The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants  is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions. Cyan and yellow fluorescent proteins  are very frequently used in a great variety of experiments employing fluorescence microscopy, because they can be distinguished quite easily by appropriate filter sets. Especially the original mammalian optimized versions, also known as enhanced cyan and yellow fluorescent proteins (ECFP and EYFP) are commonly applied, as they have been among the first spectrally distinct variants of green fluorescent protein We applied the FRET microscopy technique that is based on bleaching of the acceptor to cells transfected with interacting CFP- and YFP-tagged proteins or a positive CFP-YFP FRET probe. Interestingly, we noticed a striking difference in results obtained with living cells and those generated with fixed cells after mounting them on coverslips with a commercial mounting fluid . While tNext, we wanted to test whether the particular cellular environment influences the observed differences in bleaching kinetics. To that end, we repeated the experiments in a different cell type, which resulted in a very similar bleaching behavior . This inDA/FD, where FDA is donor fluorescence in presence of acceptor (before acceptor bleaching) and FD is donor fluorescence in absence of a functional acceptor  or Thermo Scientific (Ultramount). CFP- and YFP-constructs (pECFP-C1 and p-EYFP-N1), as well as the monomeric DsRed (mDsRed) were from Clontech Inc .-AAA AAG ATC TGC TAG CGT CGA CAC CAT GGG TCG GGA TCT GTA C containing a Bgl II site and the reverse primer 5′-AAA AAA GCT TGC GGC CGC ATT TGT ATA GTT CAT CCA TGC C containing a Hind III site. The PCR product was cut with Bgl II and Hind III and cloned into a pEGFP-C1 vector (Clontech Inc.) that had been cut with the same restriction sites, resulting in a pEGFP-YFP fusion construct. Subsequently, the EGFP part was cut out with Age I and Bgl II and replaced by ECFP . This resulted in a CFP-YFP fusion construct with a 27-amino acid spacer in between the CFP and the YFP-tag.For generating the CFP-YFP fusion construct, we first cloned a yellow fluorescent protein coding sequence into a pEGFP-C1 vector (Clontech Inc.). This was done by PCR-amplification of the YFP , and the monomeric teal fluorescent protein, mTFP1, was obtained from Allele Biotechnology Inc . The monomeric Orange fluorescent protein construct (mOrange) was from Roger Tsien's group and the HcRed1 construct from Karel Drbal. 293 cells (HEK-293) and the prostate cancer cell line DU145 were from the ATCC cell culture collection.The alternative cyan fluorescent protein Cerulean was obtained from the Addgene plasmid repository . Non-linear curve fitting was performed with GraphPad Prism 5.0™ using a single exponential decay equation.This study was conducted according to the principles expressed in the Declaration of Helsinki.Figure S1Bleaching kinetics of CFP and YFP is not significantly altered by the fixation. 293 cells were transfected with CFP (A) or YFP (B) and bleaching was recorded by conventional epifluorescence microscopy in live cells in medium; in 4% paraformaldehyde-fixed cells in PBS; in fixed cells mounted with Dako Fluorescent Mounting Fluid and in fixed cells mounted in PBS/glycerol  as indicated.(TIF)Click here for additional data file.Figure S2Bleaching characteristics is very similar in different cellular environments. 293 or DU145 cells were transfected with CFP (upper panel) or teal fluorescent protein  as indicated and bleaching was recorded for live cells or for cells fixed with 4% paraformaldehyde in PBS and mounted in PBS/glycerol 1∶7 (PBS-Gly); in Dako fluorescent mounting fluid or in Thermofisher Ultramount.(TIF)Click here for additional data file.


In addition, the ZnO-NF structures demonstrate vast surface area that allow high enzyme loading which results provided a higher sensitivity. The proposed ZnO-NF array-based sensor exhibited a high sensitivity of ∼66 mV/ decade in test electrolyte solutions of uric acid, with fast response time. The sensor response was unaffected by normal concentrations of common interferents such as ascorbic acid, glucose, and urea.In the present work zinc oxide nanoflakes (ZnO-NF) structures with a wall thickness around 50 to 100 nm were synthesized on a gold coated glass substrate using a low temperature hydrothermal method. The enzyme uricase was electrostatically immobilized in conjunction with Nafion membrane on the surface of well oriented ZnO-NFs, resulting in a sensitive, selective, stable and reproducible uric acid sensor. The electrochemical response of the ZnO-NF-based sensor  The production of excess uric acid may precipitate in the kidney and the lower extremities. One problem caused by the metabolism of the uric acid is gout . Severaletc. Due to their unique advantages in combination with immobilized enzymes, these ZnO nanosensors offer some significant advantages owing to their small size and high surface area to volume ratios allowing larger signals, better catalysis and the more rapid movement of analyte through sensors, thus showing higher sensitivity and a lower limit of detection (LOD) as compared to those prepared from bulk ZnO devices. ZnO nanoflake (ZnO-NF) structures possess lots of interesting unique properties such as porous structures and large surface areas and there have been reports on the use of ZnO-NF structures as sensors with improved performance and higher sensitivity compared to ZnO nanorods/nanowires  and 0.025 M hexamethylenetetramine [C6H12N4] that was kept in preheated an oven at 90 °C for 2–4 hours. After the growth process, the fabricated ZnO-NFs were cleaned in de-ionized water and dried at room temperature. A typical AFM image of ZnO-NPs arrays grown on the gold coated plastic electrode using this procedure are shown in In the second step, a small part of the gold coated glass was covered with plastic in order to make a contact area, and then the 10 nm of Aluminum (Al) thin film was deposited on the remaining selective portion of the gold coated substrate. To fabricate the ZnO-NFs on the prepared electrode, a low temperature hydrothermal approach was adopted . First tThe morphological and structural studies were performed by using Scanning Electron Microscopy (SEM). The SEM images of the ZnO-NFs with as fabricated, after enzymes immobilization and after measurements are shown in It can be clearly seen that the wall thickness of the grown ZnO-NFs are 50–100 nm in diameter with uniform density. These ZnO-NFs were well oriented on the surface of the electrodes. The morphological and structural characteristics of the fabricated ZnO-NFs arrays can be controlled by adjusting the growth parameters.2.3.To immobilize the uricase enzyme on the fabricated ZnO-NFs, first we have prepared an uricase solution in 10 mM PBS pH 7.4. Uricase was electrostatically immobilized by dipping the ZnO-NF-based electrode into the enzyme solution for 15 minutes at room temperature and then for drying, it was left in air for 60 min. After drying, Nafion solution  was applied onto the electrode surface to prevent possible enzyme leakage and eliminate foreign interferences. All enzyme electrodes were stored in dry conditions at 4 °C when not in use. After completing all these steps, the prepared sensors were checked potentiometrically in uric acid solutions with an Ag/AgCl reference electrode purchased from Metrohm. A pH meter  was used to measure the potentiometric output voltage of the ZnO-NFs based sensors presented here. For the time response measurements, a model 363A potentiostat/galvanostat  was used. Atomic force microscopy images were acquired using a Dimension 3100 Scanning Probe Microscope  in tapping mode with Si cantilevers.3.3.1.versus an Ag/AgCl reference electrode was measured at room temperature (23 ± 2) °C. The sensor as fabricated is sensitive to the concentration changes of uric acid in PBS. An electrochemical response from ZnO-NFs sensor in the 100 μM uric acid solution was observed around 200 mV. The response stayed around 200 mV regardless of the analyte solution volume. During all experiments the ZnO-NFs sensor followed the Nernst’s expression:The electrochemical measurements were carried out using a two-electrode configuration consisting of the ZnO-NF-based sensor as the working electrode and an Ag/AgCl one as a reference electrode. The electrochemical response of the ZnO-NFs sensor It is very important to note that ZnO-NFs are relatively stable around a neutral pH of 7.4 and this gives these sensors much more bio-compatibility in biological fluids and species since the pH of most biological fluids is around 7.4. The sensing mechanism of most electrochemical uric acid sensors is based on an enzymatic reaction catalyzed by uricase as described in When uric acid is oxidized in the presence of uricase it is turned into allantoin along with carbon dioxide and hydrogen peroxide. Due to the presence of water (H-OH), it is a high probability that allantoin will accept a proton from (H-OH) converting it to allantoinium ion, which in turn will interact with the ZnO-NFs and produce a potential change at the electrode. As the concentration of ions changes in surrounding the ZnO-NFs and the electrode potential will change . The potvs. the logarithmic concentrations of the uric acid with sensitivity around 66 mV/decade as shown in The tested sensor configuration showed large dynamic ranges with an output response (emf) that was linear 3.2.etc. The reproducibility is an important characteristic for the performance evaluation of a sensor. To evaluate reproducibility and long term stability of the proposed ZnO-NFs based sensors, we independently fabricated six sensor electrodes under the same conditions; the relative standard deviation of the fabricated sensor electrodes in standard uric acid solutions was less than 5%. The sensor to sensor reproducibility in 100 μM uric acid solution is shown in To evaluate the performances of the proposed sensor, we have checked the parameters like reproducibility, measuring range, detection limit, response time and selectivity, The measuring range of the proposed sensor was obtained from the linear part of the calibration graph as shown in 3.3.Selectivity is the most important characteristic which describes the specificity towards the target ion in the presence of other ions (interfering ions). There are different methods to determine selectivity of the potentiometric sensors . These mThis was repeated several times on new, independently prepared sensors and continued to show negligible signal response to interferences. In practical measurements, however these changes in sensor response can be neglected.4.In conclusion, we have successfully demonstrated a simple fabrication procedure for a highly sensitive electrochemical uric acid sensor based on ZnO nano-flake-based structures. The proposed electrochemical nanosensor demonstrates immense surface area to volume ratios which provide a suitable microenvironment for enzyme loading because of its porosity that allows for very good sensitivity as compared to other ZnO nanostructures as shown in The uricase sensor retained its enzymatic activity due to strong electrostatic interaction between zinc oxide and uricase. Moreover, the developed ZnO-nanoflake-based nanosensor showed excellent performance regarding sensitivity, stability, selectivity, reproducibility and resistance to interference when the sensor was exposed to uric acid test solutions. These results revealed that electrochemical sensors based on ZnO nanoflakes have the potential to perform measurements biologically relevant to on-spot clinical diagnosis. They are also convenient to assemble into portable chip based sensing devices suitable for unskilled users.


