Second harmonic generation (SHG) microscopy is a recently developed technique which allows examination of fibrillar collagen structures with a high degree of specificity and resolution. The goal of this study was to examine the potential utility of SHG and multiphoton excitation fluorescence (MPEF) microscopy in understanding the relation between tenocytes and their surrounding collagenous matrix in early tendon overuse lesions.Tenocytes have been implicated in the development of tendinosis, a chronic condition commonly seen in musculoskeletal overuse syndromes. However, the relation between abnormal tenocyte morphology and early changes in the fibrillar collagen matrix has not been closely examined Histological preparations of tendon were prepared from adult male Sprague-Dawley rats subjected to an Achilles tendon loading protocol for 12 weeks (Rat-A-PED), or from sedentary age-matched cage controls. Second harmonic generation and multiphoton excitation fluorescence were performed simultaneously on these tissue sections in at least three different areas.SHG microscopy revealed an association between abnormal tenocyte morphology and morphological changes in the fibrillar collagen matrix of mechanically loaded Achilles tendons. Collagen density and organization was significantly reduced in focal micro-regions of mechanically loaded tendons. These pathological changes occurred specifically in association with altered tenocyte morphology. Normal tendons displayed a regular distribution of fibre bundles, and the average size of these bundles as determined by Gaussian analysis was 0.47 μm ± 0.02. In comparison, fibre bundle measures from tendon regions in the vicinity of abnormal tenocytes could not be quantified due to a reduction in their regularity of distribution and orientation.SHG microscopy allowed high resolution detection of focal tendon abnormalities affecting the fibrillar collagen matrix. With ongoing repetitive loading, these tenocyte-associated focal collagen defects could predispose to the progression of overuse pathology. SHGI) scales as SHG I≈ N2 Here N is the effective number of radiating molecules. Likewise, isotropic macromolecules generate MPEF signal due to their endogenous fluorescence. Multiphoton excitation fluorescence intensity (MPEFI) emitted by these molecules undergoing multiphoton excitation can be expressed as, MPEF I≈ N. While SHG signal intensity scales as a square of number of collagen molecules, the MPEF intensity scales linearly with the number of other endogenously fluorescent molecules. Since MPEF and SHG involve different principles and contrast mechanisms, SHG and MPEF can be captured simultaneously to visualize structural changes of biological macromolecules. Particularly, SHG signals originating from the collagen structures can be used to generate high resolution images of individual collagen fibre bundles and quantify collagen density in a given tissue. Hence, SHG provides the potential to investigate precise spatial relations between collagen organization and underlying tenocyte biology.Multiphoton and associated microscopy methods have been widely used for imaging dynamic interactions in cells and tissues with submicron resolution -3. AmongTendinosis is a histological entitity characterized by collagen remodeling and angiofibroblastic hyperplasia. Tendons which rupture often demonstrate evidence of pre-existing tendinosis . HoweverResearch into the early pathogenesis of tendinosis has suggested that tenocytes may initiate degradation and remodelling of the load-bearing collagen matrix in response to mechanical loading. This model runs counter to a traditional "wear and tear" hypothesis of collagen microtrauma . Dudhia in vivo model of mechanical overload. We hypothesized that collagen density and fibrillar organization would be significantly reduced in highly localized areas associated with abnormal tenocyte morphology.The goal of the current study was to determine whether SHG microscopy could be used to examine a potential association between abnormal tenocyte morphology and local reductions of collagen density in a relevant Twelve male Sprague-Dawley rats aged 4 months, weight 448.7 ± 10 grams were obtained locally and housed singly in standard cage conditions. Following a 48 hour acclimatization period, rats were divided into controls , or runners , providing 12 tendons per group. Ethic approval for the study was obtained from the UBC animal care committee (A07-0274).Runners were subjected to a previously published mechanical loading protocol (Rat-A-PED) using a dedicated rat treadmill to induce Achilles tendinosis  . Rats we2, and whole bilateral Achilles tendons were removed with the muscle still attached, oriented longitudinally in Tissue Tek embedding medium , then frozen in isopentane chilled in liquid nitrogen. Tendons were cryosectioned onto charged glass slides at 5 micron thickness and processed for H&E , Alcian blue / fast nuclear red (stains sulphated glycosaminoglycans and cell nuclei), or Picrosirius red (enhances birefringence of collagen when viewed under polarized light).Rats were euthanized with COxyR) values follow: 20X/0.7 NA (xyR≈0.50 μm); 63X/1.2 NA (xyR≈0.292 μm). Upon entering the Leica microscope system, the laser beam was directed to the scanning mirrors, then through a 670 nm long pass dichroic mirror  and subsequently focused on the specimens through the objective lens. The backscattered emission from the sample was collected through the objective lens. Leica Confocal Software TCS SP2 was used for the image acquisition. Non-descanned detectors and spectral scanning mode both in the reflection geometry were used for capturing the MPEF images as well as for the spectral signal characterizations respectively. In the non-descanned PMT detectors , a 700 nm short pass filter  was used to prevent the scattered IR laser radiation from reaching the detector and a 455 long pass dichroic beam splitter  was used to separate SHG signal from the MPEF signal. SHG signal in forward direction was captured using a non-descanned detector in the transmission geometry equipped with a 440/20 nm band pass filter  and high NA condenser. All SHG and MPEF spectral data were generated using the de-scanned PMT detector  located inside the scan head where the emission signals were delivered through the AOBS detection system with the maximum confocal pinhole setting at 600 μm via the spectral dispersion prism. The width of the slits in front of each PMT could be software adjusted such that each PMT could detect spectral regions spanning from a 5 nm bandwidth up to the overall spectral capacity of the system (400-800 nm). With this instrument configuration, a series of individual images were collected using a narrow detection window with a width of ~5 nm, and each image detected at this specific emission wavelength band (i.e. ~ 5 nm) provided a data point in the spectral graph. The gain and the offset of the PMTs were adjusted for optimized detection using the color gradient to avoid pixel intensity saturation and background. Images (8 bit) acquired at slow scan speed i.e. 10 sec per 512 X 512 pixels.The microscope system used in our present experiments is the same as described previously . SpecifiLateral line profiles of collagen fibers were generated using Leica comprehensive image processing software from 2D spectrally clean SHG images. The sizes of the collagen fibers and their size distribution were plotted by fitting the lateral line profiles to a Gaussian curve using Origin Lab Software.A Mann-Whitney U test was used to detect differences in collagen density and blood flow. Values are expressed as means ± SE. Results were considered statistically significant if α was less than 0.05.In order to determine the microregional properties of collagen surrounding abnormal tenocytes in mechanically loaded tendon, we assessed standard histological preparations using multiphoton and second harmonic generation (SHG) microscopy. Initially, normal tendon was analyzed. Representative images originating from spectrally clean SHG signal originating from collagen (1A) and the MPEF signal originating from endogenously fluorescent tissue components Figure  are showAfter confirming the specificity of collagenous structures visualized with SHG in normal tendons, we next examined the collagen organization in sedentary and mechanically loaded tendons. All Achilles tendons analyzed from animals subjected to the mechanical loading protocol demonstrated microregions of abnormal tenocyte morphology, typified by the appearance of tenocytes with prominent cytoplasm and rounded morphology Figure . These mSHG signals emitted from the Achilles tendon proper region in normal tendon vs tendinosis lesions were next characterized by spectral analysis. The wavelength scans of both normal tendon from sedentary rats, and of tendon surrounding abnormal tenocytes from rats subjected to the mechanical loading protocol, showed a highly specific SHG signal manifested by a narrow peak at 440 nm Figure . The res2) to yield a mean collagen density value. The collagen density from micro-regions corresponding to abnormal tenocytes in rats subjected to the overuse protocol was 729 ± 90.3 AU, compared to the value of 1135 ± 60.1 in normal tendon (p < 0.05).We then applied a noise removal filter to SHG images to define the boundary between foreground and background, and the lower threshold in the histogram was set to 10% of the highest pixel intensity value. The total SHG signal intensity values thus generated were normalized by the cropped collagen area (μmSecond harmonic generation (SHG) resulted in high resolution, spectrally clean signals originating from fibrillar collagen. The collagen matrix surrounding abnormal tenocytes demonstrated an abnormal organization and a reduced density compared to sedentary cage controls. This reduction in SHG signal occurred specifically in association with the appearance of abnormal tencoytes in the tendons subjected to mechanical loading. These results provide support to an emerging model of tendinosis where degradation and/or remodelling of the load-bearing collagen matrix in response to exercise is mediated by the local activity of tenocytes, rather than being due to a passive accumulation of fatigue damage ,14.increased collagen staining using this overuse tendinopathy model based on assessment of H&E preparations [reduced SHG signal observed in the current study accurately reflects a loss of collagen density in the tendinosis samples. Indeed, H&E evaluation of tendinosis samples may be misleading as tendinosis lesions can be highly eosinophilic, but we have found these eosinophilic regions to generate very weak or absent SHG signals indicating that they may comprise provisional matrix with a higher component of non-collagenous elements. We are therefore able here to refine and further validate the description of early pathology which occurs in this model.Glazebrook et al. recently reported arations . HoweverIn the current study, SHG was used to estimate the collagen fibre bundle diameter with submicron resolution in normal tendons, but not in tendinosis samples. The longitudinal alignment of collagen fibres was reduced in the tendinosis tendons, in association with a reduction of SHG signal intensity. The technique of measuring fibre bundle diameter relied on the fibres demonstrating an orderly, longitudinal orientation, which was disrupted in areas of tendinosis.in vivo are accompanied by highly localized foci of reduced collagen density which may predispose to the progression of overuse tendinopathy.Second harmonic generation analysis of mechanically loaded tendon provides the ability to examine collagen organization in standard histological preparations, providing insight into mechanisms of early load-induced tendon changes. Tenocyte responses to mechanical loading The authors declare that they have no competing interests.AS and TA conceived of the study. AS supervised the tendon mechanical loading protocol and carried out the imaging and histological examination. GF assisted with imaging analysis. TA and AS conducted the image analysis. TA designed and oversaw the SHG and MPEF microscopy. All authors read and approved of the final manuscript.Alex Scott is a physical therapist engaged in full time research and teaching. Gloria Fong is a Research Assistant with a BSc in Medical and Laboratory Sciences. Thomas Abraham is a specialist in optical physics engaged in full time research.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/26/prepubFigure 1S Glycosaminoglycan distribution in mechanically loaded and sedentary tendon. Alcian blue staining in tendinosis (A) and normal healthy (B) tendon. Tendinosis tendon demonstrates increased sulphated glycosaminoglycan intra- and peri-cellularly, particularly in chains of abnormal-appearing tenocytes. Mast cells also stain blue with this technique in the paratendon regions (not shown). 20× objective lens.Click here for file


