The present study is devoted to the feasibility of expressing the single-domainmini-antibody (nanoantibody) selected from the library of sequences of thevariable domains of special single-stranded antibodies derived from an immunizedcamel, a gene of which was introduced into eukaryotic cells within a recombinantadenoviral vector. A vector bearing the gene of a single-domain nanoantibody wasobtained using the AdEasy Adenoviral Vector System (Stratagene). This method ofdelivering the nanoantibody gene facilitates efficient expression of this geneand functional activity of the nanoantibody. The results obtained can be used toproduce passive immunizing tools against pathogens or new-generationimmunobiological antitoxic medication. Antibodies are the primary tools of the immune system, which participate in theprotection of the organism against pathogenic microorganisms. The significance ofantibodies is growing as researchers become aware of their potential not only astools to be used in diagnostics, but in therapy as well . AntibodA significant breakthrough in this field has been the detection of non-canonicalantibodies in members of the Camelidae biological family. These antibodies do notcontain light strands and represent a dimer of shortened heavy strands , 6. AniThanks to their structure, mini-antibodies can be used to reveal epitopes that arehidden for the conventional immunoglobulins. The expression from a single genesimplifies genetic engineering procedures and, therefore, the work with thelibraries containing the sequences of variable domains. Low immunogenicity(conditioned by the high homology of the sequences of mini-antibodies with avariable domain of heavy strands of human IgG3) and the relative simplicity of thehumanization procedure open broad opportunities for the application ofmini-antibodies in the design of novel pharmaceutical agents .These features of the structure of mini-antibodies and the simplicity with whichtheir genes can be manipulated enable efficient and economical production of largeamounts of a mini-antibody, using various expression systems . The use of the prokaryotic expression system to produce mammalian proteins has to dowith the possibly low functional activity of the proteins obtained, due to theabsence of a system for post-translational modification in prokaryotic cells.Moreover, no matter how thorough the purification, the final product can still becontaminated with pyrogenes.One of the promising methods for delivering genetic material to target cells is theuse of viral vectors. Expression constructions bearing one or several recombinantgenes are incorporated into the viral genome using methods of genetic engineering.Vectors based on the genome of the adeno-associated virus have been proposed in anumber of studies , 11 for Adenoviral vectors are among the most universal tools used for delivery andexpression of recombinant genes in mammalian cells. It is known that recombinantadenoviruses efficiently transfer the genes of bacterial and viral antigens,cytokines, growth factors, and other proteins to the target cells, ensuring a highlevel and duration of target gene expression . Adenovi in vitro in special celllines, which ensures the vector’s safety [The production of recombinant adenoviruses is characterized by the following feature:the virus is capable of reproducing only s safety . The fact that recombinant adenoviral vectors can be used efficiently for theexpression of antigen-binding fragments of antibodies is borne out by the example ofmini-antibodies to the cell epitope and anthrax toxin component) , 17.The aim of the present work is to examine how recombinant adenoviral vectors can beused for delivery and efficient expression of single-domain mini-antibodies(nanoantibodies) obtained using the novel technology of generation of specialsingle-stranded antibodies extracted from camel. The nanoantibody earlier obtainedand characterized to the cell cytokeratine-8  was seleEnzymesIn this study, restriction endodeoxyribonucleases, T4 DNA ligase, alkalinephosphatase (CIAP) purchased from Fermentas MBI (Lithuania), and Taq-polymerasepurchased from Promega (United States) were used.Cell linesThe HEK-293 cell line (human embryonic kidney cell culture transformed by theE1-region of human adenovirus serotype 5) and Н1299 cell line (human lungcancer cells) were used. The cells were cultured in a DMEM medium containing 10% offetal bovine serum (FBS) purchased from HyClone (United States).Production of the cDNA clone encoding the single-domain mini-antibody(nanoantibody) which specifically recognizes the endogenous mousecytokeratin-8H H, which specifically recognizes mouse cytokeratin-8,was obtained earlier by S.V. Tillib’s research group  in collaboration with the laboratory headed by S. Muyldermans(Vrije Universiteit Brussel) and used (via binding to the fluorescent proteinsequence) to obtain fluorescent nanoantibodies (or chromobodies) aimed atdemonstrating the new method for tracing antigens in a living cell. It should benoted that the aCyK-V H H nanoantibody was one of the first antibodies toendogenous structural eukaryotic proteins. The first stage of its productioncomprised immunization of the Bactrian camel ( Camelus bactrianus )with a protein extract from mouse soft tissue cells (predominantly from the liver).The subsequent selection procedure, based on the phage display method, was performedas described in the online supplement to the article [H H recognized cytokeratin-8, a fact attested to via theimmunofluorescent staining of С2С12 (mouse myoblast cell line)with these antibodies, revealing the characteristic distribution of cytokeratinintermediate filaments in the cytoplasm.Antibody aCyK-V  article . The funH H produced in the bacterial periplasm wasmodified by binding an antigen-recognizing sequence of two additional smallfragments, epitope of influenza virus haemogglutinine (HA-tag) and six histidineresidues (His 6 -tag), in order to purify it and simplify itsdetection.The nanoantibody aCyK-V Obtaining recombinant adenovirusH HaCyK-V  . The nucleotidesequence encoding the nanoantibody was obtained by chemical synthesis in“Evrogen” JSC. The AdEasy Adenoviral Vector System  was used in order to construct the рAd-aCyK-V HH plasmid vector containing the genome of the recombinant adenovirus with E1 regiondeletion, and a transgene expression cassette incorporated instead of it viahomologous recombination in E. coli  cells. The recombinantadenovirus was obtained via transfection of HEK-293 cell lines with theрAd-aCyK-V H H plasmid construct linearized on the PacI site.Lipofectamine 2000  was used for the transfection,according to the manufacturer’s recommendations. The recombinant humanadenovirus of serotype 5 with E1 region deletion and an incorporated transgene-freecassette expression (Ad-null) inserted instead of it was used as thecontrol.Plasmids and the recombinant adenoviral vector were obtained using the gene ofantibody to cytokeratin 7 PFU of the virus per Petri dish with a diameter of 15 cm) was coated to the HEK-293cell monolayer with 50–70% confluence. The infected cell suspension wasdestroyed by three freeze-thaw cycles and clarified by centrifuging .To accumulate adenoviral preparations, an infected cell suspension (10 H H and Ad-null (10 8 PFU/ml) were determined by the plaque formation technique in the HEK-293 cellculture.The titres of the specimens Ad5-aCyK-V Infection of cells with a recombinant adenovirus6  cells of the H1299 line were infected with recombinantadenoviruses. The cells were seeded to ~ 70% of the monolayer, cultivated for 24 h,and infected with the recombinant adenovirus (the multiplicity of infection being100 PFU/cell) in a DMEM medium containing 2% of FBS. Two hours after the viralpreparation was introduced, the medium was collected, the cell culture was washed,and a fresh DMEM medium was added. The medium from the infected cells was collected72 h after infection and concentrated by centrifuge ultrafiltration through amembrane with a nominally intercepted molecular weight of 10 kDa. After thickeningby a factor of 10, the supernatant was fractioned in a 10% polyamide gel and usedfor immune blotting analysis.Approximately 10 Antigen preparationHomogenized mouse liver lysate (BALB/c line) was obtained via extraction with the useof a RIPA buffer ). The concentration of thetotal protein in the specimens was determined by the Bradford method . The specimens with an equal protein concentration were applied tothe gel to be separated by electrophoresis.Polyacrylamide gel electrophoresis and immunoblottingCellular proteins were separated by polyacrylamide gel electrophoresis by the Laemmliprocedure under denaturing conditions in the presence of sodium dodecyl sulphate.Protein Test Mixture 4  was used as the molecular weight standard.After the gel electrophoresis, the proteins were placed onto a Hybond-P PVDFmembrane  using a TE70 Semi-Dry Transfer Unit  in accordance with the manufacturer’srecommendations. The nanoantibodies were detected using the MonoclonalAnti-HA–Peroxidase antibody . Theimmobilized proteins were detected using ECL Plus Western Blotting DetectionReagents  in accordance with themanufacturer’s recommendations. The chemilumenscent radiation was recordedwith the aid of an Amersham Hyperfilm ECL X-ray film .As a result of the earlier performed selection of the phage library of theantigen-binding domains of single-stranded antibodies, DNA from the pHEN4 phagemidwith an insertion encoding the nanoantibody, which has a high affinity towards thestructural cytoplasmatic mouse protein, cytokeratin-8, was collected. The data onthe structure of the target protein were obtained via mass spectrometricidentification. The nucleotide sequence encoding the nanoantibody was cloned in therecombinant adenoviral vector.Fig.1 shows the scheme of the resulting construct.A leading peptide of the mouse immunoglobulin κ-chain was bound to itsN-terminus, in order to ensure efficient extracellular expression of a nanoantibody.The HA-tag, which is effectively recognized by commercial antibodies, a requirementfor confirming nanoantigen expression at the protein level, was bound to theC-terminus of the nanoantibody. H H antibody was cloned in the shuttleplasmid vector pShuttle-CMV . This vector containsterminal fragments of the human adenovirus serotype 5 genome, the expressioncassette containing the human cytomegalovirus promoter (CMV) and polyadenylationsignal. The presence of the insertion and its orientation were confirmed byrestriction mapping.In order to construct the adenoviral vector, the sequence encoding theHA-tag-labelled аCyK-V E. coli  cells. The plasmid constructobtained contained the replication initiation site ori, the gene of antibodyresistance, and a cassette with the target gene within the adenoviral genome. Themain advantage of this method is the potential utilization of E. coli cells as the main tools for cloning, recombination, and production ofadenoviral DNA in preparative amounts. The opportunity to perform the homologousrecombination in E. coli  cells makes it possible to deal with theindividual clones containing plasmid constructs only with recombinant adenoviruses,which eliminates the possibility of contamination with a wild-typeadenovirus.The