Heart failure with ejection fraction (HFpEF) is a syndrome resulting from several co-morbidities in which specific mediators are unknown. The platelet proteome responds to disease processes. We hypothesize that the platelet proteome will change composition in patients with HFpEF and may uncover mediators of the syndrome.Proteomic changes were assessed in platelets from hospitalized subjects with symptoms of HFpEF (n = 9), the same subjects several weeks later without symptoms (n = 7) and control subjects (n = 8). Mass spectrometry identified 6102 proteins with five scans with peptide probabilities of ≥0.85. Of the 6102 proteins, 165 were present only in symptomatic subjects, 78 were only found in outpatient subjects and 157 proteins were unique to the control group. The S100A8 protein was identified consistently in HFpEF samples when compared with controls. We validated the fining that plasma S100A8 levels are increased in subjects with HFpEF (654 ± 391) compared to controls (352 ± 204) in an external cohort (p = 0.002). Recombinant S100A8 had direct effects on the electrophysiological and calcium handling profile in human induced pluripotent stem cell-derived cardiomyocytes.Platelets may harbor proteins associated with HFpEF. S100A8 is present in the platelets of subjects with HFpEF and increased in the plasma of the same subjects. We further established a bedside-to-bench translational system that can be utilized as a secondary screen to ascertain whether the biomarkers may be an associated finding or causal to the disease process. S100A8 has been linked with other cardiovascular disease such as atherosclerosis and risk for myocardial infarction, stroke, or death. This is the first report on association of S100A8 with HFpEF.The online version of this article (doi:10.1186/s12967-016-0774-3) contains supplementary material, which is available to authorized users. The platelet proteome is an untapped resource for identifying proteins that may reflect a disease process. Platelets are easily accessible and free from major highly abundant proteins making them an attractive model for proteomic studies. Platelets change the composition of their proteins in diseases such as Alzheimer’s, cancer, diabetes, coronary artery disease and acute coronary syndrome –4. PlateHeart failure with preserved ejection fraction (HFpEF) affects almost 50 % of patients with heart failure and is increasing in prevalence , yet theWe established a novel bed-to-bench translational system to identify potential mediators of HFpEF using both platelet proteome analysis and mechanistic studies in induced pluripotent stem cell-derived cardiomyocytes. The broad utility of this strategy is to incorporate bioactivity studies into guiding the selection of proteins from proteomic studies for further investigation. We sought to compare the platelet proteome among subjects with HFpEF in the uncompensated  state, compensated (outpatient) state, and controls combined with validation in plasma samples from an external cohort and bioactivity studies using human induced pluripotent stem cell (iPSC)-derived cardiomyocytes. We hypothesized that  platelet2. Subjects were excluded if they had a clinical condition that potentially changed the platelet or plasma proteomic profile independent of HFpEF such as uncontrolled diabetes, an active infection or inflammatory disorder, chronic renal failure requiring dialysis, severe liver disease, malignancy, acute myocardial infarction, chronic obstructive pulmonary disease requiring steroids, or recent surgical or invasive cardiac procedures. Subjects were excluded if they had other cardiac causes for their symptoms such as severe valvular disease, amyloidosis, or hypertrophic cardiomyopathy. Blood was drawn from the nine subjects enrolled in the study . Five of these subjects (HFpEF outpatient group) returned ≥2 weeks after discharge for second blood draw. Subjects with an LVEF ≥50 % and without evidence of increased LV filling pressures served as the control group.For the discovery phase, subjects ≥50 years old presenting with New York Heart Association class II–III heart failure symptoms, a left ventricular ejection fraction (LVEF) >50 %, echocardiographic evidence of diastolic dysfunction and increased LV filling pressure were evaluated at the Medical College of Wisconsin between June 2012 to December 2013 for participation in this study. Increased LV filling pressures were defined as E/e′ ≥ 15, or E/e′ ≥ 8 and ≤ 15 with either a BNP ≥ 200 pg/ml or a left atrial (LA) volume index > 40 ml/mFor further biomarker validation, an additional set of 25 HFpEF subjects and 18 age and co-morbidity matched control subjects were recruited from Northwestern University. All subjects gave written informed consent to participate in the study. The Institutional Review Board at the Medical College of Wisconsin and Northwestern University approved the respective study protocols, which conformed to the principles of the Declaration of Helsinki.Supplies and other reagents were purchased from Sigma-Aldrich  unless specified. Recombinant S100A8 was purchased from Creative BioMart .d-glucose). During all steps, care was taken to avoid activation of platelets. Flow cytometry with anti-CD41  and anti-P-selectin  was performed to assess for platelet activation  in 25 mM NH4HCO3 for 30 min at room temperature followed by trypsin digestion overnight. Peptides were extracted with 0.1 % trifluoroacetic acid (TFA) and 70 % acetonitrile/5 % TFA in water, respectively. Extracts were dried in a Speedvac and subsequently acidified to 0.1 % TFA. The samples were desalted using a ZipTip (C18).Platelets from individual samples were resuspended in lysis buffer . After determining protein concentration, the protein sample was separated by 1-dimensional SDS-PAGE gel (Bis-Tris 4–12 %) with internal DNA markers as described in our earlier publication . The gel2O, 0.1 % formic acid) to buffer B . The gradient program began with 2 min at 98 % A, followed by a 3 min ramp to 95 % A, a 115 min ramp to 60 % A, a 15 min ramp to 2 % A, 3 min at 2 % A, 2 min ramp to 98 % A, then a 10 min equilibration in 98 % A. MS1 scans were detected in the FTMS section of the Orbitrap Velos in profile mode at a resolution of 30,000 . The ten most abundant parent ions from each MS1 scan were selected for fragmentation via collision induced dissociation. Results of SEQUEST searches against UniProt human database (version April 2013) and all nine runs of each sample were combined using Visualize software. Visualize software was also used to generate comparison data i) were measured using the ratiometric Ca2+ microfluorometry technique with Fura-2-AM fluorescent dye. The [Ca2+]i were monitored in spontaneously beating cells. The sample trace  at certain time points (indicated by the red arrows) using a microperfusion system. Of particular note is the recovery of the spontaneous Ca2+ transient following each depolarizing pulse. In the absence of rS100A8, the recovery was relatively fast. In contrast, the recovery was considerably slower in the presence of rS100A8. Following a third depolarizing pulse, recovery was not evident until the washout of rS100A8; this observation also suggests that rS100A8 effects are mediated through a membrane receptor. In summary, rS100A8 adversely affected the calcium handling of iPSC-derived cardiomyocytes.Intracellular Caace Fig. c shows aThe key finding of this study was that it was possible to derive platelet protein data sets specific for HFpEF patients. These proof-of-concept findings suggest that the platelet proteome might provide a useful tool for screening for HFpEF-associated biomarkers. Although several platelet proteins were identified in HFpEF subjects; their exact connection to HFpEF has yet to be determined. Though our data is limited by the small size, our discovery cohort has similar characteristics of larger HFpEF cohorts reported in the literature –31. By cThe platelet proteome in healthy individuals is remarkably stable with only minor differences in protein expression patterns . VeitingS100A8 is a member of the S100 calcium-binding family of proteins, which exhibit increased levels in a number of inflammatory states. S100A8 is commonly mentioned with its binding partner, S100A9. Even though S100A8 is found in the plasma , it is kS100A8 and S100A9 are not normally expressed in cardiomyocytes  although2+ handling profiles of human induced cardiomyocytes suggesting that S100A8 is acting through a membrane receptor. S100A8 interaction with RAGE affects calcium flux in neonatal rat ventricular cardiomyocytes and HL-1 cardiomyocytes [2+ handling profiles resulting from S100A8 treatment of human induced cardiomyocytes; validates our bedside-to-bench translational screen as an approach to identify bioactive proteins that may contribute to the disease mechanisms in HFpEF.Increased platelet S100A8 mRNA and plasma protein levels were present in patients with acute myocardial infarction . Plasma myocytes , 53. TheWe also considered the possibility that subjects progress to HFpEF through loss of cardioprotective proteins. Therefore, we searched amongst our control group and were able to identify four proteins that could potentially have protective qualities against the development of heart failure. Cyclic nucleotide phosphodiesterase 3A1 (PDE3A) regulates β-adrenergic signaling to effect physiological cardiac performance. Furthermore, PDE3A protects the heart against angiotensin II-induced cardiac remodeling in mice . Copper The present study must be interpreted within the context of its limitations. First of all, this was a discovery effort and not designed as a quantitative proteomic analysis. Therefore, we cannot determine if specific proteins are up- or down-regulated. In addition, it is unlikely that one protein is responsible for a complex disease as HFpEF, but our findings offer new perspectives regarding HFpEF and further confirmation of the platelet proteins identified in this study will need to be validated in a larger cohort. In addition, combining proteomics with functional bioactivity assessments may be a strategy to complement and strengthen the search for biomarkers by combining protein identified with biological activity in a relevant in vitro model system.In conclusion, from the discovery set in HFpEF patients, we derived a panel of platelet proteins that may be specific for HFpEF. Furthermore, this set distinguished a set of platelet proteins which are consistent in HFpEF subjects whether they are decompensated and hospitalized or compensated after discharge. We further established a bedside-to-bench translational system that can be utilized as a secondary screen to ascertain whether the biomarkers may be an associated finding or causal to the disease process.