We generated computational 3D pharmacophores based on data derived by both acoustic and tip-based transfer. The computed pharmacophores differ significantly depending upon dispensing and dilution methods. The acoustic dispensing-derived pharmacophore correctly identified active compounds in a subsequent test set where the tip-based method failed. Data from acoustic dispensing generates a pharmacophore containing two hydrophobic features, one hydrogen bond donor and one hydrogen bond acceptor. This is consistent with X-ray crystallography studies of ligand-protein interactions and automatically generated pharmacophores derived from this structural data. In contrast, the tip-based data suggest a pharmacophore with two hydrogen bond acceptors, one hydrogen bond donor and no hydrophobic features. This pharmacophore is inconsistent with the X-ray crystallographic studies and automatically generated pharmacophores. In short, traditional dispensing processes are another important source of error in high-throughput screening that impacts computational and statistical analyses. These findings have far-reaching implications in biological research.Dispensing and dilution processes may profoundly influence estimates of biological activity of compounds. Published data show Ephrin type-B receptor 4 IC There have been many studies which have evaluated aspects of biological assays and the tools involved which could result in errors and erroneous data. Processes like tip-based and acoustic dispensing have a profound influence on estimates of compound activity. Several independent studies of high-throughput screening (HTS) show that the two techniques generate conflicting results 50 values derived using tip-based serial dilution and dispensing tend to be greater  than IC50 values derived using acoustic dispensing. Some compounds appeared hundreds of times more active with the acoustic process Previous research has impugned tip-based techniques because they can generate errors due to leachates from the plastic that may profoundly affect biological assays 50 results in which larger number of molecules are used but the molecular structures are not provided for computational analysis While we are limited by the number of compounds available with data in tip-based and acoustic dispensing, this study suggests a significant impact on drug design, especially when coupled with other reports of poorly correlating IC50 values determined by AstraZeneca scientists 50 values using both serial dilution facilitated by tip-based dispensing  and direct dilution 50 values obtained with acoustic transfers suggested that the compounds were 1.5 to 276.5 times more active than when tip-based techniques were used This paper is based on the published comparisons of ICWe used these published data 50 values  with minimum features (3) and maximum features (6) as are described elsewhere 50 values obtained via tip-based serial dilution and dispensing versus acoustic dispensing with direct dilution is poor  when acoustic transfer was used. Upon statistical analysis of the 14 IC50 values for the two dispensing techniques . This would suggest that there is no statistically significant correlation or ranking between these two measures. That is, the data generated from the two techniques would lead researchers in completely different directions.The correlation between the 14 Ephrin type-B receptor 4 log ICchniques , calcula50 values for the pharmacophore derived via acoustic processes was higher than that for the pharmacophore derived from the tip-based processes  for these additional 12 compounds. We used the data to create a 3D multiple conformer database. This database was searched by the two pharmacophores in order to test whether they could discriminate between those with high and low affinity . That is, the two pharmacophores developed on the comparative data of 14 compounds were used to test an additional 12 compounds to see whether either of the two pharmacophores developed could predict which compounds in the second set of 12 were most active.The pharmacophores specific to the tip-based and acoustic-based processes were used to predict affinity to EphB4 .When the physical properties of the additional 12 compounds Receptor-ligand pharmacophores were created in 8 out of 10 cases and all consisted of hydrophobic and hydrogen bonding features .The pharmacophores derived for the tyrosine kinase EphB4 are dramatically different based upon the process used to set up the dose-response experiments. The pharmacophore derived from the acoustic dispensing data suggests the importance of specific regions of hydrophobicity as well as hydrogen bonding features. The pharmacophore from the tip-derived data suggests only hydrogen bonding features as leading to binding, with no hydrophobic interactions.in vitro data of the original articles and not using the crystal structures of the latter work.In order to further understand the impact of these models, a series of another 4 papers published by AstraZeneca describing structure-based design of tyrosine kinase EphB4 inhibitors were reviewed in vitro data ligand pharmacophores further. Receptor-ligand pharmacophores could be created in 8 out of 10 cases and all consisted of hydrophobic and hydrogen bonding features .A graph of the log IC(DOCX)Click here for additional data file.Results S1Showing pharmacophore model information for acoustic-based liquid handling with direct dilution and tip-based liquid handling with serial dilution.(DOCX)Click here for additional data file.Table S150 data and descriptors.Molecule structures, IC(PDF)Click here for additional data file.Table S2Test set data for searching with ‘acoustic dispensing’ pharmacophore.(DOCX)Click here for additional data file.Table S3Test set data for searching with ‘tip-based dispensing’ pharmacophore.(DOCX)Click here for additional data file.


Hot flashes are a significant clinical problem for many women. Currently there are limited options to hormone replacement therapy as non-hormonal pharmacological agents are associated with only modest activity and many adverse side effects. Hypnosis is one mind-body therapy that seems particularly promising for treating hot flashes and was investigated in the present study. This study examined the efficacy of hypnosis in reducing both self-reported and physiologically determined hot flash frequency and severity among post-menopausal women.One-hundred and seventy post-menopausal women with moderate to severe hot flashes were randomly assigned to either a 5-session hypnosis intervention or a 5-session structured-attention control condition. All sessions were provided consistent with a treatment manual and all therapists were trained to criteria for consistency and treatment fidelity. Primary outcome measures were self-reported hot flash frequency and severity (determined via daily diaries) and physiologically monitored hot flashes . Physiological assessment of hot flashes were made using 24-hour recordings of sternal skin conductance. Measures were obtained at baseline, at the end of the five weeks intervention, and at 12 week follow-up.Results demonstrated that hot flash scores (self-report of frequency and severity of hot flashes) for the participants that received the therapist delivered hypnosis intervention decreased by approximately 70% at 5 weeks and continued to decline to approximately 80% at the 12 week follow-up. Physiologically assessed hot flashes demonstrated a 50% reduction at 5 weeks and approximately 60% reduction at 12 weeks for participants in the therapist delivered hypnosis condition.To our knowledge, this is the first study to demonstrate a clinically significant reduction in physiologically measured hot flashes using a hypnosis intervention. This study has important implications for women experiencing hot flashes who are contraindicated for hormone replacement therapy.


Sepehrvand et al. demonstrated a considerable seroprevalence rate of anti-HEV in Iranian kidney transplant recipients . The impIn otherwise healthy kidney transplant recipients, HEV might be considered the etiological agent for the development of hepatitis in those who live in endemic regions . Viral hDecreasing the numbers and doses of immunosuppressive drugs remains the first approach toward controlling viral hepatitis E in renal transplant recipients. A prolonged follow-up period might be required to assess the eventual outcome . In addi


Patients with ischemic cardiomyopathy have an increased risk for ventricular arrhythmia, since myocardial infarction can be the substrate for re-entrant arrhythmias. Contrast-enhanced cardiac magnetic resonance imaging (CMR) has proven to reliably quantify myocardial infarction. Aim of our study was to evaluate correlations between functional and contrast-enhanced CMR findings and spontaneous ventricular tachy-arrhythmias in patients with ischemic cardiomyopathy who underwent implantable cardioverter-defibrillator (ICD) therapy.Forty-one patients with ischemic cardiomyopathy and indication for ICD therapy were examined in a 1.5-T whole-body CMR system. Cine images for quantification of left ventricular volumes and function and late gadolinium enhancement images for quantification of myocardial scar extent were acquired in all patients covering the entire left ventricle.During a follow-up period of 1184 ± 442 days 68 monomorphic and 14 polymorphic types of ventricular tachycardia (VT) could be observed in 12 patients. Patients with monomorphic VT had larger scar volumes  than patients with polymorphic VT. Moreover myocardial infarction involved more segments in the LAD perfusion territory  than in patients with polymorphic VT.Patients with spontaneous monomorphic VT during the long-term follow-up period had more infarcted tissue, which was more often present in the LAD perfusion territory than patients with polymorphic events. These data strengthen the diagnostic benefit of CMR in patients with ischemic cardiomyopathy. CMR may be used for comprehensive risk stratification in patients with ischemic cardiomyopathy undergoing ICD therapy.


