued that the current results reveal more about the long term effects of the RA disease or the treatment rather than its origin. As this project proceeds, the aim is to include patients with recent onset RA.

Halobacteria (henceforth: Haloarchaea) are predominantly aerobic halophiles that are thought to have evolved from anaerobic methanogens. This remarkable transformation most likely involved an extensive influx of bacterial genes. Whether it entailed a single massive transfer event or a gradual stream of transfers remains a matter of debate. To address this, genomes that descend from methanogen-to-halophile intermediates are necessary. Here, we present five such near-complete genomes of Marine Group IV archaea (Hikarchaeia), the closest known relatives of Haloarchaea. Their inclusion in gene tree-aware ancestral reconstructions reveals an intermediate stage that had already lost a large number of genes, including nearly all of those involved in methanogenesis and the Wood-Ljungdahl pathway. In contrast, the last Haloarchaea common ancestor gained a large number of genes and expanded its aerobic respiration and salt/UV resistance gene repertoire. Our results suggest that complex and gradual patterns of gain and loss shaped the methanogen-to-halophile transition.

Hydrogenosomes are H2-producing mitochondrial homologs found in some anaerobic microbial eukaryotes that provide a rare intracellular niche for H2-utilizing endosymbiotic archaea. Among ciliates, anaerobic and aerobic lineages are interspersed, demonstrating that the switch to an anaerobic lifestyle with hydrogenosomes has occurred repeatedly and independently. To investigate the molecular details of this transition, we generated genomic and transcriptomic data sets from anaerobic ciliates representing three distinct lineages. Our data demonstrate that hydrogenosomes have evolved from ancestral mitochondria in each case and reveal different degrees of independent mitochondrial genome and proteome reductive evolution, including the first example of complete mitochondrial genome loss in ciliates. Intriguingly, the FeFe-hydrogenase used for generating H2 has a unique domain structure among eukaryotes and appears to have been present, potentially through a single lateral gene transfer from an unknown donor, in the common aerobic ancestor of all three lineages. The early acquisition and retention of FeFe-hydrogenase helps to explain the facility whereby mitochondrial function can be so radically modified within this diverse and ecologically important group of microbial eukaryotes.

Joran Martijn , Anders E. Lind, Max E. Schön, Ian Spiertz, Lina Juzokaite, Ignas Bunikis, Olga V. Pettersson and Thijs J. G. Ettema 1,3* Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, SE-75123, Uppsala, Sweden. Science for Life Laboratory, Uppsala University, SE-75185, Uppsala, Sweden. Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Stippeneng 4, 6708WE, Wageningen, The Netherlands.

SummaryAmplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost‐effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read‐curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full‐length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high‐throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full‐length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost‐effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.

BACKGROUND:Rheumatoid arthritis (RA) is an autoimmune disorder characterized by bone and joint destruction, but other organ systems can also be involved. Recent studies have suggested that the disease may start in the lungs. Exhaled nitric oxide (FENO) is a marker of inflammation. The aims of the study were to compare the NO parameters between subjects with RA and healthy control subjects, and to examine whether the NO parameters correlated with lung function and disease activity in the subjects with RA. METHODS:Subjects with RA (n = 35) were recruited during their regular outpatient visits to the rheumatology department. The nitric oxide (NO) parameters: alveolar NO concentration (CANO), airway compartment diffusing capacity of NO (DawNO), and tissue concentration of NO in the airway wall (CawNO), were algorithmically estimated. Healthy subjects (n = 35) matched by age, gender and height were used as controls. Data are given in median, (quartile 25, 75). Wilcoxon Matched Pairs test was used for group comparisons. Mann-Whitney U test was used to make comparisons between any two groups and for pairwise comparisons. Correlations were tested with Spearman rank order correlation. RESULTS:CANO was significantly lower in the RA subjects compared with healthy subjects; 1.1 (0.5, 1.8) ppb versus 2.4 (2.0, 3.0) ppb, (p < 0.001). CawNO was significantly lower in the RA subjects with 51 (22, 87) ppb versus 120 (76, 162) ppb in the control group. DawNO was significantly higher at 25 (15, 36) mL/s in the RA group versus the control group's 7.7 (5.3, 10.7) mL/s. CONCLUSIONS:There are significant differences between subjects with RA and matched healthy control subjects regarding the exhaled NO parameters. It is unclear if this can be explained by the pathogenesis of RA, consequences of long-term disease, and/or due to drug treatment.

