Although partnerships between park and recreation agencies and health agencies are prevalent, little research has examined partnership characteristics and effectiveness among communities of different sizes. The objective of this study was to determine whether park and recreation leaders’ perceptions of partnership characteristics, effectiveness, and outcomes vary by community size.A web-based survey was completed in 2007 by 1,217 National Recreation and Park Association members. Community size was divided into 4 categories: very small, small, medium, and large. Questions measured agencies’ recognition of the need for partnerships, their level of experience, and the effectiveness and outcomes of partnerships.Larger communities were significantly more likely to recognize the need for and have more experience with partnerships than smaller communities. Very small and large communities partnered significantly more often with senior services, nonprofit health promotion agencies, and public health agencies than did small and medium ones. Large and small communities were significantly more likely than very small and medium communities to agree that their decision making in partnerships is inclusive and that they have clearly defined goals and objectives. Large communities were significantly more likely than very small communities to report that their partnership helped leverage resources, make policy changes, meet their mission statement, and link to funding opportunities.Community size shapes partnership practices, effectiveness, and outcomes. Very small communities are disadvantaged in developing and managing health partnerships. Increasing education, training, and funding opportunities for small and rural park and recreation agencies may enable them to more effectively partner with organizations to address community health concerns. Community park and recreation agencies are stakeholders in addressing health issues such as physical inactivity, obesity, and chronic disease and are partners in the healthy communities’ movement. Interorganizational partnerships can facilitate health behavior change that prevents chronic disease. Kernaghan  defined Health partnerships in parks and recreation have become more prevalent in the past 20 years as agencies try to optimize resources with limited budgets. Although the importance of health partnerships is widely recognized ,6 littleThis study is part of a larger cross-sectional project that consisted of an online survey and in-depth interviews of park and recreation managers. Data were collected by using a web-based survey, and Pennsylvania State University’s institutional review board approved this study. The National Recreation and Park Association (NRPA) membership database was used to identify directors or senior managers . Investigators reviewed the database to ensure that only 1 person from each member agency was invited to participate in the survey. NRPA is a professional organization whose mission is to advance parks, recreation, and environmental conservation efforts that enhance the quality of life for all people.A presurvey letter was e-mailed to the sample on October 1, 2007, to announce the online survey. Ten days later, a second e-mail was sent with a cover letter and a link to the survey. After the first survey invitation, follow-up e-mails were sent to nonrespondents every 10 days for 1 month. Within the list of 4,338 NRPA members, 558 e-mails were undeliverable, reducing the sample to 3,780. Of these recipients, 1,217 completed the questionnaire, a response rate of 32%. Individuals contacted could opt out of the survey, take it and stop at any time, or complete the survey. Respondents concluded their participation when they finished the survey (which took 10–15 minutes) and were entered into a drawing for a small retail gift card. Because the focus of this study was local, public community park and recreation agencies , state and national parks, military recreation, and nonprofit agencies  were excluded from subsequent analysis, resulting in 1,089 usable surveys. To assess the potential threat of nonresponse bias in the sample, a random sample of 100 nonrespondents was contacted by telephone and e-mail and answered several questions. Results indicated that respondent characteristics and agency partnership practices reflected those reported by nonrespondents regarding agency type, community size, partnership participation, and effectiveness.Community size consisted of 4 categories based on the population size served by the agency: 1) very small , 2) small , 3) medium , and 4) large . Partnership involvement was assessed by asking, “Within the past five years, has your agency/organization partnered with an outside organization  to specifically promote health, wellness or physical activity within your community” (yes/no)? The number of partnerships was assessed by asking “With how many different health/wellness partnerships is your organization currently involved?” Two items developed by Hasnain-Wynia et al  were usePartnership types were measured by a list of 13 partner types represented in the National Physical Activity Plan . Types wPerceptions of partnership effectiveness were measured with 12 statements