Oral cancer survival rates increase significantly when it is detected and treated early. Unfortunately, clinicians now lack tests which easily and reliably distinguish pre-malignant oral lesions from those already transitioned to malignancy. A test for proteins, ones found in non-invasively-collected whole saliva and whose abundances distinguish these lesion types, would meet this critical need.To discover such proteins, in a first-of-its-kind study we used advanced mass spectrometry-based quantitative proteomics analysis of the pooled soluble fraction of whole saliva from four subjects with pre-malignant lesions and four with malignant lesions. We prioritized candidate biomarkers via bioinformatics and validated selected proteins by western blotting. Bioinformatic analysis of differentially abundant proteins and initial western blotting revealed increased abundance of myosin and actin in patients with malignant lesions. We validated those results by additional western blotting of individual whole saliva samples from twelve other subjects with pre-malignant oral lesions and twelve with malignant oral lesions. Sensitivity/specificity values for distinguishing between different lesion types were 100%/75% (p = 0.002) for actin, and 67%/83% (p<0.00001) for myosin in soluble saliva. Exfoliated epithelial cells from subjects' saliva also showed increased myosin and actin abundance in those with malignant lesions, linking our observations in soluble saliva to abundance differences between pre-malignant and malignant cells.Salivary actin and myosin abundances distinguish oral lesion types with sensitivity and specificity rivaling other non-invasive oral cancer tests. Our findings provide a promising starting point for the development of non-invasive and inexpensive salivary tests to reliably detect oral cancer early. Oral cancer develops in stages, transitioning from a normal oral epithelium, to a pre-malignant, dysplastic oral lesion, to a malignant lesion, most commonly in the form of oral squamous cell carcinoma (OSCC). For those who develop OSCC, the overall 5- year survival rate is approximately 50%, unchanged over the last 30 yearsUnfortunately, pre-malignant and malignant lesion types cannot be distinguished simply by visual inspection; instead invasive tests are used. The current gold standard for characterizing lesions, histological analysis of tissue biopsiesNon-invasive, inexpensive and accurate tests, ones which foster early and frequent screening, would, if available, transform the early detection of oral cancer. Whole saliva collection is non-invasive, providing inexpensive collection of ample amounts of sample for analysis in an on-demand mannerSo far there are only a few proteomic studies relevant to developing such protein-based tests. Analyzing biopsied tissues, two such studies were recently published by Siu and colleagues, resulting in the identification of several promising biomarkers. One study compared protein abundance levels between healthy tissue and pre-malignant dysplastic tissueSadly, given their great potential for discovering promising biomarkers for early detection of oral cancer, no proteomic studies in saliva have been undertaken comparing subjects with pre-malignant and malignant lesions. Despite the urgent need for such studies, these are not easily undertaken. A main reason for this is the difficulty in collecting even modest numbers of samples from subjects with pre-malignant oral lesions, as most clinicians see these subjects sporadically and lack coordinated infrastructure for sample collection and analysis.In this first-of-its-kind study, we have overcome this difficulty, collecting saliva samples from subjects with pre-malignant oral lesions and using advanced mass spectrometry-based proteomics to discover salivary protein abundance differences between these subjects and those with malignant oral lesions. Via bioinformatic analysis and biochemical validations, we have identified myosin and actin as promising salivary biomarkers capable of distinguishing between subjects with pre-malignant and malignant lesions. Our results provide a basis for the potential development of a salivary-based test for the early detection of oral cancer.The study was done with informed written consent of all sample donors using a human subjects protocol approved by the Institutional Review Board at the University of Minnesota (IRB study number 0001M34501).Unstimulated whole saliva was collected from two groups: subjects with pre-malignant dysplastic lesions diagnosed by scalpel biopsy and histology, and subjects with invasive malignant lesions, diagnosed as primary OSCC by scalpel biopsy and histology. All OSCC patients were non-metastatic, and had either T1 or T2 stage tumors. Subjects with either lesion type  had not undergone any treatment or surgery (including tissue biopsy) at the lesion site prior to saliva collection. Unstimulated whole saliva was obtained using a standard, controlled protocolFor proteomic discovery studies and western blot validation studies, samples from subjects falling in the two groups were prepared identically. Whole saliva was centrifuged at 3000 x g at 4°C, the supernatant containing the soluble fraction of saliva proteins removed, and the cell pellets washed with PBS and lysed to obtain cellular proteins, as previously describedFor proteomic discovery experiments, 200 µg of denatured and reduced protein from the soluble portion of saliva from each of four subjects with pre-malignant lesions was pooled together; likewise, 200 µg of denatured and reduced protein from the soluble portion of saliva from each of four subjects with pre-malignant lesions was pooled together. Each separate sample pool was digested overnight with trypsin as previously describedMass spectrometry was done using an LTQ linear ion trap mass spectrometer  using Xcalibur version 2.0 operating software. In order to detect low mass reporter ions from the iTRAQ reagent, the instrument was operated using pulsed Q dissociation (PQD) mode. We have described in detail the procedure used for PQD analysis of iTRAQ reagent labeled peptides previously, as well as the HPLC and LTQ operating conditions for tandem mass spectrometry (MS/MS) analysisMS/MS spectra were searched using SEQUEST(15)  against a non-redundant human protein sequence database from the European Bioinformatics Institute  comprised of both the forward and reversed sequences, using parameters as previously described2 scale: normalized ratio  = 2[log2(protein ratio) - log2(median ratio)].Relative protein abundance ratios between the two subject groups were calculated from iTRAQ reagent reporter ion intensities, using an in-house developed automated software program, as we have previously describedTo determine proteins showing significant abundance differences between the different subject groups, mean and standard deviation for each ratio was calculated across all matched MS/MS spectra, and used to determine proteins showing significant differences in abundance between the two groups. Proteins with abundance differences greater than one standard deviation from the mean value were deemed to be significantly changing.Proteins from the soluble fraction of whole saliva showing significant abundance differences between the two subject groups were analyzed using Ingenuity Pathway Analysis (IPA) software  were mapped to canonical pathways. For each dataset analyzed, a corresponding control dataset was also analyzed containing proteins not showing abundance differences between the two stages of oral cancer development. A relative abundance window centered at unity was used to generate a random dataset of control proteins numbering the same as the proteins showing differences in relative abundance. The significance of the association between proteins within each dataset and the canonical pathway was measured in two ways: 1) A ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules that map to the canonical pathway is displayed. 2) Fisher's exact test was used to calculate a p-value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone.Commercial antibodies used for western blot validation experiments were as follows (product numbers in parentheses): myosin regulatory light chain, monoclonal antibody , and actin, monoclonal anti-actin Ab5 .Thirty µg of protein from each individual subject analyzed in validation experiments , along with thirty µg of control protein from a control saliva pool, and molecular weight markers was loaded in separate lanes and separated via SDS-PAGE on a Bio-Rad minigel apparatus. The proteins were transferred to a PVDF membrane and the membrane blocked with 1xPBS containing 2.5 mg/ml BSA and 0.25% (v/v) Tween 20, followed by incubation with the primary antibody, diluted in PBS at concentrations suggested by the antibody manufacturer. After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP).For detection, membrane-bound proteins recognized by the HRP conjugated secondary antibody were visualized by chemilumenescence using the SuperSignal West Pico ECL Substrate (Thermo Scientific). Developed films were imaged on a Bio-Rad GS-700 Imaging Densitometer, and quantification performed on background-subtracted bands with Molecular Analyst (v. 21) Software. Densitometric values of all individual samples were normalized against the signal from the common control. All blots from soluble saliva were performed in triplicate, and the mean values were used for statistical analyses.Image files generated from densitometry detection were imported to Adobe Photoshop version 9.0.2. Images were cropped to show detected bands at correct, expected molecular weight regions, and then converted to grayscale and subjected to automated contrast adjustment using the default program settings.For each candidate biomarker, the statistical difference between the pre-malignant and malignant samples was compared using the two-sample t-test with the Welch-Satterwaite method for samples of different variances. If the difference is statistically significant at the 5% level, an optimal cut-point is determined at which the Youden's Index is maximizedSupplementary Table S1 contains relevant information on all of the subjects whose saliva was analyzed in our studies.For biomarker discovery, we employed a strategy implementing several parts Figure 1Supplementary Table S2) was rigorously filtered ensuring