Euglena gracilis is riddled with introns that interrupt almost every protein-encoding gene. The atypical group II introns and twintrons (introns-within-introns) found in the E. gracilis plastid have been hypothesized to have been acquired late in the evolution of euglenids, implying that massive numbers of introns may be lacking in other taxa. This late emergence was recently corroborated by the plastid genome sequences of the two basal euglenids, Eutreptiella gymnastica and Eutreptia viridis, which were found to contain fewer introns.Photosynthetic euglenids acquired their plastid by secondary endosymbiosis of a prasinophyte-like green alga. But unlike its prasinophyte counterparts, the plastid genome of the euglenid Monomorphina aenigmatica, a freshwater species occupying an intermediate phylogenetic position between early and late branching euglenids. The M. aenigmatica UTEX 1284 plastid genome  contains 53 intron insertion sites, of which 41 were found to be shared with other euglenids including 12 of the 15 twintron insertion sites reported in E. gracilis.To gain further insights into the proliferation of introns in euglenid plastids, we have characterized the complete plastid genome sequence of The pattern of insertion sites suggests an ongoing but uneven process of intron gain in the lineage, with perhaps a minimum of two bursts of rapid intron proliferation. We also identified several sites that represent intermediates in the process of twintron evolution, where the external intron is in place, but not the internal one, offering a glimpse into how these convoluted molecular contraptions originate. Pyramimonas parkeaeEuglena gracilis, which has been used as a model organism for numerous studies in the past decades Euglenids are a morphologically diverse group of unicellular freshwater and marine eukaryotes displaying various feeding behaviours, including phagotrophy, osmotrophy, and phototrophy (reviewed in E. gracilis plastid genome (cpDNA) is the presence of over 150 introns  that interrupt almost all of its protein-coding genes , which are introns inserted within other introns. These intricate nested introns must be removed sequentially to result in accurate splicing.One of the most remarkable features of the E. gracilis introns have been found in other Euglena species Euglena (formerly Astasia) longaP. parkeae did not contain any highly-reduced group II introns Eutreptiella gymnasticaEutreptia viridisThe unorthodox Monomorphina aenigmatica (strain UTEX 1284), a freshwater species that branches between Euglena and the earlier-diverging photosynthetic euglenids M. aenigmatica plastid genome  lends further support to the intron-late hypothesis, but also suggests that at least two distinct and independent intron acquisition bursts occurred in the euglenid plastid genomes. We also identify a number of insertion sites with an intermediate stage in the emergence of twintrons, showing how such molecular structures might arise.To better understand intron evolution in photosynthetic euglenids, we determined the complete plastid genome sequence of Monomorphina aenigmatica strain UTEX 1284 maps as a circular molecule of 74,746 bp with an overall 70.6% A+T content  and the gene coding for the small 5S rRNA, which are present in E. gracilis. The 5S rRNA-encoding gene is present in Eta. viridis but absent from Etl. gymnastica and, interestingly, from the prasinophytes P. parkeae and Pycnococcus provasoliirpoA gene reported in some Euglena species psbZ and trnS(uga) does not appear to be present in M. aenigmatica. This location of the genome does not contain an ORF, and while the sequence shares barely-detectable similarity with the alpha subunit of bacterial DNA-directed RNA polymerases, it shares none with the reported euglenid RpoA sequences. The M. aenigmatica cpDNA contains only a single rRNA operon, which contrasts with the three copies found in tandem in the E. gracilis cpDNA. The 1∶1 relative coverage ratio between this operon and the other genomic regions in M. aenigmatica cpDNA assembly excludes the possibility that the single copy observed is the result of an assembly artefact that would have collapsed adjacent operons. Like its E. gracilis counterpart, however, the M. aenigmatica plastid genome features a number of tandem repeats restrained to a single locus, which are likely variable within the population. Interestingly, these variable numbers of tandem repeats (VNTR) are located in the same position, between trnL(caa) and rrs, as those reported in E. gracilis. The two genomes display a high level of synteny, with the 85 genes that they share distributed between four blocks of colinear genes.The chloroplast genome of  content , Table 1M. aenigmatica genome is roughly half the size of that of E. gracilis  with E. longa that is absent from E. gracilis, and which likely has been lost in the latter genome after the split with E. longa. Only one insertion site (psbC.Maen.1) was found to be shared between the four photosynthetic euglenids investigated to date (the psbC gene was lost in E. longa). The remaining 12 insertion sites in M. aenigmatica f′e′d′ A* c′b′a′ (4)-3′ consensuses described in M. aenigmatica genome shares only 6 twintrons with E. gracilis , probably reflecting intermediate stages of twintron evolution, although the possibility that the additional internal introns found in E. gracilis were lost in an ancestor of M. aenigmatica cannot be completely excluded. The six remaining insertion sites where E. gracilis encodes a twintron  were found to encode only a single intron in M. aenigmatica, and in all five cases the intron was orthologous to the E. gracilis external intron.Interestingly, 12 of the 15 sites reported to contain twintrons in gracilis . The psbM. aenigmatica