representing factors contributing to partnership effectiveness as established in prior partnership studies ,10,11. ILocal and municipal park and recreation agencies were most of the sample (892 [81.9%] organizations). There were also 96 (8.8%) county park systems and 101 (9.3%) special districts represented. Regarding community size, 409 (40%) agencies serve very small communities, 221 (21.6%) serve small communities, 139 (13.6%) serve medium communities, and 253 (24.8%) serve large communities; 67 respondents did not answer the question. Most respondents indicated that their organization engaged in at least 1 health partnership in the last 5 years, with an average of 5 partnerships.F = 13.64, degrees of freedom [df] = 4, P < .001). Also, as community size increased, experience with partnerships increased . Very small communities  had significantly less experience with partnerships than both medium  and large communities . Moreover, very small communities  were significantly less likely than large communities  to acknowledge the need to develop health partnerships .Results indicated that as community size increased, the mean number of health partnerships increased significantly  our health partnership decision making is inclusive, 2) our health partnerships are empowered to make decisions, 3) our health partnerships have clearly defined goals and objectives, and 4) our health partnerships have a strong vision of their mission and purpose . RegardiFour partnership outcomes varied significantly by community size: 1) the extent to which the agency leveraged additional resources because of the partnership, 2) the ability to meet the mission statement, 3) linking to funding opportunities, and 4) making policy changes . For allAs community size increased, the number of partnerships and experience with health collaborations also increased. As community size increases, the number of available partners  is likely to increase as a function of population density; this is especially relevant for rural areas that comprised more than 70% of the respondents who reported working in very small communities. These findings are consistent with those of Beatty, Harris, and Barnes’  partnersVery small and large communities were more likely than small and medium communities to partner with 7 organization types . According to Gazley and Brudney , “The moLarge communities had the highest ratings of effectiveness for all 4 items ; medium communities had the lowest ratings for 3 effectiveness items. Evidence supports the idea that clearly defined goals are particularly important to effective partnerships –19. For Large communities were significantly more likely to leverage additional resources and meet their mission statement as a result of their participation in partnerships. This may be attributed to their involvement with more partnerships than smaller communities and their ability to leverage the additional resources available as a result of those partnerships. Very small communities were significantly less likely to link to funding opportunities because of their limited partnership involvement. An ongoing challenge for rural areas is that little funding is channeled to them compared with urban and suburban communities. Only 0.2% of foundation funding in the United States is awarded to small rural communities . Often, The differences in effectiveness and outcomes related to partnerships might also be about leadership. According to Bryson et al , “the leOne of the limitations of this study is that we did not explicitly define the concept of “partnership” for respondents. Therefore, various partnership types  were likely reported on. Nevertheless, Cousens et al  reportedDespite these limitations, some differences regarding partnership practices, effectiveness, and outcomes were revealed among different sizes of communities. These findings indicate that smaller communities  are disadvantaged regarding their ability to develop and implement partnerships with health organizations. This is especially troubling since poverty, obesity, chronic disease, and uninsured rates are higher in rural areas than suburban and urban areas ,27. Not


Temora longicornis. Production of DMS can rapidly increase following microzooplankton grazing on phytoplankton. Here, we investigated whether grazing-induced DMS elicits an increase in foraging behavior in the copepod Calanus helgolandicus. We developed a semi-automated method to quantify the effect of grazing-mediated DMS on the proportion of the time budget tethered females allocate towards slow swimming, typically associated with feeding. The pooled data showed no differences in the proportion of the 25 min time budget allocated towards slow swimming between high (23.6 ± 9.74%) and low (29.1 ± 18.33%) DMS treatments. However, there was a high degree of variability between behavioral responses of individual copepods. We discuss the need for more detailed species-specific studies of individual level responses of copepods to chemical signals at different spatial scales to improve our understanding of chemical interactions between copepods and their prey.Chemical interactions play a fundamental role in the ecology of marine foodwebs. Dimethyl sulfide (DMS) is