Drosophila requires the activation of Wingless (Wg) in a small stripe along the boundary of Fringe (Fng) expressing and non-expressing cells (FB), which coincides with the dorso-ventral (D/V) boundary of the wing imaginal disc. The expression of Wg is induced by interactions between dorsal and ventral cells mediated by the Notch signalling pathway. It appears that mutual signalling from dorsal to ventral and ventral to dorsal cells by the Notch ligands Serrate (Ser) and Delta (Dl) respectively establishes a symmetric domain of Wg that straddles the D/V boundary. The directional signalling of these ligands requires the modification of Notch in dorsal cells by the glycosyltransferase Fng and is based on the restricted expression of the ligands with Ser expression to the dorsal and that of Dl to the ventral side of the wing anlage. In order to further investigate the mechanism of Notch signalling at the FB, we analysed the function of Fng, Ser and Dl during wing development at an ectopic FB and at the D/V boundary. We find that Notch signalling is initiated in an asymmetric fashion on only one side of the FB. During this initial asymmetric phase, only one ligand is required, with Ser initiating Notch-signalling at the D/V and Dl at the ectopic FB. Furthermore, our analysis suggests that Fng has also a positive effect on Ser signalling. Because of these additional properties, differential expression of the ligands, which has been a prerequisite to restrict Notch activation to the FB in the current model, is not required to restrict Notch signalling to the FB.Wing development in  Delta/Serrate/Lag 2) ligand, the Notch receptor itself and a transcription factor of the CSL (CBF1/Su(H)/Lag1) family. In Drosophila two ligands, Delta (Dl) and Serrate (Ser), exist together with one DNA binding CSL factor, Suppressor of Hairless (Su(H)). The binding of a ligand to Notch elicits a proteolytic cascade that results in the release of the intracellular domain of Notch (NICD). The cleavages are mediated by the metalloprotease Kuzbanian (Kuz)/ADAM10 and the γ-secretase complex with Presenilin (Psn) at its catalytic centre H or Su(H) function results in the weak de-repression of several target genes in the absence of Notch signalling in DrosophilaThe Notch signalling pathway is an evolutionary conserved short-range signalling pathway that is involved in numerous developmental processes and diseases Drosophila wing development, the Notch pathway mediates interactions between cells across the dorso-ventral (D/V) compartment boundary that activate and maintain the expression of several genes essential for the establishment, growth and patterning of the wing primordium, notably wingless (wg) and vestigial (vg) Ser appears to be restricted to the dorsal and that of Dl is up-regulated at the ventral side of the wing anlage. Their expression is maintained by Notch activity induced through the initial regulatory loop (Dl/Ser loop) at these sides During The mutual directional signalling of the ligands requires the activity of the glycosyltransferase Fringe (Fng) After establishment of the Wg expression domain, a second regulatory loop is initiated and maintains the activity of Notch in boundary cells The current model suggests that Notch signalling at the D/V boundary respectively FB is symmetric throughout wing development Fng domain differs.In order to further investigate the mechanism of Notch signalling at a FB, we analysed the function of Fng, Ser and Dl during early stages of wing development at an ectopic FB. With help of this information, we re-examined the situation at the D/V boundary. We find that Notch signalling is initiated in a sequential fashion at both boundaries. During an initial asymmetric phase, only one ligand is required. The ligand required as well as the location of Notch activation relative to the fng with ptcGal4. ptcGal4 activates expression of UAS fng in a broad stripe of cells at the anterior side of the antero-posterior compartment boundary (A/P boundary) with increasing expression towards the posterior (see fng coincides with the antero-posterior (A/P) compartment boundary , which express Fng . Hence, the result indicates that also Dl contributes to the activation of Notch within Fng expressing ABCs.Next, we tested the behaviour of y stages . As expey stages . Howevery stages . AnterioSer function results in the loss of most wing structures including the margin and the pouch underlining its important function in wing development 94c/SerRX106Ser mutant wing imaginal discs during early third larval instar RX106Ser and the Gbe+Su(H)-lacZ construct, which faithfully detects the activity of the Notch pathway in imaginal discs Ser null phenotype, see Loss of ap expressing dorsal wing cells and appears to have spread over the whole dorsal wing anlage -lacZ is lost during later stages of wing development in the Ser mutant wing discs. This finding confirms that Ser has an additional function in maintenance of the activity of Notch.We found that Gbe+Su(H)-lacZ was expressed at the D/V boundary in wing imaginal discs of this null mutant during early stages of third larval instar –D, G–Q. ptcGal4 in RX106Ser null mutants, we found that Gbe+Su(H)-lacZ, but not Wg was ectopically expressed in the ventral side of the remaining wing anlage -lacZ was lost  and Su(H) are central members of the repressor complex that forms in absence of Notch signalling to silence the expression of target genes continue . SerRX10halteres . In the ar discs -E.ap; Ser H triple mutant wing imaginal discs -lacZ was abolished in al discs , indicatnt discs , suggestSer H double mutants, Wg expression at the ectopic FB is restricted to Fng expressing anterior cells, even in the late third instar stages  –lacZ are expressed throughout the fng domain in H Ser mutant discs. This suggests that Ser suppresses the activity of Notch throughout the fng domain during normal development.We found that in r stages -N. HenceDl in the wing anlage that were stained with an anti-Dl antibody  null mutant clones do not violate the D/V boundary Su(H) clones abutting both sides of the D/V boundary. The presence of a Ser-lacZ construct that captures the early Ap dependent expression of Ser -RNAi with apGal4. We found that these wing discs looked remarkably normal: the diameter of the two ring-like domains of wg expression is similar to wildtype and wg is expressed along the D/V boundary -RNAi within DBCs and VBCs resulted in a Su(H) loss of function phenotype that is comparable to the null allele Su(H)Δ47 Δ47 . This reh apGal4 .Su(H) function developed until the adult stage and displayed the expected phenotypes. Flies where apGal4 was used had notae devoid of nearly all bristles, but had rather normal looking wings with broadened dorsal, but normal ventral wing veins and mild distal notches  function or over-expression of H causes a restriction of expression of Wg to VBCs, but not its abolishment. Thus, the Dl/Ser loop is probably mainly required for the later occurring patterning of the future wing margin, but not for the establishment of the wing primordium.At the ectopic FB, we found that eliminating the activity of the Notch pathway in PBCs does not result in the loss of expression of Wg in ABCs. It only prevents the late symmetric phase. This indicates that establishment of a Dl/Ser feedback loop in A/P boundary cells is not essential for reaching sufficient high levels of Notch signalling to induce Wg expression during the asymmetric phase. We observed the same for the D/V boundary: Depletion of Dl is expressed throughout the wing anlage in early discs and 2. Dl signals to DBCs at the D/V boundary in absence of Ser function. This holds true also for the ectopic FB: here Dl signals to the ABCs in the absence of Ser. However, in both cases Dl signalling is not strong enough to initiate Wg expression, which is a crucial event for wing development.A further addition is that the basic expression of Dl is independent of Ser signalling. This is indicated by the observation that 1. Ser the activation by Dl from PBCs appears to fail to reach the threshold level in a fraction of discs. In agreement with this notion it has been shown that expression of Ser is broadly induced upon ectopic expression of Fng Our results also reveal an unanticipated requirement of Ser in ABCs for the expression of Wg. This requirement could be explained through Ser signalling from the ABCs to PBCs to up-regulate Dl there, which in turn signals back to ABCs (Ser/Dl loop). This explanation would imply that the levels of Notch activation required for the induction of Dl expression are lower than that for Wg. Otherwise, the expression of Wg would not stay asymemmetric as it is observed. However, we found that Notch signalling is not required in PBCs during the early asymmetric phase. This excludes the mentioned explanation and suggests that Ser must activate Notch in the Fng expressing ABCs. This assumingly weak activation contributes to the total activity of Notch in these cells and guarantees levels of Notch signalling above the threshold required for expression of Wg. In the absence of Ser and Dl function in ABCs always abolishes the expression of Wg at the ectopic FB. Hence, Dl must also signal in Fng expressing ABCs and contribute to Notch signalling in these cells. The results suggest that the total amount of Notch activity in ABCs is the sum of signalling by Dl