chloroplast genome contains 3 putative reverse transcriptases/maturases. The first two are located within the intron insertion sites psbC.Maen.1  and psbC.Maen.2 , whereas the third (orf240) is free-standing, potentially unique to M. aenigmatica, and highly degenerated . The mat1/mat2 intron-encoded maturases are not unique to M. aenigmatica and are also found in the other euglenid introns that are inserted at cognate sites , the genome of the early branching euglenid Etl. gymnasticapsbC intron found in all four photosynthetic euglenids, as proposed by Doetsch et al.M. aenigmatica insertion sites are shared with E. gracilis , but 21 out of the 23 insertion sites found in Eta. viridis are currently unique to that species, and only one intron is common to all four species  featured only one intron, which eventually became the external intron in a twintron when a second intron was inserted within it. However, no such intermediate stage has ever been described, until now.  viridis , Table 2Thermosynechococcus elongatusDrosophilaE. gracilis plastid genome contain so many of them? The first explanation is perhaps a simple matter of density and mobility: in genomes with high intron mobility, more and more introns are expected to be found. This, in turn increases the likelihood of having an intron insert itself inside another intron. That being said, the psbC twintron cannot be explained in this way since it is present in all four euglenid genomes and likely arose before any intron proliferation. A second potential explanation is that group II intron mobility is often not random. Instead, they propagate at cognate and ectopic sites by processes known as ‘homing/retrohoming’ and ‘transposition/retrotransposition’, respectively. These processes are usually quite stringent, and while the endonucleases or reverse transcriptases involved allow for some polymorphism to occur, the target sites are usually highly conserved. If the target site is contained within a duplicated or repeated sequence however, then a given intron can, in theory, quickly move to occupy most of these loci. It is also possible that the transposition/retrotransposition processes in euglenid genomes are somehow relaxed, thus helping with the intron invasion. A third potential explanation is that in the euglenid plastid genetic system the selection for accurate splicing has been relaxed for some reason, allowing the proliferation of what might otherwise be slightly deleterious introns. It would be interesting to investigate whether the transcription is generally messy in these plastid genomes, and if the mRNAs for intron-interrupted genes form a population of viable and defective molecules. Since previous analyses found a number of splicing variants in E. gracilisGroup II introns are mobile genetic elements known to interrupt target sequences, coding or non-coding. Introns that are inserted in intergenic regions are often transient owing to the relaxed selective pressure resulting from their not interrupting a coding sequence. In contrast, however, introns that are inserted within genes must be properly spliced for the gene to retain its function and any mutation affecting the splicing efficiency of the intron could have a deleterious effect. In twintrons these conditions are compounded, adding an extra layer of complexity to an already complex mechanism. It is therefore not surprising that twintrons are quite rare - other than euglenid plastids, a few isolated cases have been described  using the protocol provided by the manufacturer. Polysaccharides were removed from the extracted DNA as follows. Total DNA (50 µl) was incubated in the presence of 500 µl CTAB buffer  at 60°C, cleaned with 500 µl of chloroform, precipitated with 500 µl of isopropanol, washed with 500 µl of EtOH 80% (vol/vol), then resuspended in water. Total RNA was extracted from liquid NO2-ground and pelleted cells with Trizol (Invitrogen) using the protocol provided by the manufacturer.M. aenigmatica illumina TruSeq library  was prepared from total DNA and sequenced on one lane of illumina HiSeq by the Beaty Biodiversity Research Centre sequencing facilities . Adapters and indexes were removed from the resulting reads and the reads filtered using the CASAVA 1.8.2 pipeline (illumina) under the default parameters. The first 320,000,000 reads  of the filtered reads were assembled in parallel with Ray version 2.0.0 rc4 M. aenigmatica plastid genome were identified as follows. The 500+ bp-filtered contigs were converted to a BLAST Euglena gracilis [EMBL:X70810] and Pyramimonas parkeae [GenBank:NC_012099] were both queried independently against the M. aenigmatica database with TBLASTN from the BLAST package using variable cut-offs to detect potential chloroplast contigs. The potential contigs of interest were pulled out from the multifasta file with the faOneRecord/faSomeRecords command line utilities from the University of California, Santa Cruz (UCSC), and searched reciprocally against the entire NCBI non-redundant database to ascertain their origin.The M. aenigmatica chloroplast contigs were further assembled and polished with CONSED 23 The E. gracilis genome as input queries. Protein-coding genes and open reading frames were annotated with Artemis 14.0 using the built-in tools. Then, each predicted M. aenigmatica protein-coding gene sequence was aligned at the amino acid level against orthologs from euglenids and green algae using the L-INS-i algorithm from MAFFT 6.925 M. aenigmatica plastid genome were searched for specifically by BLAST homology searches using the closest known orthologs. The predicted exon-intron junctions were annotated to preserve homology between orthologs whenever possible. The gene map shown in M. aenigmatica UTEX 1284 plastid genome