The risk of acquisition of antibiotic resistant-bacteria during or shortly after antibiotic therapy is still unclear and it is often confounded by scarce data on antibiotic usage.Staphylococcus aureus and extended spectrum beta-lactamase-producing Enterobacteriaceae in hospitalised patients, after starting antibiotic therapy.Primary objective of the study is to compare rates of acquisition of methicillin-resistant Staphylococcus aureus and extended spectrum beta-lactamase-producing Enterobacteriaceae will be obtained at hospital admission, discharge, at antibiotic start  and at the following intervals: day 3 (t1), 7 (t2), 15 (t3), and 30 (t4). Two nested case-control studies will be performed. The objective of the first study will be to define individual level of risk related to specific antibiotics. Patients acquiring methicillin-resistant Staphylococcus aureus and extended spectrum beta-lactamase-producing Enterobacteriaceae (cases) will be compared with patients not acquiring antibiotic-resistant strains after starting antibiotic therapy . To define the impact of antibiotics on new acquisition of target antibiotic-resistant bacteria, a second nested case-control study will be done (ratio 1:4). Control group will be selected among patients not receiving antibiotics, admitted in the same ward on the day of the corresponding case, with negative cultures at admission. Epidemiological, clinical and microbiological data will be prospective collected.The study, running in three European hospitals, is a multicenter, prospective, longitudinal, observational cohort study funded from the European Community's Seventh Framework Programme [FP7/2007-2013] within the project 'Impact of Specific Antibiotic Therapies on the prevalence of hUman host ResistaNt bacteria' (acronym SATURN). Nasal and rectal screening for methicillin-resistant The rationale of this study is to better understand the impact of antibiotic use on acquisition, selection and transmission of antimicrobial resistant-bacteria in European hospitals.NCT01208519.ClinicalTrials.gov  Staphylococcus aureus (MRSA) was increased by 1.8-fold in patients who had taken antibiotics. Antibiotic exposure was determined in the 126 days preceding MRSA isolation [Despite many efforts, the control of nosocomial antibiotic-resistant infections is still a permanent and unresolved issue in healthcare institutions worldwide facing increased morbidity, mortality and hospital costs -3. Selecsolation . Such risolation .The likelihood of acquisition of nosocomial ARB during or shortly after therapy with different antibiotic agents is still unclear and is often confounded by scarce data on antibiotic usage at the individual patient level. Study design, selection bias, definition of control groups and failure to control for confounding variables might account for conflicting results . No prosWe designed a 2-year observational cohort study (WorkPackage 4 of the EU-funded SATURN project) in order to clarify and determine the role of antibiotic therapy in the acquisition of ARB in hospitalized patients. The study will run in three European hospitals, ranging from 700 to 3,500-bed capacity: Università Cattolica Sacro Cuore "Policlinico A. Gemelli" , Institute of Infectious Diseases "Matei Bals" , and Clinical Center of Serbia . Overall, 6 medical and 6 surgical wards will be included. All centers obtained ethical approval from the local institution review board.per os and/or intravenously will be sampled at antibiotic start  and at the following intervals: day 3 (t1), 7 (t2), 15 (t3), 30 (t4)  will be excluded from follow-up cultures and analysis. Patients will be followed during the hospitalisation and afterwards for a total of 30-day from the inclusion in the study. Screening will be performed in outpatient clinics after patients' discharge from the hospital, if possible.All adult patients > 18 years) starting intravenous and/or oral antibiotic treatments during hospitalisation will be included in the study. Written informed consent will be obtained by every patient before study inclusion. Exclusion criteria will be pregnancy and recent nose surgery. Patients starting antibiotic therapy 8 years sThe primary outcome will be the rate of acquisition of target antibiotic bacteria by 1,000 antibiotic-days in hospitalised patients. The rate will be also defined according to antibiotic class and single drug. Patients will be stratified by class of risk  and rate of acquisition will be defined. The secondary outcome will be the rate of nosocomial infections by 1,000 days of hospitalisation in newly colonised patients after undergoing antibiotic therapy.The following variables will be identified in cases and controls: age; gender; reason for admission; inter and intra-wards transfers; admission from home or other acute or long-term care institutions; previous hospitalisation; surgical procedures; Intensive Care Unit (ICU) stay (previous 12 months); residency in long-term care facility; transfer form another hospital; nursing and/or medical domiciliary assistance; urinary catheter; central venous catheter ; underlying conditions ; room sharing with a ESBL or MRSA patient; previous MRSA or ESBL carrier; consciousness at admission; antibiotic therapy (previous 30 days).Antibiotic therapy will be reported as for daily dosage, frequency of dosage(s), route of administration, type of antibiotic(s), reason for starting and stopping, duration of therapy. At every clinical follow-up the following variables will be also detected: presence of urinary catheters; CVC; surgery; room sharing with a ESBL or MRSA positive patient; MRSA or ESBL-producing gram-negative infections ; albumin, creatinine and blood urea nitrogen levels.S. aureus will be confirmed through the use of a classical DNase assay or automated phenotypic methods  [Dedicated personnel will be trained in performing nasal samples. Both nostrils will be sampled. Specimens obtained with a dry swab  will be incubated overnight in 2 ml of Mueller-Hinton broth supplemented with colistin 32 μg/mL and 2.5% NaCl (CS) at 35°C in room air. Then 10 μl of broth sample will be plated onto chromogenic medium   and willton, NC) . OxacillKlebsiella species, Escherichia coli and Proteus species will be detected by confirmatory double disk diffusion test (DDT). Ceftazidime (30 μg), cefotaxime (30 μg), or cefepime (30 μg) discs will be placed on Mueller-Hinton agar plates, 20 mm apart (centre to centre) from a clavulanate disc (clavulanic acid 10 μg). The test will be considered as positive when a decreased susceptibility to cefotaxime is combined with a clear-cut enhancement of the inhibition zone of cefotaxime in front of the clavulanate-containing disk, often resulting in a characteristic shape-zone referred to as 'champagne-cork' or 'keyhole' [Rectal specimens will be obtained with a dry swab  and will be inoculated onto plates of ESBL chromogenic medium . After the direct plating, 1 ml of trypticase soy broth (TSB) with 0.5% NaCl will be added to the transport tube and the selective broth will be incubated overnight at 37°C in air. The selective medium will be plated on the ESBL chromogenic medium following the same procedure as for direct plating. Colonies will be identified according to manufacturers' recommendations and subcultured for confirmatory testing. The ESBL phenotype of identified colonies of keyhole' ,13. Plat0) will be included in the longitudinal analysis. The length of exposure to antibiotic therapy for patients newly colonised by ARB will be defined by the number of days between the inclusion in the study and the date of the first positive sample. The length of exposure to antibiotic therapy for patients not colonised will be defined by the number of days between the inclusion in the study and the date of antibiotic discontinuation. The colonisation pressure will be calculated for each center and defined as the average point of prevalence for antibiotic-resistant bacteria for all days until acquisition or discharge. Time-to-event analysis (in days) between the inclusion in the study and the acquisition of colonisation will be done using Kaplan-Meier curves and Cox proportional hazard regression modeling in which the number of days until acquisition will be the dependent variable. Two nested case-control studies will be performed:The assessable population for each target organism will include all treated patients from whom baseline screening and at least one follow-up screening will be available. The incidence of colonisation will be defined by the number of new cases of colonisation for 1,000 days of antibiotic therapy. Only patients with a negative baseline . The control group will be selected among patients not receiving antibiotics, admitted in the same ward on the day of the corresponding case, with negative cultures at hospital admission. Matching criteria will include: age (± 5 years), sex, and total length of hospitalisation.To define the impact of antibiotics on new acquisition of MRSA and ESBL-producing Enterobacteriaceae will be compared with patients not acquiring ARB after starting antibiotic therapy (ratio 1:4). Previously known risk factors or clinically relevant significant variables from the univariate analysis will be considered for inclusion in multivariate logistic regression analysis. The Hosmer and Lemeshow goodness-of-fit test will be used to assess model fit.To define individual level of risk related to specific antibiotics, patients acquiring MRSA and ESBL-producing Enterobacteriaceae: 48/95, 121/201, and 88/147 for UCSC, IDMB, CCS, respectively.Since the rate of acquisition of ESBL-producing gram-negative bacteria in patients receiving antibiotic therapy has not been established in prospective cohort studies, prevalence proportion was derived from retrospective case-control studies and defined as 5%. Sample size calculation for the rate of new acquisition of MRSA has been derived by a previous study where the acquisition rate, after starting antibiotic therapy, was 3% . To be ahttp://www.saturn-project.eu provides the information and details concerning the different WPs and the entire project.SATURN project is a European multicentre study that aims to provide an in-depth knowledge of ARB acquisition through the analysis of antibiotic classes, duration of treatment and dosage in the nosocomial setting by a multidisciplinary approach. The SATURN project website Several outcomes will be obtained at the end of the study : the rate of acquisition of target ARB by 1,000 antibiotic-days according to different classes of antibiotics, duration of therapy, and antibiotic combinations (monotherapy versus combinations); risk factors associated with new colonisation by target ARB; and risks for nosocomial infections due to target ARB after a cycle of antibiotic therapy adjusted by length of hospitalisation and ward colonisation pressure.The WP4 study is currently recruiting patients in all clinical centers. The accomplishment of the study will be pursued thanks to the great strive of all clinicians, nurses and microbiology staff in every study step. An advertising campaign  was performed in the selected wards in order to thrill and sensitise the recruited staff.The results of the study should lead to the design of specific infection control measures for preventing spread of ARB in hospitalised non-ICU patients undergoing antibiotic therapy. A potential benefit for selective screening in high-risk patients during antibiotic therapy or shorter duration of antibiotics in such patients might be hypothesised. Readily available colonisation information could impact empiric therapy for nosocomial infections or, for example, the potential need of mupirocin prophylaxis for patients colonised by MRSA during hospitalisation. Assessment of the usefulness of the recommendations derived from the results of the SATURN study should be of valuable help for those in charge of infection control, for hospital administrators, and for those managing budgets of large healthcare organisations.We do expect that SATURN project results will have a direct impact on clinical practice, improving the appropriateness of antibiotic prescribing policy, the prevention of antibiotic-resistant infections and the heightener of patients' quality care.The authors declare that they have no competing interests.GDA participated in the elaboration of clinical protocol and drafted the manuscript. GR and SV participated in the design of the study. RC reviewed the manuscript. SMK, HG, and JS elaborated the microbiological protocol. ET conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/74/prepub