and Ser in the Fng domain and Dl signalling from PBCs to ABCs. Whereby the signal from posteriors to anterior is the more important one, since its loss always abolishes expression of Wg. A similar requirement at on both sides of the boundary for Dl had been described for the D/V boundary We further observed that concomitant loss of fng during early stages of wing development abolishes expression of Wg at a time where it is solely dependent on Ser signalling. This observation indicates that Fng has a positive effect on Ser signalling in addition to its known negative one: it enhances Ser-signalling from Fng expressing dorsal to non-expressing ventral cells. This enhancement is required to activate expression of Wg in cells across the boundary. This finding is in good agreement with previous work that reports that Fng can enhance the ability of Ser to induce ectopic wing margins upon their co-overexpression Another addition to the current model is that Fng has two antagonistic effects on each ligand. The modification of Notch by Fringe is known to polarise signalling at the D/V-boundary by enhancing Dl- and suppressing Ser-signalling wg expression, which is located in Fng expressing cells at the ectopic FB, but in non-expressing cells at the DV boundary. We believe that the events observed at the ectopic FB represents the more general mode of interactions, since the interactions occur solely in the ventral pouch where the cells differ mainly with respect to the expression of Fng. In contrast, at the D/V boundary dorsal cells differ from ventral cells by expression the selector Ap, which might modify the outcome of the interactions.A difference between the ectopic FB and the D/V boundary is that the roles of the ligands are reversed: at the ectopic FB, Dl signalling is required for the establishment of the initial asymmetric phase and Ser to establish the symmetric phase and to maintain expression of Notch activity at a high level. In contrast, Ser signalling is required for the initial asymmetric phase at the D/V boundary and Dl for the establishment of the later symmetric phase and probably maintenance during later stages. Thus, activation of Notch-signalling at a FB can be initiated by both ligands. Another difference is the location of the asymmetric stripe of fng with ptcGal4, creates a band-like Fng domain in the ventral pouch. During early stages Dl and Ser (probably induced by Dl) activate Notch signalling throughout the Fng domain at low levels that are not sufficient for activation of Wg. At the sharp posterior FB, Dl signalling from PBCs to ABCs, enhanced by Fng, raises the levels of Notch in ABCs beyond the threshold required for expression of Wg. The asymmetric phase is established. Dl signalling also up-regulates expression of Ser in ABCs. Over time Ser signalling from ABCs to PBCs (enhanced by Fng), induces Wg expression in PBCs. After the solid induction of symmetric expression of Wg, the Dl/Ser/Wg loop takes over and maintains Notch signalling at the D/V boundary. We observed that the expression of Gbe+Su(H)-lacZ is broader upon ectopic expression of Fng in Ser mutant early third instar discs. Moreover, Wg is ectopically expressed throughout the ventral ptc domain upon ectopic expression of Fng by with ptcGal4 in Ser H, but not in H mutant discs. Thus, Ser probably contributes to keeping the Notch activity in the Fng domain at low level. It is known that the expression of Ser can contribute to the suppression of Notch activity in a cell-autonomous manner through cis-inhibition On the basis of our results, we first summarise the events at the ectopic boundary. During early stages of wing development, Dl is ubiquitously expressed throughout the wing anlage. Ectopic expression of Ser Su(H) FRT40A RX106Ser is a 9k b deletion within the transcription unit that leads to a complete loss of expression E31HUG035apvgBE-lacZ Ser-lacZ fngFlpvgSerDlUAS Hmib1and UAS GFPUAS GFP-GPiSu(H)-RNAi (DGRC 3497R-1), UAS Ser-RNAi (VDRC 27172), UAS Dcr2sdGal4, ptcGal4, dppGal4, and apGal4.UAS hsFLP construct during the first larval instar (24–48 h after egg laying).Clones were induced with an Following antibodies have been used: αNotch raised against the extracellular domain, αβ-Gal, αWg, αDl and αSer. The αNotch, αWg, αDl and αHnt antibodies were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. αβ-Gal was purchased from Cappel. αSer was a gift of E. Knust Figure S1ptcGal4 (green) is always expressed in a band-like domain in the centre of the wing anlage. (D–F) Expression of Dl in a wildtype disc  and a disc where fng is ectopically expressed with ptcGal4. The late expression pattern of Dl is shown.  Dl is expressed in two bands adjacent to the D/V boundary which is revealed in (E) through the expression of the notch target Gbe+Su(H)-lacZ. Similar bands are recognisable in the region of ectopic FB (arrowheads in F).(A–C) Expression of ptcGal4 and Wg throughout the third larval instar stage. (A) Wg is initially expressed in a ventral domain that defines the wing anlage. (B) This ventral domain results in a stripe-like domain along the D/V boundary (arrow) and a proximal ring-like domain (arrowhead). At late stages a second ring-like domain appears (arrowheads). (TIF)Click here for additional data file.Figure S2ptcGal4 UAS fng in different genetic backgrounds. (A-C) Early third instar discs. (D-F) Late third instar discs. For further explanation, see Range of the ectopic Wg expression domain induced by (TIF)Click here for additional data file.Figure S3RX106Ser mutants. RX106Ser mutant flies lack a wing and halteres. They bear either a small wing rudiment (A) or a wing to notum duplication (A’).  Expression of Wg and Gbe+Su(H)-lacZ in a late (B–D) and early  third larval instar wing imaginal disc.  Expression of Wg in RX106Ser mutant wing discs. The arrows highlight the area of the wing anlage. In mutant discs either the outer ring-like domain of Wg is left (E) or a second band-like domain characteristic for the notum can be observed (F). Expression of Wg along the D/V boundary (arrows in G–I) is absent in early third instar disc mutant for RX106Ser (G–J). However, expression of Gbe+Su(H)-lacZ is present . (M–Q) This situation is maintained also in later discs.  A RX106Ser mutant discs of the mid third instar at higher magnification showing expression of Gbe+Su(H)-lacZ. Arrowheads in  mark the extents of the dorsal side of the rudimentary anlage. Note, that the expression of Gbe+Su(H)-lacZ in dorsal cells disappears in late discs (see panel (T)). (R–T) Expression of UAS fng in RX106Ser mutant wing discs during early third instar with ptcGal4 results in the ectopic expression of Gbe+Su(H)-lacZ . Note, that the expression of Gbe+Su(H)-lacZ is restricted to the ptc domain. (T) The ectopic expression of Gbe+Su(H)-lacZ is lost during later stages of development.The Analysis of (TIF)Click here for additional data file.Figure S4RX106HE31Ser double mutants. Pharate adults have halteres and wings with a proper margin, indicated by the innervated bristles characteristic for the margin (shown in higher magnification in (A’)). (B-I) Analysis of RX106HE31Ser double mutant wing imaginal discs. Expression of Gbe+Su(H)-lacZ (B), Wg (C) along the D/V boundary. (D) Expression of the two Notch targets in relation to that of Ap. The magnification of the region highlighted with the arrow in (D) accentuates the fact that expression of the Notch targets is restricted to Ap-expressing DBCs (E). (F) Expression of Gbe+Su(H)-lacZ at the D/V boundary is extinguished in UG035SerRX106HE31ap triple mutant wing imaginal discs. (G) Expression of Dl in RX106HE31Ser double mutant wing imaginal discs.  Expression of Gbe+Su(H) and Ap in a early third instar RX106HE31Ser double mutant wing imaginal disc. Expression of Gbe+Su(H)-lacZ is expands throughout the Ap-expressing dorsal half of the disc (arrows). (J-N) Expression of UAS fng in RX106HE31Ser double mutant wing imaginal discs results in the ectopic expression of Gbe+Su(H)-lacZ (J) and Wg (L) (arrow in J–M). The merge of (J–L) reveals that the ectopic expression of the marker is restricted to the ptc domain, even in late third instar. The expression of the markers in PBCs seen in wt and E31H mutant discs (arrow in N) is missing. Note that the expression of Wg can be observed in a broad band throughout the ptc domain and is not restricted to the FB as in the wildtype.Analysis of the (TIF)Click here for additional data file.Figure S5H with sdGal4 (A) and apGal4 (B–D). Expression with sdGal4 results in a loss of Wg expression along the D/V boundary, while the expression with apGal4 restricts the expression along the D/V boundary to VBCs (arrow in B–D). (E) Expression of UAS Ser-RNAi only in dorsal wing cells with apGal4 results in a severe truncation of the wing (arrow).  In contrast, expression of UAS Su(H)-RNAi in the same manner allows the formation of a nearly normal wing with broadened dorsal wing veins (arrow). (H) Wing disc of a fly where UAS Su(H)-RNAi was expressed with apGal4. The arrow points to the supernumerary sensory organ precursor cells formed on the dorsal side of the wing pouch in the absence of Su(H). (I) Expression of UAS Su(H)-RNAi in dorsal and ventral cells results in a severe truncation of the wing (arrow).(A-D) Expression of UAS (TIF)Click here for additional data file.Text S1RX106Ser flies.The phenotype of (DOCX)Click here for additional data file.