recombinant plasmid adenoviral vector bearing the target gene was obtained viahomologous recombination in E. coli BJ5183 cells by electroporation. Recombinant clonesobtained by homologous recombination were collected on the selectivekanamycin-containing medium (50 µg/ml). The presence of recombinant clones ofnucleotide sequences encoding the aCyK-V H H antibody and humanadenovirus serotype 5 fibre in plasmid DNA was analyzed by PCR with specific primersand via restriction mapping, using HindIII restrictase, which enables one to obtaina restriction pattern that is typical for the human adenovirusgenome.The shuttle plasmid construct bearing the expression cassettes with the nanoantibodygene was linearized on the PmeI site and introduced along with pAd-EASY (Stratagene)to H H was analyzed by PCR using the primer pair thatwas complementary to the target gene, the hexon gene of human adenovirus serotype 5,and the E1 region of the adenovirus in order to control the possible presence ofreplication-competent viral particles. HEK-293 cells were transfected with a plasmid cleaved at the PacI site and containingthe recombinant adenovirus genome with E1 region deletion and the expressioncassette with a transgene inserted instead of it. The resulting recombinantadenovirus Ad5-aCyK-V H HDetection of the expression of the nanoantibody gene within therecombinant adenovirus Ad5-aCyK-V H H was analyzed at the level of the mRNA. With this purposein mind, the cells of the HEK-293 line that are permissive for human adenovirusserotype 5 were infected with the recombinant virus Ad5-aCyK-V H H. Thetotal RNA of infected cells was used to produce DNA, which was analyzed by PCR withprimers specific to the sequence of the nanoantibody gene to mouse cytokeratin 8, toviral DNA, and the constitutively expressed gene of glyceraldehyde-3-phosphatedehydrogenase (GAPDH). HEK-293 cell lines infected with Ad-null virus  were used asthe negative control. RT PCR was used to demonstrate that the recombinant adenoviralvector expresses mRNA of the nanoantibody gene to cytokeratin and can be used toanalyze protein production.The expression of the target gene within the recombinant human adenovirus serotype 5Ad5-aCyK-V H H tagged with HA-epitope of the influenza virus (Ad5-aCyK-VH H), and the recombinant adenovirus containing the transgene-freeexpression cassette (Ad-null). The presence of a nanoantibody in the culture mediumcontaining the infected cells was measured by immunoblotting with antibodies toHA-epitope of the influenza virus conjugated with horseradish peroxidase .Nanoantibody expression at the translational level was analyzed in H1299 cellsinfected with the recombinant adenovirus carrying the gene of nanoantibody to aCyK-VBiological activityE. coli  periplasm. The specificity of a nanoantibody expressed by the adenoviral vector to cytokeratinwas confirmed by means of comparison of the interaction between the antigene and theproteins of the cultural fluid from the cells infected with recombinant adenovirus,and the interaction between the antigene and the antibody purified fromH H. The expressed nanoantibodyserved as the primary antibody to the target protein detected in the total lysate. The membrane was simultaneously incubated withantibodies aCyK-V H H produced in E. coliperiplasm.The lysates of mouse liver and cerebrum cells were fractioned in a polyacrylamidegel, transferred to the PVDF membrane, which was incubated with the cultural mediumof the cells infected with Ad5-aCyK-V Fig. 3 shows the results of anelectrophoresis of protein lysates in polyacrylamide gel and the data obtained byimmunoblotting with nanoantibodies aCyK-V H H after development bysecondary antibodies to the HA-epitope of the influenza virus conjugated withhorseradish peroxidase.E. coli periplasm, its gene being cloned in the recombinantadenovirus.Immunoblotting results attest to the fact that the antibody expressed by theadenovirus has the same specificity as the antibody synthesized in At the time of writing, there were a number of technologies capable of producingmini-antibodies with a predetermined specificity. Only quite recently was itrevealed that, in addition to the canonical antibodies, functionally activenoncanonical single-stranded antibodies were produced in relatively large amounts inmembers of the Camelidae family. Therefore, it is now possible to obtainmini-antibodies on the basis of libraries containing the antigen-recognizing domainsof single-stranded antibodies of immunized animals. Noncanonical antibodies consistof a dimer with a single shortened heavy immunoglobulin chain (containing no lightchains). Single-domain mini-antibodies (nanoantibodies) are genetically engineeredderivatives of the antigen-recognizing domains of these noncanonical antibodies. Theselection of clones of a mini-antibody with the predetermined specificity from thelibrary of sequences of the entire repertoire of antigene-recognizing domains ofnoncanonical antibodies obtained from immunized camel is based on the highlyefficient procedure of functional selection of filamentous phage particlescontaining both an exposed mini-antibody on the surface, and the DNA encoding itwithin the phage particle (phage display).Mini-antibodies produced by this technology are characterized by high stability,solubility, and low immunogenicity. Mini-antibodies can be produced (selected) toany antigens and any antigen epitopes, including conservative ones, which oftencannot be produced using the conventional procedure. Since the encoding nucleotidesequence is known for each mini-antibody, it is possible to produce thecorresponding protein in any of the known expression systems (prokaryotic andeukaryotic).E. coli  cells, yeast, or CHO cells. When injecting thesepreparations to experimental animals (or patients), their very short lifetime in theorganism (less than 24 h) should be taken into account. The period of therapeuticaction of preparations based on mini-antibodies can be increased using the vectorsystems, providing that the synthesis of the active agent takes place immediately inthe infected cells of the organism. Recombinant adenoviruses are the optimalexpression system for solving such problems. Their safety and efficiency has beenproved in a number of clinical trials performed globally; the time needed to producea target protein is approximately 20 days. It is economically viable to produce protein preparations of mini-antibodies inThe potential application of recombinant adenoviral vectors for the expression of thegenes of the antigen-recognizing fragments of single-stranded antibodies obtainedfrom Bactrian camel was studied in this work. It was demonstrated that expression ofthe nanoantibody gene using the adenoviral vector is possible. Transgene expressionwas confirmed at the level of the RNA transcript and protein product. The specificinteraction of the nanoantibody secreted by eukaryotic cells with a target proteinattests to the fact that its functional activity is retained. Further studies arenecessary for a qualitative estimation of the efficiency of nanoantibody expressionusing a recombinant adenovirus.The delivery of the gene of a single-domain mini-antibody (nanoantibody) selectedfrom the library containing sequences of the variable domains of specificsingle-stranded antibodies of immunized camel to eukaryotic cells using therecombinant adenoviral vector provides efficient expression and functioning of thenanoantibody. The results of this study can be used for the production of passiveimmunization agents for protection against pathogens, or for the design ofnew-generation immunobiological antitoxic preparations.


Sunitinib, an oral multitargeted tyrosine kinase inhibitor, is widely used in the treatment of renal cell carcinoma and gastrointestinal stromal tumor and has had a variety of adverse events. However, sunitinib-related acute cholecystitis has been reported in only two patients with gastrointestinal stromal tumor and renal cell carcinoma (clear cell subtype).A 75-year-old Japanese woman with a right sided abdominal swelling was referred to our hospital. Computed tomography (CT) showed a hypervascular bulky tumor in her right kidney, suggesting right renal cell carcinoma in clinical T4N0M0. Although sunitinib therapy was started as neoadjuvant chemotherapy, during the fourth week of the first cycle, she developed acute acalculous cholecystitis and disseminated intravascular coagulation associated with sunitinib. Sunitinib therapy was discontinued immediately and she recovered after subsequent treatment with antibiotics and gabexate mesilate followed by percutaneous cholecystostomy. Cholecystectomy and right radical nephrectomy were performed and pathological examination showed that her renal tumor was a chromophobe renal cell carcinoma (pT2) with necrosis. Inflammation and ischemia were observed in the gallbladder wall, which was compatible with acute acalculous cholecystitis. There has been no evidence of disease recurrence for more than six months.We described the third case of sunitinib-related acute cholecystitis in a patient with chromophobe renal cell carcinoma. Attention is required to sunitinib-related acute cholecystitis which, while uncommon, could be life-threatening. Sunitinib, an oral multitargeted tyrosine kinase inhibitor, is widely used in the treatment of metastatic renal cell carcinoma (RCC) and gastrointestinal stromal tumor (GIST) and has been administered in the perioperative period . Althoug®) followed by percutaneous cholecystostomy.A 75-year-old Japanese woman with a right sided abdominal swelling was referred to our hospital. She had no history of medication or smoking and was a social drinker. Computed tomography (CT) showed a hypervascular bulky tumor in her right kidney with suspected liver invasion without distant metastasis Figure , suggestAfter three months, a follow-up contrast-enhanced computed tomography (CT) revealed a marked shrinkage of the gallbladder and a 21% reduction in the size of the renal tumor with decreased enhancement of its center. Cholecystectomy and right radical open nephrectomy were performed. Adhesions thought to be due to cholecystitis made the operation difficult although common bile duct stenosis or retraction by the tumor that could lead to cholecystitis was not observed. Pathological examination showed that her renal tumor was chromophobe RCC (pT2) with necrosis occupying more than half of the tumor Figure . InflammWe present the third case of sunitinib-related acute cholecystitis which developed in a patient with a chromophobe RCC. In the literature, sunitinib-related acute cholecystitis has been reported in only two other patients, one with GIST  and the A common clinical feature of sunitinib-related acute cholecystitis in three cases including our patient and the previous two cases ,3 was acWe described the third case of sunitinib-related acute cholecystitis in a patient with chromophobe RCC. Attention needs to be given to sunitinib-related acute cholecystitis which could be life-threatening albeit uncommon.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.KN prepared the manuscript. KS and TM interpreted the patient data and revised the manuscript. All authors reviewed and approved the final version of the manuscript.