Erythropoietin (EPO) has tissue-protective properties, but it increases the risk of thromboembolism by raising the haemoglobin concentration. New generation of EPO derivatives is tissue protective without the haematopoietic side effects. Preclinical studies have demonstrated their effectiveness and safety. This paper summarizes the development in EPO derivatives with emphasis on their potential use in critical limb ischaemia. Critical limb ischaemia (CLI) causes considerable morbidity and mortality. Surgical and endovascular revascularization are the main treatment options. One-fourth of patients presenting with CLI undergo a major amputation, primarily because they are not suitable for revascularization . Pharmac165) [More recently, the possibility of using recombinant formulations of growth factors to augment the development of collaterals and new capillaries from pre-existing blood vessels has been explored. This is otherwise known as therapeutic angiogenesis . Promine165) . Other t165) . However165) .Tissue protection may be an alternative therapeutic strategy for CLI where reducing tissue damage and maintaining tissue viability may improve outcome of current treatments and contribute to limb salvage. It has been shown previously that limb ischaemia causes apoptosis of skeletal muscle . TherefoErythropoietin (EPO) has tissue protective properties that have been described extensively , 21. We Erythropoietin (EPO) belongs to the class-I cytokine family. It is a glycoprotein hormone with a molecular mass of 30.4 kDa . The proβ-chain of IL-3/IL-5/GM-CSF receptor  belongs to the same super-family. The molecular weight of β-common receptor (βcR) is approximately 130 kDa [2) to bring about the haemopoietic action. In contrast, the EPO molecule binding to two βcR subunits sandwiching two EPOR subunits mediates the tissue protective function [βcR and EPOR) induces a conformational change in the receptor, activating a downstream signalling pathway that leads to the transcription of new proteins that determine the fate of the cell [Human EPO receptor (EPOR) is a cell surface protein containing 508 amino acids and has a molecular weight of 55 KDa . It belo 130 kDa . The EPOfunction . The fouthe cell .in vitro and in vivo. Clinical trials have also confirmed its beneficial effects in stroke and myocardial infarction [Endogenous EPO is essential for the maturation and survival of erythrocytes and thus maintaining the normal haemoglobin concentration. In chronic renal failure, the kidneys cease to make EPO, and commercially available preparations of recombinant human EPO (rHuEPO) are mainly used to treat anaemia secondary to chronic renal failure . In addifarction , 31.However, the ability of rHuEPO to bond with EPOR in extrahaematopoietic tissues including vascular endothelium is about 50 times less than that in the erythroid cells . Hence, Due to the potential side effects, most research on the tissue protective effects of EPO in CLI and other tissues has been limited to preclinical models. However, the latest nonhaemopoietic EPO derivatives may overcome the unnecessary side effects and permit the translation of preclinical results into clinical trials .  CEPO is formed by the chemical modification of the amino acid lysine to homocitrulline. It changes the receptor-binding capability of the EPO molecule .Asialo EPO is formed by decreasing the sialic acid content of rHuEPO by a technique known as the total enzymatic desialylation. Remarkably, the molecule retains its tissue protective property but loses the haemopoietic ability. This also results in the reduction of the half-life of the Asialo EPO to minutes .2. However, helix B that is hydrophilic points away from the receptor and has wide-ranging tissue-protective properties. Isolating the helix B which is devoid of the primary sequence resulted in a low molecular weight peptide. The spontaneous cyclization of Helix B surface peptide (HBSP) gives rise to pyroglutamate HBSP (pHBSP), which is also known as ARA 290. The effective dose of ARA 290 peptide that showed evidence of tissue protection was comparable on a molar basis to those studied for EPO and was higher than that required for EPO-mediated erythropoiesis. For example, in the renal ischaemia model of mice, 0.08 nmol/kg of bw  was ineffective, whereas a 10-fold higher dose elicited strong tissue protection [ The EPO molecule, a glycoprotein that contains 165 amino acids is folded in such a way that the hydrophobic areas-helixes A, C, and D face away from the surface and are embedded in the extracellular domains of (EPOR)otection . The stuin vivo.Receptor-initiated cell signaling by EPO and its derivatives occurs via multiple molecular cascades, which differ in importance depending on the specific tissue or cell type, as well as the type of injury . As in eIt is now known that EPO receptors are present in human skeletal muscle and their expression is elevated in CLI. It has also been shown that a nonhaematopoietic derivative of EPO (ARA 290) decreases inflammation and apoptosis of ischaemic myotubes and may thus be used to selectively enhance the tissue-protective activity of EPO whilst avoiding the haematopoietic side effects in CLI.in vivo studies have treated animals immediately following ischaemic injury [The timing of administering EPO derivatives depends on their half-life. CEPO has a half-life of 4–6 hours while Asialo-EPO and ARA 290 have a half-life of about 2 minutes. Most c injury , 23. In EPO receptors are present in human skeletal muscle and their expression is elevated in CLI. Nonhaematopoietic derivatives of EPO decrease inflammation and apoptosis of ischaemic myotubes whilst avoiding haematopoietic side effects. They may thus reduce tissue damage and provide a unique therapeutic alternative for CLI.


Endothelin-1 participates in the pathophysiology of heart failure. The reasons for the lack of beneficial effect of endothelin antagonists in heart failure patients remain however speculative. The anti-apoptotic properties of ET-1 on cardiomyocytes could be a reasonable explanation. We therefore hypothesized that blocking the pro-apoptotic TNF-α pathway using pentoxifylline could prevent the deleterious effect of the lack of ET-1 in a model for heart failure.We performed transaortic constriction (TAC) in vascular endothelial cells specific ET-1 deficient (VEETKO) and wild type (WT) mice (n = 5–9) and treated them with pentoxifylline for twelve weeks.TAC induced a cardiac hypertrophy in VEETKO and WT mice but a reduction of fractional shortening could be detected by echocardiography in VEETKO mice only. Cardiomyocyte diameter was significantly increased by TAC in VEETKO mice only. Pentoxifylline treatment prevented cardiac hypertrophy and reduction of fractional shortening in VEETKO mice but decreased fractional shortening in WT mice. Collagen deposition and number of apoptotic cells remained stable between the groups as did TNF-α, caspase-3 and caspase-8 messenger RNA expression levels. TAC surgery enhanced ANP, BNP and bcl2 expression. Pentoxifylline treatment reduced expression levels of BNP, bcl2 and bax.Lack of endothelial ET-1 worsened the impact of TAC-induced pressure overload on cardiac function, indicating the crucial role of ET-1 for normal cardiac function under stress. Moreover, we put in light a TNF-α-independent beneficial effect of pentoxifylline in the VEETKO mice suggesting a therapeutic potential for pentoxifylline in a subpopulation of heart failure patients at higher risk. A and ETBA receptors.Vascular endothelial cells are the main source of the vasoactive peptide endothelin-1 (ET-1) but cardiomyocytes, endocardial cells, and cardiofibroblasts produce ET-1 as well as its both receptors ETA as well as dual ETA/ETB antagonists demonstrated beneficial effects in several animal models of acute and chronic heart failure Treatment with selective ETDespite its adverse effect on the heart, overexpression of ET-1 in mice can prevent diastolic dysfunction in eNOS deficient mice We performed transaortic constriction in ET-1 deficient mice to further examine the impact of ET-1 on the heart subjected to increased afterload. Treatment with pentoxifylline (PTX) was aimed to reduce TNF-α synthesis and by doing so to demonstrate the influence of ET-1 on the TNF-α signalling.flox/flox, Cre recombinase positive: VEETKO) and their wild type littermates  We used non-ovariectomised female mice with vascular endothelium specific ET-1 deficiency .Blood pressure and heart rate were measured in awake mice by the tail-cuff method  between 9 a.m. and noon. Mice were trained to the procedure on the first day and measurements were recorded on the second day. An average of ten consecutive measurements was used.Left ventricular end-diastolic (EDD) and end-systolic dimension (ESD) were measured by echocardiography . Two-dimensional parasternal short-axis images were obtained, and targeted M-mode tracings at the level of the papillary muscles were recorded. Fractional shortening (FS) was calculated using the formula (EDD-ESD)/EDDx100. Examinations were performed within ten minutes of light isoflurane anaesthesia.Paraffin embedded heart were cut into 3 µm and 1 µm sections and submitted to Sirius-red and Hematoxylin-eosin staining. Interstitial fibrosis and myocyte diameter was quantified using a computer-aided image analysis system.T method using actin expression as reference.Total RNA was extracted from cardiac tissue using Trizol reagent  following manufacturer's protocol. Complementary DNA was obtained by reverse transcription using oligo-dT primers and the ReverTra Ace kit . Real time PCR was performed using the Thunderbird SYBR qPCR mix  on a Rotor-Gene Q thermocycler . The primers for the PCR reaction are presented in the Values were presented as mean±sem. Differences between groups were analysed