Ischemia-modified albumin is an altered serum albumin that forms under conditions of oxidative stress, a state also associated with doxorubicin-induced myocardial injury.The aim of this study was to better assess diagnostic and prognostic significance of ischemia-modified albumin in patients with breast cancer undergoing doxorubicin chemotherapy.Blood samples were collected from 152 breast cancer patients before and after each cycle of doxorubicin chemotherapy to measure the serum levels of ischemia-modified albumin, cardiac troponin T and creatine kinase-MB. We also monitored cardiac function during a 12 month follow-up.r = 0.212, P < 0.05). The combination of ischemia-modified albumin with cardiac troponin T and creatine kinase-MB increased the sensitivity to 0.920 and the specificity to 0.830 in the diagnosis of doxorubicin-induced myocardial injury. The optimal cutoff for ischemia-modified albumin concentration was 112.09 U/ml. The rate of change for ischemia-modified albumin levels correlated negatively with the rate of change for left ventricular ejection fraction at one year .There was a significant difference in ischemia-modified albumin levels before and after each cycle of chemotherapy and the ischemia-modified albumin concentration positively correlated with the cumulative dose of doxorubicin (Ischemia-modified albumin may be a clinically potential new marker for diagnosing doxorubicin-induced myocardial injury, and is helpful to predict long-term impairment of cardiac function. Streptomyces peucetiusvar.caesius in 1969 post-chemotherapy) / [mean IMA concentration]pre-chemotherapy.The rate of change for LVEF before chemotherapy and at the one year follow-up was calculated in the same way. Correlation analysis revealed that the rate of change for IMA correlated negatively with the rate of change for LVEF . There was significant difference for serum IMA concentrations before and after each cycle of DOX chemotherapy. Then the serum samples of patients after each cycle of chemotherapy were divided into myocardial injury group and no myocardial injury group according to the diagnosis criteria of myocardial injury. We found that there was significant difference of IMA concentration in serum samples between myocardial injury group and no myocardial injury group in each cycle of chemotherapy (Table 1), And the serum IMA concentration positively correlated with the cumulative dose of DOX . No previous studies have examined the relationship between changes in serum IMA concentration and DOX-induced myocardial injury. So we are ready to measure serum IMA in patients with breast cancer before and after each cycle of DOX chemotherapy and calculated the cumulative dose of DOX. However, albumin cobalt binding assay determines IMA concentration based on the unbound cobalt to albumin, IMA concentration prediction can’t be accurate unless it is determined with respect to total albumin concentration in the patients before and after DOX treatment. So the 152 serum samples of 152 patients before chemotherapy and serum samples of patients after each cycle of chemotherapy were measured for serum albumin concentration, the serum albumin concentration of patients before DOX chemotherapy and the serum albumin concentration of patients after each cycle of DOX chemotherapy  were analyzed before detecting the IMA levels. There was no significant difference for serum albumin concentration before and after each cycle of DOX chemotherapy (data not shown). Then excluding the serum albumin concentration factor, we detected the IMA levels. The results showed that the pre-chemotherapy serum concentration of IMA was (59.2 ± 10.9) U/ml, which was nearly consistent to the serum concentration of IMA for the control reference population in a previous study  . After follow-up at 1 year, we found that the impairment in cardiac function was most pronounced in patients whose IMA increase exceeded the cutoff value (Table 3). Moreover, rate of change for IMA was negatively correlated with the rate of change for LVEF .In order to further evaluate the clinical value of IMA, we compared the serum IMA concentrations with cTnT and CK-MB. After 1-year follow-up, when serum IMA were combined with cTnT and CK-MB levels, the sensitivity and specificity(with a sensitivity of 0.920 and a specificity of 0.830 )for diagnosing DOX myocardial injury were improved, and the AUC was largest 2 and 3. AfterAs an anthracycline antibiotic, DOX exhibits a high affinity for membrane phospholipids , neutral phospholipids , and cardiolipin, resulting in a very long elimination half-life from cardiac tissue . Our obsIn conclusion, we found that IMA is a valuable indicator of diagnosing DOX-induced myocardial injury and is helpful to predict long-term impairment of cardiac function for DOX-induced myocardial injury patients with breast cancer. However, whether IMA can really work as a marker of chronic toxicity (long term over years) from DOX in clinical, further studies are needed by broaden numbers of patients and a randomized control study. In addition, we studied serum IMA only in patients with breast cancer, and caution must be exercised when extrapolating our findings to patients receiving DOX for other indications. And our conclusion based on clinical observation rather than laboratory-based mechanism research, further studies also are needed to broaden the research scope to include other tumor types and to explore the mechanism of IMA generation in DOX-induced myocardial injury.


To evaluate the feasibility and value of first-pass contrast-enhanced dynamic and post-contrast 3D CMR in patients after transcatheter occlusion of left atrial appendage (LAA) to identify incorrect placement and persistent leaks.7 patients with different occluder systems  underwent 2 contrast-enhanced (Gd-DOTA) CMR sequences (2D TrueFISP first-pass perfusion and 3D-TurboFLASH) to assess localization, artifact size and potential leaks of the devices. Perfusion CMR was analyzed visually and semi-quantitatively to identify potential leaks.All occluders were positioned within the LAA. The ACP occluder presented the most extensive artifact size. Visual assessment revealed a residual perfusion of the LAA apex in 4 cases using first-pass perfusion and 3D-TurboFLASH indicating a suboptimal LAA occlusion.By assessing signal-to-time-curves the cases with a visually detected leak showed a 9-fold higher signal-peak in the LAA apex  than those without a leak . In contrast, the signal increase in LAA proximal to the occluder showed no difference .This CMR pilot study provides valuable non-invasive information in patients after transcatheter occlusion of the LAA to identify correct placement and potential leaks. We recommend incorporating CMR in future clinical studies to evaluate new device types. Atrial fibrillation is the most common sustained cardiac arrhythmia and affects 5 percent of people older than 65 years and 10 percent older than 75 years . AnticoaHowever, long-term anticoagulation is frequently associated with problems of safety and tolerability, such as increased risk of bleeding. Occlusion of the left atrial appendage could be a potential alternative strategy for prophylaxis of embolism. In comparison to surgical amputation, the percutaneous transcatheter occlusion of LAA is a minimally invasive technique and yields promising results in animal  and humaCurrently, following the transcatheter procedure, a chest X-ray is performed to confirm the correct placement of the occluder and transesophageal echocardiography allows for the evaluation of possible thrombotic appositions on the device. To date, it has been still challenging to assess directly and non-invasively the residual LAA perfusion indicating a persistent leak due to an insufficient design, a mismatch of device and LAA anatomy or a failure of complete coverage of the atrial-facing surface of the device with neoendothelial-like cells . A persiThe purpose of this pilot study was to evaluate the feasibility and value of first-pass contrast-enhanced dynamic and post-contrast 3D CMR in patients after transcatheter occlusion of LAA to identify incorrect placement and persistent leaks.7 adult patients  with non-rheumatic atrial fibrillation received transcatheter occlusion of LAA due to warfarin contraindications . 4 patients received a PLAATO device , 2 patients a Watchman device  and one patient an ACP device . Our study population for this pilot study was drawn from subsequent patients undergoing transcatheter occlusion of LAA with clinically suspected device malposition or residual leaks who consented to partake in this CMR study. The different designs of the devices are displayed in Figure The patients underwent CMR median 102 days (range 91-229 days) after the LAA occlusion procedure after written informed consent had been obtained. None of the patients had suffered a stroke between the transcatheter occlusion of LAA and the date of the CMR scans. The study was approved by the local ethics committee.Magnetic resonance imaging was performed on a 1.5-T CMR system . For signal detection, the combinations of a six-channel body matrix coil and six elements of a spine matrix coil were used. The ECG-signal was received via an external system .3) served to determine LAA anatomy and for detection of the occluder system.ECG-gated segmented steady-state-free-precession (SSFP) cine-sequences  were performed simultaneously planned from the optimal views on cine studies in the oblique axial and sagittal long axis. For each slice 40 consecutive images were acquired during the administration of a bolus of Gadoterate Meglumin  followed by 30 ml saline, both at 4 ml/second into an antecubital vein.Two slices of a saturation-recovery SSFP sequence  was acquired in the same optimized planes.After the administration of another 0.1 mmol/kg bodyweight Gd-DOTA  an ECG-gated 3D-TurboFLASH sequence  were evaluated by consensus of 2 observers experienced in cardiovascular radiology. The readings were performed blinded to any clinical information. In every patient the readers evaluated qualitatively for occluder size , occluder localization  and persistent leaks .Additionally, contrast-enhanced perfusion studies were analyzed generating signal-time-curves . Two different regions of interest (ROIs) were placed in the left atrium and in the LAA apex distal to the occluder device. Due to heart movement ROIs were manually fitted to every image without changing the size. The signal-time-curves were normalized to baseline signal  for each ROI . The sinStatistical analysis was performed using SPSS 18.0. Quantitative data are presented as mean and standard deviation or median and range when appropriate. The independent samples Mann Whitney U Test (non-parametric test) was performed to assess for differences in time to the signal peaks in the left atrium and the LAA apex and signal peaks as a percentage to baseline signal. A p value less than 0.05 was considered statistically significant.There was no dislocation of the devices outside the LAA. In 4 cases the atrial-facing device border was localized at the ostium of LAA whereas in 3 cases the device was localized more deeply within the LAA body. All of the 3 cases with device localization more deeply within the LAA presented a leak but only one localized at the ostium of LAA. In 5 cases the occluder-related signal voids show a slight bulging (4.2 ± 3.6 mm) into the left atrium. The mean size of the occluder device including surrounding artefacts was 28.6 × 25.3 × 25.4 ± 3.9 × 3.3 × 3.1 mm.Baseline characteristics of each patient are presented in table Visual evaluation of contrast-enhanced first-pass perfusion CMR revealed contrast enhancement at the apex of the LAA beyond the device indicating a residual leak in 4 cases.Patients with visually detected residual perfusion showed 9-fold higher peaks in the LAA apex distal to the device  than those without a leak . In contrast, the signal peaks in the left atrium body showed no difference in patients with compared to patients without leaks .The time to signal peaks was similar in patients with and without leaks in the left atrium  or in the LAA .According to the anatomical size the median pixel size of ROIs in the left atrium  was larger than in the left atrial appendage .Cases of visually detected residual leaks after transcatheter LAA occlusion are presented in Figure Our pilot study highlights the potential role of contrast enhanced CMR to evaluate the localization of LAA occluder systems and to detect residual leaks after implantation.Atrial fibrillation affects 3-5% in patients older than 65 years and is responsible for 15-20% of strokes . This unCurrently, chest X-ray and echocardiography are used after transcatheter procedure to verify correct placement of the device and to exclude thrombotic appositions or residual leaks. But chest X-ray is only able to detect extracardiac displacement and echocardiography is limited to evaluate residual flow since flow velocities within LAA in patients with atrial fibrillation are highly variable even before the procedure . Pre-proThere are age- and sex-related differences in LAA dimensions as well as various anatomical variants ,20. AccuOver- or undersizing, device migration or displacement could contribute to unsuccessful device deployment. Incomplete