estimated spectral false positive rates below 1% and minimal redundancy  tool to group into functional pathways those proteins showing abundance differences between the subjects with pre-malignant lesions and those with malignant lesions. We hypothesized that such grouping would reveal proteins within our catalogue from functional pathways with possible ties to cancer progression, which we would assign high priority. As a control, 198 randomly selected proteins showing no significant abundance difference between the two subject groups were also analyzed. Through this analysis pathways associated with the transition from pre-malignancy to malignancy would be significantly represented in the dataset of proteins changing in abundance, but not in the dataset of control proteins.Our analysis showed the three most significantly represented pathways to be acute phase response, followed by actin cytoskeleton signaling and tight junction signaling Figure 2−35, Conversely, the next two most significant categories, actin cytoskeleton signaling and tight junction signaling, were assigned high priority given their role in transforming cells from a pre-malignant state to an invasive, malignant stateIn addition to the myosin and actin, we also prioritized proteins from our catalogue via manual inspection seeking to select other candidate biomarker proteins warranting validation. Standing out were 28 different keratin proteins identified and quantified in our proteomics analysis, with the majority of these showing significant increases in abundance between the pre-malignant and malignant sample pools. Among the keratins, we selected keratin 19 for validation, given past descriptions of its increased expression in OSCC tissuesCandidate biomarkers  were validated via two rounds of quantitative western blotting. Initially, we screened each protein in a small number of independent samples from three pre-malignant and three malignant subjects, seeking to confirm our proteomic results and determine those showing the most significant and consistent quantitative differences, which would then be selected for validation in a larger number of patient samples. From this screen, S100A7, catalase and keratin 19 did not show consistent differences between pre-malignant and malignant subjects to merit further validation (data not shown). Actin and myosin on the other hand showed clear and consistent increases in their abundance between samples from individuals with pre-malignant and malignant oral lesions. We therefore selected these two proteins for validation and statistical analysis in a larger number of patient samples.Supplementary Table S1 for information on subjects). None of these subjects were used in our initial quantitative proteomics analysis. In order to quantitatively compare western blotting results measuring actin and myosin abundance levels in pre-malignant or malignant subjects run on different gels, we used a common reference sample on all blots, prepared by pooling saliva from eleven healthy donors. For all western blots, the same amount of protein from this reference sample and each subject was loaded on the gel, and the signal intensity from either the actin or myosin band from each subject normalized against the signal intensity derived from the reference. To guard against potential errors in sample loading and ensure the veracity of our findings, blots for actin and myosin were performed in triplicate, and normalized intensities for each protein averaged for each subject across the three blots. Supplementary Figure S1 shows all the images of all western blotting results for myosin and actin.For actin and myosin validation, we analyzed by western blot saliva samples collected from twelve subjects with pre-malignant oral lesions and twelve subjects with malignant oral lesions  shows plots of normalized actin and myosin western blot intensities averaged across the pre-malignant and malignant subjects. Both proteins showed overall increases in normalized abundance levels in subjects with malignant oral lesions compared to those with pre-malignant lesions. These findings support the assertion that our observations for actin and myosin in soluble saliva are a direct consequence of differences in their cellular abundances.To test our hypothesis, we performed a western blot for actin and myosin on proteins from exfoliated cells contained in the saliva of the same pre-malignant and malignant subjects used for validations described above. Again, we normalized the intensities of actin and myosin in each subject sample against a common reference sample of proteins derived from exfoliated cells in a saliva sample from pooled healthy donors. In this study we sought to discover and validate protein biomarkers of oral cancer in the soluble fraction of whole saliva. Our study is the first to use advanced quantitative proteomics to compare saliva collected from subjects with pre-malignant oral lesions to saliva collected from subjects with malignant oral lesions. One recent proteomic studySupplementary Table S2) may serve as a valuable guide to others seeking to characterize salivary protein biomarkers of oral cancer. Given the time and expense limitations, we were only able to validate a relatively small number of the almost 200 differentially abundant proteins in our catalogue. It is very possible that other promising biomarkers exist among this group of differentially abundant proteins which we were unable to include in validation studies. These may be identified in ongoing studies by others, and when compared to our catalogue, bolster confidence as candidates for further validation.Owing to the uniqueness of our study, our protein catalogue , suggesting that the observed increases in protein abundance are due to increased actin and myosin expression within the exfoliated cells with the onset of invasive oral malignancy, and not simply an increase in the amount of cells in saliva.in vitro tumor cell migration studiesIn addition to our empirical evidence, previous descriptions of the role of actin and myosin in the mechanism underlying the transformation of pre-malignant epithelial cells to a malignant state also support our findings. Actin and myosin are key cytoskeletal proteins enabling cell motility and invasion, behavior central to epithelial tumorigenesiset al. critiques the methods currently used by cliniciansOur calculated sensitivity and specificity values respectively of 100% and 75% for actin, and 67% and 83% for myosin, are at least comparable to published values of other non-invasive detection methods currently used for distinguishing pre-malignant and malignant oral lesions. A recent review by Lingen In conclusion, via a proteomics study we have identified actin and myosin as promising salivary biomarkers of oral cancer. More rigorous determination in a larger number of samples of sensitivity and specificity values for both proteins is necessary to better understand their potential as biomarkers for widespread clinical testing. However, our results illustrate their promise for development into a simple and cost-effective test for oral cancer, with potential implementation using emerging point-of-care nano-scale devices for clinical diagnosticsTable S1Information on subjects used in discovery and validation studies.(0.09 MB PDF)Click here for additional data file.Table S2Quantitative Proteomic Catalogue of Whole Saliva Soluble Fraction.(1.22 MB XLS)Click here for additional data file.Figure S1Western blotting images from validation studies. Lanes marked with “R” denotes the reference sample; each numbered lane is a sample from a different individual with either a pre-malignant or malignant oral lesion. Approximate positions of the molecular weight standards, in kDa, are included.(2.04 MB TIF)Click here for additional data file.


Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra) and the Abelson (Abl) cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abl's role in attractive signaling, we over-expressed Abl in Fra mutants anticipating rescue of commissures.In the fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P-motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS.The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous Taken together, these data suggest that Fra activity is required to correctly regulate Abl-dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border. Attractive and repulsive guidance cues originating at or near the midline help guide the formation of the Drosophila embryonic nervous system. Receptors for these cues initiate intracellular signaling pathways that govern axon outgrowth and steering as well as formation of dendritic branching patterns fra and Dscam interact to further reduce commissure formation Netrin null embryos, the presence of an additional Netrin-independent attractive system has been proposed turtle as an important cell adhesion molecule that interacts with mutations in Netrin or fra to further reduce commissure formation In Drosophila, Netrins are midline attractants detected by Frazzled, a receptor expressed on most CNS neurons and in its absence many posterior commissures fail to form commissureless (comm.) prevents commissures from forming, and Comm appears to play a major role in attenuating Robo-dependent repulsion At the midline, Slit-mediated repulsion prevents ipsilateral axons from crossing the midline and commissural axons from re-crossing the midline once on the contralateral side in vivoLike most cell surface receptors, both Robo and Fra use conserved motifs within their cytoplasmic domains to signal information to the cytoskeleton. Fra has three evolutionarily conserved P-motifs  in its cytoplasmic domain and it appears that P3 is particularly important for Fra signaling abl display mild defects in the axon scaffold including ectopic midline crossing errors robo itself is decreased or if mutations in genes known to interact with Robo are introduced abl mutants are combined with mutations in a variety of genes implicated in the regulation of cytoskeletal dynamics  and the 1407-Gal4 driver on chromosome II were described V12 and Cdc42V12tau-myc P-element , the Gal4 driver and/or UAS transgenes in the same fly. Stocks were confirmed by complementation tests and/or PCR screening for P-elements as described previously Stocks were raised on conventional cornmeal and molasses based media at room