TrkB-T1 is a BDNF receptor lacking a tyrosine kinase domain that is highly expressed in astrocytes and regulates BDNF-evoked calcium transients. Previous studies indicate that downregulation of TrkB-T1 in frontal cortex may be involved in neurobiological processes underlying suicide.In a microarray screening study (N = 8), we interrogated all known microRNA in the frontal cortex of suicide completers with low expression of TrkB-T1 and normal controls. These findings were validated and followed up in a larger sample of cases and controls (N = 55). Functional analyses included microRNA silencing, microRNA overexpression and luciferase assays to investigate specificity and to validate interactions between differentially expressed microRNA and TrkB-T1.nominal: 9.10−5 and 1.8.10−4 respectively; FDR-corrected p = 0.031). Bioinformatic analyses revealed five putative binding sites for the DiGeorge syndrome linked microRNA Hsa-miR-185*in the 3′UTR of TrkB-T1, but none for Hsa-miR-491-3P. The increase of Hsa-miR-185* in frontal cortex of suicide completers was validated then confirmed in a larger, randomly selected group of suicide completers, where an inverse correlation between Hsa-miR-185* and TrkB-T1 expression was observed . Silencing and overexpression studies performed in human cell lines confirmed the inverse relationship between hsa-mir-185* and trkB-T1 expression. Luciferase assays demonstrated that Hsa-miR-185* binds to sequences in the 3′UTR of TrkB-T1.MicroRNAs Hsa-miR-185* and Hsa-miR-491-3p were upregulated in suicide completers with low expression of TrkB.T1 (PThese results suggest that an increase of Hsa-miR-185* expression levels regulates, at least in part, the TrkB-T1 decrease observed in the frontal cortex of suicide completers and further implicate the 22q11 region in psychopathology. Suicide is a leading cause of death in many regions of the world, with prevalence estimates ranging from 10–20 deaths per 100,000 people NTRK2 gene located at 9q21.3 have been identified and each varies in length at the 3′ end. While each of these three transcripts, referred to as TrkB-T1, TrkB-T2 and TrkB-FL, code for receptors capable of ligand binding, only the full-length 24 exon isoform (FL) has a domain with tyrosine kinase activity. TrkB-T1 and TrkB-T2 have stop codons at exons 16 and 19, respectively. Expression patterns in brain differ across NTRK2 isoforms, in particular, with the exclusive expression of TrkB-T1 in astrocytes Neurotrophic tyrosine kinase receptor type 2 (NTRK2) and ligand brain-derived neurotrophic factor (BDNF) are the subject of intense investigation in psychiatry and have been associated with anxiety, suicide, and depression, amongst other disorders TrkB-T1 is markedly downregulated in the frontal cortex of a sub-group of individuals who died by suicide TrkB promoter. While promoter sequencing revealed no variants that could explain the finding (unpublished observations), we found that epigenetic marks could, in part, explain the decrease in TrkB-T1 expression. We reported that two CpG islands in the NTRK2 promoter were hypermethylated in the suicide brain and that these findings significantly correlated with TrkB-T1 expression levels TrkB-T1 expression levels in the suicide brain TrkB-T1 downregulation in suicide, they do not fully account for the total variance in TrkB-T1 expression changes. Therefore, additional regulatory processes may be underlying the TrkB-T1 downregulation observed in suicide.Recently, we reported that TrkB-T1 observed in frontal cortex of suicide completers. MicroRNAs are non-coding RNAs of an average length of 21 bp that function to inhibit the translation of messenger RNA and induce its destruction by binding to its 3′UTR sequence MicroRNAs, best described as post-transcriptional regulators, are excellent candidates for the regulation of specific gene transcripts, and as such, could contribute to the down-regulation of TrkB-T1 expression in the suicide brain. Through a series of complementary experiments beginning with a global microRNA microarray screening, we show that the expression of Hsa-miR-185* is significantly increased in suicide cases with low TrkB-T1 cortical expression. After replicating the association between Hsa-miR-185* and TrkB-T1 brain expression in an independent and significantly larger sample, we conducted a series of functional studies showing that Hsa-miR-185* binds to TrkB-T1 3′UTR sequences and specifically affects its expression.In this study, we explored the potential role of micro-RNAs in the regulation of www.douglasrecherche.qc.ca/suicide). The initial microRNA screening was conducted in a sample selected according to TrkB-T1 levels. This sample consisted of eight subjects, including four suicides with low levels of TrkB-T1 expression, and four normal controls. The replication sample used comprised a total of 55 individuals, including 38 suicides and 17 controls. All subjects were male and of French Canadian origin, a homogeneous population with a well-defined founder effect. All subjects died suddenly and could not have undergone any resuscitation procedures or other type of medical intervention, and thus did not experience prolonged agonal states. The majority of suicide completers had a history of major depressive disorder , followed by schizophrenia  and bipolar disorder . Six (15.8%) did not have evidence of psychopathology and all controls were psychiatrically normal.This study was approved by the Douglas Institute Research Ethics Board, and all participating families signed consent forms. Brain tissue was obtained from the Quebec Suicide Brain Bank . Each sample was labelled with Hy3 and hybridized with a reference sample labelled with Hy5 using miRCURY™ LNA Array Power labeling kit . Reference sample was made as a pool of equal quantities of 8 total RNA samples used in this experiment. Hybridization, scanning and normalization were performed according to the manufacturer’s standard protocol . A background correction was conducted according to Normexp with offset value 10 The microRNA Reverse transcription kit  and specific primers  were used to generate cDNA from microRNA enriched fraction previously extracted. Reverse transcriptions were done on total RNA fraction in order to obtain cDNA in 40 µl volume containing 1 µg of total RNA; 0,5 µg oligoDT(16) primers, 0.5 mM dNTPs, 0,01 M DTT and 400 U M-MLV RT .http://cbcsrv.watson.ibm.com/rna22_targets.html database was used to characterize differential expressed microRNAs that could bind TrkB-T1 3′UTR sequence The RNA22 20 qsp 20 µl.Relative quantification of MicroRNAs: The reactions were performed in a total volume of 20 µl, on 384 well plates using an Applied Biosystems 7900 HT. We used mix and specific primers commercialized by Applied Biosystems. We used a reference pool of cDNA to generate a standard curve for RNU6B quantification. RNU6B, which is the U6B small nuclear RNA, is a commonly used internal control (reference to adjust for sample fluctuations ) used for quantification of microRNA. In order to quantify Hsa-miR-185* microRNA, a cDNA was generated from a synthetic RNA made by IDT. Serial dilutions provided amounts ranging between 0.78125 ng and 25 ng for Hsa-miR-185* and between 0.012207 ng and 50 ng for RNU6B. For each well, PCR mix included 10 µl of 2X NoAmperaseUNG mastermix (Applied Biosystems), 1 µl of primers/probe mix, 2 µl of cDNA, HmRNA quantification: These reactions were conducted as described above, but in a total volume of 12 µl. Serials dilution provided amounts ranging between 0.003052 ng and 50 ng. Each well included 6 µl of 2X gene expression master mix, 0.6 µl of 20X primer mix, 3.4 µl of RNase free water and 2 µl of cDNA. βactin was used as internal control for normalization.TrkB-T1 3′UTR sequence containing putative binding sites for Hsa-miR-185* were obtained by PCR  and neuroblastoma cell line (CRL2137). HEK293 cells were a gift from Dr Nicolas Cermakian CRL2137 cells were seeded in 6 well plates and transfected with an antagomir. RNA extraction procedures began eight hours post-transfection. Antagomirs MIN0004611 and #1027271 (Qiagen), respectively for Hsa-miR-185* and a manufacturer supplied negative control were transfected individually at 25 nM , in CRL2137 cells using Hiperfect reagent (Qiagen) as recommended by the manufacturer. HEK293 cells were transfected with Mimics (Double-stranded oligonucleotide RNAs that mimic mature endogenous miRNAs) 24hours after seeding in 6 well plates - MSY0004611 and #1027280 (Qiagen), respectively for Hsa-miR-185* and negative control, at 10 nM  and RNA extraction procedures began 24hours post-transfection.TrkB-T1 vector, (ii) 8 ng of pRL-Null vector, used to normalize the efficiency of transfection, (iii) and a mimic or a negative control at a final concentration of 10 nM. Proteins were extracted 24 hours after transfection and the luciferase activity was quantified using a luminometer berthold and dual luciferase assay kit (Promega).For luciferase assays cells were transfected 24hours after seeding with (i) 60 ng of construct with or without Post-normalized data from the microarray study were treated with Partek Genomic Suite Software. T tests were performed on each probe in between group, giving nominal Pvalues. Then corrections for multiple testing were made using False Discovery Rate (FDR 0.05) TrkB-T1 in the frontal cortex of suicide completers, we performed an exploratory global microarray expression screen for all available microRNA’s in the brains of suicide completers with low levels of TrkB-T1 and controls matched for age, gender, Post-Mortem Interval (PMI), and Potential of Hydrogen (pH)  To understand how microRNA’s affect