a ubiquitous marine trace gas that acts as a bioactive compound by eliciting foraging behavior in a range of marine taxa including the copepod  Since copepods can effectively regulate phytoplankton and microzooplankton populations  their theiret s  and the diatom treatment . However, variation in the proportion of the time budget spent slow swimming was high [C. helgolandicus feeding on the heterotrophic dinoflagellate Oxyrrhis marina that was pre-conditioned with either a high or low DMS-producing strain of the phytoplankton Emiliania huxleyi.Detection of copepod responses to prey stimuli has previously been measured in two ways, including through changes in the frequency of appendage beating or the proportion of the time budget allocated towards slow swimming. Gill and Harris  observedEmiliania huxleyi, a high DMS-producing strain (CCMP 373) and a low DMS-producing strain (CCMP370) were purchased from the National Centre for Marine Algae and Microbiota (NCMA) and maintained in batch culture in ESAW medium [−2 s−1 during a 12 h light: 12 h dark cycle. The heterotrophic dinoflagellate Oxyrrhis marina (CCAP 1133/5) was cultured in filtered (0.2 μm poresize) and autoclaved seawater (FASW), and maintained on a diet of non-DMS producing Dunaliella tertiolecta (grown in ESAW as above). Following grazing by O. marina, CCMP 373 produces 15-fold higher DMS concentrations than CCMP 370 [O. marina was placed in the dark for 24 h to eliminate prey. Copepods  were collected weekly by trawling with a coarse mesh cod-end at the Western Channel Observatory sampling site L4 off Plymouth, U.K. . Copepods were transported to the laboratory in insulated cool boxes before adult females (average prosome length = 2.21 ± 0.119 mm) were microscopically identified and transferred into 20 L buckets containing FASW in a dark walk-in room at 15 °C. They were maintained on a diet of Rhodomonas salina and O. marina at approximately 200 μg C L−1.Two non-calcifying strains of the coccolithophore W medium  at 15 °CCCMP 370 . Prior tO. marina. Tethered individuals typically survived for several weeks and copepods used in experiments were maintained for a minimum of 24 h after the experiment to ensure survival before freezing at −80 °C for storage and body measurements.Adult females were tethered to a hair 24 h prior to experiments. Hairs were treated with acetone and washed with Milli-Q water to remove natural odors. The hair was glued to the end of a glass Pasteur pipette which had been heat molded into a position allowing copepods to be oriented in a natural swimming position. During tethering, copepods were placed ventral side down in a concaved microscope slide with the feeding appendages and antennae immersed in water. The dorsal prosome was gently dabbed dry with microscope cleaning tissue and the hair glued to its base using instant adhesive (3M Scotch-Weld CA4OH). Tethered copepods were transferred into filtered seawater and fed Filming was carried out in a dark controlled-temperature room at 15 °C. Copepods were attached to a micromanipulator and placed in 1 L polycarbonate bottles filled to the neck with FASW. A CCTV camera  with a 60 mm macro lens (Nikkor) was connected to a computer to record copepod behavior. Illumination was provided using collimated light from an infrared light-emitting diode (peak wavelength 940 nm).E. huxleyi CCMP370 (low DMS) or CCMP373 (high DMS) to achieve a density of 6000 cells mL−1. Oxyrrhis marina was added to achieve a final density of 350 cells mL−1. Bottles were placed on a plankton wheel at 1 rpm and incubated for 5 h to allow grazing interactions to occur and the build-up of DMS. A total of 16 copepods were exposed to each treatment for 45 min using the following treatments in sequence: FASW, initial treatment diet, FASW, alternate treatment diet. The order of treatment (low DMS and high DMS diet) was randomized to prevent potential bias. The experimental design was based around a repeated measures approach allowing comparisons of individual copepods behavior between high and low DMS treatments.Polycarbonate bottles were inoculated with either DMS measurements were taken from 5 mL samples, gravity filtered through a 25 mm GF/F filter into a narrow glass vial minimizing diffusive losses. Depending on the expected DMS concentration in the sample, 1 to 4 mL was immediately transferred from the bottom of the vial to a purge-and-trap system for cryogenic enrichment of DMS before quantification using gas chromatography (Varian 3800) with a pulsed flame photometric detector (PFPD) . Each exi, across the matrix obtained from this subtraction. Since copepods were tethered, no movement (breaking) led to small changes in pixel intensity, continuous rhythmic feeding appendage movements (slow swimming) led to intermediate changes in pixel intensity and rapid movements of the body (fast swimming) led to large values in δi. Subsequent analysis of δi time series was conducted in the R programming language [i, we smoothed these time series by averaging δi over a five-frame window. By visual comparison of the δi time series to observed behavior in our video recordings, we determined two thresholds for each film: one to distinguish breaking from swimming and another