annotation has been deposited in GenBank [GenBank:JX457480].Genes coding for tRNAs were identified with tRNAscan-SE 1.21 rpoC1 gene is not well conserved, we performed RT-PCR experiments using primers specific to M. aenigmatica to ascertain a number of intron-exon boundaries. The RT-PCRs were performed on total RNA with the Superscript III One-Step RT-PCR kit (Invitrogen) and the resulting products cloned with the StrataClone PCR cloning kit (Stratagene), both using the protocols provided by the manufacturers. The clones grown on LB-ampicillin (50 µg/ml) plates with X-galactose (20 mg/ml) at 37°C overnight were PCR-screened for inserts using the T3/T7 primers flanking the insertion site and the EconoTaq PLUS GREEN (Lucigen) and plated again on LB+ampicillin (37°C/8 hours). Selected clones were cultured overnight at 37°C in 2 ml of liquid LB medium+ampicillin (50 µg/ml) and the plasmids purified with the FastPlasmid Mini kit (5 Prime) using the default protocol. The products were then Sanger-sequenced using the T3/T7 primers flanking the insertion site. Note that we could not produce a reliable RT-PCR covering the insertion site located at position 4 in M. aenigmatica’s rpoC1, and believe that a small unannotated exon might be present within it.Because the M. aenigmatica [GenBank:JX457480], E. gracilis [EMBL:X70810], E. longa [EMBL: AJ294725], Eta. viridis [GenBank:JN643723], Etl. gymnastica [EMBL:HE605038]) were determined as follows. Orthologous protein sequences were aligned with their green algal counterparts using the L-INS-i algorithm from MAFFT and the insertion sites mapped manually on the corresponding sequences. Insertions located within the same amino acid but at different sites within the corresponding codon were considered distinct. We also corrected a number of annotations from the Eta. viridis and Etl. gymnastica accessions numbers  aligning each of the external and internal E. gracilis intron sequences against the target sequence. For M. aenigmatica sequences, we also scanned for the 5′-NUNNG and abcdef(3–8)f′e′d′ A* c′b′a′ (4)-3′ consensuses described in Cognate intron insertion sites between euglenid genomes . The rpoC1.Maen.4 insertion could not be verified by RT-PCR and may contain a short additional exon.(XLSX)Click here for additional data file.Data S1Euglenid plastid genomes EMBL annotations files. The Eta. viridis and Etl. gymnastica original annotations were modified to include additional introns and/or revised insertions sites. The modifications are also indicated by miscellaneous features annotation tags in the revised files for quick localization with Artemis.(ZIP)Click here for additional data file.


A 36-year-old man visited our hospital because of blurred vision and redness of the conjunctiva. Slit-lamp examination showed panuveitis. Two days later, he suddenly experienced dizziness, speech disturbance, paralysis of his right extremities, and gait disturbances. Neurological examinations suggested that his symptoms were caused by a left lateral medullary lesion. He also had erythema mainly on his trunk. Magnetic resonance imaging (MRI) of his brain demonstrated a small infarct on the left side of the medulla oblongata. Clinical presentation and MRI findings were consistent with the diagnosis of a Wallenberg's syndrome. He also had bilateral hilar lymphadenopathy. A skin biopsy showed granulomatous nodular dermatitis compatible with sarcoidosis. He was treated with steroid pulse therapy and his neurological and ocular symptoms immediately improved. Only seven similar cases of intracranical sarcoidosis have been reported, but none had been treated with steroid pulse therapy. We recommend that steroid pulse therapy be considered to treat patients with sarcoidosis with signs of lesions in the central nervous system. Sarcoidosis is characterized by noncaseating granulomas, and in 5% of the patients, the lesions are located in the central nervous system . HoweverChronic posterior ocular sarcoidosis is more difficult to manage, and it can lead to sight-threatening complications, for example, cataracts, glaucoma, and cystoid macular oedema. Eyes with posterior uveitis with chronic sarcoidosis have a poor visual prognosis despite immunosuppressive therapy .Corticosteroids, usually combined with immnosuppresive agents, remain the mainstay therapy for ocular sarcoidosis although refractory cases do occur . The refWe described a patient with ocular sarcoidosis and a cerebral infarction, who had an excellent response to steroid-pulse therapy.A 36-year-old man reported blurred vision and redness of the conjunctiva in both eyes. He was referred to our hospital in January 2009, and our examination showed that his best-corrected visual acuity (BCVA) was 1.2 (20/20) in both eyes. Slit-lamp examination showed ciliary injection and keratic precipitates. Fundus examination demonstrated vasculitis in both eyes Figures .Physical examination showed exudative erythema nodosum on his trunk and both thighs. Chest X-rays and CT scans demonstrated bilateral hilar and mediastinal lymph node swelling. Laboratory tests, including antibodies for rheumatoid factor, Sjögren syndrome, double-stranded DNA, and antinuclear antibodies, were negative. In addition, the serum antiotensin-converting enzyme level was high at 27.0 U/L .Two days after his first visit, he reported dizziness, speech disturbance, clumsiness of his right extremities, and gait disturbances. Neurological examinations showed left ptosis and miosis, hoarseness, dysphagia, right mild hemiparesis, left ataxia of his extremities, and truncal ataxia. He also had hypalgesia/hypotemperature on his right side. These findings suggested a left Wallenberg syndrome. Brain magnetic resonance imaging (MRI) showed an infarct on the left