This paper describes a case of early (7 months after transplant) cardiac allograft vasculopathy. This-43-year-old  female patient underwent an orthotopic heart transplant with a  donor heart. She had a history of herpes zoster infection and postherpetic neuralgia in the past. The patient's panel reactive antibodies had been almost undetectable on routine surveillance testing, and her surveillance endomyocardial biopsies apart from a few episodes of mild-to-moderate acute cellular rejection (treated adequately with steroids) never showed any evidence of humoral rejection. The postoperative course was complicated by multiple admissions for upper respiratory symptoms, and the patient tested positive for entero, rhino, and coronaviruses serologies. During her last admission (seven months postoperatively) the patient developed mild left ventricular dysfunction with an ejection fraction of 40%. The patient's endomyocardial biopsy done at that time revealed concentric intimal proliferation and inflammation resulting in near-total luminal occlusion in the epicardial and the intramyocardial coronary vessels, suggestive of graft vasculopathy with no evidence of rejection, and the patient had a fatal ventricular arrhythmia. Cardiac transplantation is a well-defined therapy for end-stage heart failure. Following transplantation, median survival is 10 years rising to 13 years for those who survive the first year [We present a case of early (7 months after transplant) cardiac allograft vasculopathy (CAV) in a cytomegalovirus (CMV) positive patient with a history of herpes zoster infection and multiple other viral infections in the postoperative course possibly contributing to graft vasculopathy.A 43-year-old Caucasian female with a history of nonischemic dilated cardiomyopathy with left ventricular ejection fraction (LVEF) of 10–15% status following Thoratec Heart-Mate II left ventricular assist device (LVAD) (implanted 2 years prior as a Bridge to Transplant) was transferred to our tertiary care facility for management of unresolving pseudomonas driveline infection. The patient secondary to persistent pseudomonas bacteremia despite adequate treatment with intravenous antibiotics underwent LVAD removal with reimplantation with another VAD. The patient also underwent an AICD lead extraction with generator change secondary to questionable vegetation on the defibrillator lead on transesophageal echocardiogram. The patient did well following that and remained home for 4 months while awaiting a cardiac transplant. Her past history was significant for hypertension, dyslipidemia, recurrent pulmonary embolism, history of herpes zoster infection with postherpetic neuralgia, and intracerebral hemorrhage.2) and elevated right sided pressures on right heart catheterization while the EMB remained negative for cellular or humoral rejection. An echocardiogram at the time revealed a mildly depressed left ventricular ejection fraction at 40% with mild right ventricular dysfunction. The patient's panel reactive antibodies were undetectable. Four months later the patient was electively admitted for transplant evaluation. Her panel reactive antibody (PRA) levels were found to be low at 4% as measured by flow cytometry using HLA class I Luminex-coated beads. The patient (CMV positive) finally underwent a CMV negative, Epstein-Barr virus (EBV) positive orthotopic heart transplant without the need for desensitization. The patient's immediate postoperative course was complicated by multiple failed attempts at extubation secondary to fluid overload that required tracheostomy and acute kidney injury requiring temporary hemodialysis . The patient after 4 weeks, on routine surveillance endomyocardial biopsy (EMB), was found to have ISHLT grade 2R acute cellular rejection which was successfully treated with intravenous pulsed steroids and mycophenolate mofetil. The patient was eventually discharged home 2 weeks later and was followed as an outpatient. Three months subsequent to transplant the patient started to develop signs and symptoms of upper respiratory tract infections manifesting as unremitting cough. The patient admitted was found to have viral infection with positive serologies for entero, rhino, and coronaviruses, and the EMB was negative for rejection. The patient was managed conservatively without any antiviral treatment except prophylactic ganciclovir for CMV prophylaxis and discharged home. The patient did present again with similar respiratory symptoms a month later at which time it was decided to treat the patient with a course of oseltamivir (Tamiflu) for a clinical suspicion of influenza. The patient was discharged only to be readmitted 2 months later (6 months after transplant) for symptoms of exertional dyspnea, nausea, and abdominal pain. The patient was found to have low cardiac index  inhibitors (everolimus and sirolimus), and possibly diltiazem  .Our case is an addition to a list of very limited number of cases of early allograft vasculopathy (within the first year after transplant) that aims to highlight its poor clinical outcome and survival compared to the form that develops after the first year. The lack of data on the patients viral titres is an important limitation of this case. Allograft coronary disease remains one of the greatest obstacles to long-term recipient survival after cardiac transplantation. This process has been extensively studied, yet remains poorly understood. Although it typically becomes clinically significant years after transplantation, it can also occur early and result in recipient death as early as 6 months following transplant. Why a given patient develops an aggressive form of the disease early after transplantation and another patient may live ten or even twenty years without evidence of clinically significant disease is unknown.


Aortic cusps originating arrhythmias are rare; they have special electrocardiogram features that help to locate the site of origin. We report on a 20-year-old male patient without structural heart disease presenting with accelerated idioventricular rhythm; electrocardiogram analysis was typical of left coronary cusp origin. Outflow tract ventricular arrhythmias (VAs) usually have a benign course when occurring in the setting of normal structural heart. Left coronary cusp (LCC) originating arrhythmias have special electrocardiogram (EKG) characteristics; these are well established in the literature ; recogniWe report on a 20-year-old male patient presenting with palpitations; his EKG  showed accelerated idioventricular rhythm with EKG characteristics compatible with a LCC origin . The patient had no relevant past medical history; physical examination was normal, cardiac echogram showed no structural heart disease, and laboratory parameters including cardiac markers were normal. Reversion to sinus rhythm occurred with a 75 mg bolus of Xylocaine. Given the refusal of any electrophysiological procedure (diagnostic or therapeutic), the patient was discharged home on day 3 with verapamil. Holter and stress test on days 10 and 30 after discharge showed no VA and were normal.Supravalvular left ventricular outflow tract arrhythmias originate usually from the aortic sinus of valsalva (aortic cusps) . VA origLCC-VA is known to be the most common form of coronary cusp arrhythmia; it exhibits an (r) wave in V1 [LVOT-VA displays earlier precordial transition zones (median V3 versus V5) compared to RVOT-VA ; this feThe absence of (S) wave in left precordial leads is also typical of left VA originating from coronary cusps, in contrary to VA originating in the left ventricular myocardium resulting in late right ventricular activation and typical right bundle branch pattern with prominent (S) wave in V5-V6 .The presence of rS pattern in D1  is also When invasive electrophysiological studies could not be performed, surface EKG becomes of utmost importance to diagnose this type of VAs and accordingly to differentiate it from other VAs, especially non-outflow tract VA which have a more critical prognosis if they are not treated adequately . OutflowLCC-VAs have special EKG characteristics and accordingly they can be diagnosed with sufficient accuracy based on surface EKG.in this presented case, there was no invasive electrophysiological procedure performed due to patient denial and so intracavitary mapping of the arrhythmia is lacking.The authors declare that there is no conflict of interest.According to our institutional Ethics Committee, a written consent was obtained from the patient before proceeding with this paper.Figure 1. Legend: EKG showing accelerated idioventricular rhythm with EKG characteristics compatible with a left coronary cusp origin.Figure 2. Legend: EKG (s) of different types of Coronary cusps ventricular arrhythmias. ).Click here for additional data file.Click here for additional data file.


Eucalyptus camaldulensis seedlings derived from three natural populations. Physiological and growth traits were measured and gene and allelic expression in leaves was examined by RNA sequencing (RNA-seq). Water stress had a significant impact on all the physiological and growth traits, while differences between the populations were not significant. Genes differentially expressed in leaves were identified by de novo assembly and by ab initio transcriptome mapping using the Eucalyptus grandis reference genome sequence. Gene ontology (GO) enrichment tests with 2,500 significantly differentiated genes revealed 128 stress-related gene categories were up-regulated while 28 gene categories belonging to photosynthesis and other metabolic processes were down-regulated under stress treatment. More than 190,000 single nucleotide polymorphisms (SNPs) and small indels were detected and 4,053 of these revealed differential allelic expression between control and drought stressed seedlings. Allelic expression of 70% of these variants was correlated with total gene expression. These variants may be cis-acting variants or in high linkage disequilibrium with such variants. The SNPs and indels identified in this study form a useful resource for further testing in association studies.Water stress limits plant survival and production in many parts of the world. Identification of genes responding to water stress conditions will underpin efforts to breed plants better adapted to drought. We studied the effect of water stress on


Alcohol based hand rubs (ABHRs) are extremely effective at reducing microbial contamination and are central to established best practices for hand hygiene. Modern dispensing systems have brought benefits such as hygienically sealed cartridges with integral pumps for dosing liquids, gels or foams. A remaining issue concerns the measured efficacy of such products and their use in practice as dictated by pump volume.ABHRs for professional use must demonstrate efficacy through standard in-vivo tests; either EN1500 or ASTM E1174. Products typically pass such tests using standard doses not necessarily related to real use by healthcare workers. In this study we set out to identify the optimal dose for an ABHR product based on observation of real behavior.Data from the DebMed GMS Hand Hygiene Monitoring System was used to establish product dose applied in clinical settings via the number of dispenser presses per hand hygiene event. In a separate study healthcare workers applied ABHR using best-practice methodology to establish product drying time as a function of dose. Hand coverage was assessed using a laboratory method based on product coverage. In-vivo efficacy was established using both ASTM E1174 and EN 1500 methods.Healthcare workers intuitively calibrate ABHR dose based on drying time, hand coverage and product ergonomics. In our studies using Deb dispensers, over 90% of healthcare workers used a single pump of ABHR, even when multiple pumps are indicated. Our studies established that 1.5ml of ABHR in foam format dries in approximately 30 seconds and fully covers most hands. Both ASTM 1174 and EN 1500 tests confirmed that 1.5ml of Deb ABHR in foam format with 30 seconds contact time was sufficient to provide effective hand hygiene.Product drying time, hand coverage and measured efficacy data are considered to determine the optimal dose for an ABHR in foam format dispensed from a sealed cartridge wall-mounted unit. This is combined with observed behaviour of healthcare workers to design a pump that delivers the optimal dose in a single press, connecting for the first time measured in-vivo efficacy standards with real-world use to ensure effective sanitization.None declared