expression of TrkB-T1 expression, it follows that Hsa-miR-491-3P and/or Hsa-miR 185* could affect the expression of TrkB-T1. To determine whether these microRNAs were good candidates for the regulation of TrkB-T1 expression, we sought putative binding sites in the 3′UTR sequence of TrkB-T1. In silico investigation identified five putative binding sites for Hsa-miR-185* in the 3′UTR sequence of TrkB-T1, whereas no sites were identified for Hsa-miR-491-3P  defined as the ratio of standard deviation out of mean for the three sets of normalized data. The lowest coefficient of variation was found for Hsa-miR-185* expression levels normalized with RNU6B (40.9% compared to 43.1% for Hsa-miR-26b and 112.6% for RNA5S). We also compared the expression level of RNU6B in BA10 from controls and suicide completers and found no difference between groups (t = 1.3 df = 53 p = 0.199). We consequently decided to perform all microRNA quantifications with RNU6B as endogenous control.2 = 0.6638 p = 0.014) with array results and validated a significant) increase of this microRNA among suicides  With the aim of investigating the generalizability of the array experiment results, we subsequently examined by RT-PCR the TrkB-T1 transcript obtained by quantitative PCR, grouped by class  pH  PMI or suicide methods .The differences in TrkB-T1 levels to those of Hsa-miR-185* in the 55 patients investigated, we observed a significant inverse linear correlation between these two molecules .TrkB-T1 did not show differences between groups    comparedTrkB-T1, HEK293 cells, that strongly express TrkB-T1, were cultured for 24 hours and transfected with either an Hsa-miR-185* mimic, or negative control microRNA. Following transfection, RNA was extracted and expression levels of Hsa-miR-185* and TrkB-T1 were quantified by RT-PCR. We observed a significant reduction of TrkB-T1 expression levels in the cells transfected by the mimic compared to those transfected with the negative control  (In order to confirm that an interaction exists between microRNA Hsa-miR-185* and = 0.018) .TrkB-T1 transcript, we quantified the expression levels of the TrkB-T2 and TrkB-FL transcripts in the HEK293 cells transfected by the Hsa-miR-185* mimic.No significant difference in the expression levels of TrkB-T2 and TrkB-FL was detected in the cells transfected by the Hsa-miR-185* mimetic when compared to cells transfected with a negative control. , but only one 3′ UTR sequence specific to TrkB-T1. To ensure the specificity of Hsa-miR-185* on the 3′UTR sequence of the control. .TrkB-T1  in HEK 293 cells transfected with mimic compared to cells transfected with a negative control oligonucleotide .Five putative binding sites for Hsa-miR-185* are present in the 5165 bp 3′UTR sequence of TrkB-T1 . We clonIn order to determine which of the five putative Hsa-miR-185* binding sites were contributing to the decrease in luciferase activity, we cloned each site in 130 to 330 bp fragments downstream to the luciferase coding sequence. These constructs were individually co-transfected into HEK293 cells with a Hsa-miR-185* mimic and a normalization vector in adherence to the conditions defined by Larsson et al. TrkB-T1 expression level. Through functional analyses using silencing or exogenous expression of Hsa-miR-185*, we confirmed that TrkB-T1 expression levels can be regulated by this microRNA in vitro. Finally, we characterized five potential binding sites for Hsa-miR-185* in TrkB-T1 3′ UTR sequences, three of which are functional.In this study, we presented multiple lines of evidence suggesting a role for microRNA Hsa-miR-185* in the regulation of TrkB-T1, a truncated TrkB transcript whose downregulation has been associated with suicide. The expression level of this microRNA is significantly increased in frontal cortex from suicide completers and negatively correlated with Since their discovery in C elegans in 1993 RNA interference is a process whereby organisms regulate mRNA through small noncoding RNAs, which are primarily of two types. The small interfering RNAs (siRNA) are RNA duplexes that induce sequence-specific gene silencing Hsa-miR-185* is the complementary microRNA strand form (frequently referred to as the “passenger strand”) associated with Hsa-miR-185. Two consecutive cleavages involving Drosha/DGCR8 and TRBP/Dicer complexes provide primicroRNA transcribed from genomic DNA to a RNA/RNA duplex TrkB-T1 levels, our study was designed to identify microRNAs that may regulate TrkB-T1 expression, and our results suggest that Hsa-miR-185* may play such a role; however, it is unclear why expression of Hsa-miR-185* is increased. In the current study, subjects were not assessed for micro duplications at the DiGeorge region, but this may be a worthwhile test to undertake, as this microRNA is expressed from DiGeorge critical region on chromosome 22. In addition to variation in the genomic sequences coding for microRNAs, dysregulation may be explained by a number of different factors, including changes in levels of proteins that play a role in the maturation process of microRNA. An increase of DGCR8, another gene in the DiGeorge ciritical region and which contributes to primiRNA maturation, is responsible for microRNA dysregulation in cortical regions from schizophrenic patients By focusing on individuals with extreme low We performed functional assays where several putative TrkB-T1 3′UTR. Hsa-mIR- 185* binding targets were cloned into a luciferase vector to determine if we could experimentally decrease luciferase activity by co-transfection with an Hsa-mir-185* mimic. The luciferase assays suggested that Hsa-miR-185* might induce a small decrease of TrkB-T1 expression by binding 3′UTR sites, given the subtle but significant decreases in luciferase activity. Several factors could affect the interpretation. We used HEK293 cells, where we detected 10-fold higher levels of TrkB-T1 expression compared to the neuronal cell line we used. While the choice of HEK293 cells may have been more appropriate than the neuronal cell line – any cell type that expresses modest levels of TrkB-T1 may also be expected to have relative levels of regulatory elements that modify the expression of TrkB-T1– reproducing this experiment in multiple cell lines known to express TrkB-T1 are warranted. The very subtle decrease also needs to be assessed systematically where the dynamic range of the assay can be measured; specifically, small decreases need to be detectable above basal ‘noise’ levels across replicates. Finally, the basic biology of mood disorders is not necessarily expected to produce large, high penetrance effects. The physiological implication of modest down-regulation of a particular gene is not clear – a slight decrease in expression could lead to psychiatric pathology. Even in non-psychiatry fields, several other functional studies have also shown moderate effects of microRNA on luciferase activity TrkB-T1 3′UTR is estimated to be approximately −25 kj/mol, an amount similar to the estimated folding energies of other microRNA and 3′UTR sequence interactions investigated Click here for additional data file.Figure S2TrkB-T1 in CRL2137 and HEK293 cells N = 3 for each condition.Basal expression levels of Hsa-miR-185* and (TIF)Click here for additional data file.Figure S3Analyses of Hsa-miR-185 region by sequencing on the independent replication sample composed of 17 controls and 38 suicide completers. A: Schematic representation of SNPs found in a 1.6 kb sequence comprising the 82 sequencing coding for Hsa-mir-185, 1084 bases in the upstream and 516 in the downstream region. Two tag SNPs identified: rs2078749 and rs2008591. B: Statistics summarizing the tests for an allelic association between polymorphisms at tSNPs and suicide. C: Statistics summarizing the tests a haplotypic association involving polymorphisms at tSNPs and suicide. D: graph bar of the Hsa-miR-185* expression level depending on the genotypes at tSNP rs2078749. E: graph bar of the Hsa-miR-185* expression level depending on the genotypes at tSNP rs2008591.(TIF)Click here for additional data file.Figure S4TrkB-T1 levels and controls with normal TrkB-T1 expression values (N = 8). Comparison was done by t test.Graph bar of the quantification of TrkB-FL isoform performed by western blot on the screening sample composed of suicide completers selected according to extreme low (TIF)Click here for additional data file.Figure S5TrkB-T1 3′UTR sequence in our cohort of 55 patients. A: Graph bar of the quantification of Hsa-miR-423-5p. B: Graph bar of the quantification of Hsa-miR-193a-5p. All statistical comparisons were done by t test.Graph bar of the quantification of microRNAs that could bind (TIF)Click here for additional data file.Table S1TrkB-T1 3′UTR sequence in pMIR report vector.Primers used to generate and clone fragments of (DOC)Click here for additional data file.Table S2Age, pH and PMI of matched controls and suicide completers included in the microarray study.(DOC)Click here for additional data file.Table S3List of microRNA probesets expressed in BA10 with average group expression values.(DOC)Click here for additional data file.Table S4Mean age, pH and PMI ± standard deviation for the independent larger sample of 55 individuals (17 controls and 38 suicide completers) investigated in the replication study.(DOC)Click here for additional data file.Table S5TrkB-T1 3′UTR sequence, and suicide.Statistics describing tests for allelic assciations between SNPs located in the fragment containing site 727 from (DOC)Click here for additional data file.Supporting Information S1Supporting Methods, Supporting Results, Supporting References.(DOC)Click here for additional data file.


Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology with considerable morbidity and mortality. Cigarette smoking is one of the most recognized risk factors for development of IPF. Furthermore, recent work suggests that smoking may have a detrimental effect on survival of patients with IPF. The mechanism by which smoking may contribute to the pathogenesis of IPF is largely unknown. However, accumulating evidence suggests that increased oxidative stress might promote disease progression in IPF patients who are current and former smokers. In this review, potential mechanisms by which cigarette smoking affects IPF, the effects of cigarette smoking on accelerated loss of lung function in patients with IPF, key genetic studies evaluating the potential candidate genes and gene-environment (smoking) interaction, diagnosis, and treatment with emphasis on recently closed and ongoing clinical trials are presented. Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause, occurring primarily in older adults, limited to the lungs, and associated with the histopathologic and/or radiologic pattern of usual interstitial pneumonia (UIP) . CurrentThe prevalence of tobacco use in IPF ranges from 41% to 83%, depending on the case definition used in the studies , 8. CurrThe mechanism by which smoking may contribute to the pathogenesis of IPF is unknown despite studies suggesting a strong association between the development of IPF and cigarette smoking. Because IPF is recognized as a disease of aging, it has been speculated that smoking may contribute to the development of interstitial lung disease in an age-dependent manner . Also, tAt the cellular level, IPF is characterized by fibroblast proliferation and activation, resulting in pronounced extracellular matrix (ECM) deposition . Histoloα receptors and interleukin- (IL-) 13 receptors [Fibroblasts are stromal cells that support tissue homeostasis through the generation of ECM during normal wound healing, as well as differentiating into alpha-smooth muscle actin-positive myofibroblasts. In addition to being highly synthetic, myofibroblasts are proposed to contract wound edges together, thus sealing off injured tissue and preventing damage or subsequent infection . During eceptors .Idiopathic pulmonary fibrosis fibroblasts have a lower threshold for activation and thus can have an augmented profibrotic response to mediators found in the IPF lung . FibroblAs well as being highly activated, there is an increase in fibroblast number in the lungs of IPF patients, which in part may be due to a relative resistance to apoptosis . HoweverEpithelial-to-mesenchymal transition (EMT) is a process that occurs normally during early fetal development where a seamless plasticity exists between epithelial and mesenchymal cells . During α 1, alone or in combination with other growth factors such as epidermal growth factor, begin the process of EMT by increasing the expression of MMPs [in vitro and in vivo, supporting the concept of EMT contributing to the fibrosis observed in IPF [Epithelial cells exposed to the prototypic profibrotic growth factor TGF- of MMPs , 50. The of MMPs . The ide of MMPs . Severald in IPF , 52, 53.in vitro culture, circulating CD45+ collagen I+ cells downregulate CD45 and become more adherent [β1 and Th2 cytokines [Another potential source of lung fibroblasts is the bone marrow. Fibroblasts, traditionally thought to arise from proliferation of resident stromal cells, have been shown to derive from CD45+ collagen-I-producing cells, commonly referred to as “fibrocytes” –56. Duriadherent . Moreoveadherent . Fibrocyadherent . They arytokines ; and expytokines . Human aytokines , 61; andytokines . Furtherytokines –65. Fibrytokines , 64, 65.The acute and initial recognition of these foreign mediators initiates a host defense response, resulting in clearance of the noxious agents being mediated by the innate immune system in the lung . One of M2 macrophages express elevated levels of scavenger receptors such as macrophage scavenger receptor and mannose receptor C (MRC/CD206) , 70. Assβ1 overexpression model of lung fibrosis, we have determined that depletion of lung monocyte/macrophages using liposomal clodronate reduced collagen accumulation, but this had no effect on the TGFβ-induced fibrocyte recruitment [Studies of bleomycin-induced fibrosis have assessed either M2 macrophage or fibrocyte number , 73. It ruitment .Cigarette smoke contains particulate matter as well as numerous chemicals including highly toxic RONS , 76. TheIn vitro studies assessing the effects of cigarette smoke have demonstrated that fibroblasts can produce an emphysematous response and increase MMP production [in vitro [oduction –92. The in vitro . These cin vivo cigarette smoke model in mice [Cigarette smoke also alters the way in which macrophages recognize foreign particles and pathogens . The deb in mice . TherefoDue to the plethora of mediators capable of activating cells, the combination of mediators may have a synergistic effect in the lungs of patients with IPF. Recent evidence suggests that individuals respond differently to cigarette smoke compounds in part through an altered unfolded protein response, resulting in either susceptibility or resistance to the same stimuli . As has Although the etiology is unknown, a significant percentage of patients with IPF have a familial form of the disease, suggesting genetics might play a role , 101. BaGenetic variants within the human telomerase reverse transcriptase (hTERT) or human telomerase RNA components of the telomerase gene are associated with familial pulmonary fibrosis and are present in some sporadic IPF cases. Heterozygous loss-of-function mutations in TERT have been found in up to 15% of kindreds with familial pulmonary fibrosis , 109 andα, lymphotoxin α, IL-4, IL-6, IL-8, IL-10, IL-12, and TGF-ß1 [α 1 antitrypsin and angiotensin-converting enzymes [β1), coagulation pathway genes (plasminogen activator inhibitors-1 and -2 and surfactant proteins-A and -B) [A number of candidate genes have been evaluated in search for genetic factors in sporadic cases of IPF. Among others, the following polymorphisms of genes are reported to be associated with increased frequencies in patients with sporadic IPF: genes encoding for cytokines, such as IL-1 a, tumor necrosis factor-d TGF-ß1 –123, α 1 enzymes , 124, pr and -B) , and MMP and -B)  polymorpγRIIa determines receptor affinity for immunoglobulin G subclasses and is associated with several chronic inflammatory diseases. In a case-control study, Bournazos et al. compared the distribution of FcγRIIa R131H genotypes in 142 patients with IPF and in 218 controls using allele-specific polymerase chain reaction amplification. These findings support an association between the FcγRIIa R131H polymorphism and IPF severity and progression, suggesting the involvement of immunological mechanisms in IPF pathogenesis [R131H rs1801274) polymorphism of the immunoglobulin G receptor Fc4 polymorXaubet et al. demonstrated that the carriage of double homozygote (GG/CC) at the single nucleotide polymorphism loci of COX2.3050 and COX2.8473 polymorphisms did not increase disease progression but may increase the susceptibility to IPF by approximately 1.4 fold at age 30 and by a smaller fold greater than 1 up to age 66 years . These fγRIIIb NA1/2 polymorphisms using allele-specific polymerase chain reaction amplification and demonstrated that FcγRIIIb NA1/2 polymorphisms are associated with IPF disease susceptibility. These results support a role for immunological mechanisms contributing to IPF pathogenesis [In the case-control study described above, Bournazos et al. also compared the distribution of Fcogenesis .Currently, the ATS/ERS/JRS/ALAT  committee does not recommend genetic testing in patients with either familial or sporadic IPF, as part of clinical evaluation because more functional studies that confirm their significance and studies investigating other mutations, associations, and gene-environment relationships are needed .γ-) inducible protein of 10 kDa (IP-10) in lung fibroblasts from patients with IPF. These findings suggest that epigenetic dysregulation involving histone deacetylation and hypermethylation by cigarette smoking may promote fibrotic changes in the lung disease of IPF patients.Cigarette smoking has been associated with IPF in case-control studies with patients of different ethnic backgrounds and with patients with familial pulmonary fibrosis, indicating that smoking may confer increased risk for IPF , 6. ChecDiagnosis of an IPF can be achieved with certainty only after the pulmonologist, radiologist, and pathologist have reviewed all of the clinical, radiologic, and pathologic data. The diagnostic criteria for IPF are presented in the ATS/ERS Statement . In the Idiopathic pulmonary fibrosis is an idiopathic interstitial pneumonia characterized by UIP histopathology. A confident histopathologic diagnosis of UIP either through a surgical lung biopsy or a characteristic HRCT scan of the chest must be correlated with the clinical findings. The histopathologic criterion is a heterogeneous appearance of areas of fibrosis with scarring and honeycomb change and areas of less affected or normal parenchyma . These cThe typical chest radiograph in IPF shows bilateral basal and peripheral reticular opacities. Progressive fibrosis leads to a reduction in lung volumes and honeycombing, which tends to become less frequent towards the upper lung subpleural zones. In IPF, signs of pulmonary fibrosis include traction bronchiectasis and architectural distortion such as fissure displacement, which can be detected by HRCT , 133. HiHowever, some cases of UIP are difficult to differentiate from fibrotic nonspecific interstitial pneumonia, which may exhibit honeycombing. The most useful finding when differentiating between nonspecific interstitial pneumonia and UIP at HRCT is the greater extent of honeycombing in cases of UIP . The HRCIncreasing research evidence indicates that smoking may also result in interstitial lung abnormalities on HRCT , 137. SmClinical features of IPF include gradually progressing dyspnea, chronic cough, and bibasilar inspiratory crackles . DigitalThe treatment of IPF remains a great challenge as there is no effective therapeutic option. The clinical course is gradual deterioration with a median survival of 2.5 to 3.5 years . Treatmeβin vitro [P = .0416). Pirfenidone was also studied in two additional multinational, randomized, double-blind, placebo-controlled phase 3 trials (CAPACITY 1 and CAPACITY 2) conducted in IPF patients. The CAPACITY 1 trial enrolled 344 patients and involved a daily dose of 2,403 mg of pirfenidone versus placebo. The CAPACITY 2 trial enrolled 435 patients who were randomly assigned 2 : 2 : 1, respectively, to pirfenidone 2,403 mg daily, placebo, or pirfenidone 1,197 mg daily over a 72-week treatment period. The primary endpoint of change in forced vital was met in CAPACITY 2 (P = .001) but not in CAPACITY 1 [Pirfenidone is an oral antifibrotic, anti-inflammatory, and antioxidant agent that inhibits TGF-in vitro . It was in vitro  based onPACITY 1 . LargelyGiven the high morbidity and mortality associated with IPF and the absence of FDA-approved, effective therapies, a wide array of therapeutic approaches are under active clinical investigation. Some key clinical studies evaluating the potential usefulness of the newer agents  are summarized in Recently, the ATS Guidelines Committee made recommendations of varying strength against the use of most therapies based on data from the completed studies. Treatment recommendations for specific therapies are based on the Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) approach . For exaIn the absence of longitudinal data, management of IPF is largely driven by the severity of pulmonary function impairment. A period of observation after smoking cessation is usually warranted to allow spontaneous regression of disease. Relatively good prognosis after smoking cessation with some additive effects of glucocorticosteroids distinguishes smoking-related interstitial lung disease from IPF . As descThere is a clear need for novel therapies in the treatment of IPF, as conventional therapy has limited efficacy or unfavorable safety profile and no FDA-approved therapies exist. There are a number of ongoing clinical trials targeting fibrogenic, inflammatory, and oxidative pathways. Furthermore, continued effort on better understanding the mechanisms of disease may provide better treatment options in the future. One of the key risk factors for development of IPF, accelerated loss of lung function, and survival is cigarette smoking. The only therapeutic option for these patients with IPF is observation for a period of time after smoking cessation. A search for the potential mechanisms by which cigarette smoking affects IPF and patients with IPF may provide insights on a novel therapeutic target against smoking-induced progression of IPF. A strategy to better understand the effect of smoking on IPF would be to stratify patients based on smoking status and compare the treatment responses between smokers and nonsmokers in the large clinical trials.