side of the medulla oblongata . LeptomeThe patient was diagnosed with neurosarcoidosis with a brainstem infarct. Steroid pulse therapy was prescribed initially with 1000 mg infusion for three days and then slowly tapered. During the pulse therapy, the patient had laser photocoagulation of the nonperfused areas of the retina. The patient had an improvement of his symptoms after the steroid-pulse therapy.At the last examination on February 2011, the patient was stable with no complications and no recurrence of the ocular and CNS sarcoidosis. And his BCVA was 20/20.Our findings showed that our patient had CNS sarcoidosis of the medulla oblongata, and steroid-pulse therapy was very effective in resolving the symptoms. The mechanism causing the brain infarction in neurosarcoidosis is not completely known but is thought to result from vasculitis of the small vessels, emboli, or inflammation of the large vessels , 17. GraAlthough rare, neurosarcoidosis should be considered in cases of an acute stroke with restricted diffusion of unknown etiology in a young person, even if other stigmata of sarcoidosis are not present. Despite the common finding of vasculitis and microscopic infarction at autopsy, a stroke is usually due to vascular accidents in the cerebral hemisphere and is rarely the presenting sign of neurosarcoidosis  10, 11,,11, 15. Ocular sarcoidosis is not rare in Japan, and it has been found that sarcoidosis responds well to steroid therapy. But 10% of ocular sarcoidosis required periocular corticosteroids to treat the uveitis, and 13% of ocular sarcoidosis required systemic treatment for both the ocular and systemic disease . In addiThe management of resistant uveitis associated with sarcoidosis is a serious therapeutic problem. The toxicity from steroids such as osteoporosis, femur head necrosis, infection, steroidgenic diabetes, corticosteroid-induced glaucoma, and steroid-induced psychosis is difficult to control. This is why clinicians resist the use of high-dose steroids for long periods. Thus, it is important to know that steroid-pulse therapy is effective in treating the ocular inflammation due to sarcoidosis. Because steroid-pulse therapy has fewer side-effect, we recommend steroid pulse therapy for ocular and CNS sarcoidosis.


L1 cell adhesion molecule (CD171) is expressed in many malignant tumors and its expression correlates with unfavourable outcome. It thus represents a target for tumor diagnosis and therapy. An earlier study conducted by our group identified L1 expression levels in primary gastrointestinal stromal tumors (GIST) as a prognostic marker. The aim of the current study was to compare L1 serum levels of GIST patients with those of healthy controls and to determine whether levels of soluble L1 in sera could serve as a prognostic marker.Using a sensitive enzyme-linked immunosorbent assay (ELISA), soluble L1 was measured in sera of 93 GIST patients und 151 healthy controls. Soluble L1 levels were then correlated with clinicopathological data.p < 0.001; Mann-Whitney U test) in sera of GIST patients compared to healthy individuals. Median soluble L1 levels were particularly elevated in patients with recurrence and relapse .Median levels of soluble L1 were significantly higher (These results suggest that high soluble L1 levels predict poor prognosis and may thus be a promising tumor marker that can contribute to individualise therapy. GISTs-kit CD11 gene andof GISTs . First lolerated .There is to date no prognostic serum marker for GISTs. One possible candidate is the neuronal cell adhesion molecule L1 CD171), which is expressed in primary tumors in 74% of cases . L1 is a1, which L1 overexpression has been detected in various tumor cell types of neural, mesothelial, and epithelial origin. Expression profile analysis in multiple human tumors has identified L1 as a molecular marker for differential diagnosis and targeted therapy ,22. Its The aim of this study was to evaluate whether soluble L1 could serve as diagnostic marker and prognostic factor in GIST patients by correlating L1 serum levels to long-term outcome.The study was approved by the Ethics Committee of the Chamber of Physicians in Hamburg, Germany. Written informed consent was obtained from all patients for using tissue and serum samples. For this study, 93 patients with GIST were chosen retrospectively. It was possible to evaluate a rather large number of patients suffering from this rare tumor because of the cooperation of a self-help organization for GIST patients called "Lebenshaus e.v.", Bad Nauheim, Germany, which helped to collect serial serum samples from patients across Germany, Austria and Switzerland. The blood samples were taken from patients at time of diagnosis and every 3 months thereafter. We selected patients on the basis of availability of specimens and did not stratify them due to rare occurrence and different treatment strategies. All data including sex, age, tumor size, mitotic count, tumor location, metastasis, relapse, and imatinib therapy were obtained from the clinical and pathological records. The clinicopathological data from externally treated patients participating in the self-help project were sent regularly by their attending physicians. As healthy controls, 151 blood bank donors were included in the study. Limitations in this study were that we had some missing items in the clinical and pathological data of our patients due to logistic difficulties in collecting data of patients from different hospitals. However, a sufficient number of GIST patients were included in the study.2SO4 and analysed at 450 nm using an ELISA reader. Human L1-Fc protein served as an internal standard for the assay.For the detection of soluble L1, 96-well flexible microtiter plates (Costar 9019) were