The first shunts being successfully used for the treatment of hydrocephalus were designed to lower an overpressure within the ventricles of the brain. Later the same technology was used for the treatment of normal pressure hydrocephalus, although the background of this disease can not be compared with obstructive hydrocephalus. Since then no specific device has been developed focussing on the treatment of NPH. However, the recent knowledge of NPH and the risks behind the shunting of NPH offer interesting options.For the diagnosis of NPH an often used method is the spinal tap test. The withdrawal of up to 50 cc of spinal fluid sometime leeds to impressive clinical improvement. To address this fact a new device has been developed which can be programmed to open depending on time and body position. Consequently in contrast to valves for the treatment of obstructive hydrocephalus this device does not open depending on increased baseline ICP or ICP peaks. The device can be programmed to open for two minutes intervals over 24 hours. The valve can be programmed to remain closed, to open for two minutes within 24 hours, to open the whole 24 hours or any time between two minutes and 24 hours. In addition it can be programmed whether or not the opening should be manipulated by the body position of the patient in particular to allow an only reduced flow in the standing position.The new electronic device is the first implant developed only for NPH addressing specific aspects of this decease. The valve has been investigated in vitro simulating the condition within a patient. It has not been implanted yet, because of regulatory issues. Whether or not the device improved the clinical outcome in patients with NPH, whether or not it can lower risks for these patients and introduce new treatment options can not be answered without clinical experience. However, it promises to be a tool for scientific research, which might help to improve the understanding of NPH.


Polymorphisms in the TNF-A gene have been associated with chronic obstructive pulmonary disease (COPD) in some case-control studies. Previous work has shown that COPD/chronic bronchitis subjects with alpha-1 antitrypsin deficiency with the rs361525 TNF-α single nucleotide polymorphism have 100 times more TNF-in spontaneous sputum than disease matched controls. Our objective was to determine if the presence of this polymorphism increased TNF-α production by blood monocytes from COPD subjects.Monocytes from 18 COPD/alpha-1 antitrypsin deficient subjects, with and without the rs361525 polymorphism, were cultured in the presence or absence of lipopolysaccharide. Cell-free supernatants were analyzed by ELISA and real-time PCR performed using cDNA from extracted RNA. Baseline expression of TNF-α messenger RNA was no different between the groups. No difference in messenger RNA or secreted protein was observed over time in un-stimulated cells. TNF-α messenger RNA expression and protein was not higher in lipopolysaccharide-stimulated monocytes from subjects with the polymorphism compared to cells from patients with the wild-type allele.This small pilot study did not provide an explanation for the findings of earlier observations of the association of the rs361525 polymorphism with TNF-α in airways secretions. Possible reasons for the lack of concordance include the study of blood rather than tissue cells, the use of a single stimulant rather than biological secretions and the need for far greater subject numbers to overcome intra-subject variation in monocyte TNF-α production. Chronic obstructive pulmonary disease (COPD) is a debilitating disease most commonly caused by smoking. However, only 25 % of smokers develop COPD, suggesting other contributing factors, such as genetic susceptibility . The onlet al characterised a cohort of 424 unrelated patients with COPD and AATD and determined that the rs361525 polymorphism, a G to A substitution at position -238 in the promoter region of the gene, was observed with greater frequency in subjects with a chronic bronchitis phenotype [There is a pressing need to identify new therapeutic targets in COPD, to modify disease and improve outcomes. COPD is heterogeneous and it is possible that there are many potential targets, each affecting a subset of patients. One such target may be tumor necrosis factor-alpha (TNF-α) which has been implicated in the pathogenesis of COPD in animal studies , 4 and ohenotype .Alpha-1 antitrypsin is involved in the modulation of TNF-α biosynthesis and AATD patients are known to have increased activation of the TNF-α pathway . TherefoWe hypothesised that monocytes  from subjects with AATD and COPD would produce more TNF-α if they carried the rs361525 polymorphism and that this would have an enhancing downstream effect on other monocyte functions relevant to COPD, such as phagocytosis and cytokine production. As no previous experiments had been conducted in monocytes with the rs361525 polymorphism from this patient group, it was not possible to power the current studies and hence the work presented here should be considered as a pilot study.2. Monocytes were plated at a concentration of 0.25 (for mRNA experiments) or 0.45 million per ml (in duplicate for ELISA experiments) of culture medium. Salmonella Enteritidis derived lipopolysaccharide (LPS)  was chosen as the TNF-α stimulant after conducting concentration-response and time-course experiments in healthy control monocytes to confirm it elicited the greater response, compared to a number of other stimuli, with a peak time point for TNF-α protein (6 h post LPS). An enzyme-linked immunosorbant assay (ELISA) was used to measure TNF-α in the cell-free supernatant according to manufacturer instructions . The plates were read using a Synergy HT microplate reader . All samples and standards were run in duplicate. mRNA was extracted from each cell pellet using the Isolate RNA Minikit . Median 260/280 ratio of RNA samples was 2.1 (IQR 1.8-2.6). RNA samples were reverse transcribed using a High Capacity RNA-to-cDNA Kit  in a Takara Thermal Cycler PCR machine . Real-time quantitative polymerase chain reaction (PCR) was carried out to quantify the expression of each gene of interest. Complementary deoxyribonucleic acid (cDNA) was mixed with Light Cycler 480 Probe PCR Master master mix , PCR-grade water and the relevant fluorescein isothiocyanate-labelled TaqMan gene expression assay , in each well of the plate. The reaction was run on a Roche Lightcycler 480 , for 45 amplification cycles. The Assay-on-Demand numbers for the TaqMan assays were: glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Hs99999905_m1; TNF-α: Hs00174128_m1. Where an individual normalising gene can be shown to be stably expressed in the model under study it is deemed acceptable to use only that one [-ΔCT formula was used to calculate the relative expression of mRNA [This study was conducted following ethical approval from the National Research Ethics Service Committee West Midlands, UK (reference number LREC-3359/3359-A). Patients with AATD and COPD were recruited from the UK AATD registry, held in Birmingham, UK, following the provision of written informed consent. All patients were clinically stable and exacerbation free for at least six weeks prior to recruitment. Patients were selected following careful matching for clinical criteria (described in Table that one . Stable  of mRNA . Data isTable Expression of TNF-α mRNA in freshly isolated un-stimulated monocytes was low and there was no difference between AG and GG subjects Fig. . No diffThe pilot studies presented here did not support the findings in the sputum of COPD/AATD patients with the rs361525 polymorphism, which was surprising given the magnitude of difference observed in the airways . It is cThere are other possible explanations for the lack of expected effect. Although LPS has been shown to induce transcription of TNF-α mRNA in immortalised cells of the monocyte lineage with the AG genotype using a reporter gene assay , the samIn summary our findings did not support our primary hypothesis but are consistent with the literature describing the effects of this particular polymorphism. Studies report positive, negative and no effects, in a range of cells types, employing a wide variety of techniques to measure output and in different disease states , 14–25.