Our insights using the Lifeact-EGFP mice demonstrate that F-actin coating occurs after granule fusion and is a granule-wide event.F-actin remodelling is essential for a wide variety of cell processes. It is important in exocytosis, where F-actin coats fusing exocytic granules. The purpose of these F-actin coats is unknown. They may be important in stabilizing the fused granules, they may play a contractile role and promote expulsion of granule content and finally may be important in endocytosis. To elucidate these functions of F-actin remodelling requires a reliable method to visualize F-actin dynamics in living cells. The recent development of Lifeact-EGFP transgenic animals offers such an opportunity. Here, we studied the characteristics of exocytosis in pancreatic acinar cells obtained from the Lifeact-EGFP transgenic mice. We show that the time-course of agonist-evoked exocytic events and the kinetics of each single exocytic event are the same for wild type and Lifeact-EGFP transgenic animals. We conclude that Lifeact-EGFP animals are a good model to study of exocytosis and reveal that F-actin coating is dependent on the  F-actin remodelling has long been associated with the process of exocytosis We have tested the use of Lifeact-EGFP transgenic mice using a sensitive assay for exocytosis where we can record the time-course of each individual fusion event and the single-granule kinetics of fusion In many cell types, F-actin remodelling during exocytosis appears as a coating of individual granules. A preparation of mouse pancreatic fragments from the Lifeact-EGFP transgenic animals shows strong fluorescent staining along the apical lumens and sub-cortical regions beneath the basolateral membranes. In paraformaldehyde-fixed cells, this Lifeact-EGFP fluorescence pattern was the same as that observed when phalloidin-Alexa 633 used as a counter-stain .Both patterns of fluorescence are as previously described for F-actin distribution in resting exocrine pancreatic tissue To identify and quantify exocytosis in pancreatic acinar cells, we track, in living cells, the entry of extracellular fluorescent dye into each fused granule using two photon microscopy We also use the same method of dye entry into granules to track fluorescence changes within single granules after fusion We conclude that the Lifeact-EGFP transgenic animals are a useful tool for the live study of F-actin remodelling during exocytosis. In acinar cells, it is clear that F-actin coating is specifically associated with fusing granules but the time-course of coating relative to fusion is not known. To answer this question we used isolated pancreatic fragments from Lifeact-EGFP mice bathed in extracellular solution containing the fluorescent dye sulforhodamine B (SRB) and imaged during an acetylcholine-induced response We simultaneously recorded the Lifeact-EGFP signal and extracellular dye (SRB) entry into single fusing granules, with entry of the dye as an index of the time-point of granule fusion. Single fusion events in response to 1 µM acetycholine stimulation were detected as the entry of extracellular SRB dye into the granule . From thPrevious work has indicated that F-actin coating of the granule is dependent on actin polymerisation, rather than movement of F-actin around the granule We conclude that F-actin coating occurs after fusion and is dependent on actin polymerization. Small G-proteins have been implicated in F-actin coating and it has been proposed that their activation is dependent on the mixing of components from the plasma membrane and the granule membrane Here we demonstrate that pancreatic acinar cells taken from Lifeact-EGFP transgenic animals show no differences in exocytosis measured in terms of the numbers of fusion events induced by agonist and in terms of the kinetics of fusion of single granules. Since it is well known, in these cells, that pharmacological manipulation of the F-actin cytoskeleton does affect both these parameters Using preparations of exocrine pancreatic fragments, we now directly demonstrate that F-actin remodelling occurs after granule fusion and is seen as a coating around each fused granule. Latrunculin inhibits this coating indicating that it is due to nucleation and polymerization of G-actin, rather than movement of F-actin. Finally, we show that development of F-actin polymerisation occurs with a similar time course across all regions of the granule.de novo synthesis of F-actin. Our data, with short-lasting treatment with latrunculin (10 minutes pre-treatment and continued presence during stimulation), which sequesters G-actin monomers, now shows complete block of coating. This indicates that coat dynamics require nucleation since short-lasting latrunculin treatment would not be expected to affect movement of existent F-actin filaments. This conclusion is consistent with previous observations in fixed cells These observations extend our knowledge of F-actin remodelling in acinar cells and provide new insights into the possible mechanisms. Firstly, the initial observations of F-actin coating of granules Our method has a temporal resolution of 2 frames per second and within this limitation we do not observe any difference in the timing of F-actin formation across the granule . These lDirect comparison of our data with that of Yu and Bement in oocytes There is currently no good understanding of the potential role of F-actin coating. In the pancreas, it has been suggested that it might stabilize the granule While F-actin coating may not play a role in expelling content, F-actin may play another role in the loss of content. The data of If the F-actin coat's primary role in acinar cells is stabilization we are still left with the question as to what is the purpose of this stabilization. Possibilities include stabilization of the apical region of the acinar cell and the acinus structure. This region is heavily enriched with F-actin see  and in cIn conclusion we provide a detailed analysis indicating that exocytosis in the exocrine pancreas is not affected by the presence of a Lifeact-EGFP low affinity F-actin probe. Our data show that F-actin remodelling occurs as a result of polymerisation triggered rapidly after granule fusion. The Lifeact-EGFP transgenic animals are likely to be a useful tool in unravelling the role actin plays during secretion in a wide range of cell types.Transgenic Lifeact-EGFP mice were obtained as a gift from Professor Roland Wedlich-Soldner After preparation of the pancreatic fragments and adherence to the poly-l-lysine coated coverslips, we washed the cells in extracellular solution. Cell stimulation was performed by addition of a bolus of acetylcholine stock solution to the solution surrounding the cells. In the latrunculin experiments, the drug was applied 10 minutes before cell stimulation and was present throughout stimulation with acetylcholine.Fixed specimens were imaged using an Olympus BX61 upright confocal laser scanning microscope, with a 63× objective lens (NA 1.3), and an optical slice depth of ∼1 µm. Images were collected with the appropriate filters and captured in sequential tracks to minimize crosstalk to less than 2%. Fluorescent probes were from Molecular Probes Inc. . All other compounds were from Sigma . All experiments were performed at least three times. We used Metamorph software for analysis of LSM images and Adobe Photoshop for image processing and presentation.Cell preparations were fixed in freshly prepared 4% paraformaldehyde in PBS for 30 minutes, and permeabilzed with 0.5% Triton-X 100 in PBS for 10 minutes. F-actin was visualized with phalloidin Alexa 633 .−1, Sigma) excited by femtosecond laser pulses at 850 nm, with fluorescence emission detected at 550–700 nm (SRB).We used a custom-made, video-rate, 2-photon microscope using a Sapphire-Ti laser 2, 100 pixels) over granules. Traces were rejected if extensive movement was observed.Images  were analyzed with the Metamorph program (Molecular Devices Corporation). Exocytotic event kinetics were measured from regions of interest (0.78 µmData analysis was performed on the average data obtained from regions of interest placed over the granules. In all analyses we subtracted the background fluorescent signals. For analysis of the SRB recordings, we typically normalise all records to the amplitude of the first peak response. In the case of the Lifeact-EGFP signals these are expressed in arbitrary units.To determine the time point of initiation of the signals in the experiments of All data are shown as mean ± SEM and are obtained from at least three separate animals.Images were recorded in TIF format and image panels produced within the Metamorph program.


One of the rationales behind using strength training in the treatment of adolescents with Patellofemoral Pain (PFP) is that reduced strength of the lower extremity is a risk factor for PFP and a common deficit. This rationale is based on research conducted on adolescents >15 years of age but has never been investigated among young adolescents with PFP.To compare isometric muscle strength of the lower extremity among adolescents with PFP compared to age- and gender-matched pain-free adolescents.In 2011 a population-based cohort (APA2011-cohort) consisting of 768 adolescents aged 12–15 years from 8 local schools was formed. In September 2012, all adolescents who reported knee pain in September 2011 were offered a clinical examination if they still had knee pain. From these, 20 adolescents  were diagnosed with PFP. Pain-free adolescents from the APA2011-cohort (n = 20) were recruited on random basis as age- and gender-matched pairs. Primary outcome was isometric knee extension strength normalized to body weight (%BW) and blinded towards subject information. Secondary outcomes included knee flexion, hip abduction/adduction and hip internal/external rotation strength. Demographic data included Knee Injury and Osteoarthritis Outcome Score (KOOS) and symptom duration.Adolescents with PFP reported long symptom duration and significantly worse KOOS scores compared to pain-free adolescents. There were no significant differences in isometric knee extension strength , isometric knee flexion strength  or different measures of hip strength .Young symptomatic adolescents with PFP between 12 and 16 years of age did not have decreased isometric muscle strength of the knee and hip. These results question the rationale of targeting strength deficits in the treatment of adolescents with PFP. However, strength training may still be an effective treatment for those individuals with PFP suffering from strength deficits. Knee pain is common during adolescence and up to 25% of adolescents report having knee pain We recently published data showing reduced isometric knee extension strength among adolescents with PFP aged 15–19 years compared to gender and age-matched adolescents without knee pain The latest review covering treatment of patients with PFP advocate for strength training as a keystone in the treatment of PFP Given the lack of studies specifically investigating young adolescents with PFP the purposes of this study were to investigate isometric muscle strength around the knee and hip. The primary hypothesis was that adolescents with PFP would have reduced isometric knee extension strength. Secondary hypothesises were that adolescents with PFP would have reduced isometric muscle strength in knee flexion and hip abduction/adduction and hip internal and external rotation.This cross-sectional study compared 20 adolescents diagnosed with PFP to 20 age- and gender matched pain-free adolescents. Both groups were recruited from the same population-based cohort  In September 2011 eight lower secondary schools in the community of Aalborg were invited to answer an online questionnaire and to be part of the APA2011-cohort. A total of 768 students aged 12–15 years answered the online questionnaire and 215 students (28%) reported knee pain. In September 2012, all adolescents who reported knee pain in September 2011 were contacted by telephone within a time period of two weeks, The pain-free adolescents were recruited on random basis as age- and gender matched pairs from the same cohort that the adolescents with PFP were recruited from (the APA2011-cohort). Recruitment took place in the same time period as recruitment of