coated with 50 μl per well of 10 μg/ml of capturing antibody  overnight at 4°C. Wells were blocked with 3% w/v bovine serum albumin  in PBS/T  for 45 min at room temperature and then incubated for 1 h with human sera diluted 1:5 in PBS at room temperature. After five washes with PBS/T, bound protein was detected with biotin-conjugated monoclonal antibody UJ127 - which does not cross-react with the close homolog of L1 (chL1) - followed by streptavidin-conjugated peroxidase using TMB  as substrate. The color reaction was stopped by the addition of 10 mM HTo ensure that the immunoassay was suitable for measuring clinical serum samples, reproducibility, linearity and crossreactivity were examined. The assay showed no crossreactivity with the closed homolog of L1 CHL1, excellent linearity with serial dilutions and a low coefficient of variation (CV) in intra- and inter-assay variability.4P2O7, 1 mM NaF, 1 mM EDTA, 2 mM Na3VO4, 1 mM PMSF, 1% NP-40 plus complete EDTA-free protease inhibitor mixture, pH 7.5) for 30 min at 4°C and then subjected to SDS-Page and Western blot analysis. Equal amount of protein of each sample was mixed with 2x Laemmli Buffer  and boiled at 95°C for 5 min. Protein extracts were separated by 4 - 12% SDS-PAGE  and transferred onto nitrocellulose membrane . For immunoblotting, membranes were blocked with StartingBlock (TBS) Blocking Buffer  and incubated overnight at 4°C with primary 1:1000 biotinylated antibody L1-UJ127 . UJ127 binds to a membrane proximal Fn repeat. This allows detection of both the ADAM-cleaved 180 kDa shed fragment and the 80 kDa membrane-bound fragment presumably generated by plasmin cleavage. After washes with TBST (TBS with 0.1% Tween 20), membranes were incubated with HRP-conjugated streptavidin  (1:500) for 1 h at room temperature. After extensive washes, streptavidin-reactive bands were visualized using enhanced chemiluminescent substrate  on x-ray films .For analysis of L1cam protein, PBS washed GIST882 cells were lysed in RIPA buffer   was used for statistical analysis. Statistical significance was evaluated by the Mann-Whitney U and Kruskal-Wallis test. Cross-table statistics were performed using Fisher's test. Recurrence-free survival was plotted using the Kaplan-Meier method and analysed by the log-rank test. Significance statements refer to p-values of two-tailed tests less than 0.05.SPSSA total of 93 patients suffering from GIST and 151 apparently healthy controls were included in our study. The median age of GIST patients was 61 years (range 28-81 years) and 47 years (range 30-70 years) for the healthy controls. Fifty-one (55%) of the GIST patients were male and 42 (45%) female, Ninety-eight (65%) of the healthy controls were male and 53 (35%) female. Patients` characteristics and clinicopathological data are listed in Table L1 could be detected in sera of GIST patients, representatively shown in Figure Soluble L1 (s-L1) concentrations in the sera of GIST patients were significantly higher than in healthy controls: median (25th and 75th percentiles) soluble L1 values were 1.4 (0.8 and 2.7 respectively) ng/ml in the GIST patients and 0.5 (0 and 1 respectively) ng/ml in the controls  . For additional statistical analysis, patients with high (>2 ng/ml) and low (<2 ng/ml) soluble L1 concentrations were compared by survival curves plotted by the Kaplan-Meier method for recurrence-free survival . Also mitotic count and tumor size could not be significantly correlated with recurrence .Most primary cancers develop loss of expression of adhesion molecules. This allows for a critical step in metastasis to occur: detachment of the invading cell from its neighbors ,28. SomeIn a previous study we could show that L1 expression was immunohistochemically detectable in 74% of GISTs, but not in differential diagnostic important tumors such as smooth muscle tumor, peripheral nerve sheath tumor or desmoid-type fibromatosis. Although c-kit expression is the etablished standard in the identification of GIST, additional prognostic markers are beneficial. To date, most studies regarding L1 expression have been performed at the mRNA level or by immunohistochemistry. To evaluate the role of L1 as a peripheral marker, we here examined L1 levels in serum of GIST patients and healthy controls.Using a specifically developed robust and highly sensitive immunoassay we were able to demonstrate that soluble L1 concentrations were significantly elevated in the blood of GIST patients compared to a healthy control group and other gastric tumors. Furthermore, we observed a significant association between increased soluble L1 levels and recurrence, which predicts worse outcome in GIST. Patients with high soluble L1 concentrations had a poorer recurrence-free survival than patients with lower levels. These observations are in agreement with our previous immunohistochemical study, where a trend towards worse outcome of patients with L1-positive tumors was reported ,34. ElevSoluble L1 concentration could thus become a useful marker for tumor progression and metastasis, which predicts prognosis in GIST.Our results also suggest that L1 may be an important therapeutic target in the treatment of GIST. The efficiency of L1 monoclonal antibody treatment in suppressing tumor growth has been described in nude mice, where intraperitoneal injection of an L1 antibody was found to inhibit intraperitoneal tumor proliferation and dissemination of malignant tumor cells . In thisWe provide evidence that analysis of serum concentrations of soluble L1, comprising the extracellular domain of the L1 molecule, represents a novel and useful approach to assess the malignancy and metastatic potential of GISTs. The availability of a reliable immunoassay, such as the one developed for this study, for