Plasmodium falciparum infection we have investigated whether it has an effect on the parasite asexual stage.Plasmodium has a complex biology including the ability to interact with host signals modulating their function through cellular machinery. Tumor necrosis factor (TNF) elicits diverse cellular responses including effects in malarial pathology and increased infected erythrocyte cytoadherence. As TNF levels are raised during Flow cytometry, spectrofluorimetric determinations, confocal microscopy and PCR real time quantifications were employed for characterizing TNF induced effects and membrane integrity verified by wheat germ agglutinin staining.Plasmodium falciparum Proliferating-Cell Nuclear Antigen 1, decreased after P. falciparum treatment of TNF (tumor necrosis factor) or 6-Bnz cAMP .TNF is able to decrease intracellular parasitemia, involving calcium as a second messenger of the pathway. Parasites incubated for 48 h with TNF showed reduced erythrocyte invasion. Thus, TNF induced rises in intracellular calcium concentration, which were blocked by prior addition of the purinergic receptor agonists KN62 and A438079, or interfering with intra- or extracellular calcium release by thapsigargin or EGTA (ethylene glycol tetraacetic acid). Importantly, expression of PfPCNA1 which encodes the This is potentially interesting data showing the relevance of calcium in downregulating a gene involved in cellular proliferation, triggered by TNF.Plasmodium may subvert the immunological system and use TNF for the control of its proliferation within the vertebrate host.The data show that  •P. falciparum‐infected RBCs.TNF is able to decrease parasitemia in •TNF induced rises in intracellular calcium concentration, which were blocked by the purinergic receptor agonists KN62 and A438079.•P. falciparum.Interfering with intra‐ or extracellular calcium release by thapsigargin or EGTA also block TNF‐induce calcium release in •P. falciparum Proliferating‐Cell Nuclear Antigen 1 (PfPCNA1) decreased after P. falciparum treatment with TNF or 6‐Bnz cAMP.Expression of the •The data show that Plasmodium may subvert the immunological system and use TNF for the control of its proliferation within the vertebrate host. Signal transduction inside Plasmodium has been shown as a major mechanism to control parasite development Plasmodium infection remodels the cytoskeleton network in infected erythrocyte (IE) Tumor necrosis factor (TNF) is a well-known pro-inflammatory cytokine involved in host immunological responses that elicit diverse cellular responses ranging from proliferation to activation of apoptosis  is involved in signal transmission during the inflammatory response by inducing intracellular calcium rise, activates transcription factors and leads to release of pro-inflammatory cytokines Plasmodium hepatic development is inhibited by TNF TNF was also shown to have effects on anti-microbial activities Plasmodium falciparum might also be modulated by this cytokine. Our data show that TNF is able to reduce P. falciparum parasitemia through a calcium-cAMP downstream signaling with, possibly, PCNA1 as a target. The cross-talk between calcium and cAMP is a well-known mechanism in mammalian cells and has been reported to occur in P. falciparum infected cells P. chabaudiDuring inflammation or infections the production of TNF is increased and the present work evaluated if the intra-erythrocytic stage of 22.1P. falciparum parasites  were cultured in RPMI 1640 (Invitrogen) supplemented with 37.5 mM HEPES, 7 mM D-glucose, 6 mM NaOH, 25/ml gentamicin sulphate, 2 mM L-glutamine and 10% human serum and maintained in human erythrocytes under a gas mixture of 5% O2, 5% CO2, and 90% N2. Previous to culture the erythrocyte suspensions were washed 3 times in RPMI 1640 for 30 min followed by centrifugation at 8000 rpm. The top supernatant layer was then aspirated to remove lymphocytes and platelets. Cultures were maintained, synchronized by using 5% sorbitol and experiments performed 24 h after synchronization. Procedures were approved by the São Paulo University Ethics Committee.2.2P. falciparum (3D7) were incubated for 60 min at room temperature with the fluorescent calcium indicator Fluo-4/AM (5 μM) in MOPS buffer with Ca2 + (2 mM) and 1.8 mM probenecid. The cell suspension was then washed three times 8 cells ml− 1) in a 1 ml cuvette. The fluorescence was measured continuously (acquisition rate: every 0.5 s) for 550 s at 37 °C and TNF , thapsigargin (10 μM) or control added during time course experiments. Saponin isolated parasites were incubated with KN62 (10 μM) for 30 min or A438079 (5 μM) for 2 min followed by addition of TNF (1 ng/ml). Excitation/emission wavelengths adjusted to 505/530 nm for Fluo4-AM.Isolated parasites were obtained by adding saponin (SIGMA) to a final concentration of 0.05% and the mixture was kept on ice for 5 min to lyse the erythrocytes. Following centrifugation at 10,000 rpm at 4 °C for 10 min, erythrocyte ghosts were removed and the free parasite pellets were washed twice using RPMI 1640 for 2 min at 10,000 rpm to remove any insoluble material. Isolated 2.3P. falciparum (3D7) parasites or IE after TNF (1 ng/ml) or PBS treatment for 1 h at 37 °C. Cells were fixed with paraformaldehyde 4% and glutaraldehyde 0.0075% for 30 min at room temperature, washed in PBS 3 times and incubated with DAPI (1:1000 in PBS) and washed 3 more times in PBS. WGA (Wheat Germ Agglutinin) (10 μg/ml) was added for 10 min at 37 °C followed by 3 final PBS washes. Slides were sealed with VectaShield (Vector). Image acquisitions were performed on a confocal microscope and LSM Meta software using Plan-NeoFluor 63 × objective. The samples were excited at 585–615 nm and emission at 650 nm.Immunofluorescence was performed in saponin isolated 2.4P. falciparum line 3D7 incubated for 48 h with TNF  or PBS, and medium replaced every 24 h. IE was then stained with dihydroethidine (5 μg/ml) for 20 min at 37 °C and analysed by dot plots (side scatter versus fluorescence) of 105 cells. Dihydroethidine (DE) was excited with a 488 nm Argon laser and fluorescence emission was collected at 518–605 nm. Parameters subject to adjustment of the FACSCalibur flow cytometer were forward scatter (FSC) , SSC , FL-2 . For all flow cytometry experiments initial gating was carried out with unstained erythrocytes to account for erythrocyte autofluorescence Invasion was assessed using flow cytometry analysis in 2.5ΔΔct or by the threshold cycle in absolute numbers mRNA.Total RNA from IE previously incubated for 1 h at 37 °C with 6-Bnz cAMP (20 μM), TNF (1 ng/ml) or PBS for 1 h at 37 °C were extracted by Trizol followed by DNAse I (20 U) treatment for 30 min at room temperature. RNA (0.5–1 μg) were transcribed using random primers and Superscript II RNAase H reverse transcriptase (Invitrogen). PCR was performed using Platinum Taq polymerase (Invitrogen) for 32 cycles and the visualized on agarose gels. Real-time PCR was performed in triplicate using Power SYBR-green mix (Applied Biosystems). The sequences of synthesized primers used in this work are provided in 2.66 cells per 100 μL used for flow assays.Human dermal microvascular endothelial cells (HDMEC) were obtained from commercial providers (Promocell) and maintained in a manufacturer´s recommended medium. Cells at passages 4–7 were used for all experiments. Prior to experimentation cells were stimulated with TNF at a final concentration of 20 ng/ml overnight. This procedure stimulates the induction of ICAM-1 expression on the surface of the endothelial cells. Confluent HDMEC cells were detached from T25 flask using Accutase followed by addition of HDMEC media. Next, cells were centrifuged at 1200 rpm for 3 min and 1.5 × 102.7Adhesion assays were performed as previously described 2.8ICAM-1 or CD36 (50 μg/ml) in PBS was spotted on 60-mm Petri plates for 2 h at 37 °C in a humidified chamber, and used for binding assays with parasite cultures. PBS and BSA were used as negative controls for binding. These plates were then blocked overnight with 1% BSA in PBS at 4 °C. ItG parasites at 3% parasitemia and 1% haematocrit were treated with FK506  or DMSO (0.04%) for 30 min at 37 °C and added to the spotted plates in binding buffer. The plates were carefully rotated every 10 min for 1 h at 37 °C, and unbound erythrocytes washed away with binding buffer. Bound erythrocytes were fixed in 1% glutaraldehyde for 1h at room temperature and stained with 2% Giemsa for 30 min. Bound IE was counted using a Nikon microscope at 1000 × magnification from six randomly selected distinct fields in triplicate spots from three independent experiments. DMSO treated cultures were used as controls to signify 100% binding.2.9P. falciparum (A4 line) synchronized cultures (at least 3% trophozoites) were incubated with FK506 , calyculin (2 or 10 μM), NFKKB inhibitor (1 or 2 μM) or DMSO (0.04%) for 30 min at 37 °C and washed in RPMI 1640 without serum. The pellet was incubated with Plasmagel for enrichment of trophozoites and washed again. Next, parasites were incubated with the BC6 antibody (mouse IgG mAb directed against PfEMP-1), used at a dilution of 1:50 from 310 μg/ml stock in 1% BSA/PBS for 90 min at 37 °C, washed twice in 1% BSA/PBS, centrifuged 2000 rpm for 5 min and the secondary antibody conjugated goat anti-mouse IgG-APC (1:100) (Thermo Scientific) added followed by ethidium bromide 10 mg/ml (1:1000) in 1% BSA/PBS for 90 min at 37 °C. The cells were washed twice in 1% BSA/PBS, centrifuged 2000 rpm for 5 min and fixed in 0.5% paraformaldehyde/PBS or BD CellFix (BD Biosciences) overnight at 4 °C.For flow cytometry analysis, stained infected cells were diluted in PBS and 50,000 events acquired on a Beckman Coulter FACS XL flow cytometer, gated using forward scatter and side scatter signals to acquire erythrocyte populations excluding debris and FL-1 and FL-2 fluorescence intensity measured. Analysis was performed using FlowJo software. Regions were drawn on density plots of uninfected controls to gate uninfected cells such that < 0.1% of the population fell into the region classed as ethidium bromide positive. This region was then used to create a histogram. Region R1 was created for A4 positive cells based on negative antibody controls, < 0.1% of the ethidium bromide positive population fell into region R1 when no primary antibody was included. The mean fluorescence intensity (MFI) of region R1 was used to quantitate the level of PfEMP-1 on A4 positive infected red blood cells.2.10Results are expressed as mean ± SEM of at least three independent experiments. Student’s t-test was used for comparisons between two groups, whereas for repeated measures ANOVA was used for comparisons among larger groups. P value less than 0.05 were considered indicative of a