adolescents with PFP. The inclusion criteria for the pain-free adolescents were: no current self-reported musculoskeletal pain; no self-reported prior surgery in the lower extremity; no self-reported neurological or medical conditions.During the clinical examination, the students were diagnosed with PFP if they met the following criteria 12–16 yearsInsidious onset of anterior or retro-patellar knee pain for more than 6 weeks and provoked by at least two of the following positions or functions: prolonged sitting or kneeling, squatting, running, hopping or stair walkingTenderness on palpation of the patella, or pain with stepping down or double leg squatting; andWorst pain experienced during the previous week should be reported to be more than 30 mm on a 100 mm Visual Analogue Scale (VAS).Exclusion criteria were concomitant injury or pain from the hip, lumbar spine, or other structures of the knee; i.e. tendinopathy or Mb Osgood Schlatter’s disease; previous knee surgery; patellofemoral instability; knee joint effusion; or weekly use of anti-inflammatory drugs. Adolescents who had been treated for PFP using physiotherapy during the last year were excluded. Treatment of PFP would most likely include strength training why inclusion of these adolescents could cause a differential selection bias among adolescents with PFP.Growing pains (GP) may be mistaken for PFP among young adolescents with PFP. However there are a few important differences between the two pain conditions that was used during clinical examination to distinguish the two pain conditions. Growing Pain is usually non-articular and located to the shins, calves, thighs or popliteal fossa Primary outcome were isometric knee extension strength using the best of three consecutive measurements normalized to body weight (%BW). Secondary outcomes included knee flexion, hip abduction/adduction and hip internal/external rotation. Outcome measurements were collected from the most pain-full knee in adolescents with PFP and on an identical proportion of right and left knees among pain-free adolescents. All measurements were done in September 2012 by a rater with previous experience in muscle strength testing. The rater was a physiotherapist and blinded to which of the 40 adolescents were diagnosed with PFP.The testing setup included a portable dynamometer and an examination table. Muscle strength was tested with the Mecmesin AFG2500 dynamometer that was attached to the wall through a fixed bolted connection to ensure fixation. All strength tests were isometric strength tests. The test positions were chosen based on procedures that are often applied in clinical settings. To make sure the test procedure was reliable, a pilot study was conducted two weeks prior to the testing of the adolescents (see later header “Reliability”). A total of six movement directions around the knee and hip were tested; knee flexion and extension; hip abduction and adduction; hip internal and external rotation.During all strength tests the participants were told to stabilize themselves by holding on to the sides of the examination table. A cotton cloth was placed between their lower legs and the strap from the dynamometer to allow for both standardization of the dynamometer placement and pain reduction from the pressure created by the dynamometer.After receiving instruction about the procedure, the participants were asked to perform one isometric sub-maximal trial. Then an additional practice trial was applied. Afterwards the individual test was administered three times to reduce a possible learning effect. The highest value of three consecutive measurements are presented. A 1-minute rest period was given after each trial. The standardized command given by the examiner was: “Go ahead-push-push-push-push and relax”. To allow for comparison to the latest review covering strength deficits in patients with PFP we reported strength normalized to bodyweight (BW) During knee extension and knee flexion, the strap from the dynamometer was positioned perpendicular to the anterior or posterior aspect of the tibia, 5 cm proximal to the medial malleolus , a & b. During hip abduction and adduction the participant was lying supine on the examination table , C & F. Hip external and internal rotation was tested with the participant sitting on one side of the examination table with the hip and knee flexed at 90 degrees , D & E. Secondary outcomes included the patient-reported questionnaire Knee Injury and Osteoarthritis Outcome Score (KOOS) Physical activity level was measured with The Physical Activity Scale (PAS) Before data collection, a test-retest intra-rater reliability study was performed to investigate the reliability and agreement of the isometric strength testing for the best of three measurements. The study included a convenience sample of 17 young adults who were tested twice, with 30 minutes between test and retest. A two-way random effects model (2.1), single measures, absolute agreement, intraclass correlation coefficients (ICC) were used to express intra-rater reliability. Limits of Agreements (LoA) were used to express the agreement between test and retest The sample-size was based on detecting a difference between groups of at least 20% on the normalized isometric quadriceps strength. This difference was based on quadriceps torque data from our previous study comparing adolescents aged 15–19 years with a similar methodology All data were visually inspected using a Q-Q plot. Mean values ±SD are reported if data were normally distributed. If data were non-normally distributed they were presented as median and interquartile range (IQR). Paired samples t-test was used to test the difference in isometric strength between matched pairs. All calculations were performed using Stata version 11 .Average pain duration among adolescents with PFP was 28.5 months . Self-reMean difference between groups in primary outcome, isometric knee extension strength, was 0.3% BW, p = 0.97. The difference in isometric knee flexion strength was 0.4% BW while the differences in hip strength ranged from 0.4 to 1.1% BW, p>0.35 . ConvertThis study is the first to compare isometric knee and hip muscle strength among young adolescents with PFP and compare them to age- and gender matched pain-free adolescents from the same population-based cohort. We hypothesized that adolescents with PFP would have significantly lower isometric muscle strength of the knee and hip. Despite self-report of functional limitations and long-lasting severe pain, adolescents with PFP did not have decreased isometric muscle strength of hip and knee compared to age- and gender matched pain-free adolescents.We have previously reported that adolescents aged 15–19 years showed a significantly lower isometric knee extension strength Our sample of adolescents with PFP and the pain-free adolescents were both recruited from the same well-defined population-based cohort. In addition knee pain was confirmed at two time points in the group with PFP, one year apart. The recruitment of a previously untreated population-based sample suggests the results may be generalizable to the broader adolescent population. The few previous studies on adolescents with PFP are all patient-based studies. We recruited adolescents from a closed population-based cohort, which may suggest that the adolescents with PFP have shorter symptom duration and lower pain intensity compared with patients who have already consulted their general practitioner. Only a small number of the adolescents with self-reported knee pain were excluded because they were already receiving treatment  see . Worst pThe sample-size calculation was based on the results of our previous study Different subgroups may respond differently to treatment. The different subgroups of patients with PFP have been discussed earlier but primarily in relation to the efficacy of foot orthoses Despite self-report of functional limitations, long-lasting severe pain and decreased quality of life, 12–16 year old adolescents with Patellofemoral Pain do not have decreased isometric muscle strength of the knee and hip compared to age and gender matched pain-free adolescents. These results question the rationale of targeting strength deficits in the treatment of adolescents with PFP. However, strength training may still be an effective treatment for those individuals with PFP suffering from strength deficits.Checklist S1STROBE Checklist.(DOCX)Click here for additional data file.


Its microscopic nature means it is undetectable on CT. It is an artefact attributable to 18F-FDG-tracer contamination at the injection site. This paper reports a case of a 61 year-old lady with a past history of breast carcinoma, in whom follow-up PET/CT images demonstrated an incidental intense FDG pulmonary abnormality. A follow-up PET/CT seven months later demonstrated complete resolution of the abnormality.An incidental finding of an intense focus of  Positron Emission Tomography/Computed Tomography (PET/CT) fusion imaging is a novel multi-modality technology that allows the correlation of findings from two concurrent imaging modalities in a comprehensive examination. As altered glucose metabolism is characteristic for many malignancies, FDG-PET is mostly used in oncology for staging and therapy monitoring. It can also be seen as a physiologic activity in the bowel or muscles, in benign disease such as inflammation or post-traumatic reactive changes, or simply represent artefactual uptake from inaccurate CT attenuation correction in dense objects, often mistaken for cancer –3.The experienced PET/CT reader mostly manages to differentiate malignant from non-malignant FDG uptake based on accurate interpretation of the PET data by correlating with the CT anatomic information –3. This 18F-FDG focus in the apical segment of the left lower lobe on PET without any perceptible corresponding CT lesion (This case report looks at a 61 year-old lady who has a history of nodal positive right breast cancer treated by surgery and chemotherapy in 1996. She was referred for a PET/CT in February 2007 with the query of locoregional recurrence because of recurrent chest wall pain. PET/CT study revealed an intense T lesion . By virtT lesion .18F-FDG focus in the left lower lobe is highly suggestive of thromboembolus phenomenon attributed to a tagged platelet or mixed nature rather than red blood cells [In this case, the intense od cells , 5. Thisod cells .18F-FDG-PET focus without a detectable CT abnormality may rarely be a result of an early imperceptible structural lesion [An intense l lesion . CompletIn principle, an intense FDG focus is not entirely specific for malignancy. In general, CT is more sensitive than FDG-PET in the detection of pulmonary lesions, given its superior spatial resolution . TherefoThis is in line with the fact that more than one-third of scintigraphic equivocal lesions were correctly triaged by analysis of the morphological and anatomical details on the correlative CT study .Incidence of microembolus formation involving radiotracer injection has also been reported in patients with abnormal peripheral venous system and difficult phlebotomy, following the usage of an old intravenous cannula, or the presence of a hypercoagulability state in which the FDG is mixed with blood products prior to injection . The phe18F-FDG-PET pulmonary abnormality without detectable lesions on the correlative CT study is highly suggestive of artefacts caused by FDG microembolus. This report emphasises the importance of becoming familiar with the potential pitfalls of false-positive interpretations of PET.An intense