measuring serum L1 concentrations will facilitate further studies to establish the clinical applicability of this marker in GISTs and other tumors. These studies should notably investigate L1 levels in other serum samples such as obtained pre- and post-surgery and serially during the course of therapy. Beyond clinical applicability, future work must address mechanistic questions about the functional role of L1 in the tumorigenesis of GISTs and other L1 expressing cancers.The authors declare that they have no competing interests.HZ developed the immunoassay and drafted the manuscript, TR participated in the design of the study and performed the statistical analysis, MW carried out the immunoassays, MT and MK participated in the statistical analysis and collected the clinicopathological data, GW performed the Western Blot, MB helped to recalculate the statistics for the revision, MS and JRI participated in its design and coordination and helped to draft the manuscript. JTK conceived of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/189/prepub


Ultrastructural analysis demonstrates that −/−trkb embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient ( During late embryonic development, the formation of mature and fully functional blood vessels depends on the tightly regulated association of endothelial cells and mural cells such as pericytes and smooth muscle cells (SMCs). Many growth factors, such as vascular endothelial growth factor (VEGF), regulate endothelial cell migration and survival whereas platelet derived growth factor (PDGF) is involved in the regulation of remodeling and maturation of blood vessels via actions on pericytes and smooth muscle cells pdgf or pdgfrβ result in perinatal lethality, as a consequence of vascular dysfunction caused by mural cell deficiency Pericytes/SMCs are adventitial cells located within the basement membrane of capillaries and post-capillary venules. These contractile cells play an important role in stabilizing nascent endothelial tubes by providing essential survival factors −/−bdnf ) exhibit impaired survival of endothelial cells in intramyocardial arteries and capillaries in the late gestational and early postnatal periods −/−bdnf  mice is restricted to cardiac vessels, reflecting the localized expression of BDNF and its receptor TrkB in the cardiac and skeletal muscle vasculature trkb leads to lethality during late embryonic development Numerous studies have described critical roles for neurotrophins and their receptors in non-neuronal cells, such as endothelial cells, smooth muscle cells, immune cells, and epithelial cells in different organs −/−trkb embryos exhibit a specific deficiency in pericytes/SMCs. Ultrastructural analysis shows that −/−trkb embryos have defects in endothelial cell integrity including discontinuity of the cell membrane, leading to perivascular edema. We also determined that pericyte/SMC-specific deletion of TrkB results in abnormalities of vasculogenesis that significantly contribute to the lethal phenotype observed in −/−trkb mice. Conditional TrkB deletion in SMCs results in marked reduction of pericyte/SMC density in the heart and perinatal lethality. Using cultured SMCs, we further identified that BDNF/TrkB signaling leads to cell migration and contractile force generation by actomyosin phosphorylation, to play a pivotal role in regulating pericyte morphology and migration.Here we demonstrate that TrkB is required for the development of the cardiac microvasculature, and trkb (+/−) mice  on a C57Bl/6 background were intercrossed to generate homozygous animals. For embryo analysis, the gestational age was determined by morphological criteria including limb bud, eye development, length, and weight of the embryo. The genotypes of embryos or neonates were determined using tail-derived DNA and PCR amplification as described by Jackson Laboratories. Trkb floxed (f/ftrkb) animals were generated as described previously trkb gene, which encodes the signal peptide and the first 40 amino acids of the N terminus of TrkB, was floxed. Crossing a f/ftrkb  mouse with a transgenic mouse carrying Cre driven by a smooth muscle actin promoter , generated progeny in which the expression of trkb was selectively eliminated in perivascular cells including pericytes/SMCs. The genotype of each animal was assessed using PCR of genomic DNA isolated from tail, utilizing trkB.flox forward primers 5′ TGG TGT ACT GAG CCT TCT CCA G 3′ and trkB.flox reverse primers 5′ CGA GGC TTG GAG TTG ACT GGT 3′. All animal procedures of this study were approved by the IACUC of Weill Cornell Medical College and all efforts were made to minimize animal suffering.Heterozygous Following sacrifice, embryos were immediately fixed in 3% paraformaldehyde in PBS for 18 hours. Dissected tissues were embedded in paraffin for histological analysis for hematoxylin-eosin staining, or infiltrated with 30% sucrose in PBS prior to cryoprotection in 30% sucrose:OCT  for immunohistochemical analysis. For electron microscopic analysis, embryos and newborn mice were sacrificed by decapitation and hearts were immediately removed from the chest cavity and fixed in Karnovsky’s fixative solution for 18 hours prior to embedding in Epon. One µm sections were stained with toluidine blue for initial evaluation, followed by ultra-thin sections that were counterstained with lead citrate and viewed with a JEOL electron microscope. To analyze vascular permeability, FITC-dextran  was injected as a bolus through the tail vein of 3-month-old mice. After perfusion for 10 minutes, the animals were sacrificed and hearts harvested and processed for fluorescence microscopy. FITC-dextran intensity was quantified by densitometry using ImageJ software.trkb gene was performed using PerfeCTa