to distinguish slow from fast swimming . Of the total 16 copepods filmed, the data from four copepods were discarded due to difficulties in assigning behavioral threshold values in at least one of the treatment videos. The behavioral state of 12 copepods was extracted for a period of 25 min from the beginning of recordings. Analysis over this period of time  allows observation of behaviors at the onset of each treatment and is sufficiently long enough to allow copepods to habituate to the treatment conditions [−1.A semi-automated technique was developed to assign copepod behavior into three states: breaking, slow swimming or fast swimming resulted in concentrations of 1.8 ± 0.67 nM DMS in the low DMS treatments after 5 h incubation. Grazing by O. marina on the high DMS-producing strain of E. huxleyi (CCMP373) in the high DMS treatment resulted in significantly higher DMS production with final concentrations of 13.1 ± 4.30 nM . Filtered seawater used in the experiments had a natural DMS concentration of 1.2 nM (±0.75).Grazing by t1,10 = 0.44, p = 0.667) or low DMS treatments . There were no significant differences in the average proportion of the 25 min time budget the 12 copepods allocated towards slow swimming in the high (23.6 ± 9.74%) and low (29.1 ± 18.33%) DMS treatments  with various prey items, and determined that chemically-mediated remote sensing does not occur. In addition, by modeling the boundary layer chemical concentration, Tiselius et al. [The ability of copepods to utilize chemical cues at the microscale is highly constrained by swimming strategy. For example, ambush predators such as ect prey . Cruise-currents ,40,41,42currents ,44. TiseT. longicornis [C. helgolandicus suggested that grazing-mediated DMS has no clearly defined and consistent effect on swimming behavior when considering data pooled across all 12 individuals. C. helgolandicus spent approximately a quarter of the time budget dedicated to slow swimming in the low and high DMS treatments. This is consistent with the proportion of the time budget spent slow swimming by C. helgolandicus feeding on a range of dinoflagellate diets and in filtered seawater, whilst diatom prey resulted in an increase in swimming [Oxyrrhis marina in our experiments may provide a sufficient hydromechanical stimulus indicating the presence of a prey patch, potentially overriding the additional DMS cue in the high DMS treatment. Other studies have found copepod behavior to be closely coupled to prey density, with copepods in filtered seawater spending very little time slow swimming [Temora longicornis, a typical “cruise-swimmer” , modifies its swimming speed depending on prey density in accordance with predictions of optimal foraging theory. Woodson et al. [et al. [Pseudocalanus minutus differed depending on the cue received. In the presence of prey P. minutus decreased swimming speeds and increased turning rates, whereas cell-free prey exudates elicit an increase in swimming speed combined with a decrease in turning rate [C. helgolandicus to exploit directional cues to locate high productivity prey patches. For example, swarming behaviour in response to specific dissolved amino acids has been observed in laboratory experiments with the copepods A. hudsonica and Eurytemora herdmani [At the meso- or patch scale, elevated background concentrations of infochemicals may stimulate an increase in foraging-related behaviors. This has been observed in copepods in response to amino acid additions  and celgicornis , our expswimming . The preswimming . Van Durswimming  demonstrn et al.  showed tn et al. . In a se [et al.  showed ting rate . Increasing rate . It is pherdmani . HoweverCalanus heloglandicus. Pooled data from 12 individual copepods over a 25-min period in treatments with either high or low grazing-induced DMS showed no difference in the total amount of the time budget allocated towards slow swimming.We tested the hypothesis that grazing-induced DMS production following microzooplankton herbivory on phytoplankton would result in increased feeding behavior, measured in terms of time budget allocation towards slow swimming, in the copepod Individual level responses showed a large degree of variation in between-individual behavior, both in terms of the total amount of time allocated towards slow swimming between copepods and in terms of the response of individual copepods to DMS treatments. Six of the twelve copepods showed a significant increase in slow swimming in low DMS treatments whereas only three copepods increased the proportion of slow swimming in high DMS treatments. This result highlights a cautionary note for assuming “average” copepod behavior and adds to the growing argument of the existence of individual behavioral “personalities” in invertebrates.Our experiments replicate the scenario of a copepod encountering a high productivity prey patch with the addition of either high or low grazing-mediated DMS cues. In light of previous work and our results, we suggest that copepod behavioral responses to potential grazing-induced infochemicals, such as DMS, may act over larger spatio-temporal scales. Further work to determine the scale-dependent role of DMS as a chemical cue for copepods is required.