Although it is well known that plants and animals harbor microbial symbionts that can influence host traits, the factors regulating the structure of these microbial communities often remain largely undetermined. This is particularly true for insect-associated microbial communities, as few cross-taxon comparisons have been conducted to date. To address this knowledge gap and determine how host phylogeny and ecology affect insect-associated microbial communities, we collected 137 insect specimens representing 39 species, 28 families, and 8 orders, and characterized the bacterial communities associated with each specimen via 16S rRNA gene sequencing. Bacterial taxa within the phylum Proteobacteria were dominant in nearly all insects sampled. On average, the insect-associated bacterial communities were not very diverse, with individuals typically harboring fewer than 8 bacterial phylotypes. Bacterial communities also tended to be dominated by a single phylotype; on average, the most abundant phylotype represented 54.7% of community membership. Bacterial communities were significantly more similar among closely related insects than among less-related insects, a pattern driven by within-species community similarity but detected at every level of insect taxonomy tested. Diet was a poor predictor of bacterial community composition. Individual insect species harbored remarkably unique communities: the distribution of 69.0% of bacterial phylotypes was limited to unique insect species, whereas only 5.7% of phylotypes were detected in more than five insect species. Together these results suggest that host characteristics strongly regulate the colonization and assembly of bacterial communities across insect lineages, patterns that are driven either by co-evolution between insects and their symbionts or by closely related insects sharing conserved traits that directly select for similar bacterial communities. Insects play vital roles in the functioning of both natural and managed systems that include pollinating crops, spreading disease, altering soil fertility, and plant herbivory. The specific traits and ecological attributes are, in part, related to the microorganisms found within individual insects. Symbioses between particular insect species  and their primary symbionts have been well documented Buchnera aphidicola, for example, has been explored intensively, and genome studies revealed that B. aphidicola provide essential metabolic functions for the aphid host Pseudomonas spp., Streptococcus spp. and Enterobacter spp. are common pathogens that presumably gain access to insects via their digestive tract Insect-associated bacterial communities are a mix of mutualist, pathogenic, and commensal bacteria. Insect diets are often nutrient-poor or incomplete diet, and associated bacteria can aid their survival by synthesizing essential nutrients. Some insects have specialized cells called bacteriocytes that house beneficial intracellular bacteria thought to enhance nutrient-poor diets The ecological factors regulating bacterial assemblage patterns within individual insect species have recently been explored by harnessing the power of high-throughput DNA sequencing techniques. Bacterial communities of fleas were found to differ across time and space Drosophila-associated microbial communities changed in response to diet changes and microbial communities did not differ among species when fed the same diet et al. were not able to quantify how the intra-specific variability compares to the inter-specific variability in microbial community composition across insect taxa.Recent cross-taxon comparisons of insect-associated microbial communities have aimed to disentangle host and diet effects on microbial composition. In ants, microbial communities differed among herbivorous and predatory ants, but were similar among species within the same trophic level Here we used a cross-taxon approach to determine the effects of host and diet on insect-associated bacterial communities, but have designed the study to expand upon previously-published work by collecting all samples from one location at one time, surveying the microbial community in each collected sample to an equivalent depth using the same method, and analyzing multiple specimens per insect species. We conducted our survey to characterize bacterial communities of a large number of insect species, determine richness and evenness of insect-associated bacterial communities, investigate the effects of diet and insect taxonomy on bacterial community composition, and explore cross-taxon community assemblage patterns.To minimize the effects of climate, biogeography and temporal shifts within an insect population, insects were intensely sampled over a short period from a single location. Insects were collected using a variety of techniques  from March 3– March 7, 2010 in Honolulu, Hawaii. The vast majority of specimens were collected at the Lyon Arboretum, but a few specimens were also collected from the University of Hawaii at Manoa and the Hawaii Humane Society. Upon capture, insects were immediately placed in 70% ethanol, and then stored at −20°C from the end of the collection day until DNA extraction. Only adult insects were included in the study. Most specimens were classified to species level (74.4%) and all specimens were classified to the genus level based on morphology . A vouch5′-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3′) contains the 454 Life Sciences primer B sequence, the 16S rRNA gene 27 f primer, and a two-base ‘TC’ linker; the reverse primer (5′-GCCTCCCTCGCGCCATCAGNNNNNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3′) contains the Life Sciences primer A sequence, a 12-bp error-correcting barcode, the 16S rRNA gene 338 r primer, and a two-base ‘CA’ linker Prior to DNA extraction, we rinsed insect samples with 100% ethanol to minimize the contribution of bacteria from insect surfaces. We extracted DNA from insect samples using the MoBIO PowerSoil-htp 96 Well DNA Isolation kit . For most samples, DNA was extracted from whole insects, but very large insects were frozen in liquid N2 and pulverized with a mortar and pestle to obtain a homogenous tissue sample of an appropriate size. We did not attempt to distinguish between those bacteria that are endosymbionts and those that are found within insect guts. We used barcoded pyrosequencing of a portion of the 16S rRNA gene to characterize bacterial communities. We amplified each sample (n = 137) in triplicate using 5 PRIME MasterMix with bacterial 16S rRNA gene primers that amplify the V1 and V2 hypervariable regions. The forward primer (We processed DNA sequence data using QIIME v1.5 http://rdp.cme.msu.edu) and aligned using the NAST aligner http://greengenes.lbl.gov). Aligned sequences were used as input for the generation of a neighbor-joining tree in FastTree The number of unique phylotypes detected in each sample was used as an estimate of richness in individual samples, and Shannon’s Evenness and Simpson’s Diversity were used to assess bacterial community evenness for each insect specimen. Representative sequences for each phylotype were classified according to the RDP classifier  was quantified using a metric based on phylotype abundances (Bray-Curtis Dissimilarity) and using phylogenetic metrics based upon the amount of unique phylogenetic diversity found in each sample (unweighted and weighted UniFrac) We characterized the bacterial communities of 137 individual insect samples representing 39 insect species, 28 insect families, and 8 insect orders . In totaTaxonomic classification of these phylotypes revealed that bacterial communities were dominated by only a few phyla, with greater than 94% of community membership represented by four phyla : Proteobacteria (64.6%), Bacteroidetes (14.9%), Actinobacteria (7.7%), and Firmicutes (6.9%) . ProteobThe bacterial diversity levels observed within individual insects  were relatively low . On averWolbachia phylotype that was detected in 43.6% (17/39) of insect species , insect-associated bacterial richness was low , and thiBartonella or a Rickettsiales-lineage significantly reduced overall bacterial community diversity An alternative explanation for low bacterial richness is negative interactions between established insect symbionts and invading bacteria. Commensal insect gut bacteria prevent pathogens and other bacteria from establishing Apis millifera, have a core set of bacterial taxa that are present in most individuals In some insect species, bacterial communities were remarkably similar among individual specimens; in other species, the dominant phylotypes differed among individuals . These cFew bacterial phylotypes (<6%) were detected in five or more insect species, highlighting the ability of a minority of bacterial taxa to overcome the barriers to dispersal and colonization affecting the majority of insect-associated bacteria. In general, individual insect species harbor a unique consortium of bacteria  and the et al. suggested that within-order variation among samples indicated that insect taxa did not maintain distinct bacterial communities. However, this interpretation may be biased by their focus on order-level taxonomy and lack of intra-family or intra-specific comparisons. Bacterial communities of vertebrates have also been found to be more similar among more closely related individuals than among distantly related individuals Our finding that insect hosts affect bacterial community assembly  is suppoOur finding that insect diet has little effect on bacterial community structure may seem at odds with the substantial evidence that insects often form obligate associations with bacterial symbionts in response to nutrient-poor diets Buchnera) often have profound effects on host nutrition, though in other cases (e.g. Wolbachia) they do not. Furthermore, because endosymbionts are predominantly transmitted vertically, they are under different selective pressures than environmentally acquired gut bacteria. Thus, while diet may affect both vertically transmitted and environmentally acquired bacteria, the signal from dietary effects on environmentally acquired bacteria would be obscured by vertically transmitted endosymbionts.Our research, however, has two limitations that may restrict our ability to detect the effect of diet on bacterial community composition: 1) the broad diversity of insect samples, and 2) the use of whole insects. A study focused on a less diverse group of insects with varied diets may be better suited to resolve the effects of taxonomy and diet on bacterial communities. By using whole insects, we maximized our detection of insect-associated bacteria but also included endosymbionts (i.e. intracellular bacteria) in our analyses. Endosymbionts Click here for additional data file.