temperature or 25°C. All of the stocks used in this study have been fully characterized in previous studies. The Fra deletion mutant transgenes (UAS-FraHistology: Standard methods are used to collect eggs at 25°C and stain them with mAb BP102 or mAb 1D4 against Fasciclin II (FasII) and the HRP-linked goat anti-mouse secondary antibody. The genotype of the embryos was established by X-Gal staining that identified balancer chromosomes. Mutant phenotypes in the axon scaffold were scored and at least three replicates were analyzed using a Student's t-test. Loss of commissures and ectopic crossovers were assessed as previously described -myc P-element  were described previously ΔP1 is deleted for PPDLWIHHDQMELKNIDK; pMT-FraΔP2 is deleted for TIESSKRGHPLKSFSVPGPPPTGGATPVTKHTP; pMT-FraΔP3 is deleted for ELNQEMANLEGLMKDLSAITANEFEC. All three P-motif deletion constructs contain a C-terminal HA epitope tag. To make myc-tagged versions, we first modified a commercially available S2 cell metallothionine vector  to include a hygromycin resistance cassette (herein pMT-Hygro), and then introduced a myc epitope tag and tobacco etch virus protease site (TEV) just before this vector's V5 epitope and His tags. Then, using the HA-tagged versions as templates, we PCR amplified Fra wild-type (Frawt) and P-motif deletions  and cloned them into unique SpeI and PvuI sites of this parental vector.HA epitope tagged Fra6 cells were transfected  with either myc- or HA-tagged FraWT, FraΔP1, FraΔP2, and FraΔP3 using the procedure recommended by the manufacturer. For the HA-tagged version, selection was achieved by co-transfection with pCohygro. Stable lines were selected over several weeks using 300 µg/ml hygromycin (Invitrogen). Fra protein expression was induced with 700 µM copper sulfate, and, 24 hours later, Fra was activated by adding a mixture of Netrin A and Netrin B for 30 minutes at 27°C. The cells were harvested by centrifugation at 13000 rpm and lysed in NP-40 buffer Drosophila S2 cells were maintained in Schneider's media supplemented with 10% fetal bovine serum (complete media) at 27°C as per the conditions in Drosophila Expression System (Invitrogen). Approximately 2×10abl enhance the degree of commissure loss in a fra mutant 210Abl, herein just BcrAbl) induces axons to ectopically cross the midline especially when wild-type Fra (Frawt) is co-expressed fra homozygote would partially rescue fra-dependent defects in commissure formation. Accordingly, using the GAL4–UAS system and previously characterized stocks fra mutant (3/fra4fra) embryo and assessed commissure formation using the monoclonal antibody BP102. This antibody stains the anterior and posterior commissures  as well as longitudinal connectives (LC) running on either side of the midline  of segments showing this defect  enhancement in PC defects and the emergence of AC defects  increases the frequency of thin and missing PC defects to about half that observed with over-expression of either Ablwt or BcrAbl; expression of AblKN spares the AC [One of the predominant defects in  defect . When Adefects  reduced compared to Frawt, as many segments now exhibit a distinct AC and PC  reduces the frequency of these crossovers as only 21% of embryos still show a minor crossover or two  reduced the expressivity and penetrance of the crossing errors [The fuzzy commissure phenotype is often associated with extra axons ectopically crossing the midline between the AC and PC, a result known to occur with pan-neural expression of BcrAbl  mutated . Consist mutated  arrowhea or two . Together, these immunoprecipitation assays indicate that both Abl and BcrAbl binding to Fra and the state of Fra phosphorylation are independent of any specific P-motif. This is consistent with the ability of the deletion mutants to rescue commissure formation when Abl or BcrAbl is over-expressed in a fra mutant.Forsthoefel et al. fra mutant also results in dozens of axons exiting toward the periphery (AEP defects), and, surprisingly, this defect is readily observed using BP102. Generally considered a marker of all CNS axons, BP102 clearly labels AC, PC and LC axons, and lightly stains ISN and SN nerve routes within the CNS, but it does not normally stain axons beyond the CNS/PNS boundary. Therefore, in mutant embryos, dense BP102 staining of axons beyond the CNS/PNS boundary clearly delineates a novel phenotype, herein termed an AEP defect (Axon Exiting to Periphery). Admittedly, no information on the identity of the exiting axons is provided with BP102, but this is addressed below.Expression of Abl in a 3/fra4fra mutant embryos we do not observe any AEP defects [fra heterozygote, we observe minimal AEP defects and none in the same background with Ablwt [wt (P<0.01), FraΔP1 (P<0.01) or FraΔP2 (P<0.05). A small (6%) but significant (P<0.01) frequency of AEPs is still observed if FraΔP3 is over-expressed [Consistent with the known BP102 staining pattern, in wild-type and  defects . When Bcth Ablwt . Howeverth Ablwt , arrows)th Ablwt . Like thxpressed . Therefo3/fra4fra mutant causes FasII positive longitudinal axons that normally remain in the CNS to exit. This also includes axons from the medial pCC/MP2 fascicle, which pioneers the longitudinal connective and normally does not exit the CNS the CNS  into our fra mutants expressing either BcrAbl or Ablwt. Surprisingly, decreasing Robo-dependent repulsion had no observable effect on either of the phenotypes induced by Abl activity [fra homozygote expressing Ablwt, thinning or missing AC's were seen in 60% of segments and 74% of segments had defects in the PC. Introduction of a 1robo allele did not significantly change these numbers as commissures were thinning or missing in 50% of AC's and 78% of PC's [3/fra4fra embryos expressing Ablwt had this defect compared to 26% in a 1robo heterozygote background [Both too much and too little Abl causes ectopic midline crossing errors when Robo-dependent repulsion is reduced activity . In a fr of PC's . We alsockground . A similckground . This daΔABNet mutant chromosome, which is null for both Netrin A and B genes fra mutants. Thus, the Abl gain-of-function phenotypes do not appear to be the result of Netrin dependent repulsion.In some cases, when Netrin is detected by Unc5 it can elicit a repulsive response fra mutant independent of Abl, but using gene mutations known to affect midline guidance.Besides its interactions with axon guidance receptors at the midline, Abl is a key regulator of the cytoskeletal dynamics underlying growth cone advance and steering V12) in a specific subset of FasII positive neurons leads to ectopic crossovers, the frequency of which are halved by a heterozygous fra mutation V12 in a 3fra heterozygote also causes severe errors in axon pathway formation as commissures fuse and large gaps appear in the LC  also causes FasII expressing axons to ectopically cross the midline V12 is pan-neurally expressed in wild-type embryos or a 3fra heterozygote [V12 is over-expressed in a homozygous fra embryo, it too yields a major reduction in commissure formation [The severity of these Cdc42rozygote , commissormation . While sfra mutant using a constitutively active Myosin Light Chain Kinase (U-ctMLCK) that increases myosin activity by phosphorylating the Regulatory Light Chain of myosin II V12 and Cdc42V12, are sensitive to heterozygous removal of frafra heterozygote, many segments still display fuzzy commissures  when the pan-neural elav-Gal4 driver line was utilized (data not shown). Over-expression of RacV12 in a Netrin null embryo (ΔABNet) also reduces commissure formation [Netrin due to stock health.Next, we sought to selectively elevate myosin activity in a issures .a mutant . Similarormation ; Cdc42 cV12 and Cdc42V12 do not cause AEP defects, these data also suggest that Abl contributes unique information governing which axons may leave the CNS.Together, the Rac and Cdc42 data suggest that, in the absence of Fra, unilateral perturbation of actin cytoskeletal dynamics is sufficient to reduce commissure formation. These data continue to highlight the critical role Fra plays in regulating cytoskeletal dynamics during guidance events at the midline, and implicate all three molecules, Abl, Rac and Cdc42, in this process. Moreover, because Racfra and abl activity is lost. This presumably reflects the ability of abl mutations to interact with a second Netrin receptor, Dscam, as well as Netrin independent receptors (e.g. Turtle) known to be important for commissure formation fra null embryos would improve commissure formation. However, instead of an improvement, we clearly document a major decrease in both anterior and posterior commissures and the induction of a novel phenotype whereby axons normally confined to the CNS now project into the periphery (AEP defects). It is worth emphasizing that these phenotypes occur even with the over-expression of a wild-type Abl transgene that retains its autoinhibitory domain and must be activated by endogenous mechanisms fra mutant. Taken together, we propose that during exploration of the midline, Fra is a key regulator of Abl activity and helps determine how the cytoskeletal machinery utilizes Abl. In the absence of Fra, axon outgrowth does not simply stall; but rather, axons follow a variety of aberrant trajectories away from the midline. This suggests that Fra normally competes with several other receptor systems to dictate how Abl functions to regulate the cytoskeletal machinery. While competitors undoubtedly include other midline guidance cues, the emergence of AEP defects suggests that Fra also competes with guidance systems not normally associated with the midline. In the absence of Fra, these other receptors appear to utilize the extra Abl to alter cytoskeletal dynamics at a variety of choice points, ultimately preventing commissure formation and directing some axons out of the CNS.Frazzled and Abelson Tyrosine kinase activity clearly cooperate during the formation of embryonic commissures. In the absence of Fra, detection of Netrin-dependent chemoattraction is compromised and many posterior commissures fail to form. Both anterior and posterior commissures are absent if fra is absent. That is, commissures form correctly in a heterozygous fra mutant or when partially active Fra transgenes with a single P-motif deleted are re-expressed with Abl. In S2 cell immunoprecipitation experiments, both Abl and BcrAbl