statistically significant difference. GraphPad Prism software  was used for all statistical tests.3P. falciparum infection but its role during the asexual stage was not described. The present work showed that TNF is of physiological importance in Plasmodium, as it was able to modulate Plasmodium intra-erythrocytic invasion, similarly as previously described in the liver stages TNF expression is known to be highly enhanced in Parasites where incubated with TNF . We analyzed possible changes in parasitemia and intra-erythrocytic development by flow cytometry . Data inP. falciparum parasites. For this purpose, saponin-isolated parasites were incubated with Fluo-4/AM and spectrofluorimetric analyses showed that TNF can increase free intracellular calcium concentration ([Ca2 +]i)  C and D wPlasmodium pathways 2 +/calmodulin-dependent protein kinase II inhibitor and P2X7 antagonist that also blocked [Ca2 +]i increase after TNF addition  among other organelles Extracellular calcium can also be involved in intracellular ion homeostasis so parasites were incubated with EGTA (2 or 5 mM). This was capable of decreasing the calcium ion rise at a concentration of 2 mM, and completely abolished at 5 mM . The parP. falciparum Proliferating-Cell Nuclear Antigen 1 (PfPCNA1), which may be differentially phosphorylated by TNF treatment P. falciparum and calcium is well known to cross talk with cAMP within several cells including malaria parasites P. falciparum not only with TNF but also with 6-Bnz cAMP. According to P. falciparum synchronized trophozoites treated with TNF (1 ng/ml) or 6-Bnz cAMP (20 μM) have a decrease in PfPCNA1 mRNA expression in comparison to control treatments (PBS). The data point to PfPCNA1 as a downstream target of the TNF–calcium–cAMP signaling pathway, perhaps for reducing cell cycle proliferation, but more work is needed to confirm this. Other candidates studied (e.g. PfRACK) showed no change upon TNF treatment  treatment for 1h at 37 °C. Smears are stained with WGA (10 μg/mL for 10 min 37 °C) and DAPI (1:1000 in PBS 15 min RT) and observed by confocal microscopy.Imaging of wheat germ agglutinin (WGA) staining along Fig. S1P. falciparum 3D7 intracellular cell cycle after PBS treatment for 1 h at 37 °C. Smears are stained with WGA (10 μg/mL for 10 min 37 °C) and DAPI (1:1000 in PBS 15 min RT) and observed by confocal microscopy.Imaging of wheat germ agglutinin (WGA) staining along Fig. S2P. falciparum (3D7) control (PBS), TNF (1 ng/mL) or 6-Bnz-cAMP (20 mM), after incubation for 1 h at 37 °C. Bars represent mean ± S.E.M. in 3 independent experiments (P < 0.05*).Expression of mRNA in synchronized trophozoites treated with TNF and 6-Bnz cAMP in PfRACK. Real Time PCR for Fig. S3P. falciparum (3D7) infected erythrocyte after 48 h treatment of (A) uninfected erythrocytes (RBC non), (B) IE without dihydroethidine (DE), (C) control (PBS), (D and E) TNF  and F) overlayed histogram . Gates presented here were used to define the mono (ring-trophozoites) — and multinucleated (schizonts) populations of the parasite and total parasitemia. Dihydroethidine was excited with a 488 nm argon laser and fluorescence emission collected at 518–605 nm.Representative histograms showing flow cytometry analysis of total parasitemia and intra-erythrocytic stage distribution (ring-trophozoites or schizonts) in synchronized Fig. S4Primers used in RT-PCR experiments.Supplemental Table 1The following are the Supplementary data related to this article.The authors confirm that there are no conflicts of interest.Transparency document.Image 1


The aim of the present study was to test whether gene expression of selected inflammatory genes was altered following an n-3 PUFA supplementation and to test for gene–diet interactions modulating plasma inflammatory biomarker levels. (2) Methods: 191 subjects completed a 6-week n-3 FA supplementation with 5 g/day of fish oil. Gene expression of TNF-α and IL6 was assessed in peripheral blood mononuclear cells (PBMCs) using the TaqMan technology. Genotyping of 20 SNPs from the TNF-LTA gene cluster, IL1β, IL6 and CRP genes was performed. (3) Results: There was no significant reduction of plasma IL-6, TNF-α and C-reactive protein (CRP) levels after the 6-week fish oil supplementation. TNF-α and IL6 were slightly overexpressed in PBMCs after the supplementation ), but relative quantification (RQ) within the −0.5 to 2.0 fold are considered as nonbiologically significant. In a MIXED model for repeated measures adjusted for the effects of age, sex and BMI, gene by supplementation interaction effects were observed for rs1143627, rs16944, rs1800797, and rs2069840 on IL6 levels, for rs2229094 on TNF-α levels and for rs1800629 on CRP levels . (4) Conclusions: This study shows that a 6-week n-3 FA supplementation with 5 g/day of fish oil did not alter gene expression levels of TNF-α and IL6 in PBMCs and did not have an impact on inflammatory biomarker levels. However, gene–diet interactions were observed between SNPs within inflammation-related genes modulating plasma inflammatory biomarker levels.(1) Background: A growing body of literature suggest that polymorphisms (SNPs) from inflammation-related genes could possibly play a role in cytokine production and then interact with dietary  Dietary FAs, in particular saturated FAs in addition to n-3 and omega-6 FAs could potentially modulate the expression of genes encoding cytokines, possibly altering plasma cytokine levels as well [Evidence suggests that fatty acids (FAs) can modulate adipokine production, thus accelerating and influencing an individual’s inflammatory response . Omega-3ammation . The res studies ,5. The eet al. suggested that polymorphisms (SNPs) within inflammation-related genes may interact with environmental factors, such as dietary intakes, to modulate an individual’s susceptibility to develop obesity and its comorbidities [tumor necrosis factor α (TNF-α) and interleukin 6 (IL6) may influence obesity phenotypes [n-3 FAs to modulate inflammation [A review by Joffe bidities . Interacammation .n-3 FAs are beneficial for inflammation-related diseases such as rheumatoid arthritis, inflammatory bowel diseases and asthma [n-3 FAs have been shown to decrease expression levels of inflammation-related genes as well as plasma concentrations of cytokine and C-reactive protein (CRP) [n-3 FA supplementation. Previous research from our laboratory has demonstrated changes in inflammatory pathways in human peripheral blood mononuclear cells (PBMCs) after an n-3 FA supplementation via a transcriptomic approach [et al. (2009), have examined the effects of high doses of n-3 FA supplementation on whole-genome gene expression profiles in PBMCs, and reported a decrease in expression of genes involved in inflammatory- and atherogenic-related pathways. Although, according to their results, TNF-α and IL6 gene expression levels were not underexpressed after the supplementation [d asthma . Accordiin (CRP) . Howeverapproach . Similarentation .n-3 FA supplementation and to test for possible gene–diet interactions with SNPs within these genes modulating plasma inflammatory biomarker levels. We hypothesized that a 6-week marine n-3 FA supplementation decreases gene expression of selected inflammation-related genes and also decreases plasma levels of inflammatory biomarkers such as CRP, TNF-α and IL-6 under the influence of SNPs within inflammation-related genes.The aim of the present study was to test whether gene expression of inflammation-related genes is altered following an n-3 FA supplementation (p = 0.006), but the absolute difference remains extremely low at 0.1 kg/m2, with no effects on waist circumference. After the supplementation, TG levels decreased, as expected (p < 0.0001). Population characteristics have been previously described here  for the Pre- and post-supplementation inflammatory marker levels are presented in n-3 FA supplementation (p = 0.003) as shown in p = 0.001) as a result of taking 5 g/day of fish oil supplements while saturated FA intakes were lower with an absolute difference of 3.7 g/day.Energy intakes were lower after the n-3 FA levels  from plasma phospholipids and inflammatory marker levels adjusted for baseline data (both FAs and inflammatory marker levels), age, sex and BMI are presented in n-3 FA levels negatively correlates with CRP , TNF-α , and IL-6 levels . Looking only at plasma EPA levels, a negative correlation was found with TNF-α levels  while trends were observed with CRP and IL-6 levels. Plasma DHA levels tended to be negatively correlated with CRP and IL-6 levels .Partial Spearman correlations between EPA, DHA or total CT−ΔΔ calculation method, TNF-α and IL6 were slightly overexpressed in PBMCs after the 6-week n-3 FA supplementation , but relative quantification (RQ) within the −0.5 to 2.0 fold are considered as non-significant.TNF-LTA gene cluster, 6 SNPS covered 93% of the known genetic variability, for IL6, 5 SNPS covered 100%, for IL-1β, 5 SNPs covered 100%, and for CRP, 4 SNPs covered 100%. All selected SNPs were in HWE and LD plots from Haploview v4.2 for each gene are presented in n-3 FA supplementation, as shown in n-3 FA supplementation while wild type homozygotes decreased theirs. Also, significant differences were observed in the genotype distribution of rs2229094 between positive and negative responders according to delta TNF-α levels where positive responders decreased their plasma TNF-α levels after the supplementation were an increase was observed in negative responders. There was a higher proportion of C/C HMZ that were negative responders (6.8%) vs. positive responders (2.1%) (p = 0.0003). An interaction between rs2229094 (TNF-LTA) and the 6-week n-3 FA supplementation is of particular interest because that SNP is located in an exon and is responsible for an amino acid change (Cys13Arg). Moreover, analyses with SIFT argue for potential functional effect of this SNP as the amino acid change was considered damaged using homologues in protein alignment (score = 0.04), but tolerated using orthologues in protein alignment.In a repeated MIXED model adjusted for the effects of age, sex, and BMI, several gene–diet interactions impacting inflammatory marker levels were observed following the n-3 FAs may interact with SNPs from inflammation-related genes to modulate plasma cytokine levels. There was no reduction of plasma IL-6 and TNF-α as well as CRP levels, but several gene–diet interactions with SNPs within inflammation-related genes and n-3 FAs have been found potentially modulating inflammatory marker levels. These findings are consistent with the well-known anti-inflammatory properties of n-3 FAs, but the amplitude of the results may differ according to an individual’s genotype. In addition to nutrigenetic effects, baseline