The human inferior frontal junction area (IFJ) is critically involved in three main component processes of cognitive control . As it overlaps with several areas in established anatomical labeling schemes, it is considered to be underreported as a functionally distinct location in the neuroimaging literature. While recent studies explicitly focused on the IFJ's anatomical organization and functional role as a single brain area, it is usually not explicitly denominated in studies on cognitive networks. However based on few analyses in small datasets constrained by specific a priori assumptions on its functional specialization, the IFJ has been postulated to be part of a cognitive control network. Goal of this meta-analysis was to establish the IFJ’s connectivity profile on a high formal level of evidence by aggregating published implicit knowledge about its co-activations. We applied meta-analytical connectivity modeling (MACM) based on the activation likelihood estimation (ALE) method without specific assumptions regarding functional specialization on 180 (reporting left IFJ activity) and 131 (right IFJ) published functional neuroimaging experiments derived from the BrainMap database. This method is based on coordinates in stereotaxic space, not on anatomical descriptors.The IFJ is significantly co-activated with areas in the dorsolateral and ventrolateral prefrontal cortex, anterior insula, medial frontal gyrus / pre-SMA, posterior parietal cortex, occipitotemporal junction / cerebellum, thalamus and putamen as well as language and motor areas. Results are corroborated by an independent resting-state fMRI analysis.These results support the assumption that the IFJ is part of a previously described cognitive control network. They also highlight the involvement of subcortical structures in this system. A direct line is drawn from works on the functional significance of brain activity located at the IFJ and its anatomical definition to published results related to distributed cognitive brain systems. The IFJ is therefore introduced as a convenient starting point to investigate the cognitive control network in further studies. The human inferior frontal junction area (IFJ) located at the junction of the inferior frontal sulcus and the inferior precentral sulcus had been largely neglected as a distinct region involved in cognitive control processes. Yet recent work explicitly addressing this brain area has attributed a major role to the IFJ related to three main component processes  -8. In anPeak coordinates from published human functional magnetic resonance imaging (fMRI) studies accumulated in the BrainMap database have previously been integrated in order to delineate the functional connectivity of designated brain regions based on their co-activation profiles using the activation likelihood estimation (ALE) method -14: ActiGoal of this meta-analysis was to aggregate implicit knowledge by means of coordinate representation  to characterize the functional connectivity profile of the IFJ on the basis of more than a decade of neuroimaging studies. These included a variety of studies investigating cognitive control yet there were no a priori constraints regarding the functional specialization of the IFJ. Do these aggregated data support the hypothesis of the involvement in the cognitive control network as proposed by Cole et al. ? Are theIFJ coordinates were adapted from a review article  aggregatData were retrieved from the BrainMap database using Sleuth (Version 1.2) ,21 on 30Regarding the left IFJ we identified 180 experiments/contrasts comprising 2,274 subjects in 139 articles . Consequently 2,764 of 8,301 locations reported in these papers were included in further analyses. For the right IFJ 131 experiments in 1,767 subjects reported in 111 articles (from 574 experiments in 1962 participants in these studies) matched these criteria. Thus 2,336 of 6,922 locations shown in these papers were included. A complete list of these articles is provided in the supplementary online material.The ALE random effects meta-analysis was conducted using the revised algorithm by Eickhoff et. al. in GingerALE (Version 2.0) ,23. AfteVisualizations were created using Mango (Version 2.3.2)  and an aIn order to confirm our meta-analytic results using a different approach to functional connectivity, we additionally performed a seed-based analysis of intrinsic fluctuations in a presumably independent sample.We therefore retrieved an anonymized resting-state fMRI dataset comprising 198 individuals . For each participant 119 volumes of 47 slices had been acquired with TR = 3 s using a 3 Tesla MRI-scanner . The dataset is publicly available from the 1000 Functional Connectomes Project (FCP)  and dataAs a prerequisite for further analyses, center coordinates of the meta-analytic seed as described above were converted to MNI space using the Lancaster transform  as imple3 and smoothed using a Gaussian kernel (FWHM = 6 mm).There is a range of widely accepted preprocessing steps in analyses of rs-fc data . The folResulting images were subjected to a conventional seed-based functional connectivity analysis based on Pearson linear correlation using REST (version 1.7) ,33, a MAResulting correlation maps were subjected to a one-sample t-test in SPM8. Results were thresholded at p < 0.001, corrected for multiple comparisons using family-wise error-rate (FWE). For comparison with the MACM results, peak-coordinates of rs-fc were transformed back to Talairach space. Local maxima of the correlation maps (at least 8 mm apart) were compared to meta-analytic results and assigned to brain areas as named in the results and discussion section of the meta-analysis. Additional brain regions were labeled based on the Talairach atlas ,36.The IFJ was associated bilaterally with a set of frontal and insular activations, mainly contained as separate peaks in an expansive cluster spanning anatomically distinct regions. It is exemplarily depicted in Figure There were bilateral co-activations in the PPC surrounding the intraparietal sulcus (IPS) mainly in the inferior and superior parietal lobule and partially extending into the precuneus , medial frontal cortex, anterior insula, PPC, occipito-temporal junction, premotor cortex, striatum and thalamus . The VLPFC was included in the maps of suprathreshold voxels yet it could not be identified as an independent local maximum Table . AdditioThe lateral frontal clusters of co-activation with the IFJ are formed by a set of confluent separate frontal cortical areas that have been well-characterized in the literature: A group of co-activation peaks rostral to the IFJ, predominantly assigned to the middle fontral gyrus in this analysis highly corresponds to an earlier definition of the mid-dorsolateral prefrontal cortex (mid-DLPFC) as discussed as constituent of a network subserving multiple cognitive demands  and otheObserved peak coordinates comprise a range of precentral co-activations. One of these peaks is located close to the frontal eye field. However it better corresponds to an adjacent region that has previously been associated with visuomotor hand conditional activity . Thus inIn addition to these lateral frontal areas the network observed in this meta-analysis comprises medial frontal, posterior parietal and inferior posterior cortical areas Tables . In thisOur results are highly concordant with the involvement of the IFJ in the cognitive control network (CCN) proposed by Cole et. al. . All itsA recent task-based meta-analysis of cognitive control identified a comparable fronto-parietal CCN including the IFJ. Yet it was labeled as part of the inferior frontal gyrus (IFG) based on its Talairach coordinates  in that case. The thalamus was also identified in an overall analysis across task-domains, yet it did not survive a formal conjunction analysis of different sub-domains of the construct of cognitive control .Moreover, as a rather consistent finding we observed parallel activations of the IFJ with the basal ganglia (mainly the lentiform nucleus) and the thalamus. Regarding thalamic activation there might be a pitfall in ALE analyses related to the more spherical structure of its nuclei compared to rather flat cortical areas: Thalamic activations arising from different nuclei may be concatenated to a single cluster or even lead to a common peak location near midline. This is especially problematic as the activations observed here are finally assigned to the medial-dorsal nucleus (MDN), indeed a near-midline structure. However, thalamic co-activations form rather separate sub-clusters in both thalamic hemispheres . As the VLPFC is wedged in Broca’s area in the left hemisphere and the anterior insula as well as the DLPFC in both hemispheres it might have been missed for that reason.The ACC has been extensively studied as a region crucial for cognitive control processes . TherefoFinally there were some pre- and postcentral areas of significant functional connectivity in the analysis of resting-state data that were not observed in the MACM analysis.In contrast to prior findings in resting-state fMRI analyses based on spatial independent component analyses (ICA)  the netwThere are different possible explanations for the fact that more regions were connected to the IFJ in the analysis of the resting-state dataset compared to the MACM results: Statistical power of both approaches is most likely different. In addition to some baseline-comparisons the BrainMap database contains coordinates from many well-controlled fMRI contrasts to delineate specific behavioral processes by including associated functions (e.g. stimulus-perception and motor responses) in control conditions. The additional correlations in the resting-state data may therefore also represent meaningful and necessary connections of the actual CCN components to brain areas relevant for direct interaction with the environment.Though the results of both methods are comparable the occasional differences of resting-state and MACM results point to the critical fact that current converging methods applied in the study of complex brain networks may oversimplify the actual functional organization of the human brain as they may not optimally account for the internal organization of such networks and their complex interdependences. It is a notable finding in this context that analyzing fMRI data with an increased temporal resolution using temporal ICA Smith et al. recently reported temporally-independent functional modes of spontaneous brain activity that overlapped with each other and networks known from conventional  analyses .As stated in the introduction the IFJ has been studied as a specific brain area in task-based fMRI and meta-analyses limited to a few task domains. Results can be summarized as an involvement of the IFJ in three main component processes of cognitive control  -8.The functional significance of similar fronto-parietal networks as observed in this study has explicitly or implicitly been assessed in numerous often highly specific task-based studies. As reported above, a recent meta-analysis has accumulated such findings based on the BrainMap database : In thatIn contrast to these previous meta-analytic approaches providing information about the IFJ  or the CThere is evidence in the meta-analytic results on CCN functions by Niendam et. al.  that in Taken together but potentially limited by the power to detect the involvement of certain brain regions in the different approaches reported the findings seem to support the notion that the IFJ is rather specifically involved in brain systems playing an important role in cognitive control compared to other aspects of brain function.http://www.neurosynth.org/) yields qualitatively comparable results regarding IFJ connectivity, thus at least we suppose that there is no specific bias regarding the BrainMap database and studies included.A potential limitation of the MACM approach may arise from the fact that, unlike for example in resting-state fMRI approaches to functional connectivity, results, though including several different functional domains, may be influenced by the overall distribution of tasks in the BrainMap database and correspondingly the distribution of tasks adopted by the whole functional neuroimaging community. However, as discussed above, our results are in line with recent literature regarding IFJ connectivity and a different approach to MACM  adopted Recently it has been argued that the IFJ is functionally dissociated from the directly adjacent posterior part of the inferior frontal gyrus (pIFG) . We haveThe selection of coordinates most exactly representing the IFJ in stereotaxic space is still a matter of debate. For the meta-analysis we aimed at high specificity regarding the IFJ as a distinct functional brain region without accidentally including other functionally defined areas in our seed. We therefore selected a relatively small ROI based on a large body of literature aggregated in a review based on a number of single