New bioinformatic tools are needed to analyze the growing volume of DNA sequence data. This is especially true in the case of secondary metabolite biosynthesis, where the highly repetitive nature of the associated genes creates major challenges for accurate sequence assembly and analysis. Here we introduce the web tool Natural Product Domain Seeker (NaPDoS), which provides an automated method to assess the secondary metabolite biosynthetic gene diversity and novelty of strains or environments. NaPDoS analyses are based on the phylogenetic relationships of sequence tags derived from polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, respectively. The sequence tags correspond to PKS-derived ketosynthase domains and NRPS-derived condensation domains and are compared to an internal database of experimentally characterized biosynthetic genes. NaPDoS provides a rapid mechanism to extract and classify ketosynthase and condensation domains from PCR products, genomes, and metagenomic datasets. Close database matches provide a mechanism to infer the generalized structures of secondary metabolites while new phylogenetic lineages provide targets for the discovery of new enzyme architectures or mechanisms of secondary metabolite assembly. Here we outline the main features of NaPDoS and test it on four draft genome sequences and two metagenomic datasets. The results provide a rapid method to assess secondary metabolite biosynthetic gene diversity and richness in organisms or environments and a mechanism to identify genes that may be associated with uncharacterized biochemistry. Genome sequencing has revealed that the secondary metabolite potential of even well studied bacteria has been severely underestimated Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are large enzyme families that account for many clinically important pharmaceutical agents. These enzymes employ complimentary strategies to sequentially construct a diverse array of natural products from relatively simple carboxylic acid and amino acid building blocks using an assembly line process When the challenges associated with PKS and NRPS gene assembly can be overcome, a number of effective bioinformatics tools have been developed for domain parsing Phylogenomics provides a useful approach to infer gene function based on phylogenetic relationships as opposed to sequence similarities Here we introduce the web tool Natural Product Domain Seeker (NaPDoS), which extracts and rapidly classifies KS and C domains from a wide range of sequence data. The results can be used to assess the potential for PKS and NRPS secondary metabolite biosynthesis in organisms or environments and to identify new phylogenetic lineages, which can subsequently be investigated as a source of new mechanistic biochemistry. We tested NaPDoS on four draft bacterial genome sequences and two metagenomic datasets. The results reveal a remarkable level of secondary metabolite gene diversity among closely related strains and provide a mechanism to assess secondary metabolism from poorly assembled genomic data.http://www.nii.res.in/searchall.html) KS and C domains were extracted from select PKS and NRPS genes associated with experimentally characterized biosynthetic pathways using the online program NRPS-PKS (http://www.phylogeny.fr/) The amino acid sequences of all reference KS and C domains were aligned using either MUSCLE The NaPDoS web portal identifies candidate KS and C domains through a combination of hidden markov model (HMM) searches and the basic local alignment search tool (BLAST) algorithm BLAST results are linked to a back-end MySQL relational database via CGI-scripting to retrieve and report domain classification and related pathway information. Query sequences are trimmed according to their BLAST match coordinates by a custom Perl script then aligned to each other and their database matches using MUSCLE S. arenicola strain CNH-643 (accession number PRJNA84391), S. arenicola strain CNT-088 (accession number PRJNA84269), “S. pacifica” strain CNS-143 (accession number PRJNA84389), and “S. pacifica” strain CNT-133 (accession number PRJNA84271) were obtained at 8× coverage at the J. Craig Venter Institute using 454 GS FLX pyrosequencing and 0.5× Sanger sequencing as previously described S. arenicola strains were mapped onto the complete S. arenicola strain CNS-205 genome and the S. pacifica strains were mapped to the complete S. tropica CNB-440 genome Salinispora genomes were mined for KS and C domains using NaPDoS with default settings. The metagenomic datasets  and the resulting sequences further subjected to a loose BLAST analysis with an e-value cut-off of 1 to obtain more precise coordinates and assign initial domain classifications.Draft genome sequences of http://npdomainseeker.ucsd.edu/) was created to detect and classify KS and C domains in nucleotide and amino acid sequence data. The query data can be PCR amplicons, genes, contigs, genomes, or metagenomes. The current query size limits are <30 MB and <50,000 individual sequences. The website provides a detailed tutorial on the use of this tool, which is implemented using a web interface  against the NCBI nr database is also highly recommended to check for matches that do not occur in the reference database.To generate a final classification for each domain sequence, it is highly recommended to construct a phylogenetic tree, especially in cases where the percent sequence identity to the top database match is low. If that option is chosen, a profile alignment is generated in which the query sequences are incorporated into a carefully curated reference alignment generated from the sequences in the reference database. This alignment is then used to create a phylogenetic tree, which needs to be manually interpreted to establish a final classification for each sequence. Interpreting sequences in the context of a phylogenetic tree is particularly important given that the NaPDoS pipeline is intentionally set to low stringency in an effort to detect all possible KS and C domains. Thus, homologs not involved in secondary metabolism such KSs associated with fatty acid biosynthesis are regularly detected. These sequences can be readily classified based on the phylogenetic tree.trans-AT KSs, which evolved by extensive HGT and exploit considerably greater modular architectures than the cis-AT group, which has largely evolved by gene duplication KS and C domain phylogenies form the basis of the NaPDoS classification system . KS domaIn the case of C domains, the sequences generally clade based on substrate specificity, i.e. the stereochemistry of the amino acids incorporated and the subsequent tailoring reactions they perform . Eight cStreptomyces avermitilis strain MA-4680. This analysis revealed 67 KS and 15 C domains (S. tropica (strain CNB-440) and S. arenicola (strain CNS-205) Salinispora genome sequences. These low coverage drafts were generated using pre-Titanium 454 technology (avreage read length 244 bp) and yielded poor assemblies and a large number of contigs  to those associated with the biosynthesis of salinosporamide A  genomes . While tNaPDoS was further tested on metagenomic data sets generated from a Minnesota farm soil and whale fall Rapid advances in DNA sequencing technologies are providing unprecedented opportunities to incorporate DNA sequence data into the natural product discovery process. The effective use of this information requires bioinformatic tools that can rapidly analyze large datasets in the context of a wide array of complex biosynthetic paradigms. While a number of excellent bioinformatic tools targeting secondary metabolism have been developed The Natural Product Domain Seeker (NaPDoS) is a web-based bioinformatic tool that was developed to detect and classify KS and C domains from a wide variety of sequence data. The use of domain sequence tags as proxies for the biosynthetic genes in which they reside is based on the well established and highly informative phylogentic relationships they maintain. These relationships form the foundation of the NaPDoS classification system and provide a rapid mechanism to delineate secondary metabolite biosynthetic gene richness and diversity within a genome or environmental sample. Short sequence tags (e.g. 600 bp) can be effectively analyzed using NAPDoS and thus minimum coverage, next generation sequence assemblies are well suited for this tool. The resulting estimates of biosynthetic potential can be used to guide more extensive sequencing efforts or targeted operon assembly. In cases where query sequences closely match domains derived from experimentally characterized biosynthetic pathways , it has proven possible to make accurate predictions about the structural class of the secondary metabolite(s) produced trans-AT (b) clade is comprised of KS sequenced derived from what appears to be an evolutionarily independent lineage of trans-AT sequences as well as genes associated with beta-branching There is ample evidence that the mechanistic diversity of polyketide and non-ribosomal peptide assembly is considerably greater than that currently recognized Salinispora genome sequences. Seventeen of these domains were not observed in either of the two complete Salinispora genomes providing evidence of the considerable biosynthetic variability that may occur among closely related strains. In addition, two C domains fell into the “Modified AA” clade, a lineage whose biochemical function has yet to be experimentally characterized. While the metagenomic datasets revealed similar total numbers of KS domains, the classification of those domains revealed dramatic differences in functional types. Analyses such as these provide insight into the potential significance of secondary chemistry in mediating population and community dynamics while at the same time identifying environments that can be prioritized for secondary metabolite discovery efforts.Despite poor assembly, a large number and diversity of KS and C domains were detected among the four draft Traditional natural product discovery paradigms have become increasingly inefficient At present, NaPDoS is optimized for the identification and classification of bacterial PKS and NRPS genes. Nonetheless, it is possible for NaPDoS to identify eukaryotic KS and C domains given their shared evolutionary history with prokaryotic homologs. The results obtained for non-bacterial sequences should be interpreted with caution however, as the reference database has not been adequately populated with these sequences to provide a robust classification system. Future plans include the expansion of NaPDoS to include additional eukaryotic sequences and subgroups within the FAS and PUFA lineages, the later of which were recently shown to cluster phylogenetically based on functional type Table S1NaPDoS derived KS and C domains from the S. avermitilis MA-4680 genome.(DOC)Click here for additional data file.Table S2NaPDoS results for six Salinispora genomes.(DOC)Click here for additional data file.Table S3KS and C domains detected in four draft Salinispora genomes.(DOC)Click here for additional data file.Table S4NaPDoS and antiSMASH-derived KS and C domains.(DOCX)Click here for additional data file.Table S5NaPDoS KS results for metagenomic data sets.(DOC)Click here for additional data file.Table S6KS domains detected in the whale fall metagenomic data set.(DOC)Click here for additional data file.Table S7KS domains detected in the Minnesota farm soil data set.(DOC)Click here for additional data file.