plasma EPA, DHA or total n-3 FA levels are negatively associated with plasma cytokine and CRP levels  [Ferrucci g others . They obAs only) . Our reset al. have shown in 16 healthy subjects, that the dose-response relationships between n-3 FA, phospholipid composition and cytokine production by PBMCs is U-shaped meaning that an intermediate levels of EPA within plasma and cell membrane phospholipids, resulting from an n-3 FA supplementation of <2.0 g/day, may be associated with a greater inhibitory effect on TNF-α release than higher EPA concentrations resulting from n-3 FA supplementary intakes of >2.0 g/day [et al. have suggested that there is a threshold for an anti-inflammatory effect of EPA somewhere between 1.35 and 2.7 g/day [n-3 FAs used during the protocol  is associated with plasma TNF-α levels. This SNP is of particular interest due to its exonic location and the presence of a missense mutation (Cys13Arg) that is located at a conserved residue. Recent studies have shown that rs2229094 was associated with type 2 diabetes [et al., four functional SNPs of the LTA gene, including rs2229094, may exert possible regulatory effects on gene expression and cytokine production [Moreover, besides the independent influence of dietary FAs on cytokine levels, some SNPs in the pression . In thisdiabetes , CRP levdiabetes , sepsis diabetes , Crohn’sdiabetes , and candiabetes . Accordioduction .TNF-LTA gene family was associated with inflammatory responses. Indeed, we observed an interaction between rs1800629 and n-3 FAs modulating CRP levels where carriers of the mutated allele had significantly higher CRP levels than the wild-type genotype after fish oil supplementation  with adiposity and serum lipid concentrations. The main results of these studies are that TNF −308G>A was associated with an increased risk of obesity and dyslipidaemia, and carriers of the mutated allele appeared to be more responsive to dietary fat intake [et al. reported that the production of the cytokines IL-1β, TNF-α, and IL-6 by mononuclear cells was reduced after the consumption of the low-fat, high-fish diet [Another SNPs in the entation . Song etwith LPS . Studiest intake ,22. Meydish diet .TNF-α transcriptional activity, thus playing a role in the altered TNF-α gene expression possibly leading to an increase in cytokine production [et al. have found that rs1800629 may exert direct effects on TNF-α gene regulation, potentially leading to high TNF-α phenotype  and more severe infection diseases in TNF2 homozygotes [et al. reported that carriers of TNF-α gene −308G>A were more likely to be affected by severe ischemic damage in a case-control study including elderly Italian individuals with and without coronary heart disease [TNF-α gene expression nor in plasma TNF-α levels according to the rs1800629 genotype both in dominant and additive models. Although the relative change in TNF-α levels after the supplementation was not statistically significant between genotype groups owning to the small sample size, the difference was clinically relevant and seems to be in agreement with the actual literature (delta TNF-α→G/G: 28.0% vs. A/G: 3.3% vs. A/A 1.3%). This heterogeneity observed in the response to fish oil could be partly explained by the replacement of arachidonic acids by n-3 FAs into cell membranes leading to the production of diverse eicosanoids. Accordingly, Grimble et al. have suggested that the overall effect on TNF-α production (inhibition or stimulation) probably depends on the balance among the different stimulatory and inhibitory eicosanoids produced from arachidonic acid and EPA [Evidence have shown that carriers of the mutated allele had a 2-fold increase in the oduction . Moreoveozygotes . For exa disease . However and EPA .n-3 FAs in the diet [TNF-α and IL6 genes were slightly overexpressed in PBMCs after the 6-week n-3 FA supplementation, but relative quantification (RQ) within the −0.5 to 2.0 fold are considered not significant. There was no clear effect of the 6-week n-3 FA supplementation on the expression of the two selected inflammation-related genes (IL6 and TNF-α) on a metabolically healthy, but slightly overweight population, even with the use of triplicates to ensure a better reduction in biological variance. These results failed to demonstrate changes in expression levels of TNF-α and IL6 when looking at these two genes specifically using a real-time PCR approach. However, Rudkowska et al. and Bouwens et al. have shown using transcriptomic approaches that inflammation-related pathways in PBMCs were changed to the anti-inflammatory direction after an n-3 FA supplementation [Although gene expression of inflammation-related genes is often decreased following an increase of the the diet ,28,29, sthe diet ,30. In tentation .CT−ΔΔ calculation method assuming that the endogenous control gene and target gene have both similar efficiencies. Also, this study did not allow to isolate the effect of a single FA and its potential gene–diet interactions on inflammatory markers due to the composition of the n-3 FA fish oil capsules given to participants that contained EPA and DHA.Several limitations of the present study need to be addressed. Participants were relatively young (mean age of 30.8 ± 8.7 years) and they had low inflammatory biomarker levels at baseline. The patients were healthy and we excluded participants having plasma CRP levels > 10 mg/L. We did not observe difference in gene expression of inflammation-related genes after the supplementation. This could be attributable to the use of the 22; and (4) being free of pharmacologic lipid lowering treatment and/or metabolic disorders. Subjects who had taken n-3 FA supplements six months prior to the beginning of the study were excluded. A total of 210 subjects completed the supplementation protocol. Individuals with CRP levels >10 mg/L were excluded, bringing the total down to 191 eligible individuals. This experimental protocol #C09-05-030 was approved by the CHU de Quebec ethics committee on 16 September 2009. This clinical trial was registered at clinicaltrials.gov (NCT01343342).A total of 254 unrelated subjects from the greater Quebec City metropolitan area were recruited through emails sent to University students and employees via advertisements in local newspapers. Inclusion criteria were as follow: (1) to be aged between 18 and 50 years; (2) being non-smoker; (3) having a body mass index (BMI) between 25 and 40 kg/mEating Well with Canada’s Food Guide [n-3 FA capsules in sufficient quantity for the next six weeks. They were instructed to take five capsules/d of fish oil , providing a total of 3.0–3.3 g of n-3 FAs including 1.9–2.2 g of eicosapentaenoic acid (EPA) and 1.1 g of docosahexaenoic acid (DHA). Before the run-in period, each participant completed a validated food-frequency questionnaire (FFQ) [n-3 FA supplementation period. Dietary intakes data were analyzed using Nutrition Data system for Research software v.2011 (Nutrition Coordinating Center (NCC), University of Minnesota, Minneapolis, MN, USA).In order to minimize the intra- and inter-variability in dietary intakes, a 2-week run-in period preceded the supplementation. During this run-in period, a registered dietitian gave individual dietary instructions in order to ensure that participants were in a stable condition before the beginning of the study. Participants received recommendations by a registered dietitian in order to follow the recommendations of the od Guide . Particire (FFQ)  supervis2).Body weight, height and waist circumference were measured at every visit in accordance with the Airlie Conference on the Standardization of anthropometric measurements . BMI wasBecause the study design was not intended to look specifically at inflammation, only a small subset of inflammatory biomarkers were available for this reanalysis. Blood samples were collected after a 12 h overnight fast and 48h alcohol abstinence, from an antecubital vein into vacutainer tubes containing EDTA. Plasma CRP was measured by nephelometry  using a sensitive assay, as described previously . Plasma g for 6 min and stored at −80 °C for subsequent analyses. Plasma lipids were extracted with chloroform:methanol  according to a modified Folch method [v/v) [n-6  and C22:5n-3 . Finally, phospholipids FA profiles were determined using the relative percentage areas of total FAs.FA composition of plasma phospholipids was determined by gas chromatography. Venous blood was drawn into EDTA tubes, then separated by centrifugation at 500 h method . Total ph method . Isolateh method . FA profod [v/v)  and capiTNF-LTA gene cluster (6 SNPs), IL6 (5 SNPs) and CRP (4 SNPs) were identified using the International HapMap Project SNP database, based on the National Center for Biotechnology Information (NCBI) B36 assembly Data Rel 28. phase II + III, build 126. The TNF-LTA gene cluster is made of two genes that are located very close to each other on chromosome 6 . LTA is also referred to as member 1 of the TNF-superfamily. Five hundred kilo-base pairs (kbp) downstream of each gene and 2500 kbp upstream of each gene were added to cover the 5′UTR and 3′UTR regions. Gene Tagger procedure in Haploview v4.2 was used to determine SNPs using a minor allele frequency (MAF) ≥ 5% and pairwise tagging (r2 ≥ 0.8). Subsequently, the linkage disequilibrium (LD) was examined for all SNPs using the LD Plot procedure in Haploview v4.2. The SIGMA GenElute Gel Extraction Kit  has been used to extract genomic DNA. Selected SNPs of the TNF-LTA gene family , IL6 , IL1β  and CRP  have been genotyped using validated primers and TaqMan probes  [SNPs in the MA, USA) . DNA wasIL6 and TNF-α genes was measured following the 6-week n-3 FA supplementation. Complementary DNA (cDNA) was mixed with TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) and a gene-specific primer and probe mixture in a final volume of 20 μL. All samples were run on a 7500 Fast Real Time PCR System (Thermo Fisher Scientific) using the following thermal cycling profile: 50 °C (2 min), 95 °C (10 min), followed by 40 steps of 95 °C for 15 s and 60 °C for 60 s. The real-time PCR data were imported into Expression Suite Software v1.0. These samples were analysed in triplicate and calibrated to the GAPDH gene . Relative quantification estimations were achieved using the 2CT−ΔΔ calculation method [Gene expression of n method .