using a Student's T-test for independent samples on the software SPSS. A p value less than 0.05 indicated a significant difference.Neither genotype nor TAC surgery had any influence on systolic and diastolic blood pressure. In sham-operated WT mice but not in VEETKO, PTX treatment increased SBP. SBP was thus lower in VEETKO mice compared to WT in sham-operated mice receiving PTX. In mice with TAC, SBP and DBP remained stable throughout the experiment regardless of treatment received by the mice .In sham-operated mice, neither genotype nor PTX treatment influenced heart weight to body weight ratio. TAC induced cardiac hypertrophy in WT and VEETKO mice in a similar fashion . PTX treTaken together, TAC reduced cardiac function in VEETKO mice but not in WT and this decrease could be prevented by a PTX treatment.No difference has been observed between the groups regarding the level of collagen deposition defined as Sirius red positive signal. The number of apoptotic cells counted after TUNEL assay was low and similar between the groups .In all groups , the gene expression of ET-1 was lower in VEETKO compared to WT mice. In our setting, the difference was statistically significant only in the control mice after TAC surgery . In thesThe main findings of this study are that a normal expression level of ET-1 is required to maintain cardiac function after pressure overload caused by transaortic constriction and that the adverse impact of a reduced expression of ET-1 can be prevented by a pentoxifylline treatment.A receptor in cardiomyocytes do not present a modified cardiac hypertrophic response to pharmacological stress ET-1 is known to have strong hypertrophic effects on the heart ET-1 has been held responsible for the pathophysiology of heart failure We have thus hypothesized that the reduction of cardiac function in VEETKO mice was due to the loss of anti-apoptotic properties of ET-1. In our setting, however, we could not detect changes neither in apoptotic cells number nor in caspase expression levels. This represents a major limitation of our study for which several parameters might be responsible. Apoptosis is a late event in the pathophysiology of TAC induced heart failure: Fliegner et al. (using the same protocol as ours) did not observed apoptosis nine weeks after TAC Notably, the effects observed were independent of systemic blood pressure changes. Even though previous investigations of the VEETKO mice have revealed a blood pressure lower than in the WT, we were unable to confirm this. The endothelin system is known to participate in the sex-related differences in blood pressure control Importantly, the deleterious effect of the absence of vascular ET-1 on myocardial hypertrophy and function could be prevented by PTX: fractional shortening was increased, heart weight was reduced and myocyte diameter  as well. Except from a small increase of blood pressure in the sham WT mice, for which the reasons are unknown, the effects of PTX were blood pressure independent. While some studies did not reveal improvement of cardiac structure and function in heart failure patient with PTX treatment The changes in bax and bcl2 expression must be interpreted carefully because there were independent of the genotypes and thus did not correlate with the changes in cardiac function. The PTX-induced increase of the bax/bcl2 ratio in TAC-VEETKO mice was in contradiction with the improved cardiac function. On the other hand, PTX restored this parameter to the level of the sham-operated mice, which can be seen as a beneficial effect. Beside its anti-apoptotic effects, PTX has been shown to induce apoptosis in certain conditions Finally, PTX treatment in the TAC mice induced a reduction of the expression of cardiac BNP as well, which is in line with a previous report We thus conclude that PTX prevents TAC-induced cardiac dysfunction and hypertrophy in mice with reduced ET-1 expression.In contrast to its positive impact in mice with reduced endogenous endothelin-1, PTX had a deleterious effect on cardiac function in the mice with normal level of ET-1. A clinical study have shown that PTX is effective only in a sub-population of heart failure patients, which can be identified by an elevated serum concentration of inflammatory markers Moreover, a reason for the opposite effects of PTX on cardiac function in VEETKO and WT mice may lie on the complex pharmacology of PTX: PTX is metabolized in several active compounds. In WT mice, TAC induced solely cardiac hypertrophy while an additional reduction of FS was observed in VEETKO mice, which can be considered as a worsening of the condition. The pharmacokinetics of PTX and particularly the relative concentration of its metabolites is not the same whether given to healthy humans, patients with moderate or severe heart failure Firstly, the present study confirms the essential role of ET-1 for normal cardiac function after chronic overload and participates in explaining the negative results of endothelin antagonists in heart failure trials. Secondly, our results indicate that PTX prevents cardiac failure in mice with reduced ET-1 expression. In the absence of large scale clinical trial of PTX on heart failure, it is still difficult to conclude on its therapeutic potential. Thirdly, we have shown that PTX may have opposite effects on cardiac function depending on the pathophysiological situation. Further studies should be therefore carefully designed.


Radionuclides in the environment are a major human and environmental health concern. Like the Chernobyl disaster of 1986, the Fukushima Daiichi nuclear disaster in 2011 is once again causing damage to the environment: a large quantity of radioactive waste is being generated and dumped into the environment, and if the general population is exposed to it, may cause serious life-threatening disorders. Bioremediation has been viewed as the ecologically responsible alternative to environmentally destructive physical remediation. Microorganisms carry endogenous genetic, biochemical and physiological properties that make them ideal agents for pollutant remediation in soil and groundwater. Attempts have been made to develop native or genetically engineered (GE) microbes for the remediation of environmental contaminants including radionuclides. Microorganism-mediated bioremediation can affect the solubility, bioavailability and mobility of radionuclides. Therefore, we aim to unveil the microbial-mediated mechanisms for biotransformation of radionuclides under various environmental conditions as developing strategies for waste management of radionuclides. A discussion follows of ‘-omics’-integrated genomics and proteomics technologies, which can be used to trace the genes and proteins of interest in a given microorganism towards a cell-free bioremediation strategy. However, the typical radionuclides produced through nuclear reactors via the splitting of elemental atoms are thallium-201 (201Tl), iridium-192 (192Ir), caesium-137 (137Cs) and strontium-90 (90Sr), which take a significantly long time to decay 2] can alter the solubility, transport properties and toxicity of radionuclides  reduction. The studies showed that the mutant was unable to synthesize c-type cytochrome, so it did an in vitro reduction of U(VI). The purified tetrahaem cytochrome c3 protein from Desulfovibrio vulgaris was observed to reduce U(VI) in vitro using U(VI) reductase in combination with hydrogenase as its physiological electron donor  confirmed the role of cytochrome c3 in hydrogen-dependent U(VI) reduction. Similarly, Lloyd and colleagues (c7 of the Fe(III)-reducing bacterium Geobacter sulfurreducens that may also play a role in U(VI) reduction in vitro.The oxidized forms of radionuclides are highly soluble in aqueous medium, which makes them mobile in groundwater, whereas the reduced species are highly insoluble and often precipitate from the solution. A direct enzymatic reduction of soluble U(VI) to insoluble species is presented in Fig.  Lovley, . In vivolleagues  identifi99Tc, another long-lived radionuclide  that mostly occurs in nuclear wastes, has attracted considerable interest  was quantified using a phosphorimager technique. In another study, X-ray absorption spectroscopy showed Tc(IV) as the final insoluble oxidation state after Tc(VII) was reduced by G. sulfurreducens and S. putrefaciens  with the reduced radionuclide precipitated within the cell . However, according to the model, the formate dehydrogenase component is required only if formate is supplied as an electron donor for Tc(VII) reduction in place of hydrogen. The model was authenticated by a mutant strain unable to synthesize all three components of hydrogenase to reduce Tc(VII) when either hydrogen or formate was supplied as an electron donor .Further, the biochemical basis of Tc(VII) reduction has been studied in et al., Desulfovibrio desulfuricans and related organisms were observed to utilize formate as an efficient electron donor for Tc(VII) reduction  against a high concentration of nitrate ions, which is frequently found in nuclear waste  reduction . The product can be further reduced chemically to yield separate or multi-component insoluble species  produced unidentified compounds such as organic acids, siderophores and extracellular metabolites capable of dissolving and mobilizing radionuclides into the soil. These compounds may also help to transport radionuclides within the cells  or sulfur S(IV) in the form of sulfate. The iron Fe(III) can be bioreduced into Fe(II) and S(VI) into S(II)  using the siderophores enterobactin, desferrioxamin, carboxylate amino polycarboxylate and catecholate ligands. The report conclusively showed that enterobactin siderophores are extremely effective in solubilizing actinide oxides of plutonium, among the tested other chelators.Pu(IV), Th(IV), U(VI) and Fe(III) have chemical and biochemical similarities because of the ubiquity of siderophore-producing microbes. The iron-sequestering agents produced by such microorganisms are crucial to increase the solubility and bioavailability of radionuclides. Premuzic and colleagues  reportedlleagues  solubiliSeveral microorganisms produced extracellular complexes in the presence of Pu and Th that increased the concentration of Pu and Th in soil-column elutes compared with controls. The increased mobility of Pu and Th in soil resulted from the formation of neutral and negatively charged Pu and Th complexes. In the presence of known microbial metabolites such as synthetic ligand [ethylenediamine tetraacetic acid (EDTA)] and citric acid, Pu(VI) and Th(IV) were reduced to Pu(IV) and Th(IV) respectively before complex formation, suggesting that the latter valence state would be the dominant one associated with the organic complexes in soil  and concluded that metal was most likely to bind to phosphoryl groups in the core-lipid ‘A’ of LPS, and the negatively charged side-chains affected cell hydrophobicity in Gram-negative bacteria. Similarly, Khani and colleagues  with phosphate groups in the LPS. This interaction provides nucleation sites for mineral formation and protects an outer membrane phosphatase  ; however, the resistance of these organisms to non-reducible heavy metals could possibly