SYBR Green FastMix ROX (Quanta Biosciences) and the thermocycler ABI Prism 7900 HT (Applied Biosystems). At least 5 mice of each genotype were evaluated using primers that annealed to sequences in exon 5 (5′-ACTGCCTCAATGAGAGCAGC-3′) and exon 6 (5′-GGAAAGGGTCACAGACTTTCC-3′), present in all trkb transcripts. The level of expression of trkb mRNA in gene-targeted animals was calculated as the fold change relative to the expression level of trkb in the corresponding wild type littermate. Relative mRNA expression level for each sample was normalized to the actin gene and 18S ribosomal RNA as reference genes.RNA from the hearts of 3 to 6 week old mice was extracted using RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA from total RNA was generated using random hexamers and Superscript III (Invitrogen). qPCR for the mouse http://rsb.info.nih.gov/ij/) particle analysis plug-in was used to set the lower and higher size of pixels to be considered as a countable object. Both selection thresholds were arbitrarily defined, but maintained rigorously for all the images of different conditions. Finally, objects were counted in each image using the ImageJ cell counter plug-in. NG2, PDGFRβ and PECAM intensity was analyzed by densitometry. To further ensure absence of bias in object counting or densitometry analysis, sections were coded, and the operator was blind to treatment groups. Negative controls without primary antibodies were performed for each immunodetection.For postnatal tissue, 10 micron serial frozen sections were stained with NG2 antibody  or CD31/PECAM  to detect pericytes/SMCs or endothelial cells, respectively. We used isolectin B4  to identify endothelial cells and PDGFRβ  for pericytes/SMCs in embryos. The TrkB antibody was purchased from Millipore (Cat# 07–225). The collagen IV antibody was from Abcam (Cat# Ab6586). Collagen intensity was quantified by densitometry using ImageJ software. For double-immunofluorescence, frozen sections were incubated with primary antibodies overnight, followed by incubation with either Alexa 488 or 568 conjugated secondary antibodies  at room temperature and visualized using a Zeiss microscope. Fluorescence field images were captured digitally . Counts of NG2 or PDGFRβ and PECAM objects in the heart were performed after unbiased intensity and size selection criteria. A gray level threshold was selected for all the microphotographs of all different treatments and staining. After this selection, ImageJ  was added to fresh media without FBS and cells were photographed at 6, 13 and 24 h followed by area analysis of the scratch using ImageJ. The width and area of each scratch in each condition was analyzed in 7–10 random and separate fields .Human umbilical smooth muscle cells (HuSMCs) were isolated as described previously HuSMCs were plated on glass bottom dishes (MatTek Corporation) and cultured in DMEM containing 10% FBS for 24 hours. Medium was changed to serum-free DMEM overnight. Cells were then imaged in phenol red-free DMEM (Invitrogen) supplemented with 30 mM HEPES-NaOH (pH 7.4) using an Olympus IX71 inverted microscope driven by IPLab software (BD Biosciences) and equipped with a 60X PlanApoN objective (NA 1.42), a Hamamatsu EM-CCD camera and a heated stage maintained at 37°C. Images were captured every 15 seconds. Cells were treated with BDNF (100 ng/ml). For inhibitor studies, cells were pre-treated with 10 µM Y-27632  or 10 nM K-252a  for 45 minutes, or pre-incubated with 1 µg/ml C3 transferase  for 4 hours. All movies were exported and processed using IPLab (BD Biosciences) and ImageJ (NIH) software.HuSMCs were lysed in TNE buffer  and phosphatase inhibitors (Sigma)) for 30 minutes at 4°C. 100 µg of total cell lysate protein was loaded on SDS-polyacrylamide gel and proteins were resolved and transferred to PVDF membrane. The following primary antibodies were used: TrkB , AKT , phospho-AKT , ERK , phospho-ERK , MLC , and phospho-MLC  antibodies. Anti β-actin  was used as a loading control. The respective secondary antibodies (anti-rabbit or anti-mouse IgG) were conjugated with HRP. Chemiluminescence of the blots was detected using ECL Advance Western Blotting Detection kit.AATGCTCGCAAGGACTTTCAT; a kind gift of Dr. Simon S. Murray, The University of Melbourne, Australia) and a scramble control were obtained from Open Biosystems Thermo Scientific . Lentivirus was packaged by co-transfection of shRNA constructs with packaging plasmids pMD2.G and pCMV-dR8.9 using TransIT-LT transfection reagent  into HEK293FT cells. Media was changed 24 h later, and the media supernatant was collected 48 h after transfection. Supernatants were filtered through a 0.45-mm filter and pelleted by centrifugation with PEG-it . Pellets were resuspended in medium. HuSMCs were infected, 72 h later media was changed to serum free, followed by BDNF treatment (100 ng/ml). The efficacy of TrkB knockdown was assessed by Western blot.Lentiviral plasmids containing shRNA against human TrkB (targeting sequence: −/−), consistent with a prior report in utero lethality of the embryo and early fetus −/−trkb or wild type littermates during late gestation. Histological analysis of the hearts from E15.5 to E16.5 embryos showed reduced ventricular wall thickness from the −/−trkb embryos as compared to wild type littermates, most prominently in the left ventricular wall  in mice with bdnf deficiency BDNF and TrkB are expressed in the developing cardiac vascular bed with BDNF expressed by endothelial cells and TrkB expressed prominently by pericytes and vascular smooth muscle cells (SMCs) lar wall . There wlar wall . Moreove−/−trkb embryos to survive until late gestation and to form identifiable cardiac arteries, capillaries, and veins suggest that the earlier processes of vasculogenesis