occlusion of LAA is one of the major concerns leaving the clinician in the dilemma of whether it is safe to stop warfarin therapy. CMR-based measurements could be helpful to understand the principle of residual leaks due to potential for suboptimal device deployment. All devices with residual perfusion were localized deeper into the LAA and a small rim of contrast agent could be identified around the margins of the device using 3D imaging sequences.Diagnosis of post-procedural leaks seems to be detectable using qualitative visual assessment, which obviates the need for time-consuming post-processing. However, less experienced observers may wish to confirm the visual assessment by semi-quantitative assessment and we identified 9-fold higher signal intensity peaks. A possible underlying problem of using our proposed CMR perfusion technique needs to be considered in the context of devices causing metal artifacts which can lead to spurious local increase of signal in the LAA region before contrast arrival and/or possible loss of contrast related signal after its arrival in the test region.The high incidence of residual leaks after transcatheter LAA occlusion in our pilot study result is due to a selection bias as we only included patients in whom problems of device malposition or residual leaks were suspected and does not contradict the very promising results of this therapy approach -13. HoweResidual perfusion could be a source of thromboemboli and these patients might be considered for further anticoagulation. However, despite our encouraging initial experience using contrast-enhanced CMR, this warrants further investigation regarding its predictive value and impact on treatment strategies. Given the small numbers of LAA device occlusion performed even in specialist centres, this should be done in a multi-center study.Our pilot study did not address the important question whether CMR is able to detect small thrombi on the device surface. In this pilot study we did not compare our findings to transoesophageal echocardiography.Determination of signal time curves without partial volume effect can be difficult or occasionally impossible to obtain due to the complex and variable left atrial appendage anatomy. However, leaks around the left atrial appendage occluder device are usually visible and the signal time curves are most often for confirmation only.CMR is feasible to confirm occluder placement and to detect residual LAA leaks non-invasively and without radiation. Given the variable and complex anatomy of the LAA CMR may inform device companies how to tailor and optimize the design of such devices and to test them in small pilot studies. We propose that clinical trials to test LAA occlude devices should incorporate contrast-enhanced CMR.LAA: left atrial appendage; CMR: cardiac magnetic resonance imaging; Gd-DOTA: gadolinium gadoterate meglumin; 3D: three-dimensional; FLASH: fast-low-angle-shot; FISP: Fast Imaging with Steady State Precession; SSFP: steady-state-with-free-precession; ROI: region of interest; CT: computed tomography.The authors declare that they have no competing interests.OKM - conception and design; acquisition of data; analysis and interpretation of data; drafting the manuscript; revising the manuscript critically; given final approval. NW - conception and design; analysis and interpretation of data; revising the manuscript critically; given final approval. SEP - conception and design; analysis and interpretation of data; revising the manuscript critically; given final approval. AP - conception and design; analysis and interpretation of data; revising the manuscript critically; given final approval. HUK - conception and design; analysis and interpretation of data; revising the manuscript critically; given final approval.Movie of a residual peak in sagital angulation. 67-year-old male after percutaneous transcatheter LAA occlusion using a Watchman device. The movie shows the temporal sequence of first-pass contrast-enhanced perfusion imaging in sagital angulation. The contrast-enhancement of the LAA apex beyond the device is representing a slow wash-in due to a small residual leak.Click here for fileMovie of a residual peak in axial angulation. Same patient and findings as in additional file 1 (movie 1) but in an axial angulation.Click here for file


Within the same cell, different signaling routes can signal through the same kinase. To still function in a specific manner, the kinase is localized in close proximity to its substrates and upstream signaling components. This is best described for cAMP-dependent protein kinase (PKA), which utilizes the diverse family of A-kinase anchoring proteins  for spatiotemporal control.PKA’s closest homologue is the cGMP-dependent protein kinase (PKG), however many details of its localized signaling are not well understood. Thus far, very little G-kinase anchoring proteins (GKAPs) have been identified, likely because they are lower in abundance than AKAPs. In addition, unlike as for AKAPs, common motifs in GKAPs that mediate the interaction have not been convincingly defined.Here we present a chemical proteomics approach which utilizes immobilized cAMP in combination with low concentrations of free cAMP or cGMP to specifically dissect the lower abundant PKG and its associated GKAPs, from the more prevalent PKA and interacting AKAPs. Combined with LC-MS/MS this proved a powerful screening tool for the discovery and characterization of novel protein kinase G anchoring proteins directly in several different tissues.The isolated PKG fraction contained several known GKAPs (e.g. IRAG), but also a novel putative GKAP, Huntingtin associated protein 1 (HAP1). Further characterization of the PKG-HAP1 interaction was performed with classical biochemistry, cell biology and an interaction-based proteomics approach with immunoprecipitations of GFP-HAP1 and PKG-GFP. This powerful combination confirmed the interaction and also revealed a further refinement of the cellular localization and molecular environment of the HAP1-PKG complex.Our data highlight the power of a tailored chemical proteomics approach to identify novel cGMP signaling hubs in a tissue of interest.


The intercellular transfer of misfolded proteins has received increasing attention in various neurodegenerative diseases characterized by the aggregation of specific proteins, as observed in Alzheimer’s, Parkinson’s and Huntington’s disease. One hypothesis holds that intercellular dissemination of these aggregates within the central nervous system results in the seeded assembly of the cognate soluble protein in target cells, similar to that proposed for transmissible prion diseases. The molecular mechanisms underlying the intercellular transfer of these proteinaceous aggregates are poorly understood. Various transfer modes of misfolded proteins including continuous cell-cell contacts such as nanotubes, unconventional secretion or microvesicle/exosome-associated dissemination have been suggested. Cells can release proteins, lipids and nucleic acids by vesicular exocytosis pathways destined for horizontal transfer. Encapsulation into microvesicular/exosomal vehicles not only protects these molecules from degradation and dilution in the extracellular space but also facilitates delivery over large distances, e.g. within the blood flow or interstitial fluid. Specific surface ligands might allow the highly efficient and targeted uptake of these vesicles by recipient cells. In this review, we focus on the cell biology and function of neuronal microvesicles/exosomes and discuss the evidence for pathogenic intercellular protein transfer mediated by vesicular carriers. Microparticles have been isolated from various body fluids such as urine, ascites, saliva, breast milk and blood by ultracentrifugation, ultrafiltration or immunoprecipitation  endosomal compartments by inward vagination and fission of the limiting membrane. Endosomes that are filled with these intraluminal vesicles (ILV) are termed multivesicular endosomes (MVE). ILVs can serve as storage compartments for proteins and signalling complexes and can re-enter the cytosol by backfusion with the MVE limiting membrane  are generated by shedding at the plasma membrane and include microvesicles with a heterogeneous size range from 100 nm to 1 μm and apoptotic bodies. Apoptotic bodies are released from the plasma membrane during the breakdown of apoptotic cells. They carry DNA, histones, organelles and surface markers that allow their recognition and internalization by phagocytic and other subsequent cells, thereby preventing the release of intracellular content and inflammatory reactions  subunits. MVE-mediated release could therefore be a mechanism to eliminate α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors in response to glutamatergic stimulation  has been shown to be transported trans-synaptically with vesicles resembling exosomes, followed by the binding of wg to Drosophila frizzled 2 (DFz2) receptors at the postsynapse . In infectious prion diseases, PrPsc can enter the organism by the gut, followed by the invasion of lymphoid tissue from where it spreads into the peripheral nervous system and finally the CNS. In addition to intercellular transfer by tunneling nanotubes, as discussed by Gousset et al. , bovine spongiform encephalitis (BSE) and scrapie. Prions exist in two different conformational states: the natively folded PrPt et al. , a role c to PrPsc takes place remains unclear; however, speculation that the MVE/EMV system is involved via local protein enrichment, favourable pH and the lipid environment is tempting. Macromolecular crowding has been shown to promote the conversion to β-sheet structure and the oligomerization of prions  proteins are apolipoproteins that are expressed in the liver and that circulate in the blood stream bound to high density lipoproteins. Under inflammatory conditions and interleukin-1 and -6 and tumor necrosis factor stimulation, the expression of these acute phase proteins is increased up to 1000-fold. During chronic inflammation such as rheumatoid arthritis, high concentrations of SAA eventually lead to the formation of a nucleus and polymerization of otherwise soluble SAA proteins into amyloid fibrils. Deposits of SAA fibrils can be found in the interstitial space of many organs. Similar to prion protein misfolding, this SAA fibrillation involves a conformational shift of SAA protein into a β-sheet structure followed by aggregation. Mouse models of experimental AA-amyloidosis develop systemic amyloid deposits under chronic inflammatory conditions triggered by the intravenous, intraperitoneal or oral application of SAA-containing tissue or circulating blood monocytes derived from murine SAA mouse models. This process is reminiscent of transmissible prion diseases  in amyotrophic lateral sclerosis (ALS), huntingtin in Huntington’s disease (HD) and α-synuclein in Parkinson’s disease (PD). Aggregopathies do not belong to the class of prion diseases, since infectious transmission between two individuals has never been observed. However, intra- and inter-individual spreading of disease pathology in several of these aggregopathies has led to their classification as possible prionoid disorders  transgenic mouse brains containing aggregated amyloid-β into the brains of APP transgenic mice induces the formation of amyloid-β plaques in the host brains , aggregates of misfolded superoxide dismutase 1 (SOD1) propagate in a spatiotemporal manner linking upper and lower motor neurons . In addition, tau pathology has been genetically linked to PD and LBD. Since both α-synuclein and tau have been detected in EMVs , these vesicles might represent the site in which cross-seeding occurs.In vivo evidence is needed to answer the question of whether EMVs do indeed confer toxicity and induce seeding in animal models, as has been shown in SAA amyloidosis. The study of the in vivo significance of EMV-mediated disease propagation is hampered by the lack of specific agents to interfere with EMV release or uptake; such agents would enable in vivo studies on the spread of disease pathology. The cell biology of protein sorting and EMV release is still not resolved. An interaction with the endosomal sorting complex required for transport (ESCRT) machinery has been described for mono-ubiquitinated transmembrane proteins; this machinery regulates their sorting into ILVs. Ubiquitin-interacting motifs mediate the binding of ESCRT 0 to cargo destined for sorting into MVBs. The bound cargo is sequentially transported to ESCRT complexes I and II at the late endosomal membrane from where invagination and fission into the endosomal lumen occurs with the help of ESCRT-III  and phosphatidylethanolamine (PE) and the asymmetric distribution of these lipids has been proposed as a mechanism to trigger membrane bending because of their conical shape  profiling is a common approach in the development of novel diagnostic or prognostic biomarkers, especially in oncology (for reviews, see Mathivanan et al. The potential of EMVs for the targeted delivery of therapeutic drugs is currently under investigation. This emerging concept has been boosted by a recent publication on the targeted exosomal delivery of siRNA-directed against β-secretase in an Alzheimer mouse model (Alvarez-Erviti et al.