Endosymbiosis is a widespread phenomenon in the microbial world and can be based on diverse interactions between endosymbiont and host cell. The vast majority of the known endosymbiotic interactions involve bacteria that have invaded eukaryotic host cells. However, methanogenic archaea have been found to thrive in anaerobic, hydrogenosome-containing protists and it was suggested that this symbiosis is based on the transfer of hydrogen. Here, we used culture-independent genomics approaches to sequence the genomes of two distantly related methanogenic endosymbionts that have been acquired in two independent events by closely related anaerobic ciliate hosts Nyctotherus ovalis and Metopus contortus , respectively. The sequences obtained were then validated as originating from the ciliate endosymbionts by in situ probing experiments. Comparative analyses of these genomes and their closest free-living counterparts reveal that the genomes of both endosymbionts are in an early stage of adaptation towards endosymbiosis as evidenced by the large number of genes undergoing pseudogenization. For instance, the observed loss of genes involved in amino acid biosynthesis in both endosymbiont genomes indicates that the endosymbionts rely on their hosts for obtaining several essential nutrients. Furthermore, the endosymbionts appear to have gained significant amounts of genes of potentially secreted proteins, providing targets for future studies aiming to elucidate possible mechanisms underpinning host-interactions. Altogether, our results provide the first genomic insights into prokaryotic endosymbioses from the archaeal domain of life.

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions of environmental samples and to discover previously unknown lineages. Its unique structure of interspersed conserved and variable regions is an excellent target for PCR and allows for classification of reads at all taxonomic levels. However, the relatively few phylogenetically informative sites prevent confident phylogenetic placements of novel lineages that are deep branching relative to reference taxa. This problem is exacerbated when only short 16S rRNA gene fragments are sequenced. To resolve their placement, it is common practice to gather more informative sites by combining multiple conserved genes into concatenated datasets. This however requires genomic data which may be obtained through relatively expensive metagenome sequencing and computationally demanding analyses. Here we develop a protocol that amplifies a large part of 16S and 23S rRNA genes within the rRNA operon, including the ITS region, and sequences the amplicons with PacBio long-read technology. We tested our method with a synthetic mock community and developed a read curation pipeline that reduces the overall error rate to 0.18%. Applying our method on four diverse environmental samples, we were able to capture near full-length rRNA operon amplicons from a large diversity of prokaryotes. Phylogenetic trees constructed with these sequences showed an increase in statistical support compared to trees inferred with shorter, Illumina-like sequences using only the 16S rRNA gene (250 bp). Our method is a cost-effective solution to generate high quality, near full-length 16S and 23S rRNA gene sequences from environmental prokaryotes.

Nature Microbiology 1, 16002 (2016); published 15 February 2016; corrected 6 June 2016. This Letter should have been published under a Creative Commons licence according to the Nature policy on publishing the primary sequence of an organism's genome for the first time. The editors apologize to the authors and to readers for this error.

The origin of the eukaryotic cell remains one of the most contentious puzzles in modern biology. Recent studies have provided support for the emergence of the eukaryotic host cell from within the archaeal domain of life, but the identity and nature of the putative archaeal ancestor remain a subject of debate. Here we describe the discovery of ‘Lokiarchaeota’, a novel candidate archaeal phylum, which forms a monophyletic group with eukaryotes in phylogenomic analyses, and whose genomes encode an expanded repertoire of eukaryotic signature proteins that are suggestive of sophisticated membrane remodelling capabilities. Our results provide strong support for hypotheses in which the eukaryotic host evolved from a bona fide archaeon, and demonstrate that many components that underpin eukaryote-specific features were already present in that ancestor. This provided the host with a rich genomic ‘starter-kit’ to support the increase in the cellular and genomic complexity that is characteristic of eukaryotes.

Background Detecting intracellular bacterial symbionts can be challenging when they persist at very low densities. Wolbachia , a widespread bacterial endosymbiont of invertebrates, is particularly challenging. Although it persists at high titers in many species, in others its densities are far below the detection limit of classic end-point Polymerase Chain Reaction (PCR). These low-titer infections can be reliably detected by combining PCR with DNA hybridization, but less elaborate strategies based on end-point PCR alone have proven less sensitive or less general. Results We introduce a multicopy PCR target that allows fast and reliable detection of A-supergroup Wolbachia - even at low infection titers - with standard end-point PCR. The target is a multicopy motif (designated ARM: A -supergroup r epeat m otif) discovered in the genome of w Mel (the Wolbachia in Drosophila melanogaster ). ARM is found in at least seven other Wolbachia A-supergroup strains infecting various Drosophila , the wasp Muscidifurax and the tsetse fly Glossina . We demonstrate that end-point PCR targeting ARM can reliably detect both high- and low-titer Wolbachia infections in Drosophila , Glossina and interspecific hybrids. Conclusions Simple end-point PCR of ARM facilitates detection of low-titer Wolbachia A-supergroup infections. Detecting these infections previously required more elaborate procedures. Our ARM target seems to be a general feature of Wolbachia A-supergroup genomes, unlike other multicopy markers such as insertion sequences (IS).

The Archaea represent the so-called Third Domain of life, which has evolved in parallel with the Bacteria and which is implicated to have played a pivotal role in the emergence of the eukaryotic domain of life. Recent progress in genomic sequencing technologies and cultivation-independent methods has started to unearth a plethora of data of novel, uncultivated archaeal lineages. Here, we review how the availability of such genomic data has revealed several important insights into the diversity, ecological relevance, metabolic capacity, and the origin and evolution of the archaeal domain of life.