Metadherin (MTDH) has been demonstrated as a potentially crucial mediator of various types of human malignancies. However, the expression and role of MTDH in diffuse large-B-cell lymphoma (DLBCL) have not been reported yet. This study aimed to illuminate the role of MTDH in the pathogenesis of DLBCL.A remarkable elevation of MTDH on mRNA level was detected in DLBCL tissues by quantitative polymerase chain reaction (PCR). Using Western-blot analysis we found that the expression of MTDH protein was significantly upregulated in DLBCL cell lines and DLBCL tissues compared with peripheral blood mononuclear cells (PBMCs) from healthy samples and tissues from patients of reactive hyperplasia of lymph node. The results showed high expression of MTDH in 23 of 30 (76.67%) DLBCL tissues by using immunohistochemical analysis and the over expression of MTDH was strongly correlated to the clinical staging of patients with DLBCL (P<0.05). Furthermore, the finding suggested that the increase of MTDH in DLBCL cells could distinctly enhance cell proliferation and inhibit cell apoptosis; meanwhile, inhibition of MTDH expression by specific siRNA clearly enhanced LY8 cell apoptosis. Upregulation of MTDH elevated the protein level of total β-catenin and translocation of β-catenin to the nucleus directly or indirectly. Knockdown of MTDH decreased the level of total, cytoplasmic β-catenin and reduced nuclear accumulation of β-catenin protein. This indicated that the function of MTDH on the development of DLBCL was mediated through regulation of Wnt/β-catenin signaling pathway.Our results suggest that MTDH contributes to the pathogenesis of DLBCL mediated by activation of Wnt/β-catenin pathway. This novel study may contribute to further investigation on the useful biomarkers and potential therapeutic target in the DLBCL patients. Diffuse large-B-cell lymphoma (DLBCL) is an aggressive malignancy of mature B lymphocytes and is the most common type of lymphoma in adults Metadherin  was first cloned as an HIV- and TNF-α–inducible gene in primary human fetal astrocytes (PHFAs) For the first time, our present manuscript focuses on illuminating the role of MTDH and the relationship between MTDH and Wnt/β-catenin pathway in the pathogenesis of DLBCL. We demonstrate the overexpression of MTDH and β-catenin in DLBCL and the effect of MTDH expression on biological behavior of DLBCL cell lines. We also provide evidences for the link between MTDH and Wnt/β-catenin pathway in DLBCL.Expression of MTDH was detected by Western blot, with the single band size of 75 kDa, in peripheral blood mononuclear cells (PBMCs) from healthy samples, human DLBCL cell lines LY1 and LY8, and MCL cell lines Jeko-1, Mino, and SP53. The protein level of MTDH was much lower in PBMCs from healthy samples compared with all the human DLBCL and MCL cell lines . MoreoveTo further determine whether MTDH protein overexpression is associated with clinicopathological characteristics of DLBCL, a cohort of paraffin-embedded, archived DLBCL tissues (n = 30) were examined by immunohistochemical staining with an antibody against human MTDH. As shown in Statistical analyses were done to examine the correlation between the expression of MTDH protein by immunohistochemistry analysis and the clinical features of DLBCL. As shown in 3H-TdR incorporation assay. The increase of MTDH could promote the proliferation of 2 DLBCL cell lines  supplemented with 10% fetal bovine serum  3H-thymidine was purchased from Perkin-Elmer . TNF-α was purchased from PeproTech . For studies, it was dissolved in 0.1% of BSA and aliquoted as a stock solution. To prepare working solutions, aliquots were further diluted in Iscove’s modified Dulbecco’s medium  supplemented with 10% heat-inactivated fetal bovine serum  immediately before each experiment.Rabbit anti-MTDH polyclonal antibody was purchased from Invitrogen . Rabbit anti-β-catenin polyclonal antibody and mouse anti-β-actin monoclonal antibody was from Abcam . Rabbit anti-histone 3 (H3) polyclonal antibody was obtained from Beyotime . 5′-AACAGAAGAAGAAGAACCGGA-3′ by reference to the previous study MTDH gene and negative control siRNA were cloned into the pGCL-GFP and generated using stable lentivirus expression vectors by Shanghai GeneChem. LY8 cells were plated in 96-well plates (104cells/well) and were infected with MTDH-RNAi lentivirus or negative control lentivirus with the multiplicity of infection (MOI) 80 according to the manufacturer’s instructions. The medium was changed with fresh medium after 8–12 hours. Infection efficiencies were determined by GFP fluorescence. The cells were then harvested for mRNA and protein extraction or detection of cell apoptosis. In addition, the cells were incubated in the absence or presence of TNF-α for 48 hours  and then harvested for protein extraction and detection of cell apoptosis.RNAi target sequence to human MTDH gene was −△△CT method.Total RNA was extracted from tissues using Trizol (Invitrogen). Then reverse transcription reaction was conducted by means of TaKaRa reverse transcription reagents . The reaction was incubated at 37°C for 15 minutes, and 85°C for 5 seconds. Amplification reactions were performed using SYBR Premix