Approximately one third of women diagnosed with ovarian cancer is 70 years or older. Information on the treatment reality of these elderly patients is limited.275 patients with primary epithelial ovarian cancer FIGO stage II-IV undergoing cytoreductive surgery and platinum-based chemotherapy were prospectively included in this European multicenter study. Patients <70 and ≥70 years were compared regarding clinicopathological variables and prognosis.Median age was 58 years (18–85); 47 patients (17.1%) were 70 years or older. The postoperative 60-day-mortality rate was 2.1% for elderly and 0.4% for younger patients (p < 0.001). Elderly patients were less likely to receive optimal therapy  compared to patients <70 years  and their outcome was less favorable regarding median PFS  and OS . However, in multivariate analysis age itself was not a prognostic factor for PFS while the ECOG performance status had prognostic significance in elderly patients.Elderly patients with ovarian cancer are often treated less radically. Their outcome is impaired despite no consistent prognostic effect of age itself. Biological age and functional status should be considered before individualized treatment plans are defined. Women aged 65 and above represent the fastest-growing population segment with consecutively rising incidence of malignancies . Median Irrespective of age, ovarian cancer is still regarded the most lethal gynecological malignancy with a median overall survival of approximately 44 months [In contrast to the well-established prognostic factors postoperative residual tumor load, FIGO stage at first diagnosis and lymph node involvement the role of age itself for ovarian cancer patients has been controversially discussed in the past. While several studies found higher age to be associated with poor survival -7, it coTo further understand the role of age for outcome and treatment of ovarian cancer we analyzed the cohort of prospectively enrolled patients from the Sixth Framework Programme (FP6) EU project “OVCAD – Diagnosis of a Silent Killer” with focus on age. The project was implemented in 2005 by 5 large European gynecologic cancer centers to identify and verify clinical and molecular prognostic factors for ovarian cancer. The present analysis investigates the prognostic value of age itself as well as possible confounders impairing the prognosis of elderly patients and further elaborates the need for trials specifically designed for age-related questions.Patients with advanced epithelial ovarian cancer FIGO II-IV were prospectively enrolled into the OVCAD project between February 2005 and December 2008 by five European Gynecological Cancer Centers . Data from other clinical subprojects of the OVCAD project has recently been published -11.http://www.ovcad.eu). Clinical data were anonymized and collected in a centralized online database  solely accessible by the OVCAD partners.Patients had to be 18 years or older without evidence of any other primary tumor affecting prognosis or treatment of ovarian cancer. Patients were eligible for study entry if radical surgery was performed with the intention of optimal cytoreduction and platinum-based chemotherapy was applied. Systemic treatment could be given either postoperatively or as neoadjuvant treatment. Patients were selected for neoadjuvant therapy if they were not eligible for surgery (e.g. acute thromboembolism) or initial achievement of complete tumor resection was judged to be very unlikely with upfront debulking surgery by the treating gynecologic oncologist in pre-operative evaluation. Complete surgical staging consisted of total hysterectomy and bilateral salpingo-oophorectomy, peritoneal washings, omentectomy, appendectomy, and resection of all visible tumor if possible. All histopathological reports were re-evaluated for the inclusion criteria after surgery to guarantee accurate diagnosis and staging. To measure treatment response, CA-125 levels were assessed before and after treatment completion and the diagnosis of disease recurrence or progression was established based on CA-125 variations (GCIG criteria), imaging studies, and results of performed biopsies [All statistical analyses were conducted using SPSS software version 20.0  and Stata 11.0 . P-values <0.05 were considered statistically significant.To evaluate the impact of age on treatment and prognosis patients were separated in those <70 years and those ≥70 years as previously proposed . The subA total of 275 patients met the inclusion criteria and were prospectively enrolled into the OVCAD study. Median age of the total cohort was 58 years range 18–85) with 228 patients younger than 70 years  and 47 patients aged 70 or older . The median follow up was 36 months. Detailed clinical patient characteristics are provided in Table 8–85 withDespite the non-randomized setting both patient cohorts (<70 years vs. ≥70 years) were well balanced in terms of tumor stage, grading, histological subtype and presence of ascites or peritoneal carcinomatosis Table . The majIn addition, analysis of treatment-related patient characteristics Table  revealedAll included patients received platinum-based chemotherapy according to the inclusion criteria but there were significantly more elderly patients receiving single-agent platinum instead of a combination regimen . Approximately 3/4 of the younger patients experienced clinical response to primary chemotherapy compared a significantly lower rate in the elderly cohort . Taken together only 40.4% of the elderly compared to 70.1% of the younger patients received optimal oncologic treatment as it is considered for the overall patient population consisting of complete tumor resection and platinum-based combination chemotherapy  of 12 vs. 20 months for elderly patients compared to younger patients  is currently planning a prospective trial specifically tailored for elderly women with ovarian cancer (EWOC) that evaluates optimal treatment strategies for elderly patients with the disease but will also address possible new endpoints.Elderly patients with ovarian cancer are often treated less radically than younger patients which has been confirmed in this European multicenter study. Their outcome is significantly impaired despite no consistent prognostic effect of age.Notwithstanding this treatment reality, age itself should not be a reason to withhold optimal oncologic treatment from elderly patients. Comprehensive preoperative assessment should be performed considering the biological age and functional status of the patients. With this information individualized treatment plans can be defined accounting for co-morbidities and expected complications.The authors declare they have no competing interests.FT, LW and SM were responsible for the concept and design of the presented study, analyzed the data and prepared the manuscript. CE participated in the study design and performed the statistical analysis. All authors were involved in the data acquisition and quality control for the trial. All authors supported the manuscript editing, read and approved the final manuscript.


The results of the RV144 trial support the ability of vaccine induced Antibodies to protect from HIV acquisition. The STEP trial also appears to have impacted viral load in individuals who produced strong T cell immunity. The HVTN has recently reported that our enhanced DNA (Pennvax) can drive T cell immunity as robust as Adenoviral and Pox platforms in an accelerated format in humans. We have now focused on further driving T and B cell immunity by utilizing prime boostGroups of rabbits or Indian Rhesus macaques were vaccinated with enhanced DNA vaccine plasmids encoding pGAG, pPOL + combinations of 3-5 consensus or COT designed envelopes that were either gp140 or gp160 constructs. Both IM as well as a novel minimally invasive skin delivery EP arrays (MID) were studied. DNA vaccinated animals were boosted two months following the final DNA immunization with SF162 gp120 in MF59.In rabbits, nAb titers against a broad panel of Tier 1 viruses was observed by the combination immunization approaches. Titers in the hundreds were induced by DNA and these titers were enhanced almost a log by a protein boost. Similar data was also observed when these studies were extended to the non-human primate model. Importantly, DNA-prime protein-boost had detectable ADCC activity as measured by the GTL and luciferase assay. A bias for improved antibody responses driven by the MID delivery array is also observed.Building on the success observed in HVTN 080 we report that further enhancement of DNA constructs in a prime boost setting (E-DNA prime + protein Boost) is capable of eliciting high binding, broad antibody responses and neutralization titers against a panel of tier 1 viruses. Combining improved E-DNA with other strategies shown to enhance antibody responses such new gene adjuvants are being investigated.


The recognition that HIV originated from nonhuman primates (NHP) heightens concerns associated with transmission of simian retroviruses to humans. Although recent studies document frequent infection with simian foamy virus (SFV) among primate hunters in Cameroon and primate handlers in North America, little is known about the spread and geographic distribution of SFV outside these populations.We tested 3467 plasma samples collected in 2007 in a population-based survey in 15 rural villages in central Democratic Republic of Congo (DRC). Participants completed a questionnaire about exposure to NHPs. Plasma samples were screened for SFV antibodies using a new Luminex-based EIA; reactive specimens were also tested by a second EIA utilizing recombinant SFV Gag proteins. Specimens dually reactive in both EIAS were then tested using a Western blot (WB) assay that can detect monkey and ape-type SFV reactivity. PCR amplification of DNA from buffy coats and phylogenetic analysis was used to define the NHP species origin of SFV.54 (1.6%) plasma samples were dually EIA reactive and 25 (0.72%) were WB positive. Buffy coats were available for PCR testing from 21 persons with WB positive or indeterminate results. SFV polymerase and LTR sequences were amplified from the DNA of three women 23, 50, and 57 years old. Phylogenetic analysis showed novel SFV infection in two women originating from Angolan Colobus (Colobus angolensis) and in one woman from a red-tailed guenon (C. ascanius), viral variants not previously seen in humans. Both monkeys are native to DRC. Two of these women reported exposure to NHPs via butchering and cooking, and specifically to Angolan Colobus or red-tailed guenons. One woman did not report exposure to NHPs. These women lived in three separate villages, suggesting a wide and potentially diverse distribution of SFV infections across DRC. The younger woman reported a history of monkey pox-like illness. All these women were married and had at least three children but specimens were not available from close contacts for testing. Animal exposure data were available for 3034 persons; 2413 (79.3%) reported NHP exposure.Our study documents SFV infection in the DRC for the first time and identifies human infection with new SFVs from Colobus and red-tailed monkeys. Our findings suggest that SFV is prevalent across Africa in persons exposed to NHPs inhabiting a specific region. Our results highlight the importance of determining if SFVs are transmitted from person-to-person and can cause disease. SFV infection of humans exposed to NHPs also highlights the increased potential for zoonotic transmission of other simian retroviruses with known pathogenic consequences.


A 35 years old woman presented with allergic rhinitis and asthma with cat epithelium's sensitivity (Skin prick test positive) treated with antihistamines, nasal and bronchial steroids.The patient has been maintening the cat in her apartment. After a couple of years from the onset of respiratory allergic disease, she presented an oral allergy syndrome type clinical reaction, gastric pain and diarrhea at once after eating cooked pork meat . Skin prick test (SPT) performed with commercial extracts and fresh meat were positive for pork meat according with the 'pork-chat syndrome'.Subsequently she presented the same symptoms after eating goat cheese and goat meat.A prick by prick for the culprit foods confermed the food allergy. Recently the same symptoms presented after eating not well cooked beef meat. In her personal history we focused on an anaphylactic reaction occurred at 23 y.o. after injection of antirabbit serum used for antipneumococcal vaccination. The specific IGEs dectection (microarray IMMUNOCAP ISAC system) was positive only for :bovine (Bos d6), cat (Fel d2) ,dog (Can f3), horse (Equ c3) serum albumin and cat, dog lipocalin .No other foods or respiratory allergens specific IGEs were detected.Meat allergy is a rare allergic disorder, more frequent during the first year of life but unusual in adults.Unusually the sensitization to pork meat presented before the goat and beef allergy.Interestingly no milk allergy was found associated to meat allergy (while in children is frequent ). As it's common the patient presented cat sensitivity and subsequently a crossreactivity towards dog,cat and beef serum albumins. The patient avoids pork, sheep,goat meat but she can eat well cooked beef meat.