bind to the cytoplasmic tail of Fra independent of any specific P-motif  observed with only BcrAbl also depend on Fra and specifically the P3-motif, confirming a previous report The Abl gain-of-function phenotype described herein occurs only if see also . While sin vitro translated Abl, direct binding of Abl to Fra is clearly possible. If so, these experiments suggest that Abl may bind to Fra in the regions between P-motifs, which is, in fact, where most of the tyrosine residues within the cytoplasmic domain of Fra reside in vivoΔP3 transgene is the least capable of rescuing the AEP defects caused by Ablwt or BcrAbl expression. Immunoblots of Fra phosphorylation in the absence of pervanadate pretreatment also suggest the steady-state level of tyrosine phosphorylation may be relatively low, or highly dynamic. S2 cells are known to express tyrosine phosphatases that antagonize Abl activity for some substrates The physical interaction between Fra and either Ablwt or BcrAbl in immunoprecipitation assays can reflect direct or indirect association between the two proteins. It is possible that our failure to observe P-motif dependence reflects the binding of Abl to multiple P-motifs or the use of scaffold proteins associated with more then one P-motif. Given that Forsthoefel et al. fra mutant, the loss of commissures and the exiting of CNS axons to the periphery, suggest these embryos are experiencing an excess of midline repulsion. During commissure formation, Slit dependent repulsion prevents commissural axons from crossing unless Commissureless prevents the Slit receptor Robo from accumulating on the cell surface fra mutants leading to an increase in Robo-dependent repulsion. Since increasing Robo activity in a fra embryo is sufficient to reduce commissure formation robo (Robo1) had no discernable affect on the Abl gain-of-function phenotypes, even though, in previous work, elevating Abl activity in a heterozygous robo mutant induces ectopic midline crossing errors Netrin mutant. This provides strong evidence that abnormal signaling by other Netrin receptors is not responsible for these phenotypes.Both of the phenotypes observed when Abl activity is elevated in a commissureless mutant experiencing very high levels of Slit-dependent repulsion fra Dscam double mutants, or even fra Dscam abl triple mutants sema2b commissural axons. FasII axons do not leave the CNS when midline repulsion is elevated in a commissureless mutant fra mutant also affects several different guidance decisions by Sema2b-expressing commissural axons. While the cues guiding these neurons are not well understood, the spectrum of defects observed both before and after they cross the midline suggest that these neurons are responding to more then just midline repulsion. Thus, if a repulsive mechanism is functioning, as initially suggested by the phenotype, the origin of the signal remains to be determined. Indeed, our data using activated forms of Rac and Cdc42 suggest that the primary defect lies in the ability of growth cones to properly regulate actin dynamics underlying axon outgrowth and steering. This could involve both attractive and repulsive systems.In terms of the AEP defects, which also point to excess repulsion, it is worth noting that axons do not leave the CNS in a Drosophila nervous system. Mutations in abl interact with several cytoskeletal regulators to affect axon pathway formation: kettecapuletchicadee  observed with Abl. The Cdc42 result is most intriguing as expression in a wild-type or heterozygous fra embryo results in fused commissures and gaps in the longitudinal connectives. Yet, upon complete removal of Fra, commissures do not form and the longitudinal connectives reform. Thus, in the absence of Fra, commissure formation, but not AEP defects, appears to be particularly sensitive to manipulation of actin-based processes. It is possible that our manipulation of Cdc42 and Rac activity in a fra mutant is affecting shared processes related to actin polymerization. For example, in vertebrate studies, Cdc42 and Abl work in parallel to regulate actin polymerization (e.g. fra embryos. Which guidance events are being affected has not yet been determined, but a few candidates exist. In addition to fra and Dscamabl interact with the cell-cell adhesion molecules neurotactin and amalgamfasImidline-fasciclinturtleProper axon guidance also requires concerted regulation of the cytoskeletal dynamics underlying axon outgrowth and steering ion e.g.  and Abl In summary, our data suggest a model whereby Fra activity initiates key signaling events that dictate when and how Abl activity is utilized during commissure formation. Rac and Cdc42 are probably also involved in this process, and, together with Abl, help regulate key aspects of actin dynamics underlying commissure formation. In the absence of Fra other midline guidance systems are still functioning well enough to form most commissures, but they are clearly sensitive to perturbation of intracellular signaling pathways regulating cytoskeletal dynamics. Thus, when Fra is removed, other guidance systems appear to recruit Abl, Rac or Cdc42 activity to misdirect axon outgrowth, ultimately preventing commissure formation and, with Abl, causing some axons to exit the CNS. Thus, in a normal embryo, Fra must be sending information that allows it to compete very well against these other guidance receptors to properly regulate axon outgrowth and steering during commissure formation. While an alteration in midline guidance decisions may also account for the AEP defects, the scope of guidance errors observed in neurons leaving the CNS suggest that increasing Abl activity could also be affecting other guidance systems not directly associated with the midline. While the identity and specific role of these guidance systems awaits discovery, the sensitivity of the CNS axon scaffold to Abl over-expression will be an important tool for identifying these competing pathways.


Rapid outgrowth of EM virus occurs during the first few weeks of infection. However, the rate of escape plateaued soon after, revealing a prolonged persistence of WT viremia not detectable by standard cloning and sequencing methods. The rate of escape of KP9 correlated with levels of vaccine-primed KP9-specific CD8+ T cells present at that time. Similarly, when non-KP9 responder  macaques were infected with SHIVmn229 stock containing a mixture of EM and WT virus, rapid reversion to WT was observed over the first 2 weeks following infection. However, the rate of reversion to WT slowed dramatically over the first month of infection. The serial quantitation of escape mutant viruses evolving during SIV infection shows that rapid dynamics of immune escape and reversion can be observed in early infection, particularly when CD8 T cells are primed by vaccination. However, these early rapid rates of escape and reversion are transient and followed by a significant slowing in these rates later during infection, highlighting that the rate of escape is significantly influenced by the timing of its occurrence.CD8+ cytotoxic T lymphocytes (CTL) can be effective at controlling HIV-1 in humans and SIV in macaques, but their utility is partly offset by mutational escape. The kinetics of CTL escape and reversion of escape mutant viruses upon transmission to MHC-mismatched hosts can help us understand CTL-mediated viral control and the fitness cost extracted by immune escape mutation. Traditional methods for following CTL escape and reversion are, however, insensitive to minor viral quasispecies. We developed sensitive quantitative real-time PCR assays to track the viral load of SIV Gag Immune escape from AIDS virus–specific cellular immunity is common. The driving forces behind how quickly cellular immunity forces escape are poorly understood. We developed a novel assay for a common immune escape variant of SIV in macaques. This allowed us to sensitively track the rates of immune escape even when levels of escape mutant or wild-type virus were low. We found that prior immunization of macaques resulted in very rapid immune escape during acute infection. However, when escape starts to occur later, during chronic infection, the rate of immune escape is much more gradual. Thus, both prior vaccination and timing influence the rates of immune escape and provide a fuller picture of the effectiveness of T cell immunity to HIV. CD8+ cytotoxic T lymphocyte (CTL) responses during acute HIV-1 and simian immunodeficiency virus (SIV) infection in humans and macaques, respectively, correlate with effective control of acute viremia . CTL resMane-A*10 negative pigtail macaques infected with EM challenge stock SHIVmn229, suggesting a significant fitness cost of the K165R mutation ,25mn229 Our findings have important implications when trying to infer immune selection pressure from escape rates. For example, it has been suggested that the CTL response in humans is less effective than in macaques, because of the faster escape rates observed in macaques . HoweverUnderstanding when and at what rate escape occurs at different epitopes is complex. Escape may occur late because the mutations required are rare, or because the EM virus is only weakly selected once it occurs. The strength of immune control and the fitness costs of escape mutation have been suggested as criteria for selection of optimal vaccine epitopes . HoweverAlthough we describe in this report novel sensitive qRT-PCR assays to detect minor viral quasispecies, several limitations of this assay should be noted. First, although our assay can specifically discriminate WT from EM viruses, only the EM virus that the assay was designed for can be distinguished, and other mutants that may arise will not be detected, as it is the LNA-Forward primer that drives the specificity of this qRT-PCR. Additional qRT-PCRs could be designed for rarer escape mutations if needed. For example, we have rarely detected a typically transient mutation with KP9 at position 9 (P172S) during the evolution of the canonical K165R escape ,26; undeIn summary, we have developed qRT-PCRs to measure the kinetics of CTL escape and reversion. We show that both the timing of escape and reversion and prior vaccination influence the dynamics of EM viremia. These findings facilitate a better understanding of the evolution and fitness of escape mutant viruses occurring during HIV-1 and SIV infection.