(10)-transformed, negative reciprocal or normalised by a Box-Cox transformation (TNF-α) before analysis. Subjects were categorised as positive (Δ < 0%) or negative (Δ ≥ 0%) responders based on their relative change of plasma TNF-α, CRP or IL-6 levels after the supplementation. The MIXED procedure for repeated measures was used to test for significant differences in metabolic characteristics between men and women at baseline and for differences between various nutrient intakes prior to and after n-3 FA supplementation and to test for the effects of the genotype, the supplementation and the genotype by supplementation (time) interaction on inflammatory marker levels when age, sex and BMI were included in the model. Partial Spearman correlations were calculated between EPA, DHA or total n-3 of plasma phospholipids  and inflammatory markers. The ALLELE procedure was used to verify the departure from Hardy-Weinberg equilibrium (HWE) and to calculate the minor allele frequency. Individuals with CRP levels > 10 mg/L were excluded bringing the total to 191 eligible individuals. SIFT web-based software was used to predict the effect of amino acid substitution and all tests were run under default threshold values. Finally, the FREQ procedure was used to verify differences in genotype frequency distribution between positive and negative responders to the n-3 FA supplementation. Statistical significance was defined as p ≤ 0.05.This study is a reanalysis of the Fatty Acid Sensor (FAS) Study that primarily wanted to understand how genes and environment act together on the cardiometabolic risk profile. Data were analyzed with SAS Genetics v9.3. Values that were not normally distributed were logn-3 FA supplementation with 5 g of fish oil daily did not alter gene expression levels of TNF-α and IL6 in PBMCs and did not have an impact on inflammatory biomarker levels. However, significant gene–diet interactions were observed between SNPs within inflammation-related genes modulating plasma inflammatory biomarker levels. These gene–diet interactions may potentially explain the large inter-individual variability observed in plasma inflammatory response following an n-3 FA supplementation.Overall, this study shows that a 6-week


Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6–DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6–DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6–DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6–DnaC6 complexes with excess DnaC . Thus, there are two mechanisms by which an imbalance in the replicative helicase and its associated loader protein can inhibit genome duplication.Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The   •Loading of the replicative helicase is the key step in replisome assembly.•E. coli inhibits growth.Increasing replicative helicase concentration in •Inhibition is due to helicase complexes depleted of the helicase loader protein.•Depletion inhibits replication initiation and reinitiation during replication repair.•Imbalances in replicative helicase components can prevent replication initiation. The structural complexity of the genetic material of a cell demands complex copying systems to achieve high-fidelity DNA replication. Replicative helicases are at the leading edge of replication forks, both driving strand separation and acting as a protein interaction hub at the heart of the replisome Escherichia coli, the initiation of replication starts with binding of oriC by the ATP-bound initiator protein DnaA that leads to melting of the DNA duplex and creation of a ssDNA bubble 6–DnaC6 complex that cannot translocate The replicative helicase needs to be loaded at an origin of replication before it can begin to unwind DNA. In E. coliE. coli, this minimisation of nucleoprotein-induced fork pausing by Rep is promoted by a physical interaction between the Rep C-terminus and DnaB Once a replication fork is translocating along the template DNA, then potential barriers such as DNA damage or nucleoprotein complexes are encountered frequently oriC, requires DnaC oriC to overcome this SSB-dependent barrier. In E. coli, two pathways that facilitate reloading of the replisome back onto the chromosome away from oriC exist Replisome inactivation still occurs in spite of mechanisms that reduce the probability of loss of replisome function E. coli has a modest inhibitory effect on chromosome duplication. This inhibition is alleviated upon co-overexpression of the helicase loader DnaC indicating that a reduction in the concentration of DnaB6–DnaC6 complexes, needed for DnaB loading during replication initiation and reinitiation oriC or in PriA-dependent replication reinitiation away from oriC act synergistically to increase the toxicity of DnaB overexpression. Therefore, overexpression of the replicative helicase impacts on replisome assembly at and away from oriC. However, absence of PriC does not hypersensitise cells to excess DnaB indicating that, in otherwise unstressed cells, PriA-directed fork repair predominates or that PriC-specific substrates can be targeted by PriA either with or without further processing of the forked DNA. The inhibitory effect of excess DnaB is also exacerbated in cells lacking the accessory replicative helicase Rep that correlates with the elevated replication blockage and thus replication reinitiation needed in Δrep cells 6–DnaC6 complex formation via excess DnaC 6–DnaC6 complex formation via excess DnaB as shown here. Such imbalances could conceivably result in replicative stress regardless of the organism.We have found that overexpression of DnaB in E. coli, we constructed an arabinose-inducible overexpression plasmid encoding dnaB that increased intracellular levels of DnaB in the presence of arabinose  in dnaC(R10P) downstream of dnaB failed to alleviate inhibition of growth by dnaB in Δrep cells  and rpoB(H1244Q) rpo mutations suppressed the toxicity of dnaB overexpression in Δrep cells (dnaB induction was greater for rpoB(G1260D) than for rpoB(H1244Q)  or that increase the need for initiation (Δrep) therefore hypersensitise cells to dnaB overexpression. Synergies with Δrep, priA300 or priB could potentially be explained by aberrant binding of excess DnaB onto the chromosome leading to inhibition of the elongation phase of chromosome duplication. Such DnaB binding could promote replisome blockage by forming nucleoprotein barriers and/or catalysing harmful unwinding of DNA structures. However, binding of excess DnaB to double-stranded regions of the chromosome is unlikely since DnaB cannot load onto double-stranded DNA either in the absence or in the presence of DnaC dnaB overexpression  to suppress DnaB-induced growth defects  and away from oriC (priA300 and ΔpriB)  . Partialrep cells . The PCR product was digested with HindIII and XmaI and Klenow treated before ligating into NcoI- and XmaI-digested and Klenow-treated pBAD dnaB (pJGB143) bearing kanamycin resistance and an arabinose-inducible dnaB gene.Full-length dnaBC was generated by inserting full-length E. coli dnaC downstream of dnaB into pJGB143 digested with PstI and HindIII, giving pJGB404. For this, dnaC was amplified from the genome with a forward primer containing a PstI site plus a 6-bp Shine–Dalgarno sequence up to the Kozak sequence from pBAD24 dnaC_FW.1) and a reverse primer containing a HindIII site (dnaC_RV.1). pBADdnaC (pJGB408) is derived from pJGB404 by excising dnaB via EcoRI digestion and religation of the 7.1-kb fragment.pBADDnaC was also cloned into pSK(−) as a EcoRI-BamHI fragment so that dnaC is inserted in the opposite orientation with respect to the promoter. For this, dnaC was amplified using a forward primer with EcoRI and NdeI sites (dnaC_FW.2) and a reverse primer with a BamHI site (dnaC_RV.2), creating pSK(−)dnaCinv (pPM202). pJGB412 is pSK(−) encoding dnaC(R10P)inv generated by site-directed mutagenesis of pPM202 using complementary 23-mer forward and reverse primers containing a g29c mismatch flanked by 11 bp of the dnaC wild-type sequence [dnaC(R10P)_FW and dnaC(R10P)_RV]. dnaC(R10P) from pJGB412 was PCR amplified with the same primers used for pJGB404, digested with EcoRI and HindIII and cloned into pJGB408 digested with EcoRI and HindIII to generate pBADdnaC(R10P) (pJGB415). pBADdnaBC(R10P) was generated as for pJGB404 except that the mutant dnaC from pJGB412 was used as a PCR template, resulting in pJGB418.All strains used are listed in − 1 NaCl supplemented with kanamycin (30 μg ml− 1) overnight. Growth was performed at 37 °C with the exception of the strains used in dnaA46 allele. Serial dilutions of overnight cultures were made in 56/2 salts from 10− 1 to 10− 6 and were spotted on LB kanamycin (30 μg ml− 1) agar containing 0%, 0.02% or 0.2% arabinose as indicated. The plates were incubated at 37 °C or 30 °C, as indicated, for 16 h.Strains containing pBAD derivatives were grown in liquid LB containing 5 g l− 1 kanamycin until the A650 reached 0.4. Ten millilitres of each culture was transferred to a 50-ml Falcon tube and glucose or arabinose was added to each culture to a final concentration of 0.2%. Tubes were incubated at 37 °C with shaking for 2 h then cells were centrifuged for 10 min at 3200g at 4 °C, and the supernatant was discarded. Cell pellets were resuspended in lysis buffer  and sonicated five times for 10 s. Lysed cells were then centrifuged for 15 min at 16,000g at 4 °C and the supernatant was transferred to a new tube and kept on ice overnight. Protein cell extracts were analysed using 12.5% SDS polyacrylamide gels. Protein extract (2 μg) was loaded per lane. NEB Color Prestained Protein Standard, Broad Range (11–245 kDa) and DnaB and DnaC, purified as previously described Strains containing pBAD plasmids were grown at 37 °C in LB containing 30 μg ml− 1 kanamycin with shaking at 37 °C until an A650 of 0.4 was reached. Cultures were then diluted to an A650 of 0.01 into fresh LB plus 30 μg ml− 1 kanamycin and 0.2% arabinose and growth was continued for 2 h. Afterwards, rifampicin and cephalexin were added to 100 μg ml− 1 and 15 μg ml− 1, respectively, and the cells were grown for another 2 h at 37 °C. Samples were then processed as in Ref. Culture samples for flow cytometry were grown in LB plus 30 μg mldnaB were grown in 10 ml LB supplemented with 30 mg ml− 1 kanamycin to an A650 of 0.4 at 37 °C prior to diluting to an A650 of 0.01 in 10 ml LB with 30 mg ml− 1 kanamycin without and with 0.2% arabinose. Incubation at 37 °C continued for about 2–3 h until the cultures reached A650 = 0.4, when 1 ml of culture was centrifuged. The pellet was resuspended in 400 μl 56/2 salts. The nucleoids were visualised by staining in 10 μg ml− 1 4′,6-diamidino-2-phenylindole (DAPI) for 5 min and laid on 1% agarose pads containing 56/2 salts. Microscopy was performed on a Zeiss Axioskop2 equipped with a QICAM Fast 1394 camera (QIMAGING) and a DAPI (49) filter set (Zeiss).Strains containing pBADdnaC(R10P).List of PCR primers used in this study. Sequences homologous to target DNA are underlined. Bold letters highlight the mutated base pair in Strains (TB28 derivatives).The following are the supplementary data related to this article.