In vitro studies using adult rat cardiac fibroblasts also showed that aliskiren attenuated the pro-fibrotic pattern of matrix gene and protein expression induced by D,L, homocysteine. Both in vivo and in vitro studies demonstrated that the Akt pathway was activated by homocysteine, and that treatment with aliskiren attenuated Akt activation. In conclusion, aliskiren as mono-therapy has potent and direct effects on myocardial matrix turnover and beneficial effects on diastolic function.Myocardial fibrosis, a major pathophysiologic substrate of heart failure with preserved ejection fraction (HFPEF), is modulated by multiple pathways including the renin-angiotensin system. Direct renin inhibition is a promising anti-fibrotic therapy since it attenuates the pro-fibrotic effects of renin in addition to that of other effectors of the renin-angiotensin cascade. Here we show that the oral renin inhibitor aliskiren has direct effects on collagen metabolism in cardiac fibroblasts and prevented myocardial collagen deposition in a non-hypertrophic mouse model of myocardial fibrosis. Adult mice were fed hyperhomocysteinemia-inducing diet to induce myocardial fibrosis and treated concomitantly with either vehicle or aliskiren for 12 weeks. Blood pressure and plasma angiotensin II levels were normal in control and hyperhomocysteinemic mice and reduced to levels lower than observed in the control group in the groups treated with aliskiren. Homocysteine-induced myocardial matrix gene expression and fibrosis were also prevented by aliskiren.  Heart failure with preserved ejection fraction (HFPEF) is increasing in prevalence and is associated with significant morbidity and mortality The study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures in this study were approved by the Institutional Animal Care and Use Committee of Boston University School of Medicine (Protocol # AN-15057) and Harvard Medical School (Protocol # 04783). Male C57BL6 mice (8–10 weeks old) were purchased from Charles River Laboratories  and were maintained in our institutional Division of Laboratory Animal Medicine on a 12∶12 light-to-dark cycle with free access to chow and water. Osmotic mini-pumps for delivery of aliskiren  were implanted subcutaneously in the dorsal chest under anesthesia. Either vehicle, or aliskiren in the doses of 0.5 mg/kg/day, 5 mg/kg/day, or 50 mg/kg/day, was delivered via osmotic mini-pump. Since aliskiren is designed to specifically inhibit human renin, higher doses are required to inhibit murine renin, and 50 mg/kg/day is a widely accepted dose to provide adequate inhibition of murine rennin Coronal sections of ventricular myocardium were fixed in 10% neutral buffered formalin, and serial sections (5 μm) were stained with hematoxylin and eosin for estimating myocyte size, and with Picrosirius red for estimating fibrillar collagen. Perivascular collagen, coronary arteriolar wall thickening, interstitial collagen volume fraction, and myocyte size were measured as described previously ex vivo utilizing isolated perfused Langendorff heart preparations Cardiac function was measured ΔΔCT, on the Step One Applied Real-Time PCR System Instrument (Applied Biosystems). The PCR cycling conditions were 50°C for 10 minutes, 95°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Data were analyzed using the comparative cycle threshold (Ct) method.Total RNA was extracted from frozen mouse heart sections using RNeasy Fibrous Tissue Mini-columns . High Capacity cDNA Reverse Transcription Kit  was utilized to make cDNA from 1 µg of RNA. Real-time PCR was performed using the TaqMan universal PCR mastermix  and the TaqMan inventoried FAM labeled Gene Expression assays for alpha 1 chain of type I collagen , alpha 2 chain of type I collagen , and alpha 1 chain of type III collagen . Data were normalized by multiplexing with the Taqman inventory VIC labeled gene expression assay for GAPDH  endogenous control assay and expression was compared relative to control treated samples using the formula, 2−125I-angiotensin II was then added to compete with angiotensin II present in samples and standards for the same binding sites on the anti-angiotensin II antibody. After further incubation for 6 hours, the solid phase second antibody was added to the mixture, and the antibody-bound fraction was precipitated and counted.Plasma angiotensin II level was measured in the Harvard Catalyst Central Laboratory utilizing a double-antibody radioimmunoassay using modification of the method described by Emmanuel et al Adult rat cardiac fibroblasts were isolated and cultured as described previously in publications from our laboratories Western blot analysis was conducted as described previously (15). Primary antibodies utilized for this study were: Collagen I ; phospho-Akt and Akt, ; and GAPDH .GraphPad Prism . The criterion for significance was a p value <0.05. Data are reported as means ± SEM.Data were evaluated by ANOVA with a Student-Newman-Keuls post-hoc test or by t-test as appropriate using As shown in As shown in As shown in in vitro results demonstrate that aliskiren directly attenuates the pro-fibrotic effects of homocysteine on cultured cardiac fibroblasts, and that the Akt pathway is involved in the beneficial effects of aliskiren. Cumulatively, our results suggest that direct renin inhibition with aliskiren exerts potent anti-fibrotic effects in the myocardium via both systemic effects on RAAS and direct effects on cardiac fibroblast biology, and thereby demonstrates the potential benefit of aliskiren in HFPEF.Our results show that in a non-hypertensive, non-hypertrophic model of myocardial fibrosis and diastolic dysfunction in vivo study is that we did not directly compare the effect of aliskiren to an antihypertensive that acted independent of RAAS, or to another RAAS inhibitor. Since the blood pressure and angiotensin II levels in aliskiren groups were similar in both control and Hhe treated animals exposed to the highest does of aliskiren, it is not clear whether these systemic effects may have played a role in the anti-fibrotic effects of aliskiren. The observation that the heart weight to body weight ratios and cardiomyocyte size were similar in all groups suggests that the effects were independent of any load-induced ventricular hypertrophy. In addition, the anti-fibrotic effects of aliskiren were also observed at doses that did not alter blood pressure or angiotensin II levels. Recent investigations also suggest that the beneficial effects of aliskiren may be independent of blood pressure lowering. For example, in a mouse model of post-myocardial infarction heart failure, aliskiren was shown to have better effects on ventricular remodeling compared to a dose of hydralazine that produced comparable blood pressure lowering Our results suggest that aliskiren can directly attenuate fibrotic mechanisms in the myocardium and normalize diastolic function in an established model of myocardial fibrosis. Although plasma angiotensin II levels and blood pressure were in the normal range in the Hhe group treated with vehicle, aliskiren dose dependently decreased both systolic and diastolic blood pressure and plasma angiotensin II levels on long-term treatment. A limitation of our in vitro results corroborate our in vivo findings and suggest a direct anti-fibrotic effect of aliskiren on cardiac fibroblasts. Homocysteine-induced increase in the expression of collagen types I and III genes were suppressed by aliskiren in lower doses. Similar effects were observed on collagen type I protein levels secreted into the culture medium. Multiple studies have shown similar effects of renin, prorenin and their inhibition by inhibitors including aliskiren on matrix turnover in stromal cells. For example, He and coworkers have shown that (pro)renin receptor blockade resulted in anti-proliferative and anti-fibrotic effects in cultured rat mesangial cells Our results suggest that the myocardial matrix is a major target of aliskiren's cardiovascular effects. Myocardial type I collagen mRNA and protein levels induced by Hhe were reduced by exposure to lower doses of aliskiren. Our in vivo, hyperhomocysteinemia has been demonstrated to enhance angiotensin II-induced vasoconstriction in vivo dose which did not reduce plasma angiotensin II levels or blood pressure, it is likely that the effects of aliskiren were mediated by inhibition of (pro)renin receptor mediated and angiotensin II- independent effects of renin and prorenin. Multiple studies have demonstrated effects of prorenin and renin independent of the RAAS. In the initial reports by Nguyen on the (pro)renin receptor, it was demonstrated that renin-induced DNA synthesis was independent of enzymatic activity In vivo studies on rats transgenic for the human (pro)renin receptor demonstrated that mitogen activated protein kinase activation and TGB-β expression was not attenuated by an ACEI, suggesting angiotensin II-independent MAPK activation modulated via the (pro)renin receptor Preclinical studies from our laboratories and those of others have shown that Hhe promotes myocardial fibrosis and dysfunction Multiple signal transduction pathways including Phosphoinositide-3 kinase (PI3K)/Akt pathway has been shown to be induced by prorenin in cultured vascular smooth muscle cells In conclusion, our results suggest that aliskiren exerts potent and direct anti-fibrotic effects in a clinically relevant model of myocardial fibrosis and diastolic dysfunction. Since current modes of RAAS antagonism may not provide a potent anti-fibrotic effect


Stenting of the right coronary artery stenosis caused coronary perforation and profound dye (blood) extravasation in a 69-year-old female patient. Instantaneous balloon inflation followed by implantation of a polytetrafluoroethylene- (PTFE-)covered stent sealed the coronary perforation, restored the blood flow, and perceivably caused acute occlusion of a large side branch (SB). The immediate in situ balloon inflation prevented the development of cardiac tamponade. Surprisingly, followup coronary angiography 4 and 11 months later showed spontaneous recanalization of the SB occluded by PTFE-covered stent. The SB was filled through a channel beginning at the end of the covered stent streaming retrogradely beneath it toward the SB ostium. Up to the best of our knowledge, this is the first described case of late spontaneous recanalization of as SB occluded by a PTFE-covered stent. Coronary artery perforation is a rare but life-threatening complication of percutaneous coronary intervention (PCI) , 2. AmonThe case is a 69-year-old female patient with angina pectoris. Her previous history was unremarkable apart from bilateral cosmetic implantation of breast prosthesis many years ago. Coronary angiography revealed mild changes in the left coronary artery and a significant stenosis in the right coronary artery (RCA). A relatively large SB, which was the right ventricular branch, emanated from the stenosed region of the RCA. The stenosis was dilated with an undersized balloon followed by implantation of a bare-metal stent-type driver 4.0 × 24 mm with a pressure of 14 atmospheres . This reCoronary artery perforation is a rare but life-threatening complication of PCI. It may lead to significant morbidity and mortality including cardiac tamponade and death . Ellis gThe stent balloon was still in place in our case when the coronary perforation was detected. The balloon was inflated immediately while preparing the covered stent. The instantaneous balloon inflation prevented the development of cardiac tamponade. This highlights the importance of having the stent balloon in place while checking the immediate results of stent implantation at the lesion site. Side branch occlusions have been reported during implantation of both bare-metal (7%) and drug-eluting (10%) stents. Spontaneous recanalization of an occluded SB is common after implantation of both bare-metal (67%) and drug-eluting (92%) stents . RecanalIn conclusion, we have described for the first time a case of spontaneous recanalization of a SB occluded by a blood impermeable PTFE-covered stent. We also would like to emphasize the importance of checking the coronary artery immediately after stent implantation while the stent balloon is in place. This helped us in our case to prevent development of cardiac tamponade.We would like to confirm that we have no conflict of interests regarding the paper.