studies and meta-analytical data . Yet theThough the main finding of this study is a considerably specific connection of the IFJ with brain regions previously characterized as a network engaged in cognitive control from a systems neuroscience perspective, it cannot be reliably deduced from these results that the CCN is indeed ‘only’ involved in cognitive control from a classical neuropsychological perspective. This would be some kind of problematic reverse inference . Though Using a metaanalytic connectivity modeling (MACM) approach with data from the BrainMap database of functional neuroimaging studies we characterized the functional connectivity profile of the inferior frontal junction area (IFJ) in terms of co-activations. Based on a large dataset of published peak activations our results confirm the notion that the IFJ is involved in a cognitive control network (CCN). The CCN comprises the dorsolateral prefrontal cortex, anterior insula , the medial frontal cortex/pre-SMA, parts of the dorsal premotor cortex and the posterior parietal cortex. In addition we identified connectivity of the IFJ with subcortical areas mainly pertaining to the medial dorsal nucleus of the thalamus and an inferior posterior region surrounding the cerebellar tentorium that cannot unequivocally be ascribed to the cerebellum or occipito-temporal junction based on the Talairach atlas. Assigning these locations to the CCN is compatible with prior observations mainly in resting-state fMRI and structural connectivity analyses. Other co-activations most consistently observed include language and motor-related locations. Results were largely confirmed in an additional resting-state fMRI analysis. However, using this approach, the VLPFC was less clearly identified and the CCN was complemented by the anterior cingulate cortex in line with previous observations.The CCN involving the IFJ has been proposed previously by using task-based and resting-state fMRI data in a number of subjects . Most ofStill, in light of results suggesting a role of impaired IFJ functionality in early dementia together with this information of functional connectivity of the IFJ in the CCN, the IFJ seems to be a promising starting point to investigate the cognitive control network in further studies and in particular in clinical populations.ACC: Anterior cingulate cortex; ALE: Activation likelihood estimation; BA: Brodmann area; CCN: Cognitive control network; DLPFC: Dorsolateral prefrontal cortex; FCP: 1000 Functional Connectomes Project; FDG-PET: Fluorodeoxyglucose positron emission tomography; FDR: False-discovery rate; fMRI: Functional magnetic resonance imaging; FWE: Family-wise error-rate; ICA: Independent component analysis; IFJ: Inferior frontal junction; IPS: Intraparietal sulcus; MACM: Metaanalytic connectivity modelling; MDN: Medial-dorsal nucleus of the thalamus; MNI: Montreal Neurological Institute; pIFG: Posterior part of the inferior frontal gyrus; PPC: Posterior parietal cortex; pSMA: Pre-supplementary motor area; ROI: Region of interest; rsFC: Resting-state functional connectivity; VLPFC: Ventrolateral prefrontal cortex.The authors declare that there are no potential competing interests.BS conceived the study and analyzed the data. BS and BP participated in study design, interpretation of the results and drafted the manuscript. Both authors approved the final manuscript.Table S2 – behavioral analysis of BrainMap-data on IFJ and CCN activations, List of References – 139 articles initially identified for the left IFJ, 111 articles initially identified for the right IFJ.Peak coordinates and Talairach atlas labels of brain areas exhibiting correlated activity with the IFJ in the resting-state fMRI analysis that do not conclusively correspond to the cognitive control network observed in the MACM analysis ., Click here for file


Retroviridae family, show interesting properties for CF gene therapy, since they integrate into the host genome and allow long-lasting gene expression. Proof-of-principle that LV vectors can transduce the airway epithelium and correct the basic electrophysiological defect in CF mice has been given. Initial data also demonstrate that LV vectors can be repeatedly administered to the lung and do not give rise to a gross inflammatory process, although they can elicit a T cell-mediated response to the transgene. Future studies will clarify the efficacy and safety profile of LV vectors in new complex animal models with CF, such as ferrets and pigs.Cystic fibrosis (CF) is a chronic autosomic recessive syndrome, caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, a chloride channel expressed on the apical side of the airway epithelial cells. The lack of CFTR activity brings a dysregulated exchange of ions and water through the airway epithelium, one of the main aspects of CF lung disease pathophysiology. Lentiviral (LV) vectors, of the  Furtheric cells .Staphylococcus aureus and Haemophilus influenzae are early colonizers, whereas Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) often occur later, resulting in progressive loss of lung function and premature death. Several hypotheses link mutations in CFTR to development of lung disease in CF, whose hallmarks are bacterial infection with opportunistic pathogens and a vicious neutrophil-dominated chronic inflammatory response [In vitro models of polarized human cystic fibrosis bronchial epithelial cells demonstrate an isotonic reduction in the volume of airway surface liquid, and consequently impaired movement of the overlying mucus [Lung disease in CF patients reflects chronic infection of the conducting airways with a surprisingly low number of bacterial species. response –9. Curreresponse . Reducedresponse . In vitrng mucus . The impng mucus .2.Cystic fibrosis should be an ideal candidate for gene therapy, for four main reasons: (1) it is a single gene defect; (2) it is a recessive condition, with heterozygotes being phenotypically normal ; (3) the main pathology is in the lung, which is accessible for treatment; and (4) it is a progressive disease with a virtually normal phenotype at birth, offering a therapeutic window.In vitro and in vivo studies have suggested that only 5–10% of normal CFTR function is required to reverse the chloride channel defect [in vitro and in vivo studies suggest that nearly every cell in the sample must be corrected with CFTR to reverse the excess activity of ENaC [l defect –15, alth of ENaC ,16,17. TIn clinical trials to date, two main vector systems have been harnessed to deliver the CFTR cDNA with appropriate promoter into host cells . Firin vivo, the delivery of CFTR gene therapy must be extremely efficient to overcome the physical barriers such as thick tenacious mucus and pulmonary surfactant, as well as the logistical difficulties resulting from many viral receptors which are expressed on the basolateral membrane of bronchial epithelial cells. Much of the morbidity and mortality seen in cystic fibrosis patients is related to pre-existing pulmonary inflammation; consequently, it is important that any vector system does not cause further inflammation. The ideal vector system would have the following characteristics: (1) an adequate carrying capacity; (2) to be undetectable by the immune system; (3) to be non inflammatory; (4) to be safe to the patients with pre-existing lung inflammation; (5) to have an efficiency sufficient to correct the cystic fibrosis phenotype; and (6) to have long duration of expression and/or the ability to be safely re-administered. Furthermore, to provide therapeutic benefit 3.Retroviridae family, because of their intrinsic ability to integrate into genomic DNA. However, oncoretroviral vectors have shown limited success because of the requirement of cell replication for integration. Lentiviral vectors (LV) have overcome these limitations showing the ability to transduce nondividing cells and provide a more efficient transgene expression if compared with oncoretroviral vectors. Higher efficiency of LV vectors is likely due to the stability of transcripts derived from the lentiviral provirus because of the strong 3′ poly(A) signal that causes an increased cytoplasmic accumulation of the RNA of the transgene [et al. were the first to demostrate that a LV vector derived from human immunodeficiency virus type 1 (HIV-1) could provide efficient in vivo delivery, stable integration and long-term expression of transgenes into non-mitotic cells such as neurons [Many viral vector systems have been developed for gene therapy applications, but most of these vectors are non-integrating, which limits their usefulness for transgenesis. The best characterized integrating viral vectors originate from the ransgene . Naldini neurons ,26.gag gene, viral enzymes such as a protease derived from the pro gene, Rnase H, reverse transcriptase and integrase derived from the pol gene. The virion is enveloped by the host cell membrane containing the viral protein derived from the env gene. After virus entry into the cell, reverse transcription produces viral double strand DNA which enter in the nucleus and integrate randomly and permanently in the host genome, resulting in a provirus state. In the provirus, the lentiviral genome is flanked on both sides by the long terminal repeats (LTR), which contain the U5, R and the U3 regions. The 5′-LTR can act like an RNA pol II promoter. The 3′-LTR acts to terminate transcription and promote polyadenylation. The LTR also has recognition sequences necessary for integration into the genome. Tat is an accessory viral protein which activates the 5′ LTR promoter, whereas Rev controls the amount of RNA splicing as well as the RNA export into the cytoplasm. At the end of cycle infection, viral genomes are packaged, enveloped and released from the cells. Two important cis-acting DNA elements, the central polypurine tract sequence (cPPT) and the postregulatory element (PRE), derived from woodchuck hepatitis B virus, are also included in the transfer vector to enhance the transduction efficiency and transcript stability. In particular, cPPT has been shown to facilitate nuclear translocation of pre-integration complexes, and PRE acts at the post-transcriptional level, by promoting nuclear export of transcripts and/or by increasing the efficiency of polyadenylation of the nascent transcript [Although various lentiviruses from different species  have been used to generate gene transfer vectors (reviewed in ) only HIanscript –30. Anotanscript .gag-pol genes; 2) a post-transcriptional regulator for gag and pol expression as well as nuclear RNA export encoded by the rev gene; 3) the vector carrying the transgene to be transferred to the target cells; 4) an heterologous glycoprotein i.e., vesicular stomatitis virus glycoprotein G (VSV-G) which pseudotypes the vector in order to increase its tropism. All structural proteins are HIV-1-derived, except the envelope because of the restricted host range of the HIV-1 env glycoprotein. Association of VSV-G with viral cores derived from lentiviruses, results in an high titer production and in a broad tropism of the vectors. Finally, deletion of the coding sequences for viral gene products renders the LV gene transfer vectors replication incompetent and makes 6–8 kb available for the insertion of desired genes, in this case a large transcriptional units such as an epithelial-specific promoter expressing the CFTR gene [The development of lentiviral vector system, in order to achieve a gene transfer agent, is based on modifications of a native lentivirus genome. Presently, the lentivirus-based gene delivery system is constituted by four components: 1) the packaging elements like structural proteins and enzymes involved in the formation of the viral particles, derived from the FTR gene .http://www.wiley.co.uk/genetherapy/clinical/.Lentivirus vectors only recently made their debut in human clinical trials for hematological disorders, such as β-thalassemia and sickle cell disease . In the 4.in vivo application of gene transfer vectors in the airway epithelium. Moreover, the expression of receptors for many commonly used viral vectors is more abundant on the basolateral membrane than on the apical side of the respiratory epithelium and they are hardly accessible because of the airway tight junctions [Gene therapy of cystic fibrosis