Chance and necessity in the genome evolution of endosymbiotic bacteria of insects

[EN] An open question in evolutionary biology is how does the selection¿drift balance determine the fates of biological interactions. We searched for signatures of selection and drift in genomes of five endosymbiotic bacterial groups known to evolve under strong genetic drift. Although most genes in endosymbiotic bacteria showed evidence of relaxed purifying selection, many genes in these bacteria exhibited stronger selective constraints than their orthologs in free-living bacterial relatives. Remarkably, most of these highly constrained genes had no role in the host¿symbiont interactions but were involved in either buffering the deleterious consequences of drift or other host-unrelated functions, suggesting that they have either acquired new roles or their role became more central in endosymbiotic bacteria. Experimental evolution of Escherichia coli under strong genetic drift revealed remarkable similarities in the mutational spectrum, genome reduction patterns and gene losses to endosymbiotic bacteria of insects. Interestingly, the transcriptome of the experimentally evolved lines showed a generalized deregulation of the genome that affected genes encoding proteins involved in mutational buffering, regulation and amino acid biosynthesis, patterns identical to those found in endosymbiotic bacteria. Our results indicate that drift has shaped endosymbiotic associations through a change in the functional landscape of bacterial genes and that the host had only a small role in such a shiftThis work was supported by Science Foundation Ireland (12/IP/1637) and grants from the Spanish Ministerio de Economia y Competitividad (MINECO-FEDER; BFU2012-36346 and BFU2015-66073-P) to MAF. DAP and CT were supported by Juan de la Cierva fellowships from MINECO (references: JCI-2011-11089 and JCA-2012-14056, respectively). DAP is supported by funds from the University of Nevada, Reno, NV, USA.Sabater-Muñoz, B.; Toft, C.; Alvarez-Ponce, D.; Fares Riaño, MA. (2017). Chance and necessity in the genome evolution of endosymbiotic bacteria of insects. The ISME Journal. 11(6):1291-1304. https://doi.org/10.1038/ismej.2017.18S12911304116Aguilar-Rodriguez J, Sabater-Munoz B, Montagud-Martinez R, Berlanga V, Alvarez-Ponce D, Wagner A et al. (2016). The molecular chaperone DnaK is a source of mutational robustness. Genome Biol Evol 8: 2979–2991.Alvarez-Ponce D, Sabater-Munoz B, Toft C, Ruiz-Gonzalez MX, Fares MA . (2016). Essentiality is a strong determinant of protein rates of evolution during mutation accumulation experiments in Escherichia coli. Genome Biol Evol 8: 2914–2927.Anders S, Huber W . (2010). Differential expression analysis for sequence count data. Genome Biol 11: R106.Archibald J . (2014) One Plus One Equals One: Symbiosis and the Evolution of Complex Life. Oxford University Press: Oxford, UK.Aussel L, Loiseau L, Hajj Chehade M, Pocachard B, Fontecave M, Pierrel F et al. (2014). ubiJ, a new gene required for aerobic growth and proliferation in macrophage, is involved in coenzyme Q biosynthesis in Escherichia coli and Salmonella enterica serovar Typhimurium. J Bacteriol 196: 70–79.Baumann P, Baumann L, Clark MA . (1996). Levels of Buchnera aphidicola chaperonin groEL during growth of the aphid Schizaphis graminum. Curr Microbiol 32: 7.Benjamini Y, Yekutieli Y . (2005). False discovery rate controlling confidence intervals for selected parameters. J Am Stat Assoc 100: 10.Bennett GM, Moran NA . (2015). Heritable symbiosis: the advantages and perils of an evolutionary rabbit hole. Proc Natl Acad Sci USA 112: 10169–10176.Bermingham J, Rabatel A, Calevro F, Vinuelas J, Febvay G, Charles H et al. (2009). Impact of host developmental age on the transcriptome of the symbiotic bacterium Buchnera aphidicola in the pea aphid (Acyrthosiphon pisum. Appl Environ Microbiol 75: 7294–7297.Bogumil D, Dagan T . (2010). Chaperonin-dependent accelerated substitution rates in prokaryotes. Genome Biol Evol 2: 602–608.Carbon S, Ireland A, Mungall CJ, Shu S, Marshall B, Lewis S et al. (2009). AmiGO: online access to ontology and annotation data. Bioinformatics 25: 288–289.Chen Z, Wang Y, Li Y, Li Y, Fu N, Ye J et al. (2012). Esre: a novel essential non-coding RNA in Escherichia coli. FEBS Lett 586: 1195–1200.Clark JW, Hossain S, Burnside CA, Kambhampati S . (2001). Coevolution between a cockroach and its bacterial endosymbiont: a biogeographical perspective. Proc Biol Sci 268: 393–398.Dale C, Wang B, Moran N, Ochman H . (2003). Loss of DNA recombinational repair enzymes in the initial stages of genome degeneration. Mol Biol Evol 20: 1188–1194.Deatherage DE, Barrick JE . (2014). Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol Biol 1151: 165–188.Douglas AE . (2003). The nutritional physiology of aphids. Adv Insect Physiol 31: 68.Fares MA, Barrio E, Sabater-Munoz B, Moya A . (2002a). The evolution of the heat-shock protein GroEL from Buchnera, the primary endosymbiont of aphids, is governed by positive selection. Mol Biol Evol 19: 1162–1170.Fares MA, Ruiz-Gonzalez MX, Moya A, Elena SF, Barrio E . (2002b). Endosymbiotic bacteria: groEL buffers against deleterious mutations. Nature 417: 398.Gancedo C, Flores CL, Gancedo JM . (2016). The expanding landscape of moonlighting proteins in yeasts. Microbiol Mol Biol Rev 80: 765–777.Gerardo NM, Altincicek B, Anselme C, Atamian H, Barribeau SM, de Vos M et al. (2010). Immunity and other defenses in pea aphids, Acyrthosiphon pisum. Genome Biol 11: R21.Gomez-Valero L, Latorre A, Silva FJ . (2004). The evolutionary fate of nonfunctional DNA in the bacterial endosymbiont Buchnera aphidicola. Mol Biol Evol 21: 2172–2181.Gomez-Valero L, Silva FJ, Christophe Simon J, Latorre A . (2007). Genome reduction of the aphid endosymbiont Buchnera aphidicola in a recent evolutionary time scale. Gene 389: 87–95.Gonzalez-Domenech CM, Belda E, Patino-Navarrete R, Moya A, Pereto J, Latorre A . (2012). Metabolic stasis in an ancient symbiosis: genome-scale metabolic networks from two Blattabacterium cuenoti strains, primary endosymbionts of cockroaches. BMC Microbiol 12 (Suppl 1): S5.Hansen AK, Moran NA . (2011). Aphid genome expression reveals host-symbiont cooperation in the production of amino acids. Proc Natl Acad Sci USA 108: 2849–2854.Hansen AK, Moran NA . (2014). The impact of microbial symbionts on host plant utilization by herbivorous insects. Mol Ecol 23: 1473–1496.Henderson B, Fares MA, Lund PA . (2013). Chaperonin 60: a paradoxical, evolutionarily conserved protein family with multiple moonlighting functions. Biol Rev Camb Philos Soc 88: 955–987.Humphreys NJ, Douglas AE . (1997). Partitioning of symbiotic bacteria between generations of an insect: a quantitative study of a Buchnera sp. in the pea aphid (Acyrthosiphon pisum reared at different temperatures. Appl Environ Microbiol 63: 3294–3296.International Aphid Genomics Consortium. (2010). Genome sequence of the pea aphid Acyrthosiphon pisum. PLoS Biol 8: e1000313.Kadibalban AS, Bogumil D, Landan G, Dagan T . (2016). DnaK-dependent accelerated evolutionary rate in prokaryotes. Genome Biol Evol 8: 1590–1599.Katoh K, Standley DM . (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30: 772–780.Kelkar YD, Ochman H . (2013). Genome reduction promotes increase in protein functional complexity in bacteria. Genetics 193: 303–307.Koga R, Meng XY, Tsuchida T, Fukatsu T . (2012). Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte-embryo interface. Proc Natl Acad Sci USA 109: E1230–E1237.Kuo CH, Moran NA, Ochman H . (2009). The consequences of genetic drift for bacterial genome complexity. Genome Res 19: 1450–1454.Kuo CH, Ochman H . (2009). Deletional bias across the three domains of life. Genome Biol Evol 1: 145–152.Law R, Lewis DH . (1983). Biotic environments and the maintenance of sex-some evidence from mutualistic symbioses. Biol J Linnean Soc 20: 28.Liu XD, Xie L, Wei Y, Zhou X, Jia B, Liu J et al. (2014). Abiotic stress resistance, a novel moonlighting function of ribosomal protein RPL44 in the halophilic fungus Aspergillus glaucus. Appl Environ Microbiol 80: 4294–4300.Lohse M, Bolger AM, Nagel A, Fernie AR, Lunn JE, Stitt M et al. (2012). RobiNA: a user-friendly, integrated software solution for RNA-Seq-based