Micromonas, a unicellular green alga in the Mamiellophyceae lineage, demonstrating the coexistence of several classes of introns and the occurrence of recent massive intron invasion. This study focuses on two strains, CCMP1545 and RCC299, and their related individuals from ocean samplings, showing that they not only harbor different classes of introns depending on their location in the genome, as for other Mamiellophyceae, but also uniquely carry several classes of repeat introns. These introns, dubbed introner elements (IEs), are found at novel positions in genes and have conserved sequences, contrary to canonical introns. This IE invasion has a huge impact on the genome, doubling the number of introns in the CCMP1545 strain. We hypothesize that each IE class originated from a single ancestral IE that has been colonizing the genome after strain divergence by inserting copies of itself into genes by intron transposition, likely involving reverse splicing. Along with similar cases recently observed in other organisms, our observations in Micromonas strains shed a new light on the evolution of introns, suggesting that intron gain is more widespread than previously thought.Genes in pieces and spliceosomal introns are a landmark of eukaryotes, with intron invasion usually assumed to have happened early on in evolution. Here, we analyze the intron landscape of  Micromonas strains, isolated from tropical  and coastal waters  . In common with other Mamiellophyceae, both Micromonas strains RCC299 and CCMP1545 feature two distinct classes of introns, corresponding to the unique genome heterogeneity of these picoalgae (fig. 1B). A small portion of these BOC1 introns feature noncanonical splice sites. Additionally, in Micromonas CCMP1545, Recently, several whole-genome sequences have been reported for Mamiellophyceae, eukaryotic picoalgae at the basis of the green lineage that play a major trophic role in the marine environment. Among these are the genome sequences of two CMP1545) . One str5 strain A. In comicoalgae . At mosticoalgae . Howevericoalgae . One of icoalgae   as well as the Expressed Sequence Tag (EST) libraries were obtained from the JGI portal . Metagenomic sequences containing IEs were obtained (through BlastN) from the NCBI metagenomes database (taxid: 408169) and the CAMERA portal , while C. reinhardtii intron data were derived from the latest Phytozome release (v5.3.1). When multiple isoforms were present, one representative was selected randomly.supplementary methods, Supplementary Material online). Remnants of IEs were detected using a Blast  and HMMer (v2.3.2) approach. Gene models were extensively curated through automated and manual procedures. All intron and gene information is stored in a relational database and can be accessed through the ORCAE platform   . Data seBOC1 introns are defined as short (<75 nt), AT-rich (<43 GC%) introns lying in the BOC1 region of chromosome 1 of CCMP1545  and chromosome 2 of RCC299  . CanonicMicromonas isolates. After alignment , intron positions were compared and cross-referenced against their class identifier .In total, 6,891 one-to-one orthologous pairs were identified using orthoMCL , representing 74% of the total intron content of both Micromonas genomes using a seed-and-align procedure . After quality filtering, we then compared IE positions to discover presence/absence polymorphisms (PAPs). This analysis is highly biased toward the finding of IEs that are absent in RCC299/CCMP1545 but present in the metagenomic sequences, as the reverse would require a confirmation that the read is derived from an organism that carries the specific IE. This is only the case when a sequence carries a strain identifier  or if the metagenomic sequence carries an IE up- or downstream.Metagenomic sequences were subjected to the IE prediction pipeline and aligned to the Micromonas introns can be classified into two categories, namely singleton introns, which are all unique in the sense that they do not show significant similarity to other introns in the genome, and IEs, which are a copy of or at least show partial similarity to several or many other introns. To the first category  and have a predicted NCTGAC branch-point motif at 43–52 bp upstream of the acceptor site. Comparative intron analysis revealed that 47% of all canonical intron positions are shared between CCMP1545 and RCC299 orthologs, a number that illustrates the divergence of these strains, which are members of different clades B) across the splice site aids recognition by the splicing machinery. Furthermore, 34 of these introns feature noncanonical TG or CG acceptor sites, of which the majority is validated by EST alignments. Similar noncanonical acceptor sites have been found in non-prasinophytes as well . IE-A members also have very typical splice donor sites, AG|GYGCGT or AG|GTGAGAC, with the first occurring in IE-A1 and IE-A2, while the latter is almost exclusively found in IE-A3 and IE-A4 sequences . Fifty-three percent of IE-A1 sequences contain a GC splice donor, a characteristic that was noted in earlier studies but was never linked to the presence of IEs , having lost both splice sites and all other motifs crucial for the splicing process. They are found in close proximity to coding sequences (∼UTR regions) or within canonical intron sequences, but never in coding sequences where they are counter-selected for to maintain gene functionality.Besides the IE-A introns, there are 463 IE-A-like repeats, which are positioned outside introns or inside preexisting introns (discussed later). These are remnants of IE-A introns: highly degenerated, partial copies that most often only consist of a small 50-nt motif . Their GT-TG splice sites are highly unusual but have been reported before in other species, including human . This IE-encoded protein lacks homology to other known proteins, except for a small OTU-like protease domain. As such, the function of this protein, or the reason why it is embedded within these IEs, is unknown. The IE-B class contains both the longest documented mamiellophycean intron and the first documented occurrence of a nuclear intron-encoded protein within Mamiellophyceae. A defining characteristic of these two classes is the preference for phase-2 , which contradicts the theory that newly gained introns prefer phase-0    . AlthougOstreococcus introns .As stated previously, the IE-C class (221 occurrences) exists exclusively in RCC299. The IE-C sequences (with an average length of 67 nt) are much shorter than the IEs found in CCMP1545 and feature a highly conserved branch-point motif—GACTGACG—identical to the extended branch-point sequence reported for canonical  introns  , EST evidence confirms their excision from primary transcripts. Even more, the nonexcision of IEs from the transcripts would generally lead to a premature stop codon resulting in nonsense-mediated mRNA decay .A). A second, so-called SOC, low in GC% and found in all mamiellophycean species reported so far, is also completely devoid of IEs. Other chromosomes tend to have the IEs distributed over their entire length, however with reduced densities in regions with lower GC% (B). Their tendency toward high-GC% areas even surpasses canonical introns (B).IEs are not evenly distributed in the genome and are virtually absent from low-GC% areas, such as the AT-rich fraction of the BOC A. A secoower GC% B. Their  introns B.Fig. 2We could not identify any sequence motif, both at the nucleotide level or the amino acid level that would correlate with the presence of IEs. There is also no insertion bias toward specific gene categories. On the other hand, the only functional category of genes completely lacking IEs involves genes that code for ribosomal structural components. However, it is well known that these genes are intron-poor and have a specific intron set—sometimes encoding small nucleolar RNAs—that helps to regulate the production and function of the ribosome , which cMicromonas are more often found at gene extremities  and mostly in the genic 5′ region . In total, 913 metagenomic sequences revealed 511 unique novel IE insertions. Most metagenomic sequences containing IE-A elements were highly identical to the CCMP1545 genome, while for IE-C-containing sequences, a higher degree of diversity was found . At the same time, we discovered about 13 times more metagenomic sequences with novel IE-C positions compared with IE-A or IE-B/IE-D for which we have no proof for “novel” insertions .When searching marine metagenomes (at NCBI [d CAMERA ) for IEs samples , we discMicromonas pusilla clade-I)  , for whisupplementary fig. S15, Supplementary Material online). Under this scenario, an IE can invade a transcript by reverse splicing. The resulting IE-containing transcript is subsequently reverse transcribed after which the cDNA undergoes homologous recombination with the corresponding genomic locus. The final result is that the IE is now found at a novel position in the genomic sequence.Comparison with metagenomic data thus suggests that IEs are mobile elements that can replicate themselves and transpose into new locations. IEs are only found in transcribed regions in the sense orientation, which suggests that their mobility is linked to the transcription/splicing process. The mechanism most likely to explain this scenario is known as intron transposition  . Therefore, the “mobility phase” of IEs has to occur at a stage that still features a non-spliced primary transcript and not at the mature mRNA level.An analysis of orthologous introns between CCMP1545 and RCC299 genes revealed 32 cases of IE remnants buried within conserved canonical introns. There are also several cases of nested or merged IEs, i.e., IEs inserted inside or merged with another IE  and algae (using Chlamydomonas reinhardtii as a representative) with a single or major peak of small introns and a tail or a shoulder of big introns, which usually results from the insertion of transposable elements or other repeat elements Micromonaophyceae . Canonicophyceae . It is uophyceae , the oriophyceae . Among tophyceae . The preophyceae .However, recent studies seem to imply that intron gain is more widespread than previously thought , leadingOikopleura dioica  were found out of a total of ∼75,000 introns. In this case, both NIIs in a pair were found within the same gene and were suggested to be the result of reverse splicing. In fungi, intron gain due to the insertion of near-identical introns , shares some features with IE insertions. Depending on the species, ILEs occur in a range of a few tens up to ∼500, out of a total of more than 10,000 introns. Within the Mycosphaerellaceae species, they are related to each other, suggesting the presence of ILEs predating speciation within this clade ∼100 Ma. ILEs were shown to be efficiently spliced but to share specific features compared with resident introns, such as a bigger size and a conserved secondary structure. Finally, ILEs were shown to slowly degenerate with time, loosing progressively these specific features, and were thus suggested to be ancestors of many resident introns.Peculiar intron gains were recently observed in the pelagic tunicate a dioica , the micia pulex , the dotminicola  and Cladm fulvum , and of CCMP1545 , a list Micromonas stand out is first and foremost the amplitude of intron gain, with hundreds to thousands of newly gained introns—comparable in number to an invasion of transposable elements. Because of its huge numbers, IE invasion can truly be seen as an intron-late case, in which the organisms’ intron content is significantly enriched, more than doubled in the case of CCMP1545. These IE numbers must impact the biology of Micromonas, while the other reported intron gains would likely not. The second difference lies in the genome characteristics. Micromonas, just like all other Mamiellophyceae, only contains a few resident introns, whereas the organisms listed above are intron-rich, although to a lower extent for Mycosphaerellaceae fungi, for which the number of introns lies between 1 and 2 introns per gene . This explains why M. pusilla CCMP1764, which belongs to a different clade than RCC299 . Reverse splicing, which was initially a rather wild hypothesis, nowadays turns out to fit with the current knowledge as it has recently been established in yeast that the two splicing steps are indeed reversible , both steps being documented in model eukaryotes and supported by the occurrence of intron loss for which they are required as well.Various mechanisms have been suggested to explain intron gains, such as gene duplication, insertion of transposable elements, mutational creation of novel splice sites, or splicing enhancing features. Our findings as well as other recent ones implying propagation of intron copies do favor the reverse-splicing/recombination scenario  suggesteversible . The secMicromonas spliceosome to be permissive but rather ineffective for the newcomer introns that have not yet evolved the most efficient splicing mechanism, a hypothesis previously been put forward to explain evolution of mechanisms of RNA surveillance , and different classes of IEs. These IEs have colonized the genome by copying themselves into genes, likely involving reverse splicing. The findings presented in this article further strengthen the idea that intron gain is more widespread than previously thought.The Supplementary figures S1–S17 and supplementary methods are available at Genome Biology and Evolution online .