Objective: The aim of the present study was to investigate the role of inflammation in angiogenesis of keratocystic odontogenic tumor (KCOT).Study Design: Twenty inflamed and 20 non-inflamed KCOTs were selected based on quantitative scoring of inflammation which was also applied on 20 radicular cysts. Microvessel density was assessed in all samples using CD34 antibody and angiogenesis was compared between the three groups. Statistical analysis was performed using one-way analysis of variance followed by post-hoc Scheffe test and P values less than 0.05 were considered significant.Results: A statistically significant difference in angiogenesis was found between radicular cysts and both inflamed and non-inflamed KCOTs (P < 0.001), but not between inflamed and non-inflamed KCOTs (P =0.347).Conclusion: Based on the results obtained in the present study, it seems that the effect of inflammation on angiogenesis in KCOT is minimal. However further investigation using other methods of evaluation is suggested to fully clarify the role of “inflammatory angiogenesis” in this neoplasm. Key words:Keratocystic odontogenic tumor, radicular cyst, angiogenesis, inflammation. Keratocystic odontogenic tumor (KCOT) is a cystic lesion derived from the dental lamina and differs from other odontogenic cysts in displaying an aggressive clinical behavior and repeated recurrences. These features along with its distinct histologic characteristics have been the basis of numerous investigations, leading to a change in nomenclature from odontogenic keratocyst to KCOT ,2. Both Inflammation and angiogenesis are both stromal elements which have been considered as indications of host involvement in tumor maintenance, support and progression . InflammAngiogenesis involves the formation of new microvessels from preexisting vasculature and is an essential part of growth, whether in the form of normal tissues or neoplasms . EndotheThe protocol for this study was approved by the university Research and Ethics Committee. Archival haematoxylin/eosin-stained-slides of our departments were reviewed from 2000 to 2010 and after verification of the clinical and surgical data, 20 RCs and 60 KCOTs with various amounts of inflammation were chosen and their paraffin blocks retrieved. Presence of inflammation was based on a slight modification of the method described by Hirshberg et al.  and Kapl-Inflammation density and immunohistochemical scoringFive consecutive high power fields ×400) were chosen from the connective tissue immediately adjacent to the basement membrane of the cystic epithelial lining and inflammatory cells were counted. Based on previous studies ,4, the a00 were c-Statistical AnalysisOne-way analysis of variance (ANOVA) followed by post-hoc Scheffe test was used for statistical analysis and P values less than 0.05 were regarded as significant.Our study sample consisted of 20 RCs along with 40 KCOTs, including 20 inflamed and 20 non-inflamed tumors which were chosen from the 60 initially selected cases based on the applied classification of inflammation. All endothelial cells of muscular-walled vessels showed immunostaining with CD34 antibody. In addition some inflammatory cells like mast cells and plasma cells were also positive for this antibody, which could be differentiated from individual endothelial cells by their morphologic features. Any spindle cell with or without finger-like projections was excluded during MVD counting. Mean microvessel density was 12.41 (range: 4.33-21.00) in non-inflammed KCOTs Fig. .Comparison of the three groups by one-way analysis of variance revealed a statistically significant difference (P<0.001), while according to the post hoc test, we did not observe a significant difference in angiogenesis between the inflamed and non-inflamed KCOTs (P = 0.347), but the difference between radicular cysts and both inflamed (P < 0.001) and non-inflamed KCOTs (P < 0.001) was statistically significant.In most of the inflamed KCOTs the epithelial lining demonstrated metaplasia to a non-keratinized squamous epithelium and thinning and even rete ridge formation were also observed in some specimens.Angiogenesis or neovascularization ensues as a result of imbalance between pro- and anti-angiogenic factors, which can be secreted by the tumor itself or by other cells including those residing in the host connective tissue/neoplastic stroma or related to inflammation ,19. MostThe inflammation scoring system used in the current investigation was based on that described by Hirshberg et al.  who founInflammatory cells are generally considered to be related to angiogenesis in normal and lesional tissues. An increase in inflammatory cells can result in propagation of cytokines and chemokines, some of which are angiogenic . MicroveIn conclusion our data showed a significant difference in angiogenesis among RCs and inflamed and non-inflamed KCOTs and between RCs and both types of KCOTs, but not between inflamed and non-inflamed KCOTs; which suggest that angiogenesis may be an important factor in the development and biology of these lesions. However the exact role of inflammation in the angiogenic potential of KCOTs is still unclear and would need further investigation.


Identification of historic pathogens is challenging since false positives and negatives are a serious risk. Environmental non-pathogenic contaminants are ubiquitous. Furthermore, public genetic databases contain limited information regarding these species. High-throughput sequencing may help reliably detect and identify historic pathogens.huey cocoliztli (‘Great Pestilence’ in Nahautl), an unknown disease that decimated native Mexican populations during the Spanish colonial period, in order to identify the pathogen. Comparison of these sequences with those deriving from the surrounding soil and from 4 precontact individuals from the site found a wide variety of contaminant organisms that confounded analyses. Without the comparative sequence data from the precontact individuals and soil, false positives for Yersinia pestis and rickettsiosis could have been reported.We shotgun-sequenced 8 16th-century Mixtec individuals from the site of Teposcolula Yucundaa  who are reported to have died from the False positives and negatives remain problematic in ancient DNA analyses despite the application of high-throughput sequencing. Our results suggest that several studies claiming the discovery of ancient pathogens may need further verification. Additionally, true single molecule sequencing’s short read lengths, inability to sequence through DNA lesions, and limited ancient-DNA-specific technical development hinder its application to palaeopathology. Diseases have ravaged human populations throughout history. The pathogens responsible for many historic epidemics remain either unknown or speculative . IdenMost biomolecular palaeopathological studies have been based on the polymerase chain reaction (PCR) e.g. -7). Supp. Supp7])de novo identification , the, thede non , a disease that ravaged the native Mexican population during the Spanish colonial period )Agilent 2100 Bioanalyzer profiles of the unsheared DNA extracts revealed the typical bimodal distribution of molecular lengths, with a large primary peak between 1000 and 10,000 bp and a small secondary peak around 70 bp. These peaks have previously been determined to correspond primarily to microbial contamination (large peak) and a mixture of fragmented contaminant and authentic ancient DNA  respectively ,32,56. SShearing of the DNA extracts reduced the total sequence yield drastically (5400–6800-fold reduction in the bone and 6.8-fold reduction in the soil), but yielded a concomitant 8.0–10.6-fold enrichment in the proportion of sequences mapping to the human genome in the bone samples and a 2.3-fold enrichment in human sequences in the soil. The cause of this yield reduction remains unclear. Variation between runs cannot completely explain it since the sheared and unsheared soil samples were sequenced on the same chip and samples from unrelated projects on the same chip had typical HeliScope sequencing yields . Hypothetically, oxidative products (such as 8-oxoguanine and abasic sites) near the 3′-terminus of the sheared DNA molecule could disrupt the downstream poly (A)-tailing reaction and reduce the overall sequencing yield. Costello and colleagues  noted thAn alternative explanation is that the shearing caused a decrease in the percentage of denaturable DNA molecules. DNA molecules that cannot be rendered single-stranded are unsequenceable on the HeliScope platform, thus reducing the effective input DNA concentration. One mechanism could be a relative increase in GC-rich sequences in the sheared samples, thus increasing the energy required for denaturation of these molecules. Shearing longer GC-rich DNAs into smaller and more numerous molecules would increase the apparent GC content since this would raise the relative number of available 3′-termini from the GC-rich sequences while the shorter AT-rich sequences would not shear into as many sequenceable fragments. Although the HeliScope platform is noted for its improved performance in both GC- and AT-rich regions in comparison to other high-throughput technologies, it is not impervious to these biochemical effects. This would cause an asymmetric denaturation step with only shorter and/or AT-rich molecules becoming single-stranded in the pool. An alternative, similar mechanism is that the sheared DNA molecules (which mostly derive from the high-molecular-weight peak) are more likely to be cross-linked to proteins and themselves, thus preventing denaturation and sequencing. Since the shearing raised the average length of the molecules , this indicates that a far greater percentage of the sequenced molecules in the sheared pools were derived from the high-molecular-weight ‘microbial contaminant’ peak than from the ‘ancient DNA’ peak. Many microbial metagenomes have been noted to be particularly GC-rich. Our samples have a high GC content at baseline . Shearing reduced the apparent GC content to 46.4% (standard deviation 1.0%) in the bones and a 58.1% in the soil. Moreover, there is a decrease in molecular length in comparison to their unsheared counterparts (median lengths of 26–30 bp in the sheared samples versus 32–34 bp in the unsheared samples). The AT enrichment, reduction in molecular length and increased percentage of human molecules are consistent with the denaturation hypothesis since the sequenced sheared molecules would on average have lower GC contents and be shorter, thus increasing the likelihood of sequencing endogenous molecules.Ginolhac and colleagues  reportedEnvironmental contamination is a critical issue in ancient DNA investigations of diseases. Both false positives and false negatives are a serious problem. Previous investigations of ancient pathogens using both PCR and high-throughput sequencing may need to be re-evaluated because pathogen identifications require extensive verification procedures due to the high risk of false positives. Currently, molecular enrichment to isolate molecules of interest from the complex background (via either hybridization to probes or amplification of 16S regions) is the most promising route for ancient pathogen studies.Furthermore, although the benefits of single-molecule sequencing are promising, methodological challenges remain in its application to ancient DNA research. We found that tSMS is not immune to GC-content-related biases and that the benefits of phosphatase-treatment are not universal. Analysis of these data remains complex due to the short lengths of the sequenced molecules and limitations of public databases. Bioinformatic