transcriptomics. Nucleic Acids Res 40: W622–W627.Macdonald SJ, Lin GG, Russell CW, Thomas GH, Douglas AE . (2012). The central role of the host cell in symbiotic nitrogen metabolism. Proc Biol Sci 279: 2965–2973.McClure R, Balasubramanian D, Sun Y, Bobrovskyy M, Sumby P, Genco CA et al. (2013). Computational analysis of bacterial RNA-Seq data. Nucleic Acids Res 41: e140.McCutcheon JP, Moran NA . (2012). Extreme genome reduction in symbiotic bacteria. Nat Rev Microbiol 10: 13–26.McFall-Ngai M, Hadfield MG, Bosch TC, Carey HV, Domazet-Loso T, Douglas AE et al. (2013). Animals in a bacterial world, a new imperative for the life sciences. Proc Natl Acad Sci USA 110: 3229–3236.Mira A, Ochman H, Moran NA . (2001). Deletional bias and the evolution of bacterial genomes. Trends Genet 17: 589–596.Moran NA . (1996). Accelerated evolution and Muller's rachet in endosymbiotic bacteria. Proc Natl Acad Sci USA 93: 2873–2878.Moran NA, Dunbar HE, Wilcox JL . (2005). Regulation of transcription in a reduced bacterial genome: nutrient-provisioning genes of the obligate symbiont Buchnera aphidicola. J Bacteriol 187: 4229–4237.Moran NA, McCutcheon JP, Nakabachi A . (2008). Genomics and evolution of heritable bacterial symbionts. Annu Rev Genet 42: 165–190.Moran NA, McLaughlin HJ, Sorek R . (2009). The dynamics and time scale of ongoing genomic erosion in symbiotic bacteria. Science 323: 379–382.Nakabachi A, Ishida K, Hongoh Y, Ohkuma M, Miyagishima SY . (2014). Aphid gene of bacterial origin encodes a protein transported to an obligate endosymbiont. Curr Biol 24: R640–R641.Nilsson AI, Koskiniemi S, Eriksson S, Kugelberg E, Hinton JC, Andersson DI . (2005). Bacterial genome size reduction by experimental evolution. Proc Natl Acad Sci USA 102: 12112–12116.Patino-Navarrete R, Moya A, Latorre A, Pereto J . (2013). Comparative genomics of Blattabacterium cuenoti: the frozen legacy of an ancient endosymbiont genome. Genome Biol Evol 5: 351–361.Pettersson ME, Berg OG . (2007). Muller's ratchet in symbiont populations. Genetica 130: 199–211.Price DR, Feng H, Baker JD, Bavan S, Luetje CW, Wilson AC . (2014). Aphid amino acid transporter regulates glutamine supply to intracellular bacterial symbionts. Proc Natl Acad Sci USA 111: 320–325.Reyes-Prieto M, Vargas-Chavez C, Latorre A, Moya A . (2015). SymbioGenomesDB: a database for the integration and access to knowledge on host-symbiont relationships. Database 2015: bav109 (1–8).Robinson MD, McCarthy DJ, Smyth GK . (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26: 139–140.Sabater-Muñoz B, Prats-Escriche M, Montagud-Martinez R, Lopez-Cerdan A, Toft C, Aguilar-Rodriguez J et al. (2015). Fitness trade-offs determine the role of the molecular chaperonin groel in buffering mutations. Mol Biol Evol 32: 2681–2693.Schlicker A, Domingues FS, Rahnenfuhrer J, Lengauer T . (2006). A new measure for functional similarity of gene products based on Gene Ontology. BMC Bioinformatics 7: 302.Shigenobu S, Watanabe H, Hattori M, Sakaki Y, Ishikawa H . (2000). Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407: 81–86.Supek F, Bosnjak M, Skunca N, Smuc T . (2011). REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS ONE 6: e21800.Tamas I, Klasson L, Canback B, Naslund AK, Eriksson AS, Wernegreen JJ et al. (2002). 50 million years of genomic stasis in endosymbiotic bacteria. Science 296: 2376–2379.Toft C, Fares MA . (2008). The evolution of the flagellar assembly pathway in endosymbiotic bacterial genomes. Mol Biol Evol 25: 2069–2076.van Ham RC, Kamerbeek J, Palacios C, Rausell C, Abascal F, Bastolla U et al. (2003). Reductive genome evolution in Buchnera aphidicola. Proc Natl Acad Sci USA 100: 581–586.Wernegreen JJ . (2002). Genome evolution in bacterial endosymbionts of insects. Nat Rev Genet 3: 850–861.Wernegreen JJ . (2011). Reduced selective constraint in endosymbionts: elevation in radical amino acid replacements occurs genome-wide. PLoS One 6: e28905.Williams TA, Fares MA . (2010). The effect of chaperonin buffering on protein evolution. Genome Biol Evol 2: 609–619.Yang Z . (2007). PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol 24: 1586–1591