and sprouting angiogenesis are not greatly impaired. To establish the onset of intramyocardial vessel defects, −/−trkb embryos and wild type littermates were examined from E14.5 to P0. Intramyocardial hemorrhage in −/−trkb embryos could be detected first at E16.5  and PDGFRβ antibodies, respectively. At E17.5, PDGFRβ-positivity was significantly reduced in the left ventricular wall of −/−trkb embryos as compared to wild type littermate controls  f/fTrkb-SMCCre+ allele demonstrated 20% reduction in Mendelian inheritance  and PECAM  immunodetection to determine whether deletion of TrkB results in loss of pericytes/SMCs. At postnatal day 21, rol mice . Thus, tstaining , despitestaining .f/f-trkbSMCCre+ mice, we examined the cardiac vasculature at the ultrastructural level at postnatal day 21. In wild type mice, the endothelial abluminal surface was free of cytoplasmic extensions, and the basement membrane between endothelial cells and pericytes was intact , but we detected a significant reduction in collagen IV deposition as compared with wild type-SMCCre+ mice  in 3-month-old mice, demonstrated an increase in extracellular accumulation of the dye around the pericyte/SMC-deficient vasculature of the f/f-trkbSMCCre+ hearts . HuSMCs express full length TrkB, as shown by immunohistochemical staining  have reported that the interaction of cells with growth factors in the extracellular matrix initiates ERK1/2 signaling leading to the activation of the motility machinery by enhancing phosphorylation of myosin light chain (MLC) To characterize the mechanisms by which BDNF/TrkB activation in pericytes/SMCs modulates their morphological changes and possible fast migration, we first evaluated the potential mediators of cell migration downstream of TrkB by examining the phosphoprotein profile in SMCs using the 4G10 antibody (pan-anti-phosphotyrosine). We observed phosphorylated proteins migrating at ∼42 kDda in the BDNF-treated HuSMCs as compared to untreated cells (data not shown). Reprobing of the membrane with specific antibodies, revealed that BDNF treatment induces activation of Ras/mitogen-activated protein kinases (ERK1/2) . Klemke To determine whether BDNF regulates actomyosin contractility in HuSMCs, we investigated the phosphorylation of MLC. Immunofluorescence analyses of phospho-MLC from unstimulated cells showed uniform and diffuse immunoreactivity, whereas HuSMCs treated with BDNF exhibited significant increase in phospho-MLC detection . Analysif/f-trkbSMCCre+ mice may therefore be a consequence of impaired migration of pericytes/SMCs, leading to inadequate vascular ensheathment.Together, these findings suggest that BDNF-TrkB can promote pericyte/SMC migration via activation of actomyosin contractility involving ERK1/2 and Rho/ROCK pathways . The sigin vivo and SMCs migration in vitro. The lack of pericyte investment around endothelial cells in the cardiac capillaries and arterioles of TrkB deficient mice resulted in vascular leakage and ultimately in embryonic lethality. These studies identify a novel mechanism by which a growth factor:receptor system regulates in a paracrine manner the pericyte coverage, stability and homeostasis of the cardiac microvasculature.Coronary blood vessel development requires BDNF, as mice deficient in this growth factor exhibit apoptosis of endothelial cells in the heart, leading to vessel destabilization and myocardial hemorrhage. These data raised the hypothesis that BDNF may act as an autocrine factor on endothelial cells or in a paracrine manner to induce activation of TrkB on neighboring pericytes, to stabilize nascent blood vessels. Here we establish the dependence of the cardiac vasculature on TrkB-mediated pericyte/SMC coverage of microvessels during development pdgfb and pdgfrβ knockout mice which lack desmin-positive perivascular cells −/−trkb mice may also have a role. Indeed, a previous report showed increased Tunnel labeling in the subepicardium of TrkB deficient embryos as compared to their wild type littermates at E12.5, although the identity of the apoptotic cells was not determined −/−trkb  in trkb mRNA in the f/ftrkb-SMCre+ mice heart , Erk1/2 and MLC phosphorylation and the consequently migration of the cells. Rho GTPases and MLC have been described as key regulators of pericytes/SMC shape and contractility Dysfunction in pericyte/SMC migration has been reported in many microvascular diseases, such as hypertension and diabetic microangiopathy. Therefore, it is of great importance to better understand the mechanisms by which pericytes regulate microvessel function.Figure S1TrkB mRNA analysis in f/ftrkb-SMCCre+ animals. TrkB mRNA is markedly decreased in homozygous mice lacking trkb gene specifically in smooth muscle cells (f/ftrkb-SMCCre+) compared to wild type-SMCCre+ control littermates. Bars represent mean ± s.e.m. n = 5 animals for each genotype, including mice from 3 different litters between the ages of 3 to 6 weeks old. Statistical comparisons were made by one-way analysis of variance test. *P<0.05.(DOCX)Click here for additional data file.Table S1f/fTrkb-SMCCre+ genotype analysis. Genotype analysis of 137 offsprings from f/wtrkb-SMCCre+ heterozygous intercrosses at postnatal day 21. Numbers of animals of each genotype are shown with the correspondent percentage of the total mice alive at P21 in brackets.(DOCX)Click here for additional data file.Movie S1Effect of BDNF on HuSMCs analyzed by live imaging. Increased membrane protrusions in HuSMCs in the presence of BDNF.(AVI)Click here for additional data file.Movie S2The effect of BDNF on HuSMCs was prevented by K252a. BDNF-induced membrane protrusion in HuSMCs was inhibited by K252a.(AVI)Click here for additional data file.


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.