limit their in situ activity. Martinez and colleagues (in situ immobilization of U(VI)  and nitrate had been reduced to 5 μm and 0.5 μm respectively. After 2 years of preconditioning, the level of U(VI) was reduced to 0.126 μm – below the EPA's recommended level. These studies suggested that alternation of geochemical and hydrological parameters can effectively biostimulate radionuclide-reducing bacteria in situ. Remediation of high U(VI) concentrations with ethanol-biostimulated biofilm is an alternative approach that can effectively reduce 87% of U(VI)  at low pH and high nitric acid concentrations  have been used to complex with radionuclides. These metal chelates undergo aerobic or anaerobic biodegradation and cause the precipitation of released ions as water-insoluble hydroxides or oxides, thus retarding their migration into groundwater. Citrate has been found useful as a chelating agent in decontamination because it forms highly soluble metal–citrate complexes that can be degraded by microorganisms, resulting in subsequent re-precipitation of metals. Stable complexes such as bidentate, tridentate and polynuclear complexes can be formed with citrate and radionuclides. Francis , nitrilotriacetic acid (NTA) and  Francis  reportedD. desulfuricans and facultative iron-reducing Schwanella alga reduced U(VI) complexes with citrate to U(IV) anaerobically, but little uranium was precipitated  metabolized glucose but not citrate and reduced U(VI)–citrate into U(IV)–citrate only in the presence of glucose. Another Clostridium sphenoides (ATCC 19403) metabolized bidentate Fe(III)–citrate complex. This bacterium reduced Fe(III) to Fe(II) and concomitantly metabolized citric acid. In contrast, U(VI)–citrate was reduced to U(IV)–citrate by the bacterium in the presence of electron donor glucose or uncomplex citric acid. Therefore, these results indicate that the complex of uranium with citric acid is readily available for organisms as an electron acceptor, despite their incapability to metabolize organic ligands of radionuclides.Sulfate-reducing Acinetobacter, Citrobacter and Pseudomonas from low-level radioactive waste sites to metabolize citric acid into a uranyl–citrate complex. These cultures failed to metabolize a stable binuclear complex of uranyl–citrate, possibly because the uranyl–citrate complex did not transport inside the cells, as revealed by 14C labelling analysis. Furthermore, a cell-free extract of the tested organism showed that the binuclear U–citrate complex was completely degraded. Francis and colleagues  radionuclides by altering the transporter genes nixA (Helicobacter pylori) and merTP (Serratia marcescens) respectively  and recombinant DNA technology have been employed to generate character-specific microorganisms for efficient removal of metal by sorption. Different protein constructs have been generated in which the bacterial cell surface is equipped with metal-binding polypeptides by fusion-binding domains to outer-membrane-anchored proteins that include metallothioneins , U(VI) and Tc(VII) from soil  that provides the ability to utilize carbon and energy from catabolism of toluene and mercury (radioactive contaminants) , and showing remarkable resistance against chronic irradiation (50 Gy h−1)  for protein translation. The proteome of an organism is the entire set of proteins, including enzymes, that are expressed in the organism under specific environmental conditions. In order to identify genes, proteins and enzymes involved in the bioremediation of radionuclides, it is important to study the structural and functional interactions between proteins and other metabolites. Potential genes and proteins involved in the metabolism of radionuclides can be identified and studied via advanced genomics and proteomics techniques  reduction, compared with Cr(VI) reduction, where only 83 genes were overexpressed , a glutaredoxin-like protein (tg1302) and two peroxiredoxins (tg1253 and tg1220), which allowed the organism to cope with the stress of radionuclides  are employed to enhance microbial activity; such additives might be disruptive to natural microbiota. It is still an issue to enhance the metabolic activity by maintaining the required growth conditions, such as pH, temperature, and levels of contaminants and nutrients, including diverse chemical parameters of selective microbial populations for in situ bioremediation. In addition, it is necessary to recognize the heterogeneous nature of contaminants on sites that can lead to an uneven flow of the liquid or gas containing the microbes. Due to the slow speed of achieving acceptable levels of decontamination at diverse sites, it is difficult to predict the performance of bench-scale bioremediation in field operations.Despite the progress that has been made in the field of radionuclide bioremediation using microorganisms, many challenges lie ahead. A basic concern is to optimize the conditions and procedures for sustained and effective bioremediation in the presence of competing anions, toxic metals, organic compounds and chelating agents. Another aspect that is of interest is the reoxidation and remobilization of reduced radionuclides by microbial metabolism and abiotic mechanisms. Careful consideration needs to be given to Genetically modified microorganisms have limitations in terms of liability and ethical issues from regulatory agencies such as the United States Environmental Protection Agency (USEPA). The release of GE organisms is a tedious task that requires site dependence compatibility and comparability with other organisms on site. After remediation, removal of GE microorganisms is also not an easy task. The direct immobilization of enzymes on support material could be useful for bioremediation, but it requires abundant optimization of site-specific substrates.E. coli has already uncovered potential strategies for metal detoxification (Brocklehurst and Morby, Recent developments in ‘-omics’-based technologies, i.e. genomics, transcriptomics and proteomics, are set to revolutionize many aspects of the biological sciences. A whole-genome transcription profile of Microbial transformations of radionuclides, heavy metals and minerals are a vital part of natural biosphere processes and can have beneficial consequences for the human community. As we know, the interactions between microorganisms and radionuclides are far from simple, and it is not easy to understand the wide range of environments these organisms inhabit. Study of the molecular mechanisms behind the microbial transformation of radionuclides using ‘-omics’-based approaches and exploiting them in applications such as bioremediation would assist in tracking the responsible microbial metabolic products towards cell-free bioremediation and further assist in efficient removal of radionuclides from the environment.


Up to date several authors discussed interactions between cells forming inflammatory infiltrates in the course of inflammatory bowel disease (IBD), mainly dealing with endoscopic biopsy specimens. These usually contain only mucosa. We have evaluated full bowel wall sections, which seems to be especially important in patients with Crohn's disease (CD). The purpose of our study was to evaluate the relationship between vascular density and expression of thrombospondin-1 (TSP-1) and vascular endothelial growth factor receptor 1 (VEGFR-1) in full-thickness tissue fragments of intestinal wall taken from patients after colectomy, comparing those with IBD to non-IBD control group. Histological sections were immunostained with antibodies against CD-31, TSP-1, and VEGFR-1 and analyzed by pathologists with the use of computer-assisted morphometrics. Our research showed significantly higher vascular density and vascular area percentage in all layers of bowel wall in patients with CD when compared to control. We have also demonstrated differences in vascular density distribution between ulcerative colitis (CU) and CD and between CU and control. However we have not found statistically significant correlation between those findings and VEGFR-1 or TSP-1 expression. Our results might suggest existence of different, TSP-1 independent pathways of antiangiogenesis in IBD. Ulcerative colitis (CU) and Crohn's disease (CD) are known as chronic inflammatory bowel diseases (IBD). There is still some controversy about IBD etiology. Among factors associated with inflammatory bowel disease, angiogenesis plays important role in development of clinical symptoms. It has been shown that exacerbation of IBD increases angiogenesis especially in ulcerative colitis , 2. Thisα-granule [It was reported that anti-angiogenesis in the development of IBD can be measured by detection of thrombospondin , 5. Sinc-granule . Thrombo-granule . Thrombo-granule , 5.The purpose of our study was to assess angiogenesis in correlation with expression of VEGFR-1 and thrombospondin-1 to see if they mediate in maintaining balance between angiogenesis and anti-angiogenesis in the course of IBD. We have decided to use computer-assisted morphometrics to facilitate the analysis of vascular density and immunohistochemically highlighted expression of angiogenic factors.We have analyzed full-thickness intestinal wall histological sections routinely prepared out of formalin fixed, paraffin embedded tissue samples. All samples were collected after colectomy in the course of IBD. All our IBD-affected intestines were resected because of an active phase of inflammation. CU bowels were presented with grade 5 of the disease, according to grading scale for histological assessment of inflammation in ulcerative colitis by Geboes et al. . IntestiOnly one tissue fragment containing full thickness of intestinal wall was collected from each patient. In IBD groups tissue fragments were collected from sites involved by the disease. The control group was formed with the use of fragments that were collected from macroscopically unchanged bowel wall. All tissue samples were fixed in formalin, embedded in paraffin, and sectioned into 4 micrometer sections, according to standard histopathology protocol.One section in each case was stained routinely with hematoxylin & eosin to evaluate the extent of the disease or to confirm the lack of pathology (in the control group) , thrombospondin-1 and VEGFR-1 (to show the expression of both). Antibodies and their respective dilutions are shown in Immunostained sections were subsequently photographed and analyzed.For morphometric analysis we have used Leica Quantimet workstation along with ImageJ custom macros –14.Each layer of vessel wall was analyzed separately. Vascular density was evaluated based upon two parameters—immunostaining against CD31 per square mm and percentage of section area covered by vessels. Thrombospondin expression was independently evaluated by two pathologists on a semiquantitative manner. VEGFR-1 expression was evaluated by counting the vessels that showed immunohistochemical reaction in endothelial cells. Immunohistochemical expression of VEGFR-1 has been shown in U test, Kruskall-Wallis test, correlation analysis) was performed with the use of R programming language [Statistical analysis of the results .We have also shown significant differences in both mean vessel count and mean vessel area fraction throughout the all layers between Crohn's disease and ulcerative colitis  expression and between vascular density and VEGFR-1 expression see . TSP-1 was sporadically found  is not a marker of angiogenesis—this study not revealed statistically significant reaction in normal and IBD bowels. This is contrary to Konno et al. who stated that expression of Flt-1 (among others) was increased in active CU . We beli