Arteriogenesis as a way to restore blood flow after arterial occlusion has been under investigation for the treatmentof coronary artery disease (CAD) for decades. Therapeutic approaches so far have included delivery of cytokinesand growth factors as well as mechanical stimulation such as external counterpulsation. As knowledge on the mechanismsof arteriogenesis expanded, new therapeutic approaches have emerged.This review summarizes recent attempts to stimulate the growth of the coronary vasculature and discusses their potentialin clinical application. This article also delivers an overview of current studies and trials on coronary arteriogenesis. For decades ischemic heart disease (IHD) has been the leading cause of death in industrialized western societies . MacrovaThe following review describes different approaches including inflammatory as well as mechanical stimuli but also highlighting new experimental approaches and possible downsides of an increased coronary collateral vasculature.Approaches to promote collateral artery growth have advanced from animal studies to clinical investigations studies. Arteriogenesis being an inflammatory process  it seemeVascular endothelial growth factor in Ischemia for Vascular Angiogenesis), in which vascular endothelial growth factor (VEGF) was tested versus placebo in IHD had neutral results. Similarly, the AGENT [Angiogenic GENe Therapy) and FIRST [FGF Initiating RevaScularization Trial) studies enquiring the effects of fibroblast growth factor (FGF) on capillary growth showed no benefit over placebo treatment. In the AGENT trial, an adenoviral vector containing a human fibroblast growth factor gene was applied via an intracoronary route in patients with stable IHD. Treadmill exercise testing was used to evaluate efficacy of treatment. Overall, there was a non-significant increase in exercise time between treatment and placebo group. A significant difference in the increase of exercise time was only reported in patients with baseline exercise time of less than 10 minutes. Similarly, the FIRST trial assessed single-shot intracoronary application of recombinant FGF2 (rhFGF2) for improvement of exercise tolerance in patients with coronary artery disease. Again, there was no significant difference reported in comparison to placebo treatment. The VIVA trial focused on recombinant human vascular endothelial growth factor (rhVEGF) in angina patients. Study endpoint was angina frequency and exercise time evaluated by treadmill test. The intracoronary application of rhVEGF resulted in no significant difference of angina frequency and exercise time when compared to placebo. Most of the studies first concentrated on angiogenesis as the main mechanism of blood flow restoration. The VIVA trial   was tested in swine suffering from metabolic syndrome. Treatment with NPY resulted in up-regulation of pro-arteriogenic proteins while down-regulating anti-angiogenic factors  and coulStudies involving a visual angiographic grading of collateralization and analyzing serum concentrations of inflammatory cytokines proved modulation of inflammatory cytokines in patients with good collateralization. Angiostatic ligands such as C-X-C motif chemokine 10 (CXCL10) and C-X-C motif chemokine 11 (CXCL11) were decreased, while higher concentrations of angiogenic ligands were associated with a well-developed collateral circulation .transient receptor potential cation channel family (TrpV4)  in the vessel walls which in turn induce inflammatory mediators [In accordance with the pathophysiology of arteriogenesis corroborating animal experiments, physical exercise was shown to have positive effects on collateral artery growth in the clinical setting , 30. Theediators  and concediators . Also, eediators . Inducedediators . Simultaediators  and thusediators . Consequediators . Generalediators . However1). In the MUST-EECP-study  an increase of coronary flow reserve and alleviation of angina were reported after treatment with ECP [Given the fact that mechanical factors influence arteriogenesis , there hwith ECP . This isTwo studies have directly tested the hypothesis that ECP enhances coronary collateral artery growth. Arteriogenesis Network) trial patients with stable coronary artery disease underwent a 35 1-h sessions of ECP in 7 weeks [In the non-randomized Art.Net.-2  next to an ECP-group (300 mmHg) [Another investigation by Gloekler 00 mmHg) . PatientIn an attempt to reduce angina in symptomatic CAD-patients not suitable for revascularization, the Coronary Sinus Reducer was implanted in fifteen patients. The Coronary Sinus Reducer is an expandable stainless steel stent inserted percutaneously through the internal jugular vein narrowing the coronary sinus (CS) and thus increasing coronary venous pressure. After 6 months, angina was reduced as well as myocardial ischemia as evaluated by dobutamine echocardiography and thallium single-photon emission computed tomography (SPECT) . ConsideCLaudication: Exercise Vs. Endoluminal Revascularization) has proven SE to be even superior to stent revascularization (ST) and sole optimal medical care (OMC) in patients with aortoiliac peripheral artery disease by a significant increase in peak walking distance [An active means of increasing collateral flow and shear stress, particularly in peripheral artery disease (PAD), is supervised exercise (SE). The CLEVER study (distance . Both SE2). Because of similar mechanisms a beneficial effect of exercise can be expected on the coronary circulation as well. In an individual athlete’s report, different levels of physical activity resulted in an increase on both CFI and coronary flow reserve (CFR). At baseline fitness level the healthy sportsman experienced anginal chest pain during a one minute left anterior descending coronary artery (LAD) occlusion, which he did not suffer from at a higher fitness level, suggesting a shear-stress induced arteriogenesis of pre-existing coronary collaterals . A cliniet al. for the first time increased fluid shear stress (FSS) by creation of arteriovenous (AV) side-to-side-anastomoses in the porcine peripheral circulation following femoral artery occlusion [in-vivo has been sought to find a means to translate mechanical arteriogenesis into an exogenously applicable pro-arteriogenic drug. Nevertheless, the conception of an arteriovenous shunt is potentially conceivable in the clinical situation as well, where percutaneous or surgical creation of a coronary AV-shunt in a patient with end-stage coronary artery disease and lack of therapeutic options is theoretically feasible. Pipp cclusion . Here, Acclusion  for the As mentioned above, eNOS is induced during arteriogenesis. Past studies have shown that the presence of NO is critical in arterial growth. Application of NO donating moieties has been demonstrated to stimulate arteriogenesis . SubstanAnimal studies showed increased flow restoration in a femoral artery ligation model following heart rate reduction by If-channel blockade through administration of Ivabradine. In this study, the augmented pulse amplitude is most likely the key to increased circumferential wall stress and increased arteriogenesis . HoweverIvabradine is the first substance assessed for the stimulation of arteriogenesis which has demonstrated proarteriogenic capabilities while attenuating systemic inflammation . Strong Cell therapy has gained vast interest in regenerative medicine in the past years. A prospective study on cell therapy for the stimulation of collateral artery growth using quantitative outcome measures (CFI) is currently lacking. One promising approach could lie in the application of vascular progenitor cells. A recent paper has shown the possibility of applying reprogrammed vascular progenitor cells to increase collateral artery growth , 56. ThiThere are possible downsides to a well-developed coronary collateral circulation that should be considered carefully. A meta-analysis comprising seven studies on restenosis after percutaneous coronary intervention (PCI) and drug-eluting stent (DES) implantation shows that good collateralization before intervention is associated with a 40% increase in restenosis when compared to patients with poor collateralization . In anot


We describe the case of a 50-year-old man in whom the finding of avid 18F-FDG uptake of pectoralis major muscle was encountered during investigation of metastatic melanoma.Avid functional  Nevertheless, 18F-FDG is not a tumour-specific substance as it shows non-specific affinity in other normal tissue especially in the skeletal muscle [18F-FDG uptake in muscle are also observed in diabetic patient following administration of insulin, surgical intervention or following immunosuppressive therapy.Active skeletal muscle is a common area for interpretative pitfall which is related to physiological FDG uptake . In addiMuscle uptake can be attributed to voluntary or involuntary muscle activity. Voluntary muscle activity consists of activities such as talking, chewing, and exercising-induced rhabdomyolysis. Involuntary muscle activity would include laboured breathing or stress-induced muscle spasms.Normal muscles accumulates little FDG, uptake of grade 1 or less. Muscles exercised just prior to or around the time of FDG injection can exhibit uptake of grade 3 to 4 . NotwithA 50-year-old gentleman with a small right ankle melanoma had been successfully managed with surgical removal of the melanoma in 2001. He had right inguinal nodal recurrence after 6 years disease-free progression. A PET-CT study performed in June 2007 revealed unexpected intense symmetrical uptake of the pectoralis major muscle . The pat18F-FDG uptake in pectoralis major muscle is conceptually attributed to some form of strenuous eccentric exercise 24 hours prior to PET-CT study.In our case, an avid Exercise is a well known mechanism that is capable of inducing various components of the immune system . IL-6 isThe fact is that a substantiated FDG accumulation in skeletal muscle as shown in our case cannot be entirely explained by the influence of intramuscular glucose uptake for more than 24 hours following exercise.A possible explanation of what appears to augment FDG avidity in this case might have been attributed to ongoing rhabdomyolysis following strenuous exercise. Rhabdomyolysis appears to be a relatively common sequela of strenuous exercise . Of noteIt is noteworthy that the cause of prolonged avid FDG muscle uptake in our case remains unclear as there were neither symptoms related to muscle damage i.e aching nor available biochemical analysis pertaining to the level of creatinine kinase and urine myoglobin levels.Unexpected FDG muscle uptake may occur more than 24 hours following exercise as illustrated in our case. The exact explanation is unclear but underlies the importance of adequate patient preparation and instructions before a FDG PET scan.


Transesophageal echocardiography detected atrial level hypoxemic right-to-left shunting through a patent foramen ovale (PFO). Percutaneous closure of the PFO was achieved with a PFO occluder device. After placing the PFO occluder device oxygenation increased significantly (Δ paO2 119 Torr). The patient received heart transplantation 20 weeks after BVAD implantation and was discharged from ICU 3 weeks after transplantation.We describe the interdisciplinary management of a 34-year-old woman with dilated cardiomyopathy three months postpartum on a cardiac biventricular assist device (BVAD) as bridge to heart transplantation with delayed onset of intracardial shunting and subsequent hypoxemia due to massive pulmonary embolism. After emergency surgical embolectomy pulmonary function was highly compromised (PaOAn increase in pulmonary vascular resistance in patients on BVAD can reopen a PFO resulting in atrial right-to-left shunting and subsequent hypoxemia. The case demonstrates the usefulness of transesophageal echocardiography examinations in the detection of this unexpected event. Percutaneous placement of a PFO occluder device is an appropriate strategy to stop intracardiac shunting through PFO in fixed elevation of pulmonary vascular resistance. In a literature review, few cases of atrial level right-to-left shunt in patients with left ventricular assist devices are described. All these cases were detected either intraoperatively -3 or witA 34 year old female patient was admitted to our hospital with dilated cardiomyopathy three months after birth of her third child. She had a known history of familial dilated cardiomyopathy. Recompensation was not achieved despite maximum medical therapy and insertion of an intra-aortic balloon pump. BVAD  was implanted using a bi-atrial cannulation technique as bridge to heart transplantation. Perioperative transesophageal echocardiography did not show a patent foramen ovale (PFO). Postoperative recovery was immediate and the patient was discharged from the ICU on the third postoperative day.iO2 of 1.0 was necessary to achieve sufficient oxygen saturation (paO2/FiO2 54). Modification of ventilator setting with adjustments of PEEP and peak inspiratory pressure did not lastingly improve oxygenation. Transesophageal echocardiography detected atrial level intracardial shunting . Weaning from mechanical ventilation was successful after 15 weeks.Four weeks after device implantation the patient developed fulminant pulmonary embolism despite therapeutic anticoagulation. Emergency surgical embolectomy for massive pulmonary embolism was performed since thrombolysis was not an option after recent implantation of an artificial heart Figure . Pulmona5 with activated assist device and 160 dyne•s/cm5 with deactivated assist device.After 5 weeks of therapeutic anticoagulation the residual emboli diminished and pulmonary vascular resistance was measured at 184 dyne•s/cmHeart transplantation was performed 20 weeks after implantation of the BVAD and 16 weeks after pulmonary embolism and placement of the PFO occluder device. Discharge from ICU was 3 weeks after transplantation. Informed consent for publication was obtained from the patient.The problem with PFO and left ventricular assist device leading to atrial level right-to-left shunt with consecutive hypoxemia is well described -7. PFO hTherefore, intraoperative transesophageal echocardiography with colour Doppler imaging and contrast with agitated saline is highly recommended before cardiopulmonary bypass and after LVAD activation ,13. AlteThere is only one other case of delayed onset of atrial level right-to-left shunt in patients on ventricular assist device . In thisPersisting elevation of right atrial pressure due to persisting change of the pulmonary vascular resistance in a patient with a BVAD has not been described. An etiologic reason for persisting elevation of pulmonary vascular resistance can be massive pulmonary embolism as described in our case. Our report is the first description of a patient surviving massive pulmonary embolism while on BVAD, followed by successful orthotopic heart transplantation. To the best of our knowledge there is only one other published case of pulmonary embolism in a patient with a BVAD. This patient died shortly after the event .5 to 285 ± 214 dyne•s/cm5 .Click here for file