Cirrhosis of the liver is thought to be a major cause of morbidity and mortality in sub-Saharan Africa, but few controlled studies on the etiology of cirrhosis have been conducted in this region.We aimed to elucidate the association between environmental and infectious exposures and cirrhosis in The Gambia.ser TP53 mutation.Ninety-seven individuals were diagnosed with cirrhosis using a validated ultrasound scoring system and were compared with 397 controls. Participants reported demographic and food frequency information. Blood samples were tested for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), hepatitis C virus (HCV) antibody, HCV RNA, and the aflatoxin-associated 249ser TP53 mutation in plasma, is associated with a significant increase in the risk for cirrhosis . Additionally, aflatoxin and hepatitis B virus exposure appeared to interact synergistically to substantially increase the risk of cirrhosis, although this was not statistically significant.HBsAg seropositivity was associated with a significant increase in risk of cirrhosis  as was the presence of HBeAg  and HCV infection . We present novel data that exposure to aflatoxin, as assessed both by high lifetime groundnut (peanut) intake and by the presence of the 249Our results suggest that the spectrum of morbidity associated with aflatoxin exposure could include cirrhosis. Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide , and a hWorldwide, cirrhosis of the liver is the 16th leading cause of death, responsible for hundreds of thousands of deaths each year . CirrhosHCC most commonly occurs in cirrhotic individuals . Risk faIn this study, we investigated the diagnosis of liver cirrhosis via a noninvasive, validated ultrasound scoring system  among inThe study population was recruited as a part of the Gambia Liver Cancer Study as described previously . BrieflyIndividuals without suspected liver disease were recruited from the outpatient clinics of the same three hospital sites to serve as a control group. These participants were frequency-matched to suspected liver disease patients by age (10-year groupings) and sex. Participants without suspected liver disease were administered the identical interviewer-administered questionnaire, underwent a standardized clinical examination to identify signs of underlying liver disease, and provided a blood sample. Local and international scientific and ethical review committees approved the study protocol, and informed consent was obtained from each participant before inclusion in the study.a) no evidence of space-occupying hepatic lesions by ultrasound examination; b) no evidence for alternative diagnoses ; c) complete demographic and hepatitis B surface antigen (HBsAg) information; and d) a score of 7 of a possible 11 points on the ultrasound-based cirrhosis scale developed by a) alpha-fetoprotein (AFP) levels in the normal range (< 20 ng/mL); b) no evidence of underlying liver disease on the standardized clinical exam; and c) complete demographic and HBsAg information.Individuals with suspected liver disease recruited from the liver disease referral clinics were classified as having liver cirrhosis (cases) if they had Blood specimens were processed promptly after collection and stored at either –20°C or –70°C, depending on the testing planned for each aliquot. HBsAg was determined as a marker of chronic HBV carriage by a reverse passive hemagglutination assay  with radioimmunoassay testing of negative samples . Participants positive for HBsAg were tested for hepatitis B e antigen (HBeAg) using a radioimmunoassay kit . HCV antibody status (anti-HCV) was determined by third-generation enzyme-linked immunosorbent assay . Samples that were seroreactive by ELISA to HCV were screened with a recombinant immunoblot assay  and for HCV RNA using a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay as described previously . We defiser TP53 mutation is a biomarker of the aflatoxin-associated mutational effect in the p53 gene (ser TP53 mutation in plasma have been previously described (TP53 gene was amplified. PCR products underwent restriction digestion (HaeIII) followed by automated sequencing . All samples underwent two independent analyses, and discordant results underwent a third analysis. At all steps, a series of controls without DNA template was used to rule out contamination.Previous investigations have demonstrated that the 249p53 gene ; methodsescribed . Brieflyt-tests, asymptotic chi-square tests, and Fisher’s exact tests to test for differences between study groups. Logistic regression models were used to evaluate potential risk factors for cirrhosis in cases compared with controls. To minimize any potential effect of including occult HCC within our cirrhotic cases, we performed separate analyses excluding cirrhosis cases with significantly elevated AFP levels (> 500 ng/mL); however, our findings were not substantively changed (data not shown). We evaluated markers of viral hepatitis infection alone and with adjustment for age, sex, recruitment site and date, socioeconomic status , smoking, alcohol consumption, and other viral markers.We calculated descriptive statistics for questionnaire and laboratory variables among case and control participants using ser TP53 mutations, which, although perhaps less sensitive, would be reflective of the biological effects of longer-term aflatoxin exposure and be less susceptible to changes with illness. The plasma 249ser TP53 mutation was detected more often among individuals with higher levels of lifetime groundnut consumption, but this association did not attain statistical significance (exact p = 0.10). Therefore, although related, the dietary and molecular markers represent different measures of aflatoxin exposure; we used separate models for analysis of each aflatoxin marker.Dietary and molecular markers of aflatoxin exposure, both alone and with adjustment for potential confounding factors, were evaluated as possible risk factors for cirrhosis. Self-reported dietary exposure to aflatoxin was constructed from the dietary questionnaire. Study participants were asked about their consumption of groundnuts in the past 2 months and whether this consumption was greater, less, or about the same as during the entirety of their lives. Previously, we demonstrated that persons with clinical liver disease often decrease their dietary intake as well as reduce exposure to risk factors suspected to be associated with the disease ; therefoWe evaluated interactions between aflatoxin exposure and HBV infection through participant stratification and subsequent logistic regression analysis, as well as through incorporation of first-order interaction terms into the logistic regression models. All analyses were conducted using SAS version 9.1 .After excluding liver-disease referral patients with space-occupying lesions on ultrasonography characteristic of HCC or with alternative diagnoses other than liver disease , a totalCharacteristics of the 97 cirrhosis cases and 397 controls are listed in ser TP53 mutations. ser TP53 mutations among cirrhosis cases and controls, as well as unadjusted and adjusted ORs evaluating the association between aflatoxin exposure variables and cirrhosis. Increasing lifetime groundnut intake was associated with a significant increase in the risk of cirrhosis (p-trend = 0.01). Moderate and high lifetime groundnut consumption versus low consumption was associated with an almost 2-fold and significant 3-fold increase in risk of cirrhosis, respectively. The presence of the plasma 249ser TP53 mutation conferred a statistically significant almost 4-fold increase in risk of cirrhosis in this population.Eighty cirrhosis cases and 327 controls provided information regarding groundnut consumption on the study questionnaire. In addition, 78 cirrhosis cases and 346 controls were evaluated for plasma 249ser TP53 mutation alone and > 7-fold with HBsAg positivity, but was 46-fold higher for persons expressing both compared with individuals who lacked either marker. The statistical term for the interaction between HBsAg and either lifetime groundnut intake or the 249ser TP53 mutation variables did not attain significance .Despite the postulated heavy burden of disease, remarkably little descriptive risk factor or natural history data have been published on chronic liver disease or cirrhosis from sub-Saharan African populations . In thisThe impact of aflatoxin on human health is substantial and has been documented in both acute and chronic exposure settings . Acute eAlthough standardized clinical scoring systems, such as the Child-Pugh score, have for decades been accepted for assessing the severity of liver cirrhosis, numerous recent reports have increasingly focused on the utility of noninvasive methods and clinical prediction models to diagnose fibrosis and cirrhosis . Howeverser TP53 mutation, associated with aflatoxin exposure in experimental systems (ser TP53 mutations (ser TP53 mutation in plasma exhibit seasonal variability (ser TP53 mutation represents the best biomarker currently available to assess the biological effect of cumulative aflatoxin exposure.Currently there is no gold standard methodology available to measure cumulative lifetime aflatoxin exposure. In lieu of a gold standard, we used two different approaches to assess exposure to aflatoxin during the period of time that we hypothesized would have etiologic significance for the development of cirrhosis. First, we evaluated self-reported lifetime intake of groundnuts, the primary food component contributing to aflatoxin exposure in The Gambia , 2000. Q systems , is comm systems  and is s systems , 2005. Hutations  and