Genome-Wide Functional Divergence after the Symbiosis of Proteobacteria with Insects Unraveled through a Novel Computational Approach

Symbiosis has been among the most important evolutionary steps to generate biological complexity. The establishment of symbiosis required an intimate metabolic link between biological systems with different complexity levels. The strict endo-cellular symbiotic bacteria of insects are beautiful examples of the metabolic coupling between organisms belonging to different kingdoms, a eukaryote and a prokaryote. The host (eukaryote) provides the endosymbiont (prokaryote) with a stable cellular environment while the endosymbiont supplements the host's diet with essential metabolites. For such communication to take place, endosymbionts' genomes have suffered dramatic modifications and reconfigurations of proteins' functions. Two of the main modifications, loss of genes redundant for endosymbiotic bacteria or the host and bacterial genome streamlining, have been extensively studied. However, no studies have accounted for possible functional shifts in the endosymbiotic proteomes. Here, we develop a simple method to screen genomes for evidence of functional divergence between two species clusters, and we apply it to identify functional shifts in the endosymbiotic proteomes. Despite the strong effects of genetic drift in the endosymbiotic systems, we unexpectedly identified genes to be under stronger selective constraints in endosymbionts of aphids and ants than in their free-living bacterial relatives. These genes are directly involved in supplementing the host's diet with essential metabolites. A test of functional divergence supports a strong relationship between the endosymbiosis and the functional shifts of proteins involved in the metabolic communication with the insect host. The correlation between functional divergence in the endosymbiotic bacterium and the ecological requirements of the host uncovers their intimate biochemical and metabolic communication and provides insights on the role of symbiosis in generating species diversity