Fully flexible 3D alignment method without the need for sampling the conformational space is presented. This approach offers advantages over methods that rely on multiple conformations:• Higher accuracy is achieved, since unlike discrete sampling it is not prone to missing optimal conformation.• Smaller database size is needed, pre-calculated conformers are not stored.The alignment procedure maximizes a score resembles the intersection of the volume of the molecules being aligned. The volume itself can be colored by atomic properties enabling the use of various similarity scores, like shape-, atom type or pharmacophore type similarity.Similarity scores serve as the basis for 3D virtual screening. However, the calculation of 3D similarities is computationally intensive. In order to achieve high performance fast pre-filtering by 3-dimensional molecular descriptors has been introduced. Intra molecular distance ranges are used as shape descriptors and are pre-calculated by the application of the alignment machinery itself in an initialization stage.The presentation overviews the mathematical apparatus introduced, elaborates on the implemented methods and presents results.


We have previously reported that potent, long-lasting HIV-1 Env-specific cell-mediated immune responses could be elicited in rhesus macaques and mice using plasmids encoding env DNA as the immunogen. Subsequent experiments showed that combination of DNA and protein in the form of inactivated virus particles provided significant protection from infection and high viremia. We examine a vaccine platform combining DNA and recombinant Env protein co-immunization at the same time to generate both strong cellular and humoral immune responses.Mice or macaques were immunized with HIV env gp120 DNA vaccine and/or purified gp120 protein from clade B or clade C isolates. Mice were immunized twice at 4 weeks interval with DNA only, protein only formulated in EM005 adjuvant, or DNA&protein/EM005. Macaques were immunized twice at 4 weeks interval with DNA only, DNA&protein, DNA&protein/EM005.DNA&protein co-immunization enhances the Ab responses compared with DNA or protein only in mice. DNA&protein co-immunization generated similar levels of cellular immune responses compared to mice immunized with DNA only but those levels were significantly higher than those obtained in mice immunized with protein only. The establishment of a mouse model that gives similar results with the macaque model enhances our ability to test many variations and optimize the vaccine. Importantly, in macaques this strategy elicited higher binding and neutralizing Ab responses than DNA only and the neutralizing Abs showed broad activity. The presence of the EM005 adjuvant further enhanced the Ab responses. These responses were correlated with the up-regulated activation of dendritic cells by EM005. The longevity of the Ab response was superior.The strategy of DNA and protein co-immunization has potential for development as a prophylactic HIV-1 vaccine. Our challenge studies show that DNA and protein co-immunized animals developing long-lasting Ab titers were protected from infection.


Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly. RNA-seq is a widely used tool for transcriptome analysis and gene expression estimation. Most commonly, mRNA is fragmented followed by reverse transcription into first strand cDNA primed by random hexamers. Subsequently, second strand cDNA is synthesized from first strand cDNA by DNA polymerase, again initiated by random hexamer priming. Bias in hexamer distribution and CG content affects abundance estimates and several bias correction algorithms have been developed e.g. -5. Apart from bias in hexamer priming sites, random hexamer mispriming has recently been implicated in sequence read to reference mismatches. Because mismatches mainly occur in the first seven nucleotides of first strand cDNA ,6 and arThis paper deals with sequence read to reference mismatches commonly observed in RNA-sequencing data. Mismatches are defined as any position in sequencing reads that deviate from the reference to which these reads align. Mismatches can reflect 1) true biological variation, both in genomic DNA or caused by RNA-editing, or 2) errors in the library preparation process caused by hexamer mispriming or PCR errors, and finally 3) sequencing errors caused by the erroneous identification of bases in the sequencing process. We focus on errors that most likely arise in the library preparation phase. During RNA-seq library preparation, polyA+ RNA is fragmented and reverse transcribed into first strand cDNA initiated by random hexamer priming. We refer to mismatches between the reference fragment and the observed sequence that are caused by RNA-DNA hexamer mispriming during first strand cDNA synthesis as RD-mismatches. Similarly, we use the term DD-mismatches to describe errors caused by DNA-DNA hexamer mispriming during second strand cDNA synthesis.Errors caused by random hexamer mispriming have thus far received limited attention. While read trimming to exclude error-rich first stretches of RNA-seq reads is done in some studies ,11, therTaraxacum officinale RNA mixed with ERCC RNA spikes. Twenty-three libraries were multiplexed using Illumina’s multiplex sequencing assay and pooled with a 2% ERCC spike [We conducted a RNA-seq experiment of CC spike . SubsequCC spike ; GSM5170We analyzed two independently generated RNA-seq datasets, focusing on reads mapping to ERCC spikes which are artificial RNA fragments of known sequence that are added during library preparation. ReverseFirst strand cDNA mismatches in the first seven base pairs corresponding to the hexamer binding site and the base immediately downstream of this show position-dependent and nucleotide-dependent mismatch patterns . These sThus we conclude that mispriming is non-random and can be heavily biased, as RNA binding sites with a U at positions three and four (relative to the hexamers 5’ end) misprime with hexamers having a G at that position in ~88% of the mispriming cases. These specific mismatch patterns were observed consistently in two independent data sets . The disConsistent mismatch patterns observed in the first seven nucleotides of first strand cDNA will affect downstream applications such as de novo assembly, SNP calling and RNA-editing analysis. For instance, consistent and high (20%) mismatch rates can be problematic for k-mer assembly strategies, as these erroneous k-mers cannot be effectively combined with “true” k-mers. Because mispriming rates are not random and for some positions heavily biased  they canIdentification of the hexamers that are most commonly involved in mispriming as well as the type and position of the mismatches this generates can aid in the design of strategies to counter the effects that these errors can have in downstream applications. Our results suggest that it could be useful to explore less aggressive approaches than trimming. Such approaches could include post-mapping correction of 5’ mismatches, modifications to random hexamer design to exclude commonly mispriming hexamers and specific bias correction models that mask or remove the observed mismatches in the first seven bases of reads. Our analyses shows strong and consistent bias in sequence errors in 5’ ends of RNA-seq reads, which (1) strongly supports the hypothesis that random hexamer mispriming during first strand cDNA synthesis causes the errors, and (2) highlights the risk of errors in downstream applications as well as suboptimal data use. We conclude that technical artifacts in sequencing data are insufficiently described. Further research on random hexamer mispriming will inform optimized strategies to mitigate their negative effect on downstream analysis.


The application of molecular profiling methods to a wide variety of infections suggests that a polymicrobial community is much more common than suggested by standard clinical culture . Our goaCulture and DNA extraction protocols were evaluated using synthetic bacterial communities inoculated into whole blood. Disruption of blood cells with a blood cell lysing detergent, with or without hypotonic osmotic shock, was carried out and evaluated for the ability to recover the community. Viable bacterial cells were recovered on solid media. Total DNA was examined by terminal-restriction fragment-length polymorphism (TRFLP) profiling. Efficiencies of recovery and limits of detection were determined. The optimized methodology was applied to clinical samples collected from consented patients in both the ICU and ED from two Calgary hospitals. Cultured organisms were identified by 16S rRNA gene sequencing. Molecular profiling was carried out using TRFLP and bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP).Treatment of synthetic community organisms with a 5% wt/vol detergent added at a 1:1 or 1:5 ratio did not significantly impact their viability. TRFLP analysis indicated that the DNA from these communities could be recovered from whole blood following lysis and removal of host cells. Whole blood samples were analysed from septic patients. In three case studies we identified two to 16 bacterial species in the primary infection samples using direct culture and molecular methods. Conventional diagnostics only reported one organism. Molecular profiling of blood samples from these patients also identified correlating polymicrobial communities. Blood cultures for these samples were either negative (two of three cases) or monomicrobial (one of three cases), thereby underestimating the diversity seen with the TRFLP and bTEFAP analysis. These methods have been applied to 88 adult ICU blood samples, 20 primary infection samples, 36 adult ED samples, and seven pediatric ED samples with analysis ongoing.We have successfully developed a novel method to analyse whole blood in order to characterize the microbiome of sepsis infections. Preliminary results indicate that sepsis infections are polymicrobial in nature.