thatutations  or host utations  may moduiability , suggestBoth measures of aflatoxin exposure evaluated as a part of this study were associated with a significant increase in the risk of cirrhosis. Our results also suggest that aflatoxin exposure and chronic HBV infection may interact synergistically to increase the risk of cirrhosis notably above and beyond that expected for two independent hepatotoxins. The mechanisms by which aflatoxin is activated and induces HCC-associated mutational changes have been well characterized , but lesAs expected, we found HBsAg positivity to be a significant risk factor for cirrhosis; we have extended this work to document a further increase in risk of cirrhosis associated with HBeAg seropositivity. Similarly, we found HCV infection to be a significant risk factor for cirrhosis, although the magnitude of this risk was lower than that conferred by HBsAg positivity. Notably, the average age of HCV-related cirrhosis patients was around 15 years older than HBV-related patients, consistent with the premise of a different natural history of infection between the two viruses in West Africa (for further discussion, see Cirrhosis of the liver is a major cause of morbidity and mortality in sub-Saharan Africa. Using a validated ultrasound scoring system to diagnose cirrhosis, we confirmed associations with chronic HBV and HCV infections and further provide evidence that aflatoxin may also be an etiologic factor for cirrhosis. As such, expanded access to hepatitis B vaccination programs and continued development of aflatoxin-reduction intervention programs  could su


Biological membrane fusion is a basic cellular process catalyzed by SNARE proteins and additional auxiliary factors. Yet, the critical mechanistic details of SNARE-catalyzed membrane fusion are poorly understood, especially during rapid synaptic transmission. Here, we systematically assessed the electrostatic forces between SNARE complex, auxiliary proteins and fusing membranes by the nonlinear Poisson-Boltzmann equation using explicit models of membranes and proteins. We found that a previously unrecognized, structurally preferred and energetically highly favorable lateral orientation exists for the SNARE complex between fusing membranes. This preferred orientation immediately suggests a novel and simple synaptotagmin-dependent mechanistic trigger of membrane fusion. Moreover, electrostatic interactions between membranes, SNARE complex, and auxiliary proteins appear to orchestrate a series of membrane curvature events that set the stage for rapid synaptic vesicle fusion. Together, our electrostatic analyses of SNAREs and their regulatory factors suggest unexpected and potentially novel mechanisms for eukaryotic membrane fusion proteins. In vivo, fusion is catalyzed by three families of conserved proteins collectively termed the SNARE proteins .Biological membrane fusion is a basic cellular process necessary for exocytosis, endocytosis and exchange of vesicular contents in eukaryotic cells. Fusion of membranes is highly energetically demanding since tremendous electrostatic repulsion between negatively charged lipid bilayers has to be overcome.The transient and critical molecular events during which SNAREs and auxiliary proteins control membrane fusion are still poorly understood. One reason is that, compared to extensive gain- and loss-of-function studies of the roles of fusion proteins, quantitative understanding of electrostatic and other physical interactions between membranes, SNARE and auxiliary proteins has been lacking. For instance, the synaptic SNARE core complex possesses a cluster of positively charged residues near its C-terminus.A predominant form of protein-membrane physical interactions during biological membrane fusion is electrostatic interactions. During fusion, the SNARE complex is positioned close to and sandwiched between two fusing membranes. At such a distance, electrostatic forces overcome ionic screening effect and will have a major influence on interactions between molecules.Here, we analyzed intermolecular electrostatic interactions in SNARE-mediated membrane fusion by solving nonlinear Poisson-Boltzmann equation on all-atom models of the system. We focused on interactions between fusion proteins and membranes, but not the energetics of fusion of pure lipid systems which has been studied in many other works e.g.,. SeveralWe first constructed surface potential profiles for the synaptic SNARE complex based on its crystal structure.To functionally dissect SNARE's electrostatic interactions with the membranes, we first asked whether there is a preferred lateral orientation of SNARE complex between fusing membranes. Interestingly, we found that both intrinsic structural constraints and intermolecular interactions point to a single preferred rotation for SNARE complex prior to fusion . StructuTo our surprise, the structurally preferred orientation exactly matches the orientation where SNARE complex exhibits minimal electrostatic interaction energies with fusing membranes . We init2+ sensor during neuronal transmission, can bind to both membrane and the SNARE complex during membrane fusion.2+-binding loop in synaptotagmin C2B domain points straight toward the v-membrane in the synaptotagmin/SNARE complex2+-binding loop is the motif that will insert into the membrane in response to Ca2+ influx, the signal that triggers synaptic vesicle release.2+-binding loop insert into the opposite t-membrane in response to calcium influx, the result will be turning over of the entire SNARE complex by 180° away from its resting preferred orientation. During this process, the TMDs of SNARE proteins are expected to act as a propeller and drastically distort local membrane structure, potentially enabling fusion pore formation. Indeed, membrane insertion of C2B occurs preferentially towards the PIP2-rich microdomains of the t-membrane.This putative preferred orientation immediately suggests a simple and novel “propeller” mechanism for SNARE to directly drive fast membrane fusion. Synaptotagmin, an important auxiliary fusion protein and the putative CaFollowing the identification of a structurally determined and energetically most favorable orientation, we analyzed the two sides of SNARE complex that face the v- and t-membranes, respectively, by following this putative preferred orientation. In the side facing the v-membrane (i.e. the membrane that bears the TMD of VAMP), surface electrostatic potential is mostly negative . By contTo quantitatively characterize electrostatic interactions, we determined the levels of interaction free energies between SNARE complex and membranes using the nonlinear Poisson-Boltzmann method .[15],  BecBecause some models suggest that SNARE complex assembly is not completed before the onset of fusion,In the first set of calculations , the C-tCharged residues are evolutionarily conserved on the SNARE complex . A similOur calculations show that at the final stage of fusion, the v-membrane is electrostatically repelled from the site of fusion by the SNARE complex. Meanwhile, the v-membrane is also pulled toward the site of fusion by the SNARE TMDs and by local electrostatic attractions from positive charges at the SNARE core domain C-terminus . The disAs a first step towards answering this question, we provided an estimate of the level of potential membrane bending generated by this force couple. Since the repulsion between SNARE and v-membrane is weaker for vesicles with smaller diameters which by definition have greater membrane bending, we asked: at what point will the energy penalty of membrane bending just be offset by a reduction in electrostatic repulsion exerted by SNARE complex as a result of greater bending?From Helfrich's membrane elasticity theoryNext, we systematically derived interaction energies between SNARE complex and v-membranes that bear a series of different curvatures as the “standard curve” . By plugWe argue that this rough estimate is an underestimate because: (1) In our calculations we assumed that the entire lower hemisphere of synaptic vesicles is bended to the same degree. However, in reality the effect is necessarily more local ; (2) TheComplexin and synaptotagmin are important auxiliary proteins to the SNARE complex.We first plotted the surface potential profile of the complexin/SNARE (cpx/SNARE) complex2+ sensor during synaptic vesicle release, and has been shown to bind to both membrane and the SNARE complex.2+ greatly enhances synaptotagmin binding to SNARE, it is unclear whether synaptotagmin is bound to the SNARE complex before Ca2+ influx in neurons. We thus analyzed the syt/SNARE complex with and without bound Ca2+ ions separately.Next, we addressed the electrostatics of synaptotagmin. Synaptotagmin is the putative Ca2+ is bound to C2B  sufficient for overcoming the energy barrier to lateral rotation of SNARE complex  and palmitoyl-oleyl-phosphatidylcholine (POPC) as previously described.Models of proteins include: neuronal SNARE core complex (PDB entry 1SFC), complexin/SNARE complex (PDB entry 1KIL), endosomal SNARE core complex (PDB entry 1GL2), and synaptotagmin/SNARE complex.Models for the SNARE/v-/t-membrane ternary complex were built according to current knowledge of the fusion-competent state, when trans-SNARE complex assembly was just complete . We perfThe relative lateral rotation of SNARE complex with respect to v- and t-membranes is based on considerations detailed in The distance between proteins and membranes in this study is defined as the nearest vertical distance between the van der Waals surfaces of two macromolecules.Molecular dynamics simulations were performed with GROMACSThe nonlinear Poisson-Boltzmann equation was solved numerically with parallel multigrid focusing, using the Adaptive Poisson-Boltzmann Solver (APBS).The precision of calculations was estimated by two means. First, we used a lower focusing grid resolution (1 Å/grid instead of 0.5 Å/grid) to perform the same interaction energy calculations.From Helfrich's membrane elasticity theory,