Imaging of hydrothermal altered zones in Wadi Al-Bana, in southern Yemen, using remote sensing techniques and very low frequency–electromagnetic data

© 2019, Saudi Society for Geosciences. Economic mineralization and hydrothermally altered zones are areas of great economic interests. This study focusses on hydrothermal altered zones of high mineralization potentials in Wadi Al-Bana, in southern Yemen. An azimuthal very low frequency–electromagnetic (AVLF-EM) data acquisition was conducted in search for mineralization in the study area. The study integrated observations from geophysical field data with others extracted from object-oriented principal component analysis (PCA) to better map and understand mineralization in the investigated area. This technique was applied to two data sets, ASTER and Landsat 8 Operational Land Imager (OLI) imagery. The results of PCA revealed high accuracy in detecting alteration minerals and for mapping zones of high concentration of these minerals. The PCA-based distribution of selected alteration zones correlated spatially with high conductivity anomalies in the subsurface that were detected by VLF measurements. Finally, a GIS model was built and successfully utilized to categorize the resulted altered zones, into three levels. [Figure not available: see fulltext.]

Effect of carbohydrate mouth rinsing on multiple sprint performance

BACKGROUND: Research suggests that carbohydrate mouth rinsing (CMR) improves endurance performance; yet, little is known regarding the effect of CMR on multiple sprint efforts. As many sports involve multiple sprinting efforts, followed by periods of recovery, the aim of our current study was to investigate the influence of CMR on multiple sprint performance. METHODS: We recruited eight active males (Age; 22 ± 1 y; 75.0 ± 8.8 kg; estimated VO2(max) 52.0 ± 3.0 ml/kg/min) to participate in a randomly assigned, double-blind, counterbalanced study administering a CMR (6.4% Maltodextrin) or similarly flavoured placebo solution. Primary outcomes for our study included: (a) time for three repeated sprint ability tests (RSA) and (b) the Loughborough Intermittent Shuttle Test (LIST). Time was expressed in seconds (sec). Secondary outcomes included ratings of perceived exertion (RPE) and blood glucose concentration. Tertiary outcomes included two psychological assessments designed to determine perceived activation (i.e., arousal) and pleasure-displeasure after each section of the LIST. We analysed our data using a two-way analysis of variance (ANOVA) for repeated measures, a Bonferroni adjusted post hoc t-test to determine significant differences in treatment, and a liberal 90% confidence interval between treatment conditions. Effect sizes were calculated between trials and interpreted as ≤ 0.2 trivial, > 0.2 small, > 0.6 moderate, > 1.2 large, > 2 very large and > 4 extremely large. Data are means ± SD. Overall statistical significance was set as P < 0.05; yet, modified accordingly when Bonferroni adjustments were made. RESULTS: Overall, we observed no significant difference in average (3.46 ± 0.2 vs. 3.44 ± 0.17; P = 0.11) or fastest time (3.38 ± 0.2 vs. 3.37 ± 0.2; P = 0.39) in the RSA test for the placebo vs. CMR conditions, respectively. Similar findings were also noted for the placebo vs. CMR, respectively, during the LIST test (3.52 ± 0.2 vs. 3.54 ± 0.2 sec; P = 0.63). Despite a significantly higher within group RPE during the 3rd and 4th sections of the LIST (< 0.05), no between group differences were otherwise noted. No differences were noted for blood glucose concentrations throughout the testing protocol. Lastly, from a psychological perspective, we observed no differences in pleasure-displeasure or perceived activation. CONCLUSIONS: The results of our current study suggest that CMR does not improve exercise performance, RPE or perceived pleasure-displeasure during high intensity activity requiring repeated, intermittent, sprint efforts

A Combinatorial Approach to Detect Coevolved Amino Acid Networks in Protein Families of Variable Divergence