Through binding to and activation of two receptors, TNF-R1 and TNF-R2, Tumor Necrosis Factor α (TNF) modulates various biological processes in the immune system. A growing body of evidence in the literature indicates that TNF may behave as a tumorigenic cytokine, facilitating cancer cell immune escape and tumor progression. We have recently provided evidence that host TNF signaling impairs CD8+ T cell-dependent immune response against melanoma. TNF blockade (with Etanercept) or deficiency (TNF knock out) facilitates the accumulation of CD8+ Tumor-Infiltrating Lymphocytes (TILs), thereby limiting the growth of melanoma cell lines, which express Major Histocompatibility Class 1 molecules (MHCI) at high levels. Similar findings were observed in mice lacking TNF-R1, but not TNF-R2, indicating that host TNF-R1 plays a critical role in limiting the establishment of such a CD8+ T cell-dependent immune response against melanoma under our experimental conditions [Listeria monocytogenes is not compromised in mice lacking FAN [We and others have documented that the factor associated with neutral sphingomyelinase activation (FAN), which is an adaptor protein of the TNF-R1 , enhanceking FAN .One of the main mechanisms that likely accounts for the TNF-dependent immunosuppression in our experimental mouse melanoma model is the capacity of TNF to trigger cell death of activated CD8+ T cells. Whereas TNF potently induced cell death of activated CD8+ T cells as documented by others, it remained unclear which TNF receptor is required for this process. On the one hand, activated CD8+ T cells expressed TNF-R2, which mediated activation-induced cell death . On the Our recent findings demonstrate that TNF-R1-dependent TNF signaling impairs CD8+ TIL content and likely constitutes a potent immune checkpoint in melanoma, facilitating melanoma progression. Targeting either TNF or TNF-R1 may reduce the death of activated CD8+ TILs in patients affected with melanoma and thus may constitute an emerging immunotherapy to enhance CD8+ T cell-dependent immune response against melanoma. Clinical trials are necessary to evaluate the therapeutic value of TNF blockade strategy in melanoma patients and define the eligibility criteria such as (i) TNF expression in melanoma biopsies, (ii) pre-existing CD8+ T cell infiltration, (iii) MHCI expression by melanoma cells.


Cytotoxic T lymphocytes (CTL) are a major factor in the control of HIV replication. CTL arise in acute infection, causing escape mutations to spread rapidly through the population of infected cells. As a result, the virus develops partial resistance to the immune response. The factors controlling the order of mutating epitope sites are currently unknown and would provide a valuable tool for predicting conserved epitopes. In this work, we adapt a well-established mathematical model of HIV evolution under dynamical selection pressure from multiple CTL clones to include partial impairment of CTL recognition,  Like many viruses, HIV has evolved mechanisms to evade the host immune response. As early as a few weeks after infection is initiated, mutations appear in the viral genome that reduce the ability of cytotoxic T lymphocytes (CTL) to control virus replication. However, of the many mutations in the viral genome that could potentially mediate viral escape from the CTL response, a specific subset are typically observed. This suggests that some mutations either entail too high a fitness cost for the virus, or are relatively inefficient escape mutations. A successful vaccine would target the CTL response to these regions in such a way that escape would not be possible. We use a computational model of HIV infection in order to study the factors that determine whether a given escape mutation will occur, how long it will be maintained in the population, and how these changes in the viral genome will affect the CTL response. Our analysis highlights the important role of partial recognition loss conferred by a mutation in producing the complex dynamics of escape that are observed during the course of infection. HIV replication continues for years despite a highly active immune response. Depletion of cytotoxic CD8+ T cells (CTL) in SIV infected animals causes rapid increase in viremia n). It can be inferred that escape mutations come at a cost from the observation of occasional reversion of escape mutations upon transmission between MHC mismatched individuals Mathematical models of HIV evolution in the presence of multiple CTL clones have been applied to study the emergence of late escape mutations In the present work, we address the process of antigenic escape in terms of a cost-benefit diagram. The benefit of an escape mutation is a partial CTL recognition loss and the mutation cost is a partial reduction in viral replication rate. We extend the basic model introduced by Althaus and De Boer Furthermore, the inclusion of partially effective escape mutations in the model can reproduce the diverse patterns of intra-epitope escape that are routinely observed in HIV infected patients. During the majority of escape mutations that have been studied with time-resolved viral sequencing, 2–10 distinct epitope sequences grow in number to replace the transmitted sequence and eventually one mutant epitope spreads to the entire population. Furthermore, the dominant mutated epitope sequence changes over time In order to study escape from the immune response we consider a model that includes target cells, infected cells and multiple CTL clones which recognize regions in the viral genome (epitopes) with equal avidities , as folln, and on the degree of recognition loss per mutation, The dynamics of infected cells and CTL during the escape phase (Phase 3) depends on the number of epitopes that are targeted, tation,  . In the Escape mutations begin to be selected once CTL reach sufficiently high levels  in response to CTL pressure. In In order to study intra-epitope dynamics, we use the main model  to 8 andrameter  . We consOnce an escape haplotype has reached a sufficiently high frequency, CTL clone pe rates –4, and tThe simple pattern of escape  can occuThe leapfrog pattern can occur if haplotype 01 gains the advantage over 10 before the 10 loses the advantage over 00 . In ordeThe CTL clone to the escaping epitope decays at a faster rate during the time period when variant 10 dominates the population than than during the period when 01 dominates the population. The difference is due to the relatively higher recognition loss of the mutation at the first site inset . The exaWhen both single-mutant haplotypes continue to have a positive growth rate throughout the course of infection, as in the case of constant selection pressure, the double-mutant haplotype has the highest escape rate . The nesn, the escape rate for the first haplotype, We use numeric computation to determine which pattern of escape is observed over a range of recognition and fitness losses at each of the two epitope sites. The number of epitopes is fixed, nfection  or chronnfection   The proliferation rate of CTL clones saturates with the infected cell number, but not with the total CTL level. In the original model Thus, our model has demonstrated that partial recognition losses, in addition to fitness costs and the breadth of the CTL response, dramatically affect the rate and the order of escape mutations during an HIV infection. These findings help to interpret the positive correlation between fitness costs and recognition losses observed in the Pol gene i and the relative CTL recognition of sequence i by clone j, as compared to the transmitted sequence, respectively:We model SIV/HIV evolution in the presence of multiple CTL clones . Each CTT) are replenished at a rate β, and the number of productively infected cells n epitopes, each consists of m amino acid positions (sites), giving a total of i with n epitopes denoted m sites i is reduced by mutations. A mutation in epitope j at site k contributes cost The processes described are, as follows. Highly infectable target cells . Several authors have argued against single-compartment models for the following reasons: (i) Although viremia and CTL levels vary strongly among patients, the decay rate of viremia under anti-retroviral therapy (ART)  between CD8+ depleted and control groups −1 for the total CTL response, was obtained by Wick et al invitro expanded, CD8+ T cells to 2.5% of total CD8 count. Wick et al then directly quantified the decay rate of virus-expressing cells. The estimate they obtained is 4–10 fold higher than the rate of viremia decay under ART, indicating a short virus producing phase of 2–8 hours. The estimate agrees with those obtained by modeling data from acute SIV infection and HIV dynamics during ART The total CTL killing rate in our model is given by −1, which is several-fold smaller than the CTL killing rate in steady state −1  does not change the dynamics of escape.We also introduce the virus-induced infected cell death rate, i in the epitope that have escaped. Here parameter We introduce a simplified model, which does not explicitly consider dynamics, in order to study the sequence of escape mutations for a realistic size genome. Fractional fitness costs Click here for additional data file.Figure S2Estimating the relationship between  and  approximately from three published experiments. Our model contains one parameter for CTL recognition loss caused by a mutation, (TIF)Click here for additional data file.Figure S3CTL lifetime changes the frequency with which intra-epitope escape patterns are observed. Here we show a modified version of he model  to 8 witpfrog” , , “nestedrog” , , , “nested pattern  is obser(TIF)Click here for additional data file.Text S1Steady state and the escape rate.(PDF)Click here for additional data file.Text S2Escape causes contraction of CTL clones.(PDF)Click here for additional data file.Text S3General conditions for observing the leapfrog pattern of escape.(PDF)Click here for additional data file.Text S4Simulation: Leapfrog pattern can occur in a broad range of fitness and recognition losses.(PDF)Click here for additional data file.Text S5Finding the relationship between  and HLA binding loss from three different experiments.(PDF)Click here for additional data file.