Hypnales comprise over 50% of all pleurocarpous mosses. They provide a young radiation complicating phylogenetic analyses. To resolve the hypnalean phylogeny, it is necessary to use a phylogenetic marker providing highly variable features to resolve species on the one hand and conserved features enabling a backbone analysis on the other. Therefore we used highly variable internal transcribed spacer 2 (ITS2) sequences and conserved secondary structures, as deposited with the ITS2 Database, simultaneously.We built an accurate and in parts robustly resolved large scale phylogeny for 1,634 currently available hypnalean ITS2 sequence-structure pairs.Profile Neighbor-Joining revealed a possible hypnalean backbone, indicating that most of the hypnalean taxa classified as different moss families are polyphyletic assemblages awaiting taxonomic changes. Pleurocarpous mosses, which are mainly found in tropical forests, account for more than 50% of all moss species ,2. BrothPinnatella and Taiwanobryum.The alignment and editing tool 4SALE ,16 generAfter setting the highlighted clades in Figure Meteorium and Papillaria. A maximum likelihood tree of their research (hereinafter referred to as Meteoriaceae Q) is now to be compared to the subtree "Meteoriaceae I"  the "Meteoriaceae I" strongly resemble published results [Chrysocladium retrorsum is no longer the only most basal taxon, but is joined by Papillaria deppei. Within clade P Papillaria africana is now directly related to Papillaria nitens.Quandt et al.  provided" Figure . Both tr results  and 2)  MeteoriuRigodium toxarion) of our taxon sampling. These findings strongly reflect the results of Quandt et al. in 2009 [Rigodium was placed in several diffrent families. In 1909 Brotherus originally ranked Rigodium among the Brachytheciaceae [Rigodium to the Brachytheciaceae, dispite the lack of paraphyllia and distinct leaf papillae [Rigodium among the Thuidiaceae, Crum [Morphological convergence is widespread among the pleurocarps. This is one reason for the difficulties in moss systematics. Among the order of Hypnales several families  were recently revealed as polyphyletic ,27. This in 2009 . Along teciaceae , later ieciaceae . Due to eciaceae  returnedpapillae . Whereaspapillae  counted ae, Crum  constituae, Crum ,33.Considering the distance tree Figure  and OlssWhile the distance tree could reveal phylogenetic relationships within families, Profile Neighbor-Joining successfully allowed us to gain information about the backbone of the hypnalean phylogeny Figure . The PriMoreover, there is no doubt that the "Brachytheciaceae IV" are a sister to Lembophyllaceae II + Symphyodontaceae with "Brachytheciaceae I" (including Lembophyllaceae II) being a sister to this clade. Although "Brachytheciaceae III" and "II" meet during the PNJ process, they are still only distantly related to "Brachytheciaceae I" and "IV" and thus are polyphyletic. Also the three Neckeraceae clades do not meet during the PNJ, which could be a hint for the actual polyphyly, as outlined by Olsson et al. . DespiteBy rooting the phylogenetic trees at Fabroniaceae we use the same root as Ignatov et al. . Unlike To describe the clades of the hypnalean backbone Figure  as monopThe large scale analysis of Hypnales (Bryophyta) inferred from internal transcribed spacer 2 sequence-structure data successfully accomplished one step towards the deciphering of the taxonomy of pleurocarpous mosses. The use of not only ITS2 sequences, but also structural data is a plausible method, for it provides the feature of calculating evolutionary distances both at the level of highly variable sequences and at the level of conserved structures. Although further studies are needed and asked for to confirm the phylogenetic backbone of the Hypnales, the results of this survey show evidence for the actual polyphyly of many classic pleurocarpous families, still awaiting taxonomic changes. Now it is desirable, that Bryologists revisit specific groups within Hypnales in the context of our large scale analysis. Back to back research considering morphological characters of mosses on a small scale contrasting our approach in mega-systematics is absolutely necessary in establishing a taxonomy of Hypnales. Furthermore, novel methods, like the transfer of the sequence-structure and profile approaches to a Maximum Likelihood algorithm, could provide a great leap towards the true hypnalean phylogeny in the future.1,730 hypnalean ITS2-sequences including their secondary structures were retrieved from the ITS2 Database -13,23,24http://profdist.bioapps.biozentrum.uni-wuerzburg.de/cgi-bin/index.php?section=cart2prof searches for every cartoon in a FigTree NEXUS file and converts the information into a ProfDistS compatible profile file.An automatic multiple sequence-structure alignment (MSA) was calculated with ClustalW  as impleA further sequence-structure alignment was generated containing only clade M and P of the "Meteoriaceae I". The alignment was processed by ProfDistS -20 usingAll tree files . Hypnales_filtered.xfasta contains the filtered set of sequence-structure pairs used for the final analyses including 1,634 sequence-structure pairs.Click here for fileDistance tree file. The large distance tree (Figure e Figure  as tree Click here for file


In this study we tested the hypothesis that the noble gas argon has a neuroprotective potential as well. Since traumatic brain injury and stroke are widespread and generate an enormous economic and social burden, we investigated the possible neuroprotective effect in Organotypic hippocampal slice cultures from mice pups were subjected to either oxygen-glucose deprivation or to a focal mechanical trauma and subsequently treated with three different concentrations  of argon immediately after trauma or with a two-or-three-hour delay. After 72 hours of incubation tissue injury assessment was performed using propidium iodide, a staining agent that becomes fluorescent when it diffuses into damaged cells via disintegrated cell membranes.We could show argon's neuroprotective effects at different concentrations when applied directly after oxygen-glucose deprivation or trauma. Even three hours after application, argon was still neuroprotective.in vitro models of oxygen-glucose deprivation and traumatic brain injury. Our promising results justify further in vivo animal research.Argon showed a neuroprotective effect in both  The first biological effects of argon were demonstrated as early as 1939  were theStroke and traumatic brain injury (TBI) are two very common causes of death and disability worldwide and create a significant economic and social burden -17. Whilin vitro models that involved either a focal mechanical trauma or oxygen-glucose deprivation of cultured hippocampal slices.When compared to xenon argon has some conspicuous advantages: low cost; no narcotic effects at normobaric pressures. Yet, data on argon's effects on cells are sparse. Therefore, we tested the effect of argon in All experiments were performed in compliance with the local Institutional Ethical Review Committee and have been approved by the animal protection representative at the Institute of Animal Research at the RWTH Aachen University Hospital, according to the German animal protection law §4, Section 3. Unless otherwise stated, all chemicals were obtained from PAA Laboratories GmbH .Cultures were prepared as previously reported , with so2. After baseline fluorescence imaging, oxygen-glucose deprivation (OGD) was accomplished as previously described % N2.All slices subject to traumatic brain injury were first incubated for 30 minutes at 37°C, 95% air and 5% COescribed ,19,22. IPropidium iodide is a fluorescent intercalating agent which is widely used to stain DNA ,21,23. WThe fluorescence images were digitalized at eight bit, allowing us to differentiate between a spectrum of 256 (from 0 to 255) grey scale levels. Damaged regions with a high PI uptake emitted at a high grey scale level, while vital regions showed only minor emissions. The red channel of each image was analyzed with ImageJ software  . ImageJ P ≤ 0.05 was considered as statistically significant.After measuring the induced cell damage for each slice, these results were combined for every experimental group and then normalized to the intensities of trauma in each trauma group . Mean values and standard errors of the means (SEM) were calculated with SPSS 16.0 . Statistical significance was evaluated with SPSS using the one-way analysis of variance (ANOVA) with bonferroni post-hoc analysis; Tissue damage after 14 days of incubation was negligible  otherwiTBI produced less absolute tissue damage than OGD due to the focal nature of this injury. Nevertheless the difference between traumatized and non-traumatized groups was still greater in TBI due to the very low damage found in non-traumatized TBI-slices. More than likely this can be attributed to a longer and more strenuous procedure during OGD. While there were only two medium changes necessary during TBI, OGD slices were subjected to three exchanges. Furthermore the pressure chamber had to be flushed twice for OGD, exposing to a certain extent the surface of the slices to dehydration. Therefore, the TBI control group reached only 6% of the total trauma while the OGD