methods developed for modern DNA analysis and for longer sequences are not appropriate for HeliScope ancient DNA data.Finally, further methodological development is required for the identification of ancient pathogens to become routine and reliable. Until these techniques become available, the burden-of-proof is on the researchers reporting the discovery of these disease agents to demonstrate their results’ authenticity.http://www.ncbi.nlm.nih.gov/sra].The data sets supporting the results of this article are available in the National Center for Biotechnology Information Sequence Read Archive, [SRP022977: ADNA: Ancient deoxyribonucleic acid; BP: Base pairs; DNA: Deoxyribonucleic acid; PCR: Polymerase chain reaction; RNA: Ribonucleic acid; TSMS: True single-molecule sequencing; VHF: Viral hemorrhagic fever.The authors declare that they have no competing interests.MGC conducted the molecular genetic analyses, performed the bioinformatic analysis and drafted the manuscript. NRG provided samples and conceived the project. FJR participated in the coordination and design of the project. NT performed molecular experiments and conceived, coordinated and led the project. All authors read and approved the final manuscript.GenBank accessions for the genomes utilized in this study.Click here for fileAlignment statistics of the HeliScope data sets against the rat and chimpanzee genomes.Click here for fileConcordance of the mitochondrial haplotypes between our Illumina data sets and those reported in Warinner. Click here for fileMEGAN analysis of the reads mapped to the Yersinia pestis genome. The reads are non-specific for the pathogen. Click here for fileMEGAN analysis of the reads mapped to Rickettsia genomes. The reads are non-specific for the pathogens. Instead, they correspond to common environmental organisms. Click here for fileMEGAN comparison of the BLAST hits between the Grand Plaza (blue) and Churchyard (red) populations. Significant differences in relative species prevalence are highlighted in black. The vast majority of the DNA sequences are derived from the environment, obscuring any ancient pathogen signal. Click here for file


Several gene ontology terms including transcription factor activity and biological process such as glucose metabolic process were enriched among these DETs. Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to reference pathways in the KEGG database.Fish under intensive rearing conditions experience various stressors which have negative impacts on survival, growth, reproduction and fillet quality. Identifying and characterizing the molecular mechanisms underlying stress responses will facilitate the development of strategies that aim to improve animal welfare and aquaculture production efficiency. In this study, we used RNA-seq to identify transcripts which are differentially expressed in the rainbow trout liver in response to handling and confinement stress. These stressors were selected due to their relevance in aquaculture production. Total RNA was extracted from the livers of individual fish in five tanks having eight fish each, including three tanks of fish subjected to a 3 hour handling and confinement stress and two control tanks. Equal amount of total RNA of six individual fish was pooled by tank to create five RNA-seq libraries which were sequenced in one lane of Illumina HiSeq 2000. Three sequencing runs were conducted to obtain a total of 491,570,566 reads which were mapped onto the previously generated stress reference transcriptome to identify 316 differentially expressed transcripts (DETs). Twenty one DETs were selected for qPCR to validate the RNA-seq approach. The fold changes in gene expression identified by RNA-seq and qPCR were highly correlated (RRaw RNA-seq reads have been submitted to the NCBI Short Read Archive under accession number SRP022881.All customized scripts described in this paper are available from Dr. Guangtu Gao or the corresponding author. Oncorhynchus mykiss) is not only an important aquaculture species; it is also a primary research model for fish Aquaculture is the fastest-growing animal food producing sector of agriculture. Rainbow trout  axis and release of catecholamines from chromaffin cells that induce many biochemical and physiological changes Melanotaenia duboulayi) Ictalurus spp.) High-throughput next-generation DNA sequencing technologies have revolutionized analyses of genomes and transcriptomes. Transcriptome sequencing, or RNA-seq, overcomes many inherent limitations of microarray technologies, and has emerged recently as a powerful tool to characterize gene expression profiles in both model and non-model species et al.The experimental fish and the stress challenge have been reported in our previous publication Using the standard TRIzol protocol, total RNA was extracted from the liver of each individual fish in five tanks, including three challenged tanks and two control tanks. Equal amount of total RNA from six fish was pooled by tank, and the pooled RNA was used to prepare mRNA libraries for sequencing following the standard Illumina protocol. The five RNA-seq libraries were sequenced with a read length of 75 nucleotides in one lane of Illumina HiSeq 2000. To increase the read coverage and examine the technical sequencing variation, a total of three sequencing runs were performed.Software CD-HIT The GOSSIP st-strand cDNA synthesis kit following the manufacture's protocol . The cDNA was diluted and used for qPCR in a total reaction volume of 15 ul containing 7.5 ul SYBR green supermix, 4 ul cDNA template and an optimized amount of primers. The optimized reactions amplify a single product with a single peak for the melting curve and a single band on agarose gel. The PCRs were also optimized with an efficiency value between 1.85 and 2.15. After qPCR was optimized, the qPCR was performed in duplicate for each individual liver RNA samples used for RNA-seq. The housekeeping gene β-actin has been widely used as a control for qPCR in rainbow trout Twenty-one transcripts covering the full range of gene expression fold changes were selected for qPCR. The PCR primers designed with the software primer 3 are listed in In total, 491,570,566 reads were generated and have been archived in the NCBI's short read archive, accession number SRP022881 . About 7A total of 316 DETs  were ideGO terms were assigned to 158 out of the 190 DETs that were annotated by sequence similarity to well characterized genes in the NCBI database. For GO enrichment analysis, we focused on biological process and molecular function; 36 enriched GO terms were identified. Most of these were represented by a few DETs. GO terms represented by 10 or more DETs are shown in Using the web-based server KAAS, we were able to assign 78 k numbers to 91 DETs . Based op-value <0.0001). However, qPCR only identified significant differences in expression for 14 transcripts , both the direction and magnitude of gene expression changes were similar between those measured by qPCR and RNA-seq analysis . The traAs reported in Sanchez et al. RNA-seq has revolutionized transcriptome studies in both model and non-model organisms, with an emphasis in the latter for providing an efficient approach for gene discovery, expression analyses, and identification of sequence polymorphisms. We report the use of RNA-seq to identify 316 DETs that respond to handling and confinement stress in rainbow trout. The reliability and accuracy of RNA-seq analysis was demonstrated by the 0.88 coefficient of determination between RNA-seq and qPCR.Both known and novel stress response genes in fish were among the 316 DETs identified in this study. For example, Momoda et al. Among the 21 transcripts selected for qPCR, twenty transcripts had very similar fold changes in expression based on RNA-seq and qPCR, respectively. Only one transcript (primer pair RT43) had an opposite expression fold change based on RNA-seq and qPCR. This transcript was annotated as hsp70, whose expression in liver has been observed to increase in response to elevated plasma cortisol concentrations that are associated with some stress responses but not including handling and confinement in rainbow trout Also, the qPCR results were only determined to be statistically significant for 14 transcripts . This maActivation of HPI axis and catecholamine release leads to many changes at the biochemical and physiological level. Since liver is the major organ for metabolic adjustments, it is not surprising to observe enrichment of GO terms related to glucose metabolism and transcription factors in this study. Due to the limited annotation of DETs identified in this study and the paucity of well documented pathways in rainbow trout, it is challenging to describe all of the exact pathways involved in response to handling and confinement stress in rainbow trout. However, several pathways were implicated in response to handling and confinement stress in this study. Glucose is an important fuel that is oxidized to meet the energy demand associated with stress response. Elevation of plasma glucose concentration is a well-documented metabolic stress response in rainbow trout. Activation of the gluconeogenesis pathway in response to stress has been reported in several previous studies While cortisol is a key hormone associated with the stress response, other hormones such as insulin-like growth factor (IGF), insulin, and estrogen signaling may also play important roles. Similar to the increased expression of hepatic expression of IGF binding protein-1b (IGFBP-1b), -2a, and -3 during transport in zebrafish It is clear that the immune response was altered in rainbow trout in response to handling and confinement stress. Many important elements of innate and adaptive immunity such as cytokine receptors, antigen presenting and processing molecules, immunoglobins and transcriptional factors were among the DETs identified in this study. We observed that cytokine receptors such as c-x-c chemokine receptor type 4 (CXCR4), c-c chemokine receptor 9 (CCR9) and tumor necrosis factor receptor member 9 (SF9), were up-regulated. However, cytokine receptor KIT (proto-oncogene tyrosine protein kinase) was down-regulated. Transcription factor NF-κB might play critical roles in the regulation of diverse cellular responses including cell proliferation, survival and inflammation Even though induction of acute phase response in fish is often associated with the activation of the inflammatory response, acute phase response proteins (APPs) also play a role in response to confinement stress challenge in rainbow trout. Cairns et al. Deep sequencing of the rainbow trout liver transcriptome in response to handling and confinement stress identified 316 DETs. The reliability and accuracy of RNA-seq analysis was demonstrated by the 0.88 coefficient of determination between RNA-seq and qPCR. Several GO terms including transcription factor activity and -biological process such as glucose metabolic process were enriched among the 316 DETs. Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to the reference pathways in KEGG database. The findings of this study provide a basis for further characterization of molecular response to handling and confinement stress in rainbow trout.Figure S1Comparison of normalized uniquely mapped reads among three runs. 1A, tank1; 1B, tank 2; 1C, tank 3, 1D, tank 4; and 1E, tank 5.(PPTX)Click here for additional data file.Table S1All 316 DETs in response to handling and confinement stress identified in this study.(XLSX)Click here for additional data file.Table S2Primers used for qPCR to validate transcript expression fold changes measured by RNA-seq.(XLSX)Click here for additional data file.