TrkB allele (fB) mouse designed for deletion of TrkB to determine its role in the adult striatum.The high affinity tyrosine kinase receptor, TrkB, is the primary receptor for brain derived neurotrophic factor (BDNF) and plays an important role in development, maintenance and plasticity of the striatal output medium size spiny neuron. The striatal BDNF/TrkB system is thereby implicated in many physiologic and pathophysiologic processes, the latter including mood disorders, addiction, and Huntington’s disease. We crossed a mouse harboring a transgene directing cre-recombinase expression primarily to postnatal, dorsal striatal medium spiny neurons, to a mouse containing a floxed We found that there were sexually dimorphic alterations in behaviors in response to stressful situations and drugs of abuse. Significant sex and/or genotype differences were found in the forced swim test of depression-like behaviors, anxiety-like behaviors on the elevated plus maze, and cocaine conditioned reward. Microarray analysis of dorsal striatum revealed significant dysregulation in individual and groups of genes that may contribute to the observed behavioral responses and in some cases, represent previously unidentified downstream targets of TrkB.The data point to a set of behaviors and changes in gene expression following postnatal deletion of TrkB in the dorsal striatum distinct from those in other brain regions. Ntrk2). There are two major TrkB isoforms, including the full-length TrkB.FL containing the tyrosine kinase domain, i.e. tk+, and a C-terminal truncated form, TrkB.T1, lacking the catalytic tyrosine kinase domain. TrkB.T1 is a dominant-negative receptor that inhibits TrkB.FL signaling, but has many other functions, including as a signal transducing molecule -dATP. The probes had the following sequence: for Tacr1/substance P, complementary to bases 20–67, GenBank accession no. M68909; Penk/enkephalin, bases 304–351, M13227; Pdyn/dynorphin, bases 807–854, AF026537; Ntrk2/TrkB, bases 733–780, NM 008745; Ppp1r1b/DARPP-32, bases 1253–1300, BC031129. Striatal gene expression was determined in the nucleus accumbens . In the case of comparison between genotypes, unpaired two-tailed Student t-test was used. One-way ANOVA or two- way ANOVA with sex and genotype or drug and genotype factors were used to analyze the behavioral data. Bonferroni post-hoc comparisons were used with significant ANOVAs. Unpaired t-test was used to analyze western blot, Total RNA was extracted from the striatum of fB/fB and D9ΔTrkB transgenic mice using the Nucleospin RNA kit . RNA quantity was assessed with Nanodrop (Nanodrop Technologies) and quality with the Agilent Bioanalyzer (Agilent Technologies). Total RNA (200 ng) was amplified, biotinylated and hybridized on Illumina Mouse_8_V2 Beadchips, querying the expression of ~22,000 Refseq transcripts, as per manufacturer’s protocol. Slides were scanned using Illumina BeadStation and signal extracted using Illumina BeadStudio software . Raw data was analyzed as previously described using Bioconductor packages. Quality assessment was performed looking at the inter-array Pearson correlation and clustering based on top variant genes was used to assess overall data coherence. Contrast analysis of differential expression was performed using the ‘limma’ package . After lhttp://david.abcc.ncifcrf.gov/) to understand the potential biological relevance of differentially expressed genes. Canonical pathway analysis was performed using Ingenuity Systems Pathway Analysis (http://www.ingenuity.com).Gene Ontology (GO) terms were identified using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (The authors declare that there is no conflict of interest.in situ hybridization or molecular genetic experiments, interpreted primary data and approved the final manuscript. All authors read and approved the final manuscript.EMU, MEP, TBB, EAT, HS and MEE conceived and designed the experiments, performed experiments, reviewed and interpreted primary data, and were involved in drafting and editing the manuscript. BX and LFR contributed reagents and edited the manuscript. JSM, KAP, JB, MR,and BT designed and performed behavior, Genes differentially expressed in striatum of D9ΔTrkB mice at P < 0.05.Click here for file


Long interspersed nuclear element 1 (L1) retrotransposons are the only autonomously mobile human transposable elements. L1 retrotransposition has shaped our genome via insertional mutagenesis, sequence transduction, pseudogene formation, and ectopic recombination. However, L1 germline retrotransposition dynamics are poorly understood because de novo insertions occur very rarely: the frequency of disease-causing retrotransposon insertions suggests that one insertion event occurs in roughly 18–180 gametes. The method described here recovers full-length L1 insertions by using hybridization enrichment to capture L1 sequences from multiplex PCR-amplified DNA. Enrichment is achieved by hybridizing L1-specific biotinylated oligonucleotides to complementary molecules, followed by capture on streptavidin-coated paramagnetic beads. We show that multiplex, long-range PCR can amplify single molecules containing full-length L1 insertions for recovery by hybridization enrichment. We screened 600 µg of sperm DNA from one donor, but no bone fide de novo L1 insertions were found, suggesting a L1 retrotransposition frequency of <1 insertion in 400 haploid genomes. This lies below the lower bound of previous estimates, and indicates that L1 insertion, at least into the loci studied, is very rare in the male germline. It is a paradox that L1 replication is ongoing in the face of such apparently low activity. Hum Mutat 32:1–11, 2011. © 2011 Wiley-Liss, Inc. Transposable elements are the most common class of repetitive DNA in the human genome, accounting for ∼45% of our DNA ) that can apparently insert anywhere within a large (3 Gigabase [Gb]) genome. Third, the frequency of insertion is likely to be extremely low, with the current estimates of de novo L1 activity predicting that a single insertion will occur in 1 in 9 to 1 in 186 humans, corresponding to a single de novo L1 insertion in 54 pg to 1.12 ng of germline DNA [Kazazian, Here we present the development of an L1 hybridization enrichment method capable of physically recovering complete L1 insertions into genomic targets devoid of L1 sequences. We illustrate the method's ability to recover full-length L1 insertions at the single DNA molecule level and present a study that enabled us to estimate an upper bound of the frequency of L1 retrotransposition in the human male germline at our selected loci.Sperm DNA was prepared as described previously [Jeffreys et al., http://www.repeatmasker.org), to locate nonrepetitive DNA suitable for primary and secondary polymerase chain reaction (PCR) primer design, as shown in Eight target loci known to have harbored disease-causing L1 insertions were selected for investigation, along with two additional loci (HoxD and MHC Class II) . None of2, 6.7 mM 2-mercaptoethanol, 113 µg/ml BSA, 1.1 mM dNTPs), 0.05 µM of each primer and 0.025 U/µl 20:1 Taq/Pfu polymerases . PCR cycling was performed in an MJ Tetrad PTC250 thermal cycler  at 96°C for 1 min, then at 96°C for 20 sec, 62°C for 1 min/kb of target amplicon plus 1 min for 30 cycles, followed by 61°C for 30 min. HPLC purified primers were supplied by Thermo Electron, and handled under PCR-clean conditions.A total of 20 µl PCR reactions contained 20 to 50 ng of genomic DNA (gDNA), PCR buffer  to an estimated concentration of 1 haploid genome/µl. PCRs were carried out in eight replicates of 1 µl input per dilution, then diluted 10-fold in 5 mM Tris HCl (pH 7.5) and 2 µl of the dilution used to seed nested secondary PCRs. Secondary PCR products were fractionated by agarose gel electrophoresis in the presence of 0.5 µg/ml ethidium bromide. The frequency of positive and negative reactions was used to Poisson estimate the maximum likelihood number of amplifiable molecules and its 95% confidence intervals [Jeffreys et al., Genomic DNA samples were serially diluted in 10-fold steps using single molecule diluent  to remove oligonucleotide primers and DNA polymerase that could interfere with hybridization enrichment. The aqueous phase was reextracted with chloroform and the purified DNA collected by ethanol precipitation. DNA was redissolved in 33 µl 5 mM Tris-HCl (pH 7.5) prior to hybridization enrichment.The principal stages of L1 hybridization enrichment are illustrated in 2, 6.7 mM 2-mercaptoethanol, 4.4 µM EDTA, 2 µg/ml single-stranded (heat denatured) high molecular weight herring sperm DNA). The washed beads were resuspended in a volume of 1 × DHB 1/12th that of the original volume. This working stock of beads was kept in the dark, on ice.M-280 streptavidin-coated super-paramagnetic Dynabeads  were captured with a Dynal MPC-S magnetic particle concentrator and washed three times at room temperature (resuspending each time) with 100 µl 1 × denaturing/hybridizing/binding buffer  prior to transfer to a fresh siliconized Eppendorf tube. The Dynabeads were finally captured and resuspended in 50 µl ED prior to thermal elution.Binding of the bio-oligo/DNA hybrids to Dynabeads , step 3 Single-stranded DNA was recovered from bead-bound bio–oligos , step 4 The unbound fraction collected at the first cycle of enrichment was reextracted, by adding more bio-oligos and following the annealing/binding/recovery procedure as above, to maximize DNA recovery. In total, one extraction and two reextractions were carried out per sample. The eluates from the extraction and the reextractions were pooled. A total of 33 µl of each pooled eluate was then subjected to a second round of hybridization enrichment as above, and again eluates from the secondary extraction and the reextractions were pooled. All eluates and washes were stored in the dark at 4°C.32P-labeled L1-specific oligonucleotide probe (PFLR5999). L1-sequence containing PCR products were detected by autoradiography. The remaining PCR products were fractionated by gel electrophoresis and stained with ethidium bromide (0.5 µg/ml). L1 sequence-containing bands identified by autoradiography were visualized using a Dark Reader trans-illuminator  and excised from the gel. DNA was extracted using the QIAquick gel extraction kit , cloned and sequenced (see below).The eluted DNA was subjected to nested PCR amplification using secondary target site primers Supp.  as shownE. coli cells. Plasmid DNA was recovered using the QIAprep Spin miniprep kit . A total of 20–30 ng/kb of plasmid DNA was sequenced using the Big