control group showed almost 27.3% of maximum trauma intensity. After establishing a valid measurement for tissue injury, we tested the effects of argon on traumatized slices. We treated groups of slices with 25%, 50% or 74% argon after TBI or OGD was induced. Figure Argon showed neuroprotective potential in TBI Figure , panel BTo adapt our laboratory setting more closely to a typical clinical situation, we decided to apply argon two and three hours after trauma. We incubated the slices in an atmosphere containing 50% argon because this concentration showed the best neuroprotective effect after TBI and there was no significant difference detectable between these three concentrations in OGD. As before, we could show that Argon strongly reduced cell damage in OGD and likewise in TBI  compared to xenon (20 €/l). Furthermore, argon has no anaesthetic properties at normobaric conditions . It may A receptor [in vitro and in vivo ischemia models and several potential mechanisms have been proposed [A receptor and the following chloride-based membrane hyperpolarization inhibit N-methyl-D-aspartate (NMDA) receptors [in vivo rat model, Zhang et al. could show that GABA receptor activation diminished the phosphorylation of the NR2A subunit of the NMDA receptor, thus directly attenuating the receptors functionality [While to date little is known about argon's mechanism of action, it has been proposed that argon triggers gamma-aminobutyric acid (GABA) neurotransmission by acting at the benzodiazepine binding site and possibly at multiple other discrete sites of the GABAreceptor . The actproposed ,50. Firseceptors . In an iionality . NMDA reionality ,54. Secoionality . Xu and ionality . These fNevertheless it is surprising that noble gases are able to generate an effect as it requires forming a chemical bond with another molecule. The gas molecules' outer valence shell is completely filled with electrons, making the gas inert to basic chemical reactions. However, these freely vacillating electrons can be polarized. Trudell et al. suggest that a charged element of the binding site itself can induce a bipole in the gas molecules, thus generating enough binding energy to form a bond with the binding site. Another component of the binding energy might be the London dispersion force which is generated by changes in electron density. When the distribution of electrons in one molecule fluctuates to produce an instantaneous dipole, this dipole can produce a dipole in a second molecule . Thus, tin vitro models of ischemia and traumatic brain injury. Protection was observed with three different concentrations of argon , either directly applied after the trauma or when administered at a concentration of 50% two and three hours after the injuries. Considering these promising results, despite the inherent simplifications of any in vitro model, further animal research, preferably using a whole animal model, seems appropriate.This study shows that argon bears surprisingly effective neuroprotective potential in both • We found that Argon exerts neuroprotective effects in two different types of brain lesion (oxygen-glucose deprivation and traumatic brain injury) in organotypic hippocampal slice cultures when administered after trauma.• Protection was observed with three different concentrations of argon .• Even 3 hours after trauma, argon (50%) was still neuroprotective in both models of injury.ANOVA: analysis of variance; ATP: adenosine triphosphate; EM: experimental medium; GABA: gamma-aminobutyric acid; NMDA: N-methyl-D-aspartate; OGD: oxygen-glucose deprivation; PI: propidium iodide; SEM: standard error of the mean; TBI: traumatic brain injury.MC and RR received lecture and consultant fees from Air Liquide Santé International, a company interested in developing clinical applications for medical gases, including argon and xenon. All other authors declare that they have no competing interests.PDL conducted the experimental laboratory work, performed the statistical analysis and drafted the manuscript. RR participated in the study design and coordination and helped to draft the manuscript. JR, JW, MF, AF, YR and OG helped to perform the study and draft the manuscript. MC conceived of the study, participated in the study design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


TNC arelocated in the cortex, where mature cells are rare; the occurrence of mature phenotypecells within these structures suggests that they represent a microenvironment for theselection and generation of mature T cells.Thymic nurse cells (TNC), multicellular complexes consisting of lymphoid cellsenclosed within cortical epithelial cells, were isolated from mouse thymus by a modifiedprocedure allowing immunofluorescent labeling and flow cytometric analysis of theirlymphoid contents (TNC-L). Collagenase was the only protease used for tissuedigestion, to ensure that surface antigen markers remained intact. Zonal unit-gravityelutriation was used to enrich the TNC on the basis of their high sedimentation rate,followed by immunomagnetic bead depletion to remove residual mononuclear cellcontaminants and a density separation to remove debris. The TNC-L were then releasedfrom inside TNC by a short period of culture. The measured contamination of TNC-Lwith exogenous thymocytes was around 0.5%. Three-color immunofluorescent labelingrevealed that TNC-L included, as well as a maiority of immature CD4


An asymptomatic 48-year-old man came to our institute for routine cardiac evaluation. Electrocardiogram, echocardiogram, and treadmill exercise testing were within normal limits. The patient opted for a CT angiogram (CTA) of the coronary arteries and was subsequently referred to our department. The angiogram was performed on a Light speed VCT  using a standard protocol.The study revealed an anomalous right coronary artery (RCA) originating from the left coronary sinus and coursing between the aortic root and main pulmonary artery . NarrowiThe findings described above are characteristic of a malignant RCA. It is called “malignant” because the RCA can undergo compression between the aorta and the pulmonary trunk, especially during exercise, and this carries the risk of sudden cardiac death.Coronary artery anomalies occur in about 1% of patients undergoing cardiac catheterization. Approximately 20% of coronary artery anomalies produce life-threatening symptoms, such as arrhythmias and syncope, or may cause myocardial infarction or sudden death. The causAt catheter angiography, the precise course of the anomalous vessel may be difficult to delineate, because its complex three-dimensional geometry is displayed in two dimensions fluoroscopically. DifficulMulti-detector CT (MDCT) with its cross-sectional imaging capability is a viable noninvasive modality for delineating coronary artery anomalies, particularly if findings at coronary angiography are equivocal. MDCT is


In contrast to effort-induced symptoms in obstructive coronary disease, spasm in normal coronary arteries is characterized by angina at rest. We describe a 44-year-old patient with minor coronary plaques and pure exercised-induced coronary spasm. The case questions the differential pathogenic considerations of variant of the variant as opposed to Prinzmetal's variant angina. As outlined by Maseri and coworkers, coronary artery spasm is a potential cause of angina pectoris in patients with and without atherosclerotic coronary heart disease . WhereasIn June 2008, a 44-year-old salesman started to suffer from chest discomfort during hiking and bicycle riding. However, bicycle stress testing was negative. He was nonsmoker and his LDL-cholesterol was normal (101 mg/dL). Because of recurrent episodes of effort-related thoracic pain alleviated by sublingual nitroglycerine and a family history of coronary heart diseases in both parents, the patient underwent cardiac catheterization in July 2008, demonstrating near normal vessels with minor and nonobstructive sclerotic lesions at the left anterior descending and the right coronary arteries. Because of mild hypertension during exercise, he was treated with ramipril 2,5 mg daily. As symptoms increased in intensity and frequency, another stress test in January 2009 at our institution revealed exercise induced ST-depression in leads V4–V6 accompanied by typical anginal symptoms . A seconIn the following, acetylcholine was infused into the left coronary artery resulting in complete occlusion of the LAD accompanied by angina and ST-depression  resolvinThe patient was put on long-term medication with isosorbide dinitrate and amlodipine and was free of symptoms thereafter.Obstructive atherosclerotic lesions are the leading cause of symptomatic coronary heart disease throughout the Western civilization. As opposed to effort-induced symptoms due to stenotic coronary disease, Prinzmetal reported about a special cohort of patients with angina at rest (variant angina). He speculated on an increased vascular tone at the site of coronary plaques which had been found at postmortem studies . MyocardCoronary spasm in normal coronary arteries occurs mostly at rest, in rare cases at rest and during exercise , 8. In tIt seems mandatory for the spastic vascular response to a generalized stimulus to be based on an pathologic substrate within a particular segment of the arterial wall. Although numerous articles on spasm in symptomatic patients without angiographic detectable coronary disease have been published, imaging with newer technologies such as intracoronary ultrasound and coronary tomography question whether spasm is an entity on its own (variant of the variant) or always related to the presence of atherosclerosis  . The pre