Epstein-Barr virus related lymphoproliferative disorders are a rare but potentially fatal complication of allogeneic stem cell transplantation with an incidence of 1–3% and occurring within 6 months after transplantation. The most relevant risk factors include the use of in vivo T-cell depletion with antithymocyte globulin, HLA disparities between donor and recipient, donor type, splenectomy etc. The higher the numbers of risk factors the higher the risk of developing Epstein-Barr virus related lymphoproliferative disorders. Monitoring EBV viremia after transplantation is of value and it should be applied to high risk patients since it allows pre-emptive therapy initiation at specified threshold values and early treatment. This strategy might reduce mortality which was >80% prior to the implementation of anti-EBV therapy. Treatment of EBV-LPD after allogeneic SCT may consist of anti-B-cell therapy (rituximab), adoptive T-cell immunotherapy or both. Rituximab treatment should be considered the first treatment option, preferably guided by intensive monitoring of EBV DNA while reduction of immunosuppression should be carefully evaluated for the risk of graft versus host disease. EBV is an ubiquitous lympho- and epitheliotropic gamma1-herpesvirus. Primary infection of Epstein-Barr virus (EBV) is transmitted by saliva and occurs in childhood in asymptomatic manner. EBV actively replicates in the epithelial cells of the oropharynx and it can subsequently infects recirculating B lymphocytes. In 50% of adolescents primary EBV infection may lead to acute infectious mononucleosis. This symptomatic condition is the consequence of the powerful antiviral T cell response to the EBV-driven B-cell proliferationEBV-related clonal lesions range from policlonal plasmacytic hyperplasia to the atypical monoclonal polymorphic B-cell hyperplasia until the onset of an aggressive non Hodgkin’s lymphoma. The majority of PTLD cases are EBV-positive and many show a latency III pattern of gene expression. In healthy individuals the outgrowth of EBV transformed B-cells is prevented by a cell mediated response realized by cytotoxic T-lymphocytes and MHC-unrestricted NK cells. The most important control against the proliferation of viral infected B cells involves CD8 + specific cytotoxic T cells (CTL), which recognize viral epitopes presented by MHC Class I molecules on the surface of infected B cellEBV-DNA-emia– detection of EBV-DNA in the blood. Primary EBV infection– EBV detected in a previously EBV-seronegative patient. Probable EBV disease– significant lympho-adenopathy (or other end-organ disease) with high EBV-DNA load in the blood, in the absence of other etiologic factors or established diseases. Proven EBV disease (PTLD or other end-organ disease) – EBV detected from an organ by biopsy or other invasive procedures with a test with appropriate sensitivity and specificity together with symptoms and/or signs from the affected organ Pre-emptive therapy in this setting is defined as any agent or specific cells given to an asymptomatic patient with EBV detected by a screening assay, while treatment of EBV disease is with agents or other therapeutic methods applied to a patient with EBV disease (proven or probable).The guidelines on the management of EBV infection in patients with hematological malignancies and after SCT from the Second European Conference on Infections in Leukemia have been published recentlyThe PTLD is a severe complication of prolonged immunosuppression. The first cases of these disorders have been described in the contest of immunosuppression secondary to SOT. Prior to the introduction of cyclosporine A, the occurrence of PTLD was extremely rare. The rate of PTLD has been significantly increased after the introduction of the triple therapy with cyclosporine, OKT3 antibody and antithymocyte globulin (ATG) but on the other hand the introduction of effective immunosuppression has undoubtedly changed the rate of success of SOT through a significant prolongation of survival of patients. In this setting the incidence has been reported up to 15%. EBV related PTLD following SOT develops, in most of the cases, in the first years post transplantThe majority of cases of PLTD in solid organ transplantation are of recipient origin although recently the donor origin has been consistently reported after SOTThe genetic origin of PTLD has been determined by means of different technique including microsatellite analysis, performed on DNA extracted from tumor cells, from donor biopsy specimen obtained form transplanted organ and from recipient DNA.After allogeneic SCT the overall frequencies of PLTD is about 1%. In allogeneic stem cell transplantation EBV-PTLD is nearly always of donor origin. Reddiconto et al.The major risk factors for EBV-PTLD following allogeneic stem cell transplantation are: T-cell depletion of the graft, the use of serotherapy with ATG or Campath for prophylaxis and treatment of graft versus host disease (GVHD), transplantation from an unrelated or haploidentical donor, cord blood as source of stem cells in a reduced intensity conditioning (RIC) regimen setting, HLA disparity donor-recipient, duration of immunosuppressive prophylaxis and, finally, the type of conditioning regimen.Van Esser et al.A number of studies suggest that T cell reconstitution after RIC-regimens may be delayed compared to conventional myeloablative regimensA feature of PTLD following UCBT is a longer interval for malignancy development compared to peripheral blood or bone marrow SCT, in both myeloablative and non myeloablative transplantations. The absence of EBV specific memory T cell in cord blood may account for this behaviour.In a group of patients receiving Campath in vivo, ChakrabartiIn a multivariate analysis from Sundin et al.As PTLD may evolve from a polyclonal disorder to a more aggressive monoclonal variant, an early diagnosis is relevant. In the last years, PCR monitoring of EBV viral load, a minimally invasive technique, has been increasing for the prevention and early detection of post transplant lymphoproliferative disease with the aim of export the successful paradigm of CMV pre-emptive strategy after SCT. EBV viremia was initially carried out by using qualitative PCR analysis and more recently by quantitative PCR technique.6 in healthy carriers. In transplant recipients without EBV related diseases, the circulating viral load is generally higher than those reported in healthy donors, probably as a consequence of the shift of the immune balance towards the virus.The observed EBV loads in healthy donors are very similar in all the studies and are typically very low or undetectable. This depends on the low frequency of EBV positive B cells in the circulation that are lesser than 10 per 103 copies/ml or 200 copies per 105 PBMC, empirically in many transplant centers with the aim to reduce the progression rate to PTLD.Even if the mean EBV load in the peripheral blood seems to be higher in HSCT recipients with PTLD, than in recipient without PTLD, patients with PTLD may present a broad range of PCR value within 1–5 logs. In addition, these values often overlap with the results of patients not developing PTLD. Thus we are not able to detect on the basis of viral EBV load patients progressing to EBV-PTLD. As a result pre-emptive treatment is now used in patients with increasing viral load or over the threshold of 10Another obstacle for the interpretation of EBV data after SCT is related to different PCR monitoring analysis in published studies and many data are based on retrospective analysis of single center experience. In addition there is no general consensus on which is the best specimen to adopt . In fact the determination of cell-associated EBV DNA loads could be interpreted as a marker of EBV-induced cell proliferation while plasma levels of EBV DNA could be expression of either virus production or the release of episomal DNA from apoptotic cells, or bothThe sensitivity of EBV-DNA for the diagnosis of PTLD ranges from 78% and 100% in symptomatic patients, but is only between 50% and 80% when used for preemptive diagnosis. Subclinical reactivation of EBV detectable by PCR occurs from 20% to 60% of HSCT recipients without any PTLD symptoms and the positive predictive role of PCR monitoring for EBV remains highly variableManifestations of post-transplant EBV infection, both primary and reactivation, include EBV-associated enteritis with multiple ulcers, EBV-related hepatitis, encephalitis, and fulminant EBV-associated hemophagocytosis. These complications are, however, infrequent. Chronic active EBV infection characterized by chronic or recurrent infectious mononucleosis-like symptoms such as fever, hepatosplenomegaly, persistent hepatitis, and extensive lymphadenopathy has also been described. The most severe manifestation of EBV infections following allogeneic SCTin situ hybridization for the EBER (Epstein–Barr-encoded RNA) transcripts.Diagnosis of LPD or PTLD must be based on symptoms and/or signs consistent with lymphoproliferative process developing after SCT, together with detection of EBV by an appropriate method applied to a specimen from the involved tissue. Definitive diagnosis of EBV-PTLD requires biopsy and histological examination (including immunohistochemistry or flow cytometry for CD19+ and CD20+). It is important to remember that CD20 can be downregulated on lymphoma cells after therapy. EBV detection in biopsy specimen requires detection of viral antigens or Definitive diagnosis of LPD or PTLD must be based on symptoms and/or signs consistent with PTLD together with detection of EBV by an appropriate method applied to a specimen from the involved tissue.The mortality from PTLD after HSCT was >80% prior the implementation of anti-EBV therapy. Treatment strategies for EBV-related PTLD include reduction of immunosuppression, anti-B cell monoclonal antibodies, conventional chemotherapy and radiation. No effect of antivirals drugs can be expected with respect to prevention and treatment of EBV-LPD. When PTLD is established, standard lymphoma chemotherapy is used in combination with rituximabThe reduction of immunosuppression therapy is usually the first-line approach for PTLD transplantation but this may not be feasible in patients with active GVHD.The overall success rates of EBV-related PTLD were lower than after pre-emptive therapy, reaching 63% and 88.2% of total EBV-DNA clearance with rituximab and CTL therapy, respectively. Thus, because of the progressive nature of PTLD, an early or pre-emptive treatment with either anti-B cell monoclonal antibodies or donor-derived EBV-specific cytotoxic-T lymphocytes is appealingWagner et al.Among them, eight developed symptoms of PTLD. Hence, the detection of EBV-DNA levels > 4000 