Factor V (FV) in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available.The FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma.The FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin) may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC), the FV 1-stage, 2-stage (With activation by thrombin), and total (2-stage activity - 1-stage activity) activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP). The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP.The FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in both research and clinical laboratories to provide measurement of the functional coagulation activity of FV in human plasma. The use of microplate-based assays for quantification of enzyme activity is now common practice in many laboratories for enzyme linked immunosorbant assays  and coloprothrombinase enzyme complex [FV circulates in humans at approx 12-40nM as an inactive procofactor of Mr 330kDa . In resp complex . In the  complex . The FV  complex . The FV  complex .Although methods for determining the FV 1-stage activity (Without activation by thrombin) in human plasma using manual tilt-tube assays have been described ,9, the rThe equipment required to perform automated coagulation assays is expensive. Although these analyzers are generally used in clinical laboratories to measure clot times in patient plasmas, the rate and extent of clot formation are not routinely assessed. The FV microplate assay described here provides measurement of the time, initial rate, and extent of clot formation in human plasma in a fast and convenient manner. It may be easily adapted to measure the activity of any coagulation factor in the extrinsic, intrinsic, or common pathway using the appropriate factor-deficient plasma and clot initiating reagent. This new microplate-based coagulation assay will be useful in both research and clinical laboratories to increase our understanding of the biochemistry and functional activity of FV.et al. [et al. [Normal pooled human reference plasma (NHP) was obtained from Precision Biologicals  and was stored at -80°C until use. Fresh frozen human plasma was obtained from Lakeridge Healthcare  and stored at -80°C until use. FV-deficient plasma was made from fresh frozen human plasma according to Bloom et al. , dialyze [et al. . The nin [et al. . PatientThe FV microplate assay was performed in the wells of immunomodule strips  using a multichannel pipette to make simultaneous additions of FV-deficient plasma (50 μl), followed by normal or patient plasma diluted in HBS (50 μl), and finally thromboplastin (50 μl). The samples were incubated in a Spectra Max 190 microplate reader  at 25°C for 1 minute with shaking during the 15-25 seconds of the 1 minute incubation. Calcium chloride (50 μl of 25mM) was then added to initiate clotting. The samples were then shaken for 10 seconds and the absorbance at 405nm was read every 5 seconds for 6 minutes. The 10 seconds required for shaking prior to absorbance readings were not included in the calculated clot times. The clot times were calculated as the time to reach the midpoint between the minimum and maximum absorbance at 405nm after calcium chloride addition. The initial rate of clot formation was calculated as the rate of increase in absorbance at 405nm (mUnits/minute) using the first 5 time points of clot formation in the linear portion of the absorbance vs time curve. The extent of clot formation was calculated as the difference between the maximal and minimal absorbance in Units at 405nm achieved during the clot formation event.The FV 1-stage activity of a plasma sample diluted 40-fold in HBS was determined by comparison to the FV 1-stage microplate assay standard curves of times or initial rates of clot formation vs FV activity measured upon serial dilution of NHP (0- to 1024-fold) in HBS. One unit of FV activity was defined as the activity in 1 ml of NHP prior to activation by thrombin (See below). Up to 12 samples were analyzed simultaneously using SOFTmax PRO 4.3 LS software . The above microplate assay measures the FV 1-stage activity (Without activation by thrombin) of a plasma sample. Since FV is activated with thrombin to the active cofactor, FVa [The results shown for the times and initial rates of clot formation in the FV 1-stage assay standard curves are representative of experiments conducted on greater than thirty different occasions. The intra- and inter-FV assay variability of the time, extent, and initial rate of clot formation were calculated from six samples of NHP diluted 40-fold in HBS on eight different days.A typical depiction of clot formation in the FV coagulation assay over time generated by the microplate reader is shown in Figure 2 = 0.980). Fitting of the Log-Log plot of the initial rate of clot formation vs FV 1-stage activity also demonstrated a strong linear relationship between these variables after regression analysis . The relationship of both clot time and initial rate of clot formation vs FV 1-stage activity remained linear in NHP diluted up to approx 1024-fold. Given that the FV concentration in NHP is approx 12-40nM [et al. for the FV 1-stage activity measured with an automated analyzer [In order to measure the FV activity in a plasma sample, standard curves of time and initial rate of clot formation vs FV 1-stage activity were generated upon serial dilution of NHP. Fitting the Log-Log plot of clot time vs FV 1-stage activity demonstrated a strong linear relationship between these variables after regression analysis  as well as being able to analyze multiple samples simultaneously (up to 12). These assay characteristics are especially important and advantageous when expensive reagents are required such as factor-deficient plasmas; when only small sample volumes are available such as patient plasmas or when analyzing a large number of samples during purification of FV from plasma.The results indicate that the FV in the DIC patient plasmas was functionally less active than in NHP because of prolonged clot times and decreased initial rates of clot formation in the FV 1-stage assay. However, the initial rates of clot formation in the FV 2-stage assay in the DIC patients were not largely different from NHP. The extents of clot formation in the DIC patient plasmas were also not largely different from NHP in the FV 1-stage and 2-stage assays. Since the extent of clot formation in the DIC patient plasmas was the same as observed with NHP, this was most likely determined by the fibrinogen level in the FV-depleted plasma and unrelated to characteristics of the patient plasmas.These results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP. Compared with NHP, the FV in the DIC patient plasmas was also on average, 44% less activatable with thrombin in the FV 2-stage assay. Finally, the FV total activity in the DIC patients decreased by approx 42% on average, from that observed in NHP. The decreased FV 1-stage, 2-stage, and total activities may have been due to increased FV consumption  and/or iThe results of the microplate assay indicate that measurement of FV coagulation activity in human plasma samples may be obtained from the time and initial rate measurements of clot formation from the FV 1-stage assay and the time of clot formation and total activity measurement from the FV 2-stage assay. It was however not possible to obtain a quantitative measurement of FV coagulation activity in a plasma sample based on the extent of clot formation in the FV 1- and 2-stage assays or the initial rate of clot formation in the FV 2-stage assay. Since all reactions reached approximately the same extent of clot formation at 405nm of 0.35 - 0.45 Units between the starting absorbance before and the maximal absorbance after thromboplastin and calcium chloride addition, measurement of the extent of clot formation does not strictly provide a quantitative measurement of FV coagulant activity in any given plasma sample.The new microplate assay will be useful in both clinical and research laboratories for measurement of FV activity in human plasma samples. The microplate assay may be used to measure the time, initial rate, and extent of clot formation; parameters not routinely monitored simultaneously in most automated coagulation analyzers. In its entirety, this quantitative information provides more of a complete 'picture' of the involvement of FV during the clot formation event in human plasma than has been previously reported ,7-10,21.The FV microplate assay format may be easily adapted to measure the activity of any coagulation factor in the extrinsic, intrinsic, or common pathway using the appropriate factor-deficient plasma and clot initiating reagent. The clinical and research use of this new microplate-based coagulation assay will provide further information and understanding of FV biochemistry and functional activity in the future.The authors declare that they have no competing interests.JAS supervised the study design, interpretation of results, manuscript preparation and wrote the manuscript. DT acquired and analyzed the data and helped write the manuscript. IL assisted DT in acquiring and analyzing some of the data and helped write the manuscript. All authors read and approved the final manuscript.


The purpose of this study was to evaluate the inter-rater agreement among nurses using the triage system REETS- HEV  in a Danish ED.The use of triage systems in Denmark has recently been implemented together with structural changes in hospital organization .Testing and evaluation is therefore needed. The REETS-HEV is a five scale triage system being used in the Emergency department (ED) of Herning, Denmark since May 2010. The ED is semi-large with 29,000 annual visits.Consecutive patients presenting to the ED were assessed by both a duty and study nurse using REETS-HEV. Nurses did not receive training prior to the study. 146 patients were enrolled and a blinded, paired and simultaneous triage was conducted independently to evaluate inter-rater agreement using Fleiss kappa.A total of 155 patients were triaged over a 10 day period and complete data were available for 146 patients. We found the overall agreement to be good ). The kappa estimate was higher for the group of patients needing immediate attention ).The study demonstrated good inter-rater agreement between two independent observers not receiving any new triage training prior to the study.