Communication between distant sites often defines the biological role of a protein: amino acid long-range interactions are as important in binding specificity, allosteric regulation and conformational change as residues directly contacting the substrate. The maintaining of functional and structural coupling of long-range interacting residues requires coevolution of these residues. Networks of interaction between coevolved residues can be reconstructed, and from the networks, one can possibly derive insights into functional mechanisms for the protein family. We propose a combinatorial method for mapping conserved networks of amino acid interactions in a protein which is based on the analysis of a set of aligned sequences, the associated distance tree and the combinatorics of its subtrees. The degree of coevolution of all pairs of coevolved residues is identified numerically, and networks are reconstructed with a dedicated clustering algorithm. The method drops the constraints on high sequence divergence limiting the range of applicability of the statistical approaches previously proposed. We apply the method to four protein families where we show an accurate detection of functional networks and the possibility to treat sets of protein sequences of variable divergence

Positively selected amino acid replacements within the RuBisCO enzyme of oak trees are associated with ecological adaptations

Phylogenetic analysis by maximum likelihood (PAML) has become the standard approach to study positive selection at the molecular level, but other methods may provide complementary ways to identify amino acid replacements associated with particular conditions. Here, we compare results of the decision tree (DT) model method with ones of PAML using the key photosynthetic enzyme RuBisCO as a model system to study molecular adaptation to particular ecological conditions in oaks (Quercus). We sequenced the chloroplast rbcL gene encoding RuBisCO large subunit in 158 Quercus species, covering about a third of the global genus diversity. It has been hypothesized that RuBisCO has evolved differentially depending on the environmental conditions and leaf traits governing internal gas diffusion patterns. Here, we show, using PAML, that amino acid replacements at the residue positions 95, 145, 251, 262 and 328 of the RuBisCO large subunit have been the subject of positive selection along particular Quercus lineages associated with the leaf traits and climate characteristics. In parallel, the DT model identified amino acid replacements at sites 95, 219, 262 and 328 being associated with the leaf traits and climate characteristics, exhibiting partial overlap with the results obtained using PAML

Genetic modifiers ameliorate endocytic and neuromuscular defects in a model of spinal muscular atrophy

© 2020 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.Background: Understanding the genetic modifiers of neurodegenerative diseases can provide insight into the mechanisms underlying these disorders. Here, we examine the relationship between the motor neuron disease spinal muscular atrophy (SMA), which is caused by reduced levels of the survival of motor neuron (SMN) protein, and the actin-bundling protein Plastin 3 (PLS3). Increased PLS3 levels suppress symptoms in a subset of SMA patients and ameliorate defects in SMA disease models, but the functional connection between PLS3 and SMN is poorly understood.Results: We provide immunohistochemical and biochemical evidence for large protein complexes localized in vertebrate motor neuron processes that contain PLS3, SMN and members of the hnRNP F/H family of proteins. Using a Caenorhabditis elegans (C. elegans) SMA model, we determine that overexpression of PLS3 or loss of the C. elegans hnRNP F/H ortholog SYM-2 enhances endocytic function and ameliorates neuromuscular defects caused by decreased SMN-1 levels. Furthermore, either increasing PLS3 or decreasing SYM-2 levels suppresses defects in a C. elegans ALS model.Conclusions: We propose that hnRNP F/H act in the same protein complex as PLS3 and SMN and that the function of this complex is critical for endocytic pathways, suggesting that hnRNP F/H proteins could be potential targets for therapy development.Peer reviewe

Global, regional, and national burden of low back pain, 1990–2020, its attributable risk factors, and projections to 2050: a systematic analysis of the Global Burden of Disease Study 2021