copies /μg PBMC DNA had a sensitivity of 100% for the prediction of early PTLD but a specificity of only 50% (8/16). All patients received treatment: two patients received CTLs, five patients received rituximab (1–4 dose of 375 mg/m2/die) and another one received CTLs and rituximab. Clinical symptoms associated with PTLD disappeared and EBV load decreased to a normal value in 7 out of 8 patients.In another study, Van Esser et al. employed PCR for an early diagnosis and pre-emptive treatment of the EBV related PTLD. The threshold of 1000 copies of viral DNA genome per millilitre was set as the reference level for a high predictive value of PTLD development. They studied 49 patients receiving a partial T-cell depleted transplant from HLA-matched sibling donor or matched unrelated donor. EBV reactivation with a viral load above 1000 copies/ml was detected in 17 out of 49 patients (35%). Among them, 2 patients presented an active PTLD and received two infusions of rituximab, obtaining complete and persistent clearance of EBV viral load. The other 15 patients presented a subclinical reactivation and were treated with rituximab pre-emptively. Fourteen patients have shown a complete response after a single dose of rituximab. The only non responder patient showed an increased viral load and a progression to EBV related PTLD and was treated with a second dose of rituximab and DLI and achieved a clinical response. Thus, pre-emptive rituximab selectively administered to high-risk patients, abrogates EBV reactivation and reduces the incidence of EBV-PTLDAnnels et al.We have prospectively monitored EBV reactivation in a cohort of 104 patients receiving HSCT between February 2005 and August 2009. Seventy-nine patients received an allogeneic SCT and twenty-five patients received an autologous SCT. EBV reactivation rate was of 16% (4/25) with a median time to the reactivation of 115 days in the autologous SCT group. In allogeneic transplant group EBV reactivation was of 60.76% (48/79) with a median time to reactivation of 59 days (range 3–840), and a median value of viral genome copies per ml of 1.577.017 (range 408–60.000.000) . StrikinIn fourthy-eight patients who reactivated EBV, serum protein electrophoresis was carried out. A γ-peak, indicating the presence of a monoclonal gammopathy (MG), was detected in 31 patients (64.5%). Twenty-seven patients with MG were further investigated with immunofixation on serum and urine. Monoclonal gammopathy was present in 22/27 patients and in 5 patients the Ig isotype was not identified . SubtypeSix patients developed PTLD . In five of these, PTLD followed EBV DNA detection in peripheral blood, while in the last patient PTLD was diagnosed by biopsy, in the absence of previous EBV detection. All patients with PLTD presented a γ-peak at the serum protein electrophoresis. In five patients a monoclonal gammopathy was confirmed by immunofixation: four patients presented a single monoclonal gammopathy  and one patient presented a double monoclonal gammopathy IgMk+IgGλ .Treatment with rituximab (375mg/m2/die for 1–4 doses) was administered to all cases of PTLD and to others 12 asymptomatic or symptomatic patients with an high number of EBV genome copies in order to avoid the progression to PTLD. The vast majority of patients (88.9%) treated with rituximab showed a sharp viral load clearance. In 11 out of 18 patients treated with rituximab (61%) abnormalities of serum protein electrophoresis normalized after treatment. After rituximab, immunofixation was evaluated only in 10 out 18 treated patients. Five of the these patients showed the complete resolution of the MG after treatment. Two patients with PTLD died early after treatment: one patient from multi-organ failure and the other because of progressive disease. The remaining four patients presented resolution of symptoms and a drop in EBV viremia in peripheral blood until its disappearance. They are alive at follow-up with an overall survival of 6,12, 13 and 45 months, respectively.Hence, the risk factors reported in the literature are substantially confirmed in this prospective study. Rituximab was effective for a prompt treatment of PTLD and useful as pre-emptive treatment limiting the progression of EBV reactivation to PTLD. Monoclonal gammopathy may help to define patients with EBV viremia at high risk of PTLD after SCTIn summary, treatment options include manipulation of the balance between outgrowing EBV-infected B cells and the EBV CTLs response targeting the B cells with monoclonal antibody rituximab and conventional chemotherapy. The efficacy of this strategy is greater in early PTLD than in later disease. In the case of EBV-specific CTL it should be noted that this approach is not widely available. Rituximab is useful as a pre-emptive treatment in patients with high EBV viral load and with risk factors for PTLD . Over-trWith current approaches the mortality from EBV-PTLD can be significantly reduced but preemptive treatment based on EBV viremia should be validate and diagnostic tests should be standardized in prospective trials. The search for unrecognized risk factors for EBV-related LPD after SCT is ongoing and includes biomarkers, immune-phenotype and genetic polymorphisms of both recipient and donor.


A combined quantitative trait loci (QTL) and microarray-based approach is commonly used to find differentially expressed genes which are then identified based on the known function of a gene in the biological process governing the trait of interest. However, a low cutoff value in individual gene analyses may result in many genes with moderate but meaningful changes in expression being missed.We modified a gene set analysis to identify intersection sets with significantly affected expression for which the changes in the individual gene sets are less significant. The gene expression profiles in liver tissues of four strains of mice from publicly available microarray sources were analyzed to detect trait-associated pathways using information on the QTL regions of blood concentrations of high density lipoproteins (HDL) cholesterol and insulin-like growth factor 1 (IGF-1). Several metabolic pathways related to HDL levels, including lipid metabolism, ABC transporters and cytochrome P450 pathways were detected for HDL QTL regions. Most of the pathways identified for the IGF-1 phenotype were signal transduction pathways associated with biological processes for IGF-1's regulation.We have developed a method of identifying pathways associated with a quantitative trait using information on QTL. Our approach provides insights into genotype-phenotype relations at the level of biological pathways which may help to elucidate the genetic architecture underlying variation in phenotypic traits. In the last decade, a large number of quantitative trait loci (QTL) have been mapped in various organisms, and positional information on QTL is already included in resources such as Online Mendelian Inheritance in Man , Mouse GAnalyzing expression quantitative trait loci (eQTL) in specific tissues is an alternative way to identify potentially causative genes ,10. eQTLThe present study aims to identify pathways associated with a quantitative trait of interest using information on QTL. We have developed a pathway-based QTL/microarray approach which provides candidate genes and biological insights into quantitative traits. Our approach is based on a modified GSA, that tests whether a subset of genes within a QTL region is more likely to be relevant to a trait of interest than whole pathway genes. We demonstrate that the combination of gene expression profiling, QTL mapping and GSA produced gene set scores suitable to detect trait-associated pathways.The Mouse Phenome Database (MPD)  has devep-values between four comparisons for HDL gene sets as shown in Figure p-values. Out of 10 pathways, 9 were classified in the "Metabolism" category including lipid metabolism (Arachidonic acid and Linoleic acid metabolism) and Drug metabolism - cytochrome P450 pathway in which HDL cholesterol levels increase with increasing liver microsomal P450-enzymes [Apoa2 is also known to be involved in the response to HDL cholesterol levels, but its change was relatively small in all comparisons (from 1.00 to 1.04 fold).The HDL cholesterol QTL set contained 5,938 genes and 117 pathways. We identified 10 pathways as significantly associated with the trait for four comparisons Table . The sor-enzymes ,17. The -enzymes . Apoa2 ip-value distributions of IGF-1 gene sets in four and three comparisons, respectively. Most of the pathways identified were categorized as Environmental Information Processing and Organismal Systems, related to some biological processes for IGF-1 regulation. We can roughly divide them into two groups. The first group includes gene sets related to "upstream" biological processes or genes for IGF-1. Yusta et al. (2009) [FGF21 in the MAPK signaling pathway decreases on the expression of IGF-1 via a reduction in concentrations of the active form of STAT5 [The IGF-1 QTL set consisted of 3,135 genes and 80 pathways. In all four comparisons, there were only two pathways in common (Table . 2009)  suggeste09  suggeof STAT5 , the conof STAT5 .In this study, we have developed a method of identifying pathways that are associated with quantitative traits using information on QTL. In the application of our approach to the data on mouse inbred strains, the metabolic pathways for HDL cholesterol trait and the IGF-1 related signaling pathways were clearly detected. Our approach provides insights into genotype-phenotype relations at the level of biological pathways which may help to elucidate the genetic architecture underlying the variation in phenotypic traits. The method presented here is also useful for identifying positional candidate genes in the QTL region through the detection of trait-associated pathways. Once trait-associated pathways are detected, differentially expressed genes that share the pathways would be promising candidates, even if the genes are outside the QTL region.GSA methods can be separated into a number of discrete steps enabling systematic comparison . The cur1) With the current method, the simplest and most widely used fold change is calculated as a gene-level statistic by the log-ratio (base 2) of the group means of one gene.2) In addition to an absence of transformation for the typical single direction enrichment, absolute values of per-gene test statistics were used to identify bi-directional changes, providing a more precise understanding of transcriptome regulation.3) The most common choice for the gene set statistic is the mean of local statistics, especially when the gene-level scores are assumed to follow a normal distribution. We employed Z-scores for each gene set which were calculated from (absolute) fold change values for each gene between two experimental groups, as follows:μ and σ are the mean (absolute) fold change and the standard deviation of all genes in a given microarray, respectively, and x and n are the mean of (absolute) fold change and the number of genes in the gene (sub-)set, respectively.where m gene sets (iG) in the pathway category and these p-values are computed from the Z-score. For