Diabetic cardiomyopathy is a specific disease process distinct from coronary artery disease and hypertension. The disease features cardiac remodeling stimulated by hyperglycemia of the left ventricle wall and disrupts contractile functions. Cardiac mast cells may be activated by metabolic byproducts resulted from hyperglycermia and then participate in the remodeling process by releasing a multitude of cytokines and bioactive enzymes. Nedocromil, a pharmacologic stabilizer of mast cells, has been shown to normalize cytokine levels and attenuate cardiac remodeling. In this study, we describe the activation of cardiac mast cells by inducing diabetes in normal mice using streptozotocin (STZ). Next, we treated the diabetic mice with nedocromil for 12 weeks and then examined their hearts for signs of cardiac remodeling and quantified contractile function. We observed significantly impaired heart function in diabetic mice, as well as increased cardiac mast cell density and elevated mast cell secretions that correlated with gene expression and aberrant cytokine levels associated with cardiac remodeling. Nedocromil treatment halted contractile dysfunction in diabetic mice and reduced cardiac mast cell density, which correlated with reduced bioactive enzyme secretions, reduced expression of extracellular matrix remodeling factors and collagen synthesis, and normalized cytokine levels. However, the results showed nedocromil treatments did not return diabetic mice to a normal state. We concluded that manipulation of cardiac mast cell function is sufficient to attenuate cardiomyopathy stimulated by diabetes, but other cellular pathways also contribute to the disease process. Diabetic cardiomyopathy is defined as a primary disease process distinct from coronary artery disease and hypertension Mast cells are recognized as active participants in allergic and anaphylactic reactions There is indirect evidence that cardiac mast cell activation may contribute to cardiac remodeling. Mast cell degranulation has been observed in the human heart Based on these previous studies, we propose to use an experimental diabetic cardiomyopathy model to observe if hyperglycemia is associated with cardiac mast cell activation and if mast cell activation correlated with collagen deposition and cardiac remodeling in the heart. We modulate cardiac mast cell activity by administering the mast cell-stabilizing agent nedocromil (Ned) to diabetic mice and then monitor cardiac function, cardiac mast cell activity, expression levels of proteins associated with cardiac remodeling, and the extent of collagen deposition as indirect measurements of the contribution of cardiac mast cell degranulation in diabetic cardiomyopathy.The study protocol was approved by the medical ethics committee of Shanghai Changzheng Hospital, conforms to the Principles of Laboratory Animal Care , and was conducted according to National Institutes of Health guidelines.We used previously described methods to induce diabetes in our mouse model Diabetic mice (13-week-old) were randomly divided into three groups: 1) untreated group; 2) nedocromil group, with nedocromil released at the rate of 30 mg/kg per day from a subcutaneous (s.c.) pellet implantation 2 and 5% CO2 continuously bubbled into the solution. The physiological pressure transducer was sutured to the apical area with 4-0 thread.To assess cardiac function, mouse hearts were isolated and perfused using the Langendorff system Cardiac function indicators were recorded using Powerlab multi-channel physiological recorder. The recorded indicators include maximal contractile rate (+dF/dt), maximal relaxation rate (-dF/dt), heart rate (bpm), and heart work (g.bpm). Heart work was calculated by multiplying the force (g) by the heart rate and normalized to heart weight. After the recording, the left ventricle was placed on ice and then cut transversely into three equal slices perpendicular to the long axis. The slices were either processed immediately for molecular characterization or flash-frozen in liquid nitrogen and stored at -80°C for future analysis.Histological analyses were performed as previously described Immunohistochemical analysis was performed to determine the number of mast cells and mast cell activation based on chymase expression Real time-polymerase chain reaction (RT-PCR) was used to measure mRNA expression levels of type I and III collagen, and to confirm chymase mRNA expression. Total cellular RNA was extracted using Trizol  according to the manufacturer's instructions. RNA (0.2 µg) was reverse-transcribed using High-Capacity cDNA synthesis kit . RT-PCR was performed using the QuantiFast Sybr Green PCR kit . The samples were amplified using Light Cycler 480 real-time PCR machine . GAPDH expression was used for normalization. mRNA levels were expressed as fold-change relative to untreated normal or untreated diabetic mice. Primers specific for the target genes, the primer's annealing temperature and amplicon length are listed in Western blots were used to detect the protein expression levels of type I and III collagen, MMP-2, MMP-9, tryptase, histamine, and chymase in cardiac tissues. Protein extraction was performed using a protein extraction kit  and the concentration was determined using a protein assay kit . The extracts were resolved on SDS-PAGE gels by electrophoresis and transferred onto nitrocellulose membranes . The membranes were block with 5% nonfat milk in PBS-T. The primary antibodies used were: rabbit anti-mouse collagen I and collagen III antibodies (dilution 1∶5000); rabbit anti-mouse MMP2 and MMP9 antibodies (dilution 1∶1000); rabbit anti-mouse tryptase (dilution 1∶100), rabbit anti-mouse histamine (1∶1000), and goat anti-mouse chymase antibody (dilution 1∶200). Goat anti-rabbit HRP conjugated IgG (dilution 1∶2000), and donkey anti-Goat HRP conjugated IgG (dilution 1∶5000) were used as secondary antibodies to resolve the signal. All antibodies were purchased from Abcam .Commercial ELISA kits  were purchased to determine the levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-6, IL-10, and angiotensin (Ang) II in cardiac tissue. The assays were performed according to manufacturer's instructions using extracted proteins as described above.Data are expressed as mean ± standard deviation (SD). ANOVA with Scheffe's-F test was used to determine statistical significance between treatment groups, when appropriate. A p-value of <0.05 was considered significant.Hyperglycemia has long been implicated in the development of cardiac diseases including cardiac remodeling As shown in Cardiac function was evaluated based on four parameters: maximal cardiac contractility, maximal cardiac relaxation, heart rate, and heart work. Heart work was calculated by multiplying the contraction force by the heart rate and normalized to the heart weight. Nedocromil-treated diabetic mice showed significantly improved heart function compared with controls . The conPrevious studies have shown that cardiac mast cells are activated in injured cardiac tissue to mediate cardiac remodeling We performed immunohistochemical studies on cardiac tissues from normal and diabetic mice that underwent nedocromil treatment. We examined mast cell activation by assessing chymase level because the enzyme is overexpressed and released during the degranulation process The number of chymase-positive cells was significantly higher in the cardiac tissues of diabetic mice than in normal mice. Nedocromil significantly reduced the number of chymase-positive cells in samples from diabetic mice, and had little effect on samples from normal mice . The calWe further confirmed cardiac mast cell activation by evaluating the protein levels of additional mast cell-associated enzymes, which are also induced upon mast cell activation We have observed nedocromil treatment improved overall cardiac function in diabetic mice . So, we Trace amount of collagen deposits were observed in the hearts from normal mice, while those from diabetic mice showed significant areas of collagen deposition  that corCardiac mast cell activation was reported to modulate cytokines that contribute to cardiac remodeling Hyperglycemia has long been implicated in cardiac diseases by producing metabolic stress and triggering cardiac remodeling We have presented data that showed cardiac mast cell activation and cardiac remodeling occurs following hyperglycemia. The hearts of STZ-induced diabetic mice showed significantly impaired contractile function that resulted from increased collagen deposits and initiation of extracellular matrix degradation. We also examined chymase and tryptase levels in the heart because the enzymes are known mast cell products and have been linked to increased collagen production and to induce remodeling of the extracellular matrix by cleaving pro-forms of MMP-2 and MMP-9 Many of these features were reversed upon mast cell stabilization using nedocromil. We showed the drug had protective effects against cardiac dysfunction in diabetic mice by reducing mast cell density and chymase secretion, and confirmed the expression of remodeling factors had decreased using biochemical assays.We also observed aberrant cytokine levels in diabetic mice, which may contribute to cardiac remodeling. Nedocromil treatment was able to normalize them in diabetic mice. Cardiac mast cell activation has been reported to modulate cytokines that contribute to cardiac remodeling Tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and angiotensin Ang (II) have long been associated with cardiac remodeling. They were all significantly increased in diabetic mice. TNF-α is known to increase collagen production by stimulating the angiotensin II type 1 receptor The role of IL-6 in cardiac remodeling is unclear because its expression has been shown to vary greatly at different stages of cardiac remodeling in hypertensive rats Our data also showed nedocromil had no effect on blood glucose levels in diabetic mice, indicating mast cell activation is likely correlated with blood glucose levels and not by possible nonspecific effects from nedocromil. We also confirmed the effects of nedocromil had no significant effects in normal mice. However, the drug did not completely inhibit the degranulation process, as shown by the stable tryptase and histamine levels. We speculate that heightened levels of tryptase and histamine in diabetic mice may contribute to cardiac remodeling by an unknown mechanism, which may explain why nedocromil treatment did not completely abrogate the molecular changes observed in diabetic hearts.We have shown a correlation between mast cell activation and cardiac remodeling in diabetic mice. We concluded that regulating mast cell activity was sufficient to significantly improve heart function in diabetic mice, and reduce signs of collage deposition and extracellular matrix destablization. Nedocromil treatment alone normalized the levels of several cytokines that are known contributors to cardiac remodeling. Our results propose that the manipulation of cardiac mast cells can attenuate cardiomyopathy by modulating aberrant cytokine levels. However, this mechanism is insufficient to fully restore normal cardiac functions and suggests other cellular pathways may contribute to the disease process. In addition, the molecular mechanisms that link hyperglycemia and cardiac mast cell activation remain unclear and warrant further study.Figure S1Immunohistochemical staining for chymase in cardiac tissues (400X magnification).(TIF)Click here for additional data file.


The biology of chronic migraine (CM) as a true entity or as a consequence of analgesic overuse is controversial. There are no available biological markers for CM, while CGRP, a marker of trigemino-vascular activation, has been shown to be increased during acute migraine attacks in episodic migraine .To determine CGRP levels in peripheral blood in a series of patients with CM as compared with matched subjects without a headache history.This series comprises 61 women meeting CM diagnostic criteria (IHC-II 2006 revised) with a mean age of 44 years (range 16-63) and 19 women  without any headache history. CGRP levels were determined in blood samples obtained from right cubital vein between 9-12 am with an ELISA kit from USCN following manufacturer's instructions. Acute medication had not been taken the day before.CGRP levels were increased in women with CM  as compared to controls  .CGRP levels are clearly increased in patients with CM, which is compatible with a permanent activation of trigemino-vascular system in this entity. CGRP determination may constitute the first reliable biological marker for CM.