lung disease needs an efficient delivery and a persistent expression of the CFTR transgene into the airway epithelium. LV vectors appear to be promising vehicles for gene delivery into respiratory epithelial cells due to their ability to infect nondividing cells and to integrate into the host cell genome mediating long-term persistence of transgene expression. Various physical and pathological barriers, inherent also to CF lung, including mucus plugs, glycocalyx components and mucins at the apical surface of the epithelia, represent an obstacle for viral attachment to the target cells and inhibit viral transduction from the luminal face ,35. Thesunctions –39.5.Pseudomonas aeruginosa infection on adenovirus-mediated gene transfer was evaluated by two studies [Pseudomonas. Increased adenovirus-specific CD8 cytotoxic T cell activity was considered by the authors as one of the underlying mechanisms [To mimic the persistent and progressive infection typical for CF patients, the bacteria can be encapsulated into agar beads and injected to the mouse lung via the trachea . The imp studies ,42, whicchanisms . AlthougOther studies have shown that the capacity of LV vectors to transduce a fully differentiated respiratory epithelium is lower compared with an undifferentiated one. A first generation VSV-G pseudotyped HIV vector was able to transduce wild type CFTR into poorly differentiated human bronchial xenografts and its expression corrected the chloride transport defect and reverted the bacterial killing activity; however gene transfer was less efficient when the epithelium was fully differentiated . A seconPseudotyping the vectors with heterologous envelopes and pre-conditioning of the airway tight junctions are the strategies currently used to overcome the paucity of receptors for lentivirus on the apical surface of the respiratory epithelium and to allow the access to the basolateral compartement receptors .Most of the studies on HIV-1-derived lentiviral mediated gene transfer in the airway epithelium refer to gene delivery mediated by LV vectors pseudotyped with VSV-G which is useful not only to allow virus particle concentration, but also to modulate virus interaction with the host immune system and to broaden its host range.in vivo gene transfer into a fully differentiated epithelium unless disruption of the epithelial barrier integrity obtained with calcium-chelating agents like EGTA [et al. demostrated that direct in vivo delivery of VSV-G pseudotyped HIV-1- based vector to the nasal epithelium of rats and mice failed to mediate gene transfer, but injury of the epithelium with sulfur dioxide (SO2) 1–2 hours before vector delivery strongly enhanced transduction levels of the nasal epithelium. SO2 pre-treatment also enhanced gene transfer to the tracheas of rodents [VSV-G LV vectors have been shown to be inefficient in ike EGTA , inhalatike EGTA , or modiike EGTA  is obtai rodents .in vitro transduction of fully differentiated human CF airway epithelia with a VSV-G pseudotyped FIV vector formulated with EGTA [Calcium-chelating agents may be more useful in the context of CF lung disease, since they transiently disrupt tight junctions . CFTR fuith EGTA . In the in vitro model of polarized airway epithelial cells. In particular, heparan sulfate, chondroitin sulfate A and B seem to be strongly involved in the infection of respiratory cells mediated by a VSV-G pseudotyped LV vector [The attachment and entry mechanism of VSV-G pseudotyped LV vectors into the airway epithelial cells and subsequent infection are still unknown. We demonstrated a role of glycosaminoglycans (GAGs) in gene transfer mediated by a third generation VSV-G pseudotyped LV vector in an V vector . HoweverV vector , indicatOther biological modifers of the membrane permeability have been actively searched . LPC preIn a recent work, a VSV-G pseudotyped LV carrying LacZ or CFTR gene was delivered in the nostrils of mice 1 hour after pre-treatment with 0,3 % LPC via inhalation-driven instillation. LacZ transgene expression lasted over 24 months and was restricted to the anterior regions of transitional and respiratory epithelium within the dosed nasal airway affecting primarily ciliated and nonciliated cells, but also a low number of secretory and basal cells. At 24 months when LacZ gene expression was still present, the number of cells transduced was significantly reduced compared to the initial 1 week level. Significant correction of CFTR function, measuring nasal potential, was present at 1 and 12 months compared to untreated mice . AlthougAutografa californica [Agents disrupting tight junction structure and disturbing membrane permeability may not be useful in the context of CF lung disease, since CF lungs are replenished with bacterial products and inflammatory mediators. To overcome the inability of VSV-G-pseudotyped lentiviral vectors to efficiently infect the airway epithelium in the absence of injuring agents, other heterologous envelope glycoproteins, such as those from the Marburg virus or the Zaire subtype Ebola virus ,57, the ifornica  and the ifornica , were usEx-vivo transduction of human trachea by EboZ-pseudotyped vector resulted in high levels of LacZ expression as compared to VSV-G pseudotyped vector. When the Ebo-Z pseudotyped virus was injected in the trachea of mice, 30% of the entire tracheal epithelium was transduced at day 28 and 24% at day 63. Interestingly, high expression was observed in submucosal glands, while transduction efficiency was lower in epithelia of more distal lung  cells.HIV-1-based vectors pseudotyped with the envelope from the Zaire strain of the Ebola virus (EboZ) gave efficient transduction (70% of positive cells) when applied to the apical side of polarized epithelia . Ex-vivoA. californica conferred to a FIV lentiviral vector the ability to transduce differentiated human airway epithelia from the apical surface with higher efficiency than VSV-G LV [Pseudotyping with the GP64 derived from the baculovirus VSV-G LV . Intranaet al. compared the efficiency of transduction of mice nasal airways by a VSV-G pseudotyped vector (carrying LacZ gene) after a pre-treatment with LPC, and the same vector pseudotyped with GP64 envelope without any pre-treatment. In the absence of pre-conditioning the GP64-pseudotyped lentiviral vector resulted significantly less efficient than the VSV-G pseudotyped vector after LPC treatment. The levels of transduction were similar for the two vectors when a pre-treatment with LPC was used. The cell types transduced were essentially the same with the majority of cells transduced being respiratory (ciliated cells). However, while the VSV-G LV resulted in persisting gene expression, transduction with the GP64 LV resulted in gene expression that decline to undetectable levels over six months, whether or not an LPC treatment was used. These results suggest that the GP64-pseudotyped LV vector, in contrast to the VSV-G-pseudotyped vector, was unable to transduce progenitor cells for respiratory epithelium, even when used in combination with LPC pre-treatment [Kremer reatment .in vitro and in vivo were obtained with other envelope proteins, such as those from influenza virus [et al. demostrated that an HIV-1-derived LV vector pseudotyped with influenza virus hemagglutinin could efficiently transduce, via the apical membrane, differentiated cultures of human and murine airway epithelial cells, and mouse tracheal epithelium in vivo, with a preference for ciliated cells [Different results in transducing airway epithelial cells za virus , Ross Riza virus , and Jaaza virus . For exaed cells . These v6.in vivo usually extinguishes within 2 to 3 weeks, concurrent with the development of inflammation. This is caused by the rapid activation of potent CD8 and CD4 T cell responses against both the viral antigens and the transgene. In addition, activation of B cells by viral capsid proteins, leading to the production of neutralizing antibodies, limits effective readministration of the vector. When administered to the lung, adenoviral vectors activate cytolytic transgene-specific T cells [in vivo delivery of a HIV-1-derived vector in the tracheas of C57Bl/6 mice did not induce inflammatory infiltrates post-infection [Gene therapy viral vectors are known to elicit transgene- or viral protein-specific immune responses . This is T cells . This drnfection , and thenfection . Intravenfection  and resunfection , whereasnfection –68.in vivo re-administration of lentiviral vectors to respiratory epithelia, associated with an increase expression of reporter or therapeutic transgenes, without the development of innate and adaptive responses that represent a significant limitation to clinical applications for airway diseases for other virus vectors, such as adenoviruses or adenoassociated viruses. In this study, GP64-FIV was repeatedly delivered to murine nasal epithelia, in particular four groups of mice received 1, 3, 5 or 7 doses of the vector carrying the luciferase transgene over the same number of consecutive days. Mice underwent bioluminescence imaging 1, 4, 8 and 12 weeks following the final dose demonstrating additive increases in transgene expression with repeat dosing, probably due both to an increase of percentage of cells expressing a transgene and in the number of transgene copies/cells. The levels of anti-GP64 IgG were measured in sera and in bronchoalveolar lavage (BAL) fluid one week after a booster dose of the vector, showing low levels of anti-GP64 IgG antibodies in sera for all groups of mice and higher levels in BAL fluid; importantly, the levels of inactivating antibodies measured with a neutralizing antibody assay resulted below the limit of detection indicating that though an adaptive immune response is mounted against the vector, it is insufficient to block gene transfer. Although these results are encouraging for the expression of a therapeutic protein over the life, recently published data argue against this scenario. Limberis and colleagues demonstrated that transient expression of Green Fluorescent Protein (GFP) at day 90 in alveolar epithelium following an intratracheal injection of VSV-G-pseudotyped HIV-1-derived vector to the mouse lung is due to transgene- and gag-specific T-cell activation [A GP64-pseudotyped FIV vector was also used to investigate the efficacy of repeated administration to the airways . In parttivation . These rtivation . It is ttivation .Lineage-regulated transgene expression will be necessary for effective, long-term gene therapies for disorders affecting the lung. Most gene therapy clinical trials directed to respiratory epithelium to date have used constitutive promoters such as the promoter from the cytomegalovirus (CMV) or the promoter in the retroviral LTR. However, long-term animal studies have indicated that, over time, transgene expression can be silenced from viral promoters ,74, or cet al. constructed a series of lentiviral vectors with regulatory elements to obtain lung-specific transgene expression: the surfactant protein C promoter (SPC) for alveolar epithelial type II cell (AECII) expression, the Clara cell 10-kDa protein (CC10) for Clara cell expression in the airway, and the Jaagsiekte sheep retrovirus (JSRV) promoter for expression in both cell types. Transgene expression from the SPC and CC10 vectors was restricted to alveolar epithelial type II and Clara cell lines, respectively, while expression from the JSRV vector was observed in multiple respiratory and non-respiratory cell types. After intratracheal delivery of lentiviral vector to mice, transgene expression was observed in AECII from the SPC lentiviral vector, and in Clara cells from the CC10-promoted lentivector. Transgene expression was not detected in non-respiratory tissues after intravenous delivery of CC10 and SPC lentiviral vectors [Hendrickson  vectors .in vitro in airway epithelial cells. A third-generation VSV-G LV vector did not induce those pro-inflammatory signals usually observed in human respiratory epithelial cells exposed to classical stimuli, such as TNF-α, or other gene transfer vectors previously studied, such as adenovirus-derived vectors, except for a mild and transient induction of transcription of IFN-γ in an epithelial tracheal cell line [Innate immune responses to LV vectors in the context of lung gene