the given QTL category, the sorted p-values for sub-sets are4) If a pathway is associated with the trait, we expect a sub-set of pathways within the QTL regions to have altered transcription. We intersected two gene sets, each of which belongs to the pathway and QTL category so that the intersection has composite information. Statistical tests conducted use a competitive null hypothesis. Suppose there are Q represent genes within all QTL regions for a specific trait. We are particularly interested in the intersection sets with significant expression changes for which the changes in the individual gene sets are less significant. If we want the FDR (α) [where  FDR (α)  of an eniα/m means the critical value in the Benjamini-Hochberg procedure. In the current approach, an additional filtering based on the comparison of significance between sub- and whole pathways is implemented, although the method is not testing any specific null hypothesis for comparison of sub- with whole pathways. This approach assumes that the sub-set of pathways within the QTL region is under cis-regulation and partial trans-regulation within multiple QTL regions.where We applied our methods to two sets of mouse QTL identified from a public database. We extracted 47 and 17 QTL associated with blood concentrations of HDL cholesterol and IGF-1 in mice from MGI. For HDL cholesterol, we used annotations derived from the MP term "abnormal circulating HDL cholesterol level" (MP:0000184). For IGF-1, we used annotations from the term "abnormal circulating insulin-like growth factor I level" (MP:0004700). The QTL studies, the QTL name, peak position and confidence intervals were downloaded from MGI. If QTL flanking markers were not known, we assigned an interval by peak marker ± 20 Mb. To assign base pair and cytogenetic positions to all flanking and peak markers, we downloaded the UCSC Genome Browser  positionBennet et al. (2010)  comparedEach gene is subsequently annotated with its associated biological pathways, obtained from KEGG, a collection of manually drawn pathway maps representing the molecular interaction and reaction networks for 1) metabolism, 2) genetic information processing 3) environmental information processing, 4) cellular processes, 5) organismal systems and 6) human diseases. We limited the gene set size between ten and five hundreds and the resulting number of gene sets was 151 with 4,115 genes on the microarray . If QTL information is about eQTL regions, other gene sets such as transcription factor-target genes are needed to consider the characteristic feature.eQTL: expression quantitative trait loci; FDR: false discovery rate; GEO: gene expression omnibus; GSA: gene set analysis; GWAS: genome wide association studies; HDL: high density lipoproteins; IGA: individual gene analysis; IGF-1: insulin-like growth factor 1; KEGG: Kyoto encyclopedia of genes and genomes; MGI: Mouse Genome Informatics; MPD: Mouse Phenome Database; QTL: quantitative trait locus; RMA: Robust Multi-array Average method.The authors declare that they have no competing interests.HM conceived of this study, developed and implemented the statistical analysis, drafted the manuscript. YT supported the design of the experiments and the interpretation of the results. HI coordinated the study and contributed in writing the manuscript. All authors read and approved the final manuscript.


Construction of artificial polyepitope antigens is one of the most promising strategies for developing more efficient and safer vaccines evoking T-cell immune responses. Epitope rearrangements and utilization of certain spacer sequences have been proven to greatly influence the immunogenicity of polyepitope constructs. However, despite numerous efforts towards constructing and evaluating artificial polyepitope immunogens as well as despite numerous computational methods elaborated to date for predicting T-cell epitopes, peptides binding to TAP and for antigen processing prediction, only a few computational tools were currently developed for rational design of polyepitope antigens.Here we present a PolyCTLDesigner program that is intended for constructing polyepitope immunogens. Given a set of either known or predicted T-cell epitopes the program selects N-terminal flanking sequences for each epitope to optimize its binding to TAP (if necessary) and joins resulting oligopeptides into a polyepitope in a way providing efficient liberation of potential epitopes by proteasomal and/or immunoproteasomal processing. And it also tries to minimize the number of non-target junctional epitopes resulting from artificial juxtaposition of target epitopes within the polyepitope. For constructing polyepitopes, PolyCTLDesigner utilizes known amino acid patterns of TAP-binding and proteasomal/immunoproteasomal cleavage specificity together with genetic algorithm and graph theory approaches. The program was implemented using Python programming language and it can be used either interactively or through scripting, which allows users familiar with Python to create custom pipelines.http://tepredict.sourceforge.net/PolyCTLDesigner.html.The developed software realizes a rational approach to designing poly-CTL-epitope antigens and can be used to develop new candidate polyepitope vaccines. The current version of PolyCTLDesigner is integrated with our TEpredict program for predicting T-cell epitopes, and thus it can be used not only for constructing the polyepitope antigens based on preselected sets of T-cell epitopes, but also for predicting cytotoxic and helper T-cell epitopes within selected protein antigens. PolyCTLDesigner is freely available from the project’s web site:  One of the most promising approaches to designing more efficient and safer vaccines is construction of artificial polyepitope antigens -6. TheirBoth interaction of peptides with MHC molecules and peptides binding to TAP complex are sufficiently specific for certain amino acid patterns. Proteasomal and immunoproteasomal cleavage sites are also to a considerable degree determined by the amino acid sequences of antigens and degenerate amino acid motifs that determine the efficiency of proteasomal cleavage sites are currently known ,31 as weIt has been shown that longer peptides transported into the endoplasmic reticulum (ER) undergo N-terminal trimming by ER aminopeptidases (ERAPs) to allow them to bind MHC class I molecules , and thaThe goal of this work was to develop a program intended for rational design of polyepitope T-cell antigens with a special focus on optimizing their immunogenicity via selecting amino acid spacer sequences for each pair of epitopes and choosing the optimal ordering of the epitopes within the polyepitope.A program named PolyCTLDesigner was developed. Given a set of either known or predicted CTL epitopes, PolyCTLDesigner predicts affinity of their binding to TAP and then N-termini of inefficient binders are extended with certain flanking residues using the model created by Peters et al. , implyinThen all possible pairs of obtained oligopeptides are produced and PolyCTLDesigner predicts the proteasomal and/or immunoproteasomal cleavage sites using the models developed by Toes et al. . AccordiW is the weight (rank) of spacer sequence ss between the epitopes pep1 and pep2; rankHLA is the rank of non-target junctional epitope predicted to be the most efficient binder for HLA class I allele HLA; freqHLA is the genotypic frequency of that allele within the population of interest  is the length of spacer ss; rankpr corresponds to the rank of proteasomal cleavage site predicted at the pep1 C-terminus ; rankimpr is the rank of immunoproteasomal cleavage site; rankHLA value; Neps is the number of predicted junctional epitopes and NHLA is the number of HLA alleles predicted to bind non-target epitopes with sufficient affinity , the value of 2 corresponds to high affinity (7.3 ≤ pIC50 < 8.3) and 3 corresponds to the highest affinity . Thus the optimal spacer sequence should have the least weight.where om dbMHC ); len and edges corresponding to allowed epitope matchings. Each edge has two parameters: the optimal spacer sequence and its weight which was calculated by the ranking function described above. The constructed weighted digraph is in turn transformed into a complete one by adding edges corresponding to disallowed epitope matchings; their weights are set to 5000 while the weights of allowed epitope matchings usually don’t exceed 10. The sequence of desired polyepitope antigen can be determined as the least weighted complete simple path in the constructed weighted digraph, and as one can see this task is related to the travelling salesman problem (TSP). To find optimized sequence of polyepitope antigen PolyCTLDesigner uses either greedy nearest neighbor approach (only in the case of a nondegenerate spacer sequence), or genetic algorithm-based TSP-solver implemented in PyEvolve library . The maiBuilding the graph is the most time consuming step and for a set of 40–50 peptides it can take about 6–8 hours to be completed on typical desktop personal computer. However in future we plan to implement parallelized algorithm to reduce time consumption.To illustrate the importance of poly-CTL-epitope optimization we performed a small theoretical analysis: six well-defined HLA-A*02:01-restricted HIV-1 CTL epitopes were used to produce artificial polyepitopes using our PolyCTLDesigner program. It was found that probability of selecting an optimal epitopes permutation at random was less than 0.00139 and only 17% of all possible polyepitope constructs did not contain inefficient proteasomal cleavage sites between target CTL epitopes. Besides, the choice of spacer sequences can have a great impact on proteasomal cleavage efficiency, especially for certain peptides. The detailed description of the study and its results can be found in Additional file In addition, PolyCTLDesigner is also able to assist in constructing polyepitope fragments containing T-helper epitopes. Currently T-helper epitopes can be predicted with ProPred models  : platform-independentProgramming language: PythonOther requirements: Python 2.7, TEpredict (provided with the PolyCTLDesigner), Biopython, NumPy, PyEvolve, and Python graphLicense: Creative Commons Attribution Non-Commercial License V2.0 (CC BY-NC 2.0)Any restrictions to use by non-academics: see CC BY-NC 2.0 licenseThe authors declare that they have no competing interests.SIB initiated the studies of polyepitope immunogens at the State Research Center of Virology and Biotechnology “Vector” and has developed the prototype of polyepitope rational design algorithm. DVA completed the concept, chose the models and algorithms, and implemented PolyCTLDesigner. DVA drafted the manuscript. SIB was involved in writing the manuscript and its critical revision and has given the final approval for the version to be published. Both authors read and approved the final manuscript.This file describes the short sample study of designing artificial poly-CTL-epitope antigen composed of 6 well studied HIV-1 CTL epitopes using PolyCTLDesigner.Click here for fileThis file contains the source code of PolyCTLDesigner together with a sample workflow script (designer.py).Click here for file