Ex Taq   on ABI 7500 Real-Time quantitative PCR System with cycling as follows: an initial cycle for 2 minutes at 95°C, followed by 40 bi-phasic cycles of 15 seconds at 95°C and 1 minute at 60°C. PCR products were confirmed as a single product at the desired size on agarose gels and visualized by ethidium bromide staining. Specific primers for RT-PCR were obtained from Biosune , and the primer sequences are listed in Total protein was extracted from DLBCL tumors, reactive hyperplasia of lymph node tissues, LY1, LY8, Jeko-1, Mino, SP53 cell lines and PBMCs of healthy samples using RIPA and 1% PMSF . For cytoplasmic and nuclear extracts, cells were washed with phosphate-buffered saline (PBS) and were lysed in NE-PER extraction reagent (Pierce) according to the manufacturer's protocol. The protein concentration of the samples was determined by the BCA assay (Shenergy Biocolor). Cell lysates were then electrophoresed on 10% SDS-polyacrylamide gels, while cytoplasmic and nuclear lysates were electrophoresed on 12% SDS-polyacrylamide gels, and next transferred onto nitrocellulose membranes. After the membranes were blocked with 5% skim milk in Tris-saline buffer with 0.1% Tween-20, they were subsequently probed with primary antibodies at 4°C overnight. After washings with TBST, secondary antibody conjugated with the horseradish peroxidase  was added to the membranes. After washings with TBST, proteins were detected using the chemiluminescence detection kit . Antibodies used in this study included anti-MTDH (1 µg/ml), anti-β-catenin (1∶5000), anti-β-actin (1∶10000) and anti-H3 with a dilution of 1∶1000. Western blot results were analyzed using the Las-4000 Image software and Multi Gauge Ver.3.0 software .In brief, formalin-fixed, paraffin-embedded tissue sections of 4-µM thickness were deparaffinized and hydrated. High-pressure antigen retrieval was performed using citrate buffer (pH6). Endogenous peroxidase was quenched with 3% hydrogen peroxide in methanol for 15 minutes, followed by incubation with normal serum to block non-specific staining. Rabbit anti-MTDH (1∶25) antibody was then incubated with the sections overnight at in a humidified chamber 4°C; the second antibody was from SP reagent kit . After washing, the tissue sections were treated with biotinylated anti-rabbit secondary antibody, followed by further incubation with streptavidin-horseradish peroxidase complex. Stained with diaminobenzidine Kit , the sections were counterstained with hematoxylin and mounted. Immunohistochemical staining of samples and negative controls occurred simultaneously, and the primary antibody was replaced with PBS for negative controls.Immunohistochemical stainings were assessed in a series of randomly selected 5 high-power fields, which were believed to be representative of the average in tumors at×400 magnification, by two independent observers who were blinded to all clinical data. The sections were scored according to the proportion of positively stained tumor cells. Tumors displaying staining in 30% or more of the cells were categorized as positive cases. In the meanwhile, tumors displaying staining less than 30% of the cells were categorized as negative cases.3, respectively) were seeded into 96-well plates, treated with TNF-α (250 pg/ml) and cultured for 48 hours. 3H-thymidine (1 µCi/well) was added into the cultures 16 hours before the end of the experiment. Then cell proliferation was evaluated using 3H-TdR incorporation method as described previously LY1 and LY8 cell lines . Viable cells are not stained with annexin V-FITC or PI. The necrotic cells were annexin V-FITC and PI-positive, whereas apoptotic cells were annexin V-FITC-positive and PI-negative After treatment of LY1 and LY8 cells with TNF-α (250 pg/ml) for 48 h, cell apoptosis and necrosis were determined using an annexin V- fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit , according to the manufacturer's instructions. Briefly, an aliquot of 10All statistical analyses were performed by using the statistics software SPSS 13.0 for Windows. The numerical data were statistically analyzed by 2-tailed Student’s t test t-test. Fisher’s exact test was used to analyze the relationship between the level of MTDH expression and clinicopathological features. Bivariate correlation between two independent variables was calculated by Spearman’s rank correlation coefficient. Statistically significance was defined as P<0.05.Figure S1Infection efficiency of lentivirus and knockdown of MTDH in DLBCL cells. (A) Infection efficiency of lentivirus in LY8 cells. The cells were treated with MTDH-specific lentivirus-mediated small interfering RNA (siMTDH) or with non-targeting siRNA as a negative control (siCon). Infection efficiency was observed by fluorescent microscopy: the upper row, bright field photos; the lower row, green fluorescence photos . (B) The knockdown efficiency of MTDH mRNA by siRNA was detected using quantitative PCR. The specific siRNA inhibited MTDH expression (p<0.05 versus control). (C) The specific siRNA effectively suppressed MTDH protein expression analyzed by Western blot. Expression of β-actin was used as loading control.(TIF)Click here for additional data file.