Background: Low back pain is highly prevalent and the main cause of years lived with disability (YLDs). We present the most up-to-date global, regional, and national data on prevalence and YLDs for low back pain from the Global Burden of Diseases, Injuries, and Risk Factors Study 2021. Methods: Population-based studies from 1980 to 2019 identified in a systematic review, international surveys, US medical claims data, and dataset contributions by collaborators were used to estimate the prevalence and YLDs for low back pain from 1990 to 2020, for 204 countries and territories. Low back pain was defined as pain between the 12th ribs and the gluteal folds that lasted a day or more; input data using alternative definitions were adjusted in a network meta-regression analysis. Nested Bayesian meta-regression models were used to estimate prevalence and YLDs by age, sex, year, and location. Prevalence was projected to 2050 by running a regression on prevalence rates using Socio-demographic Index as a predictor, then multiplying them by projected population estimates. Findings: In 2020, low back pain affected 619 million (95% uncertainty interval 554–694) people globally, with a projection of 843 million (759–933) prevalent cases by 2050. In 2020, the global age-standardised rate of YLDs was 832 per 100 000 (578–1070). Between 1990 and 2020, age-standardised rates of prevalence and YLDs decreased by 10·4% (10·9–10·0) and 10·5% (11·1–10·0), respectively. A total of 38·8% (28·7–47·0) of YLDs were attributed to occupational factors, smoking, and high BMI. Interpretation: Low back pain remains the leading cause of YLDs globally, and in 2020, there were more than half a billion prevalent cases of low back pain worldwide. While age-standardised rates have decreased modestly over the past three decades, it is projected that globally in 2050, more than 800 million people will have low back pain. Challenges persist in obtaining primary country-level data on low back pain, and there is an urgent need for more high-quality, primary, country-level data on both prevalence and severity distributions to improve accuracy and monitor change. Funding: Bill and Melinda Gates Foundation

Nintedanib for Systemic Sclerosis-Associated Interstitial Lung Disease

BACKGROUND: Interstitial lung disease (ILD) is a common manifestation of systemic sclerosis and a leading cause of systemic sclerosis-related death. Nintedanib, a tyrosine kinase inhibitor, has been shown to have antifibrotic and antiinflammatory effects in preclinical models of systemic sclerosis and ILD. METHODS: We conducted a randomized, double-blind, placebo-controlled trial to investigate the efficacy and safety of nintedanib in patients with ILD associated with systemic sclerosis. Patients who had systemic sclerosis with an onset of the first non-Raynaud's symptom within the past 7 years and a high-resolution computed tomographic scan that showed fibrosis affecting at least 10% of the lungs were randomly assigned, in a 1:1 ratio, to receive 150 mg of nintedanib, administered orally twice daily, or placebo. The primary end point was the annual rate of decline in forced vital capacity (FVC), assessed over a 52-week period. Key secondary end points were absolute changes from baseline in the modified Rodnan skin score and in the total score on the St. George's Respiratory Questionnaire (SGRQ) at week 52. RESULTS: A total of 576 patients received at least one dose of nintedanib or placebo; 51.9% had diffuse cutaneous systemic sclerosis, and 48.4% were receiving mycophenolate at baseline. In the primary end-point analysis, the adjusted annual rate of change in FVC was 1252.4 ml per year in the nintedanib group and 1293.3 ml per year in the placebo group (difference, 41.0 ml per year; 95% confidence interval [CI], 2.9 to 79.0; P=0.04). Sensitivity analyses based on multiple imputation for missing data yielded P values for the primary end point ranging from 0.06 to 0.10. The change from baseline in the modified Rodnan skin score and the total score on the SGRQ at week 52 did not differ significantly between the trial groups, with differences of 120.21 (95% CI, 120.94 to 0.53; P=0.58) and 1.69 (95% CI, 120.73 to 4.12 [not adjusted for multiple comparisons]), respectively. Diarrhea, the most common adverse event, was reported in 75.7% of the patients in the nintedanib group and in 31.6% of those in the placebo group. CONCLUSIONS: Among patients with ILD associated with systemic sclerosis, the annual rate of decline in FVC was lower with nintedanib than with placebo; no clinical benefit of nintedanib was observed for other manifestations of systemic sclerosis. The adverse-event profile of nintedanib observed in this trial was similar to that observed in patients with idiopathic pulmonary fibrosis; gastrointestinal adverse events, including diarrhea, were more common with nintedanib than with placebo