Dye Terminator v3.1 ReadyReaction system  with M13F or M13R sequencing primers. Excess reaction components were removed using PERFORMA DTR Gel Filtration Cartridges  and samples were analysed on an ABI3730XL capillary sequencer (Applied Biosystems).Purified amplicons were ligated into the pGEM®-T Easy plasmid vector , following the manufacturer's protocol, and transformed into ultra competent DH5α http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) and the GCG package . Assembled sequences were aligned with the human L1 element L1.3 (accession L19088) and the appropriate target site sequence  [Kazazian, 6 sperm, or ∼100 insertions per ejaculate (∼108 sperm).Ten target loci  were selBssSI allowed identification of DNA fragments derived from each target (32P-labeled target site-specific oligonucleotide probes (data not shown).Thermal cycling conditions were optimized to ensure efficient amplification of all 10 targets in a single 50-µl multiplex PCR seeded with 0.5 µg of sperm DNA, the maximum DNA input compatible with efficient PCR. Digestion of the 10-plex secondary PCR products with h target ; these fh target , indicatTo test whether multiplex PCR could also efficiently amplify molecules carrying a full-length L1 insertion, we added an extra primer pair specific for a locus containing the polymorphic AL121819 L1 insertion [Badge et al., Filled-site targets (12 kb) are much less efficiently amplified than empty sites (6 kb), as evident in 11 molecules over all 10 targets). It was therefore essential to recover de novo insertion PCR products by hybridization enrichment.To quantify the effect of this low PCR efficiency on de novo insert amplification and recovery, we analyzed the PCR product yield of an 11-plex PCR seeded with 0.5 µg of donor A gDNA, effectively containing ∼80,000 amplifiable molecules of the empty AL121819 site and ∼40,000 molecules of the filled site. This analysis indicated a gain of PCR products per cycle, from the empty and filled sites, respectively, of ∼1.8 and ∼1.6 over 20 cycles of PCR. This differential efficiency means that amplification of 0.5 µg gDNA containing a single amplifiable insertion molecule would produce 12,000 filled site molecules present in a huge excess of empty site molecules  [Jeffreys and May, −6% of empty target site molecules. This indicates a >500,000 fold enrichment of L1-containing amplicons. After multiplex PCR, a pool of DNA would contain ∼12,000 L1-containing molecules derived from each de novo insertion, plus ∼1011 empty target site molecules. Following a single round of enrichment, this ratio of filled to empty site molecules would increase from 1/8,000,000 to >1/16, allowing a single molecule of a de novo insertion to be readily detected.Following two rounds of optimized hybridization enrichment, we routinely recovered at least 2% of PCR-amplified L1-containing molecules, compared with <4.5 × 106 fold) of insert-free genomic DNA, and that hybridization enrichment was essential for their detection.To test whether DEASH could recover full-length L1 insertions at the single DNA molecule level, we mixed 24 pg sperm gDNA from donor A containing ∼2 amplifiable molecules of the AL121819 insertion (see above) with 0.5 µg of sperm gDNA from donor B, who lacks the AL121819 insertion. This DNA mixture, along with 95 additional reactions each containing 0.5 µg of sperm gDNA from donor B alone, was subjected to 11-plex PCR to amplify all ten targets plus the AL121819 locus. Amplified DNA from all 96 reactions was pooled and purified, and one-third of this DNA subjected to L1 hybridization enrichment. PCR amplification of the AL121819 target, either alone or as part of an 11-plex PCR for all targets, was followed by reamplification using primers designed to separately amplify the 5′ and 3′ junctions of the AL121819 insert . These PHaving established that hybridization enrichment could recover insertions at the single DNA molecule level, we screened 576 µg sperm gDNA from donor B for de novo L1 insertions. DNA was amplified by 10-plex PCR in 0.5 µg aliquots distributed over 12 96-well plates, and the PCR products from each plate pooled and enriched as above. Each of the 12 batches of enriched DNA was then screened for L1–containing target molecules using 10-plex (or duplex) primary PCR followed by nested PCRs as shown in A genuine de novo L1 insertion into one of the target loci should contain an L1 sequence and a poly A tail, flanked 5′ and 3′ by target site sequences. All of the 11 putative insertion amplicons showed sequence similarity to one of the target loci  most often associated with Alu elements (10/11). In 7 of the 11 cases the L1 and flanking sequence were >99% identical to regions of the genome harboring known L1 elements. Although no genuine insertions were identified, these chimaeric artifacts do provide further validation of the MP-HE approach, showing that L1 hybrid molecules generated during PCR amplification can be recovered by our strategy, but are easily identified as artifacts by sequencing.We used MP-HE to survey 576 µg of sperm DNA from a single donor for de novo L1 insertions. Eleven putative L1 insertions were identified, all containing L18 sperm, or 9.6 × 107 amplifiable molecules of each target under the assumption that single molecule PCR is 50% efficient when amplifying a 12-kb amplicon, as established for control target molecules carrying full-length L1 insertions. The 10 loci surveyed together cover 51 kb of target DNA per sperm, within which a de novo insertion could be detected. As half of the loci are on the X chromosome, and so only present in 50% of sperm, this is effectively reduced to 38 kb of DNA per sperm. We have therefore screened 38 kb × 9.6 × 107 = 3.7 × 109 kb genomic DNA for insertions. The lack of insertions places an upper bound on the L1 insertion frequency of three insertions in 3.7 × 109 kb (P = 0.05), or <1 event per 400 haploid genomes, lower than estimates of L1 retrotransposition frequency derived from the incidence of pathogenic L1 insertions in humans  [Brouha et al., This major survey of sperm DNA from a single donor failed to yield any genuine insertions. These data can be used to estimate an upper bound of the frequency of L1 insertion in this man's germline, with the caveat that this estimate only applies to the selected target loci. Indeed, because most of the target loci have accommodated pathogenic insertions in the past we may have ascertainment bias in favor of insertion-prone loci. This bias is likely to cause overestimation of the insertion rate, making even more significant the lack of insertions detected here. The DNA analysed was derived from 1.9 × 10The reason for this very low estimated frequency of de novo insertion of L1 elements in an individual male germline is unclear. It is unlikely that the chosen target loci are refractory to insertion because L1 insertion into the genome appears to be largely random [Feng et al., It is possible that donor B has an unusually low frequency of germline L1 retrotransposition due to the absence of active L1s in his genome. He does indeed lack the most active L1 identified to date, AC002980 [Brouha et al., Other factors could contribute to the very low rate of L1 retrotransposition observed here. Previously it was thought that L1 was primarily active during meiosis [Brouha et al.,


LINE-1 (L1) is the dominant category of transposable elements in placental mammals. L1 has significantly affected the size and structure of all mammalian genomes and understanding the nature of the interactions between L1 and its mammalian host remains a question of crucial importance in comparative genomics. For this reason, much attention has been dedicated to the evolution of L1. Among the most studied elements is the mouse L1 which has been the subject of a number of studies in the 1980s and 1990s. These seminal studies, performed in the pre-genomic era when only a limited number of L1 sequences were available, have significantly improved our understanding of L1 evolution. Yet, no comprehensive study on the evolution of L1 in mouse has been performed since the completion of this genome sequence.Using the Genome Parsing Suite we performed the first evolutionary analysis of mouse L1 over the entire length of the element. This analysis indicates that the mouse L1 has recruited novel 5’UTR sequences more frequently than previously thought and that the simultaneous activity of non-homologous promoters seems to be one of the conditions for the co-existence of multiple L1 families or lineages. In addition the exchange of genetic information between L1 families is not limited to the 5’UTR as evidence of inter-family recombination was observed in ORF1, ORF2, and the 3’UTR. In contrast to the human L1, there was little evidence of rapid amino-acid replacement in the coiled-coil of ORF1, although this region is structurally unstable. We propose that the structural instability of the coiled-coil domain might be adaptive and that structural changes in this region are selectively equivalent to the rapid evolution at the amino-acid level reported in the human lineage.The pattern of evolution of L1 in mouse shows some similarity with human suggesting that the nature of the interactions between L1 and its host might be similar in these two species. Yet, some notable differences, particularly in the evolution of ORF1, suggest that the molecular mechanisms involved in host-L1 interactions might be different in these two species. Long interspersed nuclear element-1 (LINE-1 or L1) constitutes the dominant category of transposable elements in mammalian genomes. L1s have accumulated in the genomes of their mammalian hosts in extremely large numbers and contribute to more than 20% of genome size in human and mouse -6. HowevThe pattern of L1 element evolution in mammals is very unusual. In most species analyzed so far, L1 evolves as a single lineage: a family of elements emerges, amplifies to hundreds or thousands of copies and then becomes extinct, being replaced by a more recently evolved family ,33-35. TMus musculus) have demonstrated the presence of multiple concurrently active L1 families with non-homologous promoters [The patterns described above result mostly from the analysis of the human genome and it is unclear how patterns of evolution in human recapitulate L1 evolution in other species. It is thus important to examine in greater detail the evolution of L1 lineages in other mammals. Pre-genomics studies in the house mouse ; (2) a 5’UTR that was not characterized before, named Mus (because it is absent from the rat genome); and (3) a 5’UTR that shows no similarity with any others, named N (for novel).A total of 20,459 L1 inserts with complete reverse transcriptase (RT) domains were identified using the Genome Parsing Suite (GPS). L1 elements were first grouped based on their 5’UTR. This was done by comparing the 5’ end of all elements with a library of previously described mouse 5’UTR using the Repeatmasker program . The A, Once elements were sorted based on their 5’UTRs, they were further categorized into families using a phylogenetic analysis of the 3’ terminus. A family is defined as a collection of elements that result from the activity of a highly homogenous group of progenitors, which are characterized by a unique combination of characters. In the first step of the phylogenetic analysis, neighbor joining trees  of elemeUsing this approach we identified 29 families and consensus sequences were derived for each of them (Table anc promoter (L1MdFanc_II). At the same time two families branched independently from the main lineage, one carrying a Mus promoter (L1MdMus_I) the other one the Fanc promoter (L1MdFanc_I). Preceding the L1MdFanc_II family a lineage made of four families with an Lx promoter was active. At two points in time the Lx promoter was replaced by the V promoter (yielding L1MdV_II and III) but these families did not persist or produce novel lineages.As L1 families have extensively recombined with each other (see below), various regions of L1 yield different evolutionary histories and it is impossible to build a single phylogenetic tree based on the entire length of the element. Figure anc promoter (L1MdFanc_II) and by the V promoter twice (L1MdV_II and III). The Fanc promoter was replaced independently twice by the Mus promoter as L1MdMus_I and L1MdMus_II do not form a monophyletic group. The Mus promoter was eventually replaced by the V promoter (L1MdV_I) and went extinct. The F promoter was then resuscitated approximately 6.4 MY ago and gave rise to families L1MdF_I to V. Approximately 4.6 MY ago the A promoter was recruited yielding the modern A lineage which extend from families L1MdA_VII to I. Within this lineage, an additional recruitment occurred resulting in the L1MdN_I family. Finally the F promoter was recently recruited twice, approximately 2.2 MY by the L1MdGf_II family and approximately 1.2 MY by the Tf/Gf_I lineage. Thus we estimate that L1 in mouse has experienced 11 replacements of 5’UTR.One of the most striking features visible on the tree is that families with similar 5’UTRs do not form monophyletic groups indicating that L1 families have frequently recruited novel 5’UTR, either from unknown sources or from ancient families. The oldest families in our study carried an Lx promoter, which was replaced three times: once by the FThe topology of the ORF2 tree indicates that mouse L1 families evolved mostly as a single lineage. This does not mean that a single family or single lineage was active at a time. In fact, the co-existence of multiple active families characterizes the evolution of L1 for the last 13MY of mouse evolution. For instance between 1 and 2.5 MY ago, six families  were active in the mouse genome as attested by the overlap in their average pairwise divergence  and L1MdF_II, near the 3’ end of the element Figure D. A 666 It should be noted that our criteria for identifying recombination events were stringent, as we only considered the recombination of large segments to be significant. Thus it is plausible that exchanges of sequences of shorter length have occurred between L1 families but were not detected due to the small number of defining characters in some conserved regions of L1, such as ORF2. The number of recombination events reported here suggests that recombination has played a significant role in the evolution of novel L1 families in mouse and can occur across the entire length of L1.The exchange of genetic information between families constitutes a significant challenge for evolutionary analyses as most phylogenetic algorithms do not allow for recombination. Thus we performed phylogenetic analyses using regions of L1 delimited by recombination breakpoints to fully assess the impact of recombination on the evolutionary history of FL L1 elements Figure . Trees AWe then examined the evolution of the protein coding sequences encoded in L1, ORF1, and ORF2. ORF2 is the most conserved region of L1. There are very few amino acid changes, in particular in the endonuclease and reverse transcriptase domains which are functionally indispensable ,58. All We examined the level of conservation of domains of ORF1 that are known to be functionally important ,59,60. TMore surprising is the degree of conservation at the amino acid level of the CC domain. Previous studies have shown that the CC domain of ORF1 has evolved under positive selection in primates ,39. In tanc_II and a 24 bp deletion in the second repeat found in L1MdFanc_I and L1MdF_IV. The 66–42 bp motif was followed by a duplication of the 42 bp unit resulting in a 66-42-42 bp structure which is found in families L1MdA_V to II, L1MdN_I, L1MdTf_III to I, and L1MdF_I, II, and V. This motif further evolved by the loss of the second 42 bp repeats in L1MdA_I and L1MdF_IV and by the addition of a third 42 bp unit in family L1MdGf_I. The ancestral 66–66 bp motif was recruited by recombination in families L1MdF_III, L1MdA_VI, and VII, and acquired a third 66 bp unit in family L1MdGf_II. These structural changes in the LPR resulted in changes in the length and structure of the CC. Coiled coils are formed from two or more α-helical peptide chains that contain a distinct arrangement of non-polar side chains. Domains that can form CC consist of heptads (or seven residue repeats) with non-polar or hydrophobic residues in the first and fourth positions [Although the CC domain is relatively conserved at the amino acid level, it shows a high level of structural variation. Previous studies have identified a region called length polymorphism region (LPR) ,61. Usinositions . The CC ositions . The shoMus and it has been proposed that some L1 families in the house mouse genome were acquired by hybridization [Finally, we examined the possibility of lateral transfer in the evolution of murine L1. In mammals, L1 is transmitted vertically and there is no evidence of lateral transfer , except dization ,64,65. IWe performed the first comprehensive analysis of L1 evolution since the completion of the mouse genome . The anaThe evolution of L1 in mouse fits the single lineage mode of evolution described previously in other mammals and particularly in human ,35,63,69anc_I (active 6.80 MY ago) to the ancestor of L1MdF_V (at 6.43 MY) and subsequently transferred from L1MdF_I (active 2.12 MY ago) to the recently active L1MdTf and L1MdGf families.The analysis of FL elements has revealed the extraordinary ability of L1 families to acquire novel motifs and to exchange sequences Figures  and 3. TThe first ORF is arguably the least understood region of L1, although it has been the subject of much attention in the past few years ,60,74-78We performed a comprehensive analysis of L1 evolution in mouse. This analysis covered the last 13 MY of mouse evolution, since the split between mouse and rat. The mouse L1 has evolved as a single lineage for most of its evolution, although co-existence between families carrying different promoter sequences was observed. L1 families have frequently acquired novel 5’UTR and have exchanged sequences over the entire length of the element. No evidence of rapid amino acid replacement in the ORF1 was detected, although it is likely that the structural instability of the CC domain is adaptive. The general pattern of evolution of mouse L1 is similar to the one in human suggesting that the nature of the interactions between L1 and its host might be similar in these two species. There are however some intriguing differences between mouse and human, particularly in the evolution of ORF1. These differences suggest that the molecular mechanisms involved in host-L1 interactions might be different in these two species.Mus musculus 2006 (mm8) genome built using the GPS [anc, V, Lx, Mus, or N; see result) followed by a roman number. The smaller the roman number, the younger the family is. For instance families L1MdA_I, L1MdA_II, and L1MdA_III are subsets of the previously described L1MdA family; family L1MdA_I is younger than family L1MdA_II and family L1MdA_III is the oldest of the three. We kept the Gf [Full-length (FL) elements were collected from the  the GPS . GPS con the GPS ,33. Thus the GPS  of eleme the GPS  based on the GPS . The modt the Gf  and Tf [t the Gf  names fohttp://darwin.uvigo.es/rdp/rdp.html) was used to search for evidence of recombination among families. RDP allows for the use of several recombination detection methods including substitution and phylogeny-based methods. Two substitution-based methods, MaxChi [NJ, maximum parsimony (MP), and maximum likelihood (ML) trees were calculated for each region of L1. Phylogenetic trees were reconstructed using the MEGA 5.01 package . The RDP, MaxChi  and Chim, MaxChi , as well, MaxChi , were us, MaxChi  which ishttp://www.datamonkey.com[a priori specification of branches in a phylogeny that may have evolved under different values of dN/dS. The dN/dS test is however not very sensitive, particularly if selection acts on a few codons. For this reason we used three methods designed to detect the action of positive or negative selection at specific sites in an alignment: Single Likelihood Ancestor Counting (SLAC), a Random Effects Likelihood (REL), and Fixed Effects Likelihood (FEL) [To test for evidence of selection in the evolution of L1 several methods implemented in the web server onkey.com of the Honkey.com were useonkey.com. A ratioonkey.com can deteod (FEL) . For eacod (FEL) ,39. CC sod (FEL) . Domainsod (FEL)  was usedMus/Rattus at 13MY [The age of each subfamily was estimated by calculating the average pairwise divergence based on the 3’UTR. CpG dinucleotides and the highly mutable polypurine tract located in the 3’UTR were removed from alignment. The average divergence between copies as well as the standard error was calculated using the maximum likelihood parameter distance (using the MEGA 5.01 software). Divergences were converted to time assuming a neutral rodent genomic substitution rate of 1.1%/MY .http://www.girinst.org/repbase/).The consensus sequences are available in Repbase .Click here for fileAlignments showing recombination break-points among L1 families. Only the parsimony-informative sites are shown.Click here for file