In this article, a scanning probe method based on nanoscale capacitance measurements was used to investigate the lateral homogeneity of the electron mean free path both in pristine and ion-irradiated graphene. The local variations in the electronic transport properties were explained taking into account the scattering of electrons by charged impurities and point defects (vacancies). Electron mean free path is mainly limited by charged impurities in unirradiated graphene, whereas an important role is played by lattice vacancies after irradiation. The local density of the charged impurities and vacancies were determined for different irradiated ion fluences. IdealD sheet oe (HOPG) . The mob methods  and in g methods .-1 in the Raman spectra is a fingerprint of defects/disorder in the crystalline lattice of graphitic materials. However, the lateral resolution of μR is limited by the laser spot size . In this article, we present a scanning probe method based on nanoscale capacitance measurements to determine locally  the electron mean free path in pristine and in ion-irradiated graphene with different ion fluences. The impurity and vacancy densities on the probed area were extracted by fitting the experimental results with models of electron scattering by Coulomb impurities and lattice defects.Recently, the intentional production of defects in selected areas of a graphene sheet has also been proposed as a method to locally modulate the transport properties. Several methods, like plasma treatments , and ele+-Si substrate covered with 100 nm SiO2 [+ ions at 500 keV. Irradiations of the samples with C+ ions were carried out under high vacuum conditions (10-6 Torr) to minimize surface contaminations. At 500 keV energy, the projected range of the C+ ions is approximately 1 μm, quite deep into the n+-Si substrate. This minimizes the damage both in the 100 nm SiO2 layer and at the interface between SiO2 and n+ Si. Infact, a quality of SiO2 and SiO2/Si interface comparable to that of non-irradiated samples is crucial for the capacitance measurements discussed later. Different C+ ion fluences, ranging from 1 × 1013 to 1 × 1014 ions/cm2, were used for irradiation [Graphene samples obtained by mechanical exfoliation of HOPG were deposited on a n nm SiO2 . Optical nm SiO2 . Some of2/n+Si stack, using scanning capacitance spectroscopy (SCS) [The lateral homogeneity of the electronic transport properties both in pristine and ion-irradiated graphene was investigated by local capacitance measurements on the graphene/SiOpy (SCS) ,15.V = Vg/2(1 + sin(ωt)), with amplitude Vg in the range from -1.2 to 1.2 V and frequency ω = 100 kHz, was applied between the Si n+ backgate and the nanometric contact on graphene represented by a Pt-coated Si tip  capacitance sensor connected to the conductive AFM tip measures, through a lock-in system, the capacitance variation ΔC induced by the modulating bias.Scanning capacitance spectroscopy (SCS) was performed at room temperature using a DI3100 AFM by Veeco equipped with Nanoscope V electronics and with the scanning capacitance microscopy (SCM) head. SCS is an extension of the conventional SCM -19. In S2 and on graphene-coated SiO2 are reported for a sample not subjected to ion irradiation. The tip positions are indicated in the AFM image in the inset of Figure 2, a typical capacitance-voltage curve for a metal-oxide-semiconductor (MOS) capacitor from accumulation (at negative sample bias) to depletion (at positive sample bias) is measured . The value of EF can be related to the applied bias as EF = ħvFkF, being kF = (πn)1/2, ħ the reduced Planck's constant, and vF = 1 × 106 m/s, the electron Fermi velocity in graphene. The induced charge n spreads over an area, Aeff, which can be thought as the tip-graphene-insulator-semiconductor capacitor effective area  and on bare SiO2 regions (|ΔCox|) [Aeff = Atip|ΔCgr|/|ΔCox|, where the tip contact area Atip can be independently determined by scanning electron microscopy (Atip = 80 nm2 in the present case). The evaluated Aeff is reported as a function of the gate bias in Figure Vg = 0, Aeff increases linearly with |Vg| both for negative and positive Vg values.In Figure (|ΔCox|) , i.e., AAeff obtained by local capacitance measurements is related to the local electron mean free path l in graphene by Aeff = πl2 [l is reported versus the evaluated Fermi energy. It can be noted that l is almost independent of EF close to the Dirac point. The behavior close to the Dirac point is consistent with the common adopted picture of the 2DEG split in a landscape of adjacent "electron-hole puddles" [EF| > 25 meV, l increases linearly with EF both in the hole and electron branches. This linear dependence gives indication on the main scattering mechanisms limiting l in our graphene samples.It has been recently demonstrated that the effective area ff = πl2 . In Figupuddles" . Close tl have been determined for the different scattering mechanisms in the framework of a semiclassical model based on the Boltzmann transport theory [lphon) can be