therapy have not been thoroughly investigated. Epithelial respiratory cells function as part of the innate immune responses and not merely only as a physical barrier. Multiple substances with pro- and anti-inflammatory as well as antimicrobial activities are secreted by airway epithelial cells –79. Airwell line . These r7.In vivo GFP gene transfer to fetal respiratory epithelium has been achieved in rabbits after direct VSV-G LV intra-tracheal or intra-amniotic administration, hovewer tracheal delivery was associated with high fetal mortality rate (75%) and an elevated degree of transgene expression in the control ipsilateral uninjected fetuses, likely due to contamination of fetomaternal circulation, compared with intra-amniotic administration [Recent studies have explored the feasibility of LV vector-mediated gene expression in the fetal airway epithelium, considering the advantage of a therapeutic intervention before clinical onset and in the absence of a functional immune response. stration .Injection of VSV-G LV carrying GFP reporter gene into the lung parenchima of fetuses of rhesus monkeys resulted in transgene expression in pulmonary vasculature and alveolar cells in term fetuses and three month aged pups . This woIn a recent study an HIV-based lentiviral pseudotyped vector with the baculovirus GP64 envelope was applied to the fetal, neonatal or adult airways. Fetal intra-amniotic administration resulted in transduction of approximately 14% of airway epithelial cells, including both ciliated and non-ciliated epithelia of the upper, mid and lower airways, with a negligible alveolar or nasal transduction. A significant transduction of the airway epithelium mainly in distal lung and alveoli was detected at 1 year after application following neonatal intra-nasal administration, while in the adult the majority of transduction was restricted to alveoli. In contrast, VSV-G pseudotyped virus transduced only after adult and neonatal application and no transduction was observed after fetal administration .Taken together, these results strongly indicate that fetal airway epithelium can be transduced by LV vectors and that the pseudotype can be fundamental as in the transduction of adult airway epithelium. The early intervention with an integrating gene transfer vector able to transduce the lung via the lumen, avoiding immune elimination of transgenic protein and probably increasing lung stem-cell transduction to result in a persistent gene expression, while ethically complex, may be a successful therapeutic approach for CF.8.in vitro cultures of human polarized airway epithelia and small conventional animal models, the apical surface of the airway epithelium poses a strenous barrier to efficient transduction from this side, due to the lack of apical receptors. Moreover, while other viral vectors have been considered in light of the extracellular barriers, such as the thick and dehydrathed mucus layer covering the airway epithelium, comprehensive studies on this aspect are still lacking. The alternative to the use of tight junction disrupting agents and membrane detergents, which could possibly enhance the leakage of bacterial products and inflammatory mediators into the submucosa and further damage the CF lung, is the pseudotyping of LV envelope with heterologous proteins from other viruses. This approach should be evaluated in terms of the safety issue, when pathogen viruses are used [LV vectors are endowed with interesting features which make them promising agents for application to gene therapy of CF lung disease. However, they present some drawbacks which need to be addressed before they can enter into the clinic. Although the issue of attachment and entry mechanisms has not been studied in detail, it seems that, at least in are used . Anotherare used ,87.i.e., the correction of the electrophysiological defect) of LV vectors have been collected in CF mouse models. For both anatomical and physiological reasons, mice do not show a respiratory syndrome that is equivalent to that found in humans [Most of the data on the efficacy (n humans . To deven humans ,90.The issue of immune response to transgene- and viral genes is a caveat in the use of viral vectors for human gene therapy. The first indications on the elicitation of innate and adaptive responses to LV vectors have been produced, although this field is in its infancy. It seems that LV vectors have a low pro-inflammatory potential, while they can stimulate a transgene-directed T cell response. Novel animal models may aid also in elucidating this issue.


Limosa limosa islandica) to quantify sexual segregation at scales ranging from the occupation of sites throughout the non-breeding range to within-site differences in distribution and resource use. Throughout the range of this migratory shorebird, there is no evidence of large-scale sex differences in distribution during the non-breeding season. However, the sexes differ in their selection of prey types and sizes, which results in small-scale sexual segregation within estuaries. The scale of sexual segregation therefore depends on the scale of variation in resource distribution, which, in this system, is primarily within estuaries. Sexual segregation in within-site distribution and resource use means that local-scale anthropogenic impacts on estuarine benthic prey communities may disproportionately affect the sexes in these migratory shorebirds.In migratory species, sexual size dimorphism can mean differing energetic requirements for males and females. Differences in the costs of migration and in the environmental conditions occurring throughout the range may therefore result in sex-biases in distribution and resource use at different spatial scales. In order to identify the scale at which sexual segregation operates, and thus the scale at which environmental changes may have sex-biased impacts, we use range-wide tracking of individually color-ringed Icelandic black-tailed godwits ( Sexual size dimorphism is a widespread phenomenon across the animal kingdom Darwin . For dimMigratory shorebirds often range over large geographic areas within their annual cycle, and the variation in form and magnitude of sexual dimorphism found in this group encompasses virtually the entire range found in birds , in which larger billed females are commoner in the southern part of the range (Nebel Limosa lapponica), with the larger females being proportionately more abundant in higher quality foraging patches within mudflats   is a sexually dimorphic shorebird , east England , Iceland (519 ringed at sites throughout the country since 1999), and west Portugal . Regular scanning of godwit flocks throughout the migratory range by a network of >2000 volunteer observers has resulted in >60% of all marked individuals being observed during winter months  or female if above 92 mm (n = 42) , when sex differences in nest attendance patterns can mean that one sex is likely to be caught more frequently; (3) during autumn migration and winter , when the sexes are less likely to differ in capture probability. Chi-squared goodness-of-fit tests were used to compare the observed number of color-ringed male godwits from each capture period wintering across the seven regions to the expected sex ratio .To account for sex differences in capture probability in relation to location and time of year, sex ratio was estimated separately for individuals caught during three distinct periods of the annual cycle: (1) arrival in Iceland during April , when males are likely to be proportionately more abundant due to pressure for early arrival on the breeding sites  and polychaetes (mostly Hediste diversicolor), although smaller items such as the gastropod Hydrobia ulvae are also occasionally consumed  were classified as polychaete or bivalve-foraging, according to the prey type being consumed by the majority of focal individuals (no flock was ever recorded in which the majority of godwits were foraging on gastropods). In addition, the bill length and overall body size of each focal individual was used to assign a sex to each bird, including color-ringed individuals of known sex, which allowed estimating error rates in assigning sex during observations. Chi-squared tests of independence were used to test for differences in the number of male and female godwits (1) present in polychaete or bivalve-foraging flocks, and (2) foraging on polychaetes or bivalves within foraging flocks. Focal observations of prey intake rates of color-ringed individuals (n = 200 observations) for which bill length had been measured on capture  were used to test for differences in prey size classes attained by godwits with differing bill lengths using Fisher's exact test of independence. In order to test for sex-specific differences in site use, the proportion of re-sightings of individually color-ringed male and female godwits on sites dominated by polychaetes was compared with a Mann–Whitney U-test. All observations were carried out by a single observer (JAA) using a ×20–60 telescope.Icelandic godwits foraging on estuarine flats on the Tagus estuary in Portugal, one of the key wintering site for this population  to give a measure of total handling time. Prey type and size class were recorded for all of these observations . Differences in extraction and handling times between prey size classes were tested with one-way analysis of variance (ANOVA) and post-hoc tests. The ash-free dry mass content of the average prey size item within each category  and H. diversicolor (22.0 kJ/g) ; breeding in Iceland ; and non-breeding season  ; breeding in Iceland 71%); non-breeding (69%). Across the seven major wintering locations for Icelandic godwits, ranging from Britain and Ireland to Portugal, the observed number of males did not differ significantly from the expected proportion : arrival in Iceland  had high densities of S. plana and the remaining two (13 and 18) comprised relatively low densities of both prey species. Accordingly, across the 18 sites at which foraging godwits were recorded, the proportion of godwits in each flock foraging on S. plana or H. diversicolor also ranges from the entire flock foraging on S. plana to more than 95% foraging on H. diversicolor (average number of flocks observed per site: 5.4 ± 0.8 SE)  males, whereas flocks foraging on H. diversicolor comprised an average of ca. 52.5% (± 2.7 SE) females . The number of males and females foraging on different prey within these flocks also differed  were foraging on S. plana while 56% of all observed females (n = 1222) were foraging on H. diversicolor. This difference was apparent regardless of the predominant prey type being consumed by the majority of individuals in the flock, with male godwits consuming proportionately more S. plana and females taking proportionately more H. diversicolor  and females on 80% ± 3.6 SE of all observations (n = 227). The locations of color-ringed females (n = 29) and males (n = 50) were recorded during fortnightly surveys of the estuarine sites used by godwits across the Tagus estuary, with each godwit being recorded at least once every month . In order to explore whether the inclusion of incorrectly sexed individuals may have influenced the observed sex differences in use of prey resources, the use of sites dominated by H. diversicolor . Moreover, only 24% of the males were ever recorded in H. diversicolor-dominated sites, while only 29% of the females were never recorded in these sites , ca. 50% are large or very large  will be within reach of even the smallest godwit bill   than on more northerly estuaries within the range  experiencing higher food demands, and Icelandic godwits wintering in Portugal require an additional 1390 kJ to complete their migration to Iceland than conspecifics wintering in England or Ireland (Alves et al. ses Fig. . Male goiet Fig. A and areiet Fig. , and verMale and female Icelandic godwits show distinct prey preferences at the estuary-scale, but do not display a clear pattern of large-scale sexual segregation across the winter range, probably because the distribution of the two main prey types varies little across this range (Piersma et al. Estuarine invertebrate populations are subject to a wide range of anthropogenic impacts. On many estuaries through Europe, extensive shellfishing (Piersma et al.