expressed as [Recently, expressions of the energy dependence of t theory . The eleessed as ρ is the graphene density (ρ = 7.6 × 10-7 kg/m2) [DA is the acoustic deformation potential (DA = 18 eV) [vs is the sound velocity in graphene [kB is the Boltzmann constant, and T is the absolute temperature.where lci) can be expressed as [The electron mean free path limited by Coulomb scattering with charged impurities , and Nci is the density of impurities.where lvac) can be expressed as [Finally, the electron mean free path for scattering by vacancies .where l on EF, far from the Dirac point, suggests that scattering with charged impurities and/or point defects, e.g., vacancies, can be assumed as the main mechanisms limiting electron mean free path.The experimentally determined linear dependence of D peak in micro-Raman spectra. Hence, charged impurities, either adsorbed on graphene surface, or located at the interface with SiO2 substrate, can be assumed as the main scattering source liming l. The density of charged impurities in the probed position can be estimated by fitting the experimental curves in Figure Nci = 49 × 1010 cm-2 both for the holes and the electron branch.In this pristine graphene sample, the density of defects is negligible, as confirmed by the absence of the characteristic l versus EF measured on an array of 5 × 5 tip positions on pristine graphene is reported. By fitting each curve of the array with Equation 2, the local density Nci for each probed position can be extracted. The histogram of the charged impurity density on the analyzed area is reported in Figure Nci〉 = 50 × 1010 cm-2 and with FWHM of 4 × 1010 cm-2.In Figure l versus EF is reported for two arrays of tip positions on graphene samples irradiated with two different ion fluences, i.e., Φ = 1 × 1013 cm-2 and Φ = 1 × 1014 cm-2. Comparing the set of curves in Figure l values increases with the irradiated fluence. However, it is worth noting that two groups of l-EF curves can be distinguished for irradiated samples: (i) a first group, with l values comparable to those in the pristine sample, (ii) a second group with reduced mean free path. We assumed that C irradiation causes the formation of point defects (vacancies), whereas the density of charged impurities adsorbed on the graphene surface or at the interface with the substrate remains almost unchanged. Hence, the first group of curves in Figure l can be fitted using Equation 2. The histograms of the Nci values determined in the probed positions is reported in Figure Nci distributions in irradiated samples are very similar to those of non-irradiated sample. For the second group of curves in Figure l is limited both by charged impurities and vacancies scattering, i.e.,In Figure Nci〉 = 50 × 1010 cm-2), and the local vacancy density was determined from Equations 2-4 using Nvac as the fitting parameter. The distributions of the vacancy densities in the probed positions are reported in Figure Nvac is higher than 2.5 × 1010 cm-2 (i.e. more than one vacancy on the probed area at Vg = 1V) on only 16% of the probed positions, in graphene irradiated with the highest fluence Nvac > 2.5 × 1010 cm-2 on more than 75% of the probed positions.For simplicity, an average value of the charged impurities density will be assumed in those positions , and ν is the vacancy generation efficiency. By linear fitting the data in Figure Nvac,0〉 = (1.59 ± 0.04) × 1010 cm-2 and νσNgr = (8.55 ± 0.06) × 10-4 are obtained. For the calculated values of the C-C scattering cross section σ, ranging from 2 × 10-17 to 7 × 10-17 cm2, a very low vacancy generation efficiency (ranging approximately from 0.3 to 1.1%) is obtained for graphene irradiation with 500 keV C+ ions. It might be associated to a dynamical annealing, e.g. vacancy-interstitial recombination, during irradiation.where 〈+ ions at fluences ranging from 1 × 1013 to 1 × 1014 cm-2 was used to introduce defects in SLG deposited on a SiO2/n+Si substrate. The local charged impurity and vacancy density distributions were determined for the different irradiation fluences, and a low efficiency of vacancy generation (approximately from 0.3 to 1.1%) was demonstrated.In summary, the authors propose an innovative method based on local capacitance measurements to probe the local changes in graphene electron mean free path, due to the presence of charged impurities or point defects, e.g., vacancies. Irradiation with 500 keV C2D: two-dimensional; HOPG: highly oriented pyrolytic graphite; SCM: scanning capacitance microscopy; SCS: scanning capacitance spectroscopy; SLG: single layers of graphene.The authors declare that they have no competing interests.FG and VR conceived the study. FG coordinated the experiment, participated to the analysis of the data and wrote the article. SS carried out the sample preparation, the measurements and participated to the analysis of the data. ER worked on the evaluation of ion-graphene interaction cross sections. All the authors read and approved the manuscript.


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 .