Cowpea chlorotic mottle virus (CCMV) particles as a scaffold for epitope presentation and identify sites for epitope fusion or insertion that would not interfere with virus-like-particle formation, chimeric CCMV coat protein (CP) gene constructs were engineered, followed by expression in E. coli and assessment of VLP formation. Various constructs were made encoding a 6x-His-tag, or selected epitopes from Influenza A virus [IAV]  or Foot and Mouth Disease Virus [FMDV] (VP1 and 2C). The epitopes were either inserted 1) in predicted exposed loop structures of the CCMV CP protein, 2) fused to the amino- (N) or carboxyl-terminal (C) ends, or 3) to a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein.Within the last decade Virus-Like Particles (VLPs) have increasingly received attention from scientists for their use as a carrier of (peptide) molecules or as scaffold to present epitopes for use in subunit vaccines. To test the feasibility of N and GC glycoproteins of the recently emerged Schmallenberg orthobunyavirus at both termini of the CP protein.High levels of insoluble protein expression, relative to proteins from the entire cell lysate, were obtained for CCMV CP and all chimeric derivatives. A straightforward protocol was used that, without the use of purification columns, successfully enabled CCMV CP protein solubilization, reassembly and subsequent collection of CCMV CP VLPs. While insertions of His-tag or M2e (7-23 aa) into the predicted external loop structures did abolish VLP formation, high yields of VLPs were obtained with all fusions of His-tag or various epitopes (13- 27 aa) from IAV and FMDV at the N- or C-terminal ends of CCMV CP or N∆24-CP. VLPs derived from CCMV CP still encapsulated RNA, while those from CCMV CP-chimera containing a negatively charged N-terminal domain had lost this ability. The usefulness and rapid ease of exploitation of CCMV VLPs for the production of potential subunit vaccines was demonstrated with the synthesis of chimeric CCMV VLPs containing selected sequences from the GCCMV VLPs can be successfully exploited as scaffold for epitope fusions up to 31 aa at the N- and C-terminus, and at a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein. Virus-like particles (VLPs) have become recognized as ideal biopolymers for various applications within Bionanotechnology , amongstBromovirus genus within the family of Bromoviridae. It consists of spherical particles, approximately 28 nm in diameter and capsid entirely composed of a single coat protein (CP) of about 20 kDa ) was fused at the N-terminus of CCMV CP. Another, negatively charged E-coil (EIAALEK[3]) sequence, able to heterodimerize to the K-coil, was fused to eGFP. Mixing of K-coil-CP in a certain ratio with CCMV CP subunits, enabled the encapsulation of 15 E-coil-eGFP molecules during reassembly of CCMV VLPs. Although this strategy allows encapsulation of very large molecules, due to volume constraints of the core, the number of these will be limited.While data presented in this study indicate that VLPs from NΔ24-CP present one of the most interesting scaffolds for epitope display, it is highly unlikely that very large sequences can be fused to its N-terminus without disrupting VLP formation. Recently another strategy has been developed  that migWhether (CCMV-derived) VLPs present the best and most widely applicable nanoscaffolds for various purposes, or others like DNA origami structures  turn outN and GC), although their real potential in the application for vaccine purposes still has to be demonstrated.In this study we have demonstrated the feasibility of CCMV-based VLPs as scaffold for epitope presentation, but limited to fusions of peptide sequences to the N and/or C-terminus of CCMV CP or the N-terminus of a mutant lacking the first 24 N-terminal aa residues (NΔ24). Several CCMV CP chimeras have been constructed that resulted in the formation of VLPs containing various  viral epitopes from IAV (HA and M2e), FMDV (VP1 and 2C) and SBV , which underlined their appropriateness and potential for medical applications .NcoI and HindIII restriction sites for feasible cloning purposes. Based on the crystal structure of the CCMV CP subunit [SpeI and SstI restriction sites, and subsequent cloning of a His-tag adaptor into the loops. Constructs containing terminal-end fusions with a His-tag were engineered using a two-step PCR  using Phusion DNA polymerase (Promega) and a primer set to introduce  subunit , four diPCR Fig.  during wrs Table . Since Ites Fig. . A T-celtes Fig.  and clonE. coli BL21 (DE3). A 100 ml culture containing 50 mg/l kanamycin was freshly inoculated with 1 ml of the overnight culture and grown at 37 °C until an OD600 of 0.6 was obtained. Bacteria were induced for protein expression with 0.25 mM IPTG and incubated at 37 °C for another 4 hours. Then, cells were pelleted by centrifugation at 5000 rpm for 15 minutes at 4 °C and stored at -20 °C until further analysis.Constructs were transformed into E. coli lysates. To this end, a disassembly-reassembly process was carried out entirely at 4 °C, as follows: E. coli cells induced and incubated overnight were pelleted and resuspended in 5 ml of disassembly buffer . Next, cells were lysed by continuous ultra-sonication  during 3 rounds of 1 min at 40 % amplitude (output 6). The lysate was centrifuged at 5.000 rpm for 15 min and the insoluble fraction resuspended in 10 ml of disassembly buffer containing 8 M urea and again sonicated but this time at 20 % amplitude using pulses of 10 seconds until the pellet was entirely dissolved. Thereafter another 15 ml of disassembly buffer was added and mixed with the sonication liquid, the sample was centrifuged at 15,000 rpm for 15 min. The supernatant was collected and dialyzed 6 times against dialysis buffer (0.01 M Tris-HCl pH7.4). The soluble CCMV CP fraction was finally reassembled into VLPs during 2 dialysis steps against reassembly buffer  and concentrated by ultracentrifugation (Beckman) on a 30 % sucrose cushion at 40,000 rpm for 3 hours. The pellet was resuspended in 1-2 ml of virus buffer  and stored at 4 °C until further analysis.CCMV VLPs were re-assembled from insoluble CP aggregates collected from Bacterial cell lysates containing expressed (chimeric) CCMV CP protein or samples of purified VLPs were resolved onto a 15 % SDS-PAGE gel. Proteins were transferred onto Immobilon P membrane  and screened with anti-CCMV polyclonal crude antiserum (1:1000), anti-His (1:1000) and anti-M2e monoclonal antisera . For VP1 and 2C epitopes, a serum from FMDV-infected guinea peg  was used (1:500 crude serum).in vitro reassembly and sucrose cushion were diluted 10x in virus buffer and 10 μl placed on 400 mesh copper grids  for 2 min followed by negative staining. For immuno-electron microscopy [The reassembly of all CCMV CP proteins into VLPs was verified by transmission electron microscopy. To this end, protein samples collected after croscopy  10 μl ofA major safety issue related to the exploitation of VLP-based systems for vaccine purposes deals with the presence/absence of genetic material within VLPs. To analyze the presence of mRNA transcribed from the pET-28-CCMV CP-chimera plasmids and encapsulated in VLPs, 200 μl purified VLP suspensions from CCMV CP, NΔ24-CP, M2e(23)NΔ24-CP, HA-NΔ24-CP, VP1-NΔ24-CP, 2C-NΔ24-CP, VP1-CP, 2C-CP, CP-HA and CP-M2e(23) was subjected to a total RNA isolation protocol using Trizol according to the manufacturer’s instructions (Invitrogen). As a negative control, a sample of IPTG-induced BL21 cells containing empty pET-28 plasmid DNA was included. To detect CCMV-CP-specific RNA molecules transcribed from pET28, RT-PCR was performed on total RNA purified from VLPs using specific primer sets  at pH 7.4 and 4.8, corresponding to buffer conditions reflecting the CCMV CP disassembly and CCMV VLP reassembly steps as described above.


It has been suggested that neuronal hyperexcitability contributes to Alzheimer’s disease (AD), so we asked how hyperexcitability develops in a common mouse model of β-amyloid neuropathology - Tg2576 mice. Using video-EEG recordings, we found synchronized, large amplitude potentials resembling interictal spikes (IIS) in epilepsy at just 5 weeks of age, long before memory impairments or β-amyloid deposition. Seizures were not detected, but they did occur later in life, suggesting that IIS are possibly the earliest stage of hyperexcitability. Interestingly, IIS primarily occurred during rapid-eye movement (REM) sleep, which is notable because REM is associated with increased cholinergic tone and cholinergic impairments are implicated in AD. Although previous studies suggest that cholinergic antagonists would worsen pathophysiology, the muscarinic antagonist atropine reduced IIS frequency. In addition, we found IIS occurred in APP51 mice which overexpress wild type (WT)-APP, although not as uniformly or as early in life as Tg2576 mice. Taken together with results from prior studies, the data suggest that surprising and multiple mechanisms contribute to hyperexcitability. The data also suggest that IIS may be a biomarker for early detection of AD. Alzheimer’s disease (AD) is a devastating and progressive disease where few treatments are successful23In this study, we investigated a widely used mouse model, Tg2576 mice, where mice overexpress human amyloid precursor protein (APP) with the mutation found in a familial form of AD in Sweden (APPSwe)Using the Tg2576 mouse at a very young age (5 week-old), long before β-amyloid deposition811We clarify mechanisms contributing to IIS, by demonstrating that a muscarinic cholinergic receptor antagonist reduces IIS, which is surprising in light of the literature suggesting cholinergic function gradually deteriorates in AD. We also show that nicotinic antagonists do not have this effect, and the antiseizure drug levetiracetam reduces IIS. Furthermore, mice with overexpression of WT-APP have some IIS or IIS-like events in their EEG, although not as uniformly or as young as 5 week-oldTg2576 mice. We suggest that multiple mechanisms contribute to IIS and that IIS could be useful as an early biomarker.We performed simultaneous video-EEG using cortical  and bilateral dorsal hippocampal electrodes in freely behaving Tg2576 and WT littermates. We began recording at 5 weeks of age, prior to the earliest behavioral deficits that have been reported , quiet wakefulness , non-REM (NREM) sleep or REM sleep . The cla18Although the Tg2576 mouse has elevated Aβ levels at young ages , the rate-limiting enzyme for ACh synthesis in neurons. ChAT-immunoreactivity (ir) was high in Tg2576 mice at 1 and 4 months of age compared to 14 months of age, which was particularly clear in the hippocampus, possibly due to the laminar organization of the primary cholinergic input, the septohippocampal projection . Based o18Interestingly, genotypes were similar in the dorsal striatum, where cholinergic neurons are intrinsic. Thus, in dorsal striatum, there were no genotypic differences in the number of cholinergic neurons or mean pixel density  in the s32Because the MS makes a major contribution to hippocampal, cortical and RSC ChAT-irConsidering the data suggesting that MS neurons were not deteriorating, but could be more active in young Tg2576 mice than WT mice, we determined whether antagonists of cholinergic receptors would reduce IIS frequency. We also examined the effect of donepezil, a cholinesterase inhibitor that is used to treat AD. Levetiracetam was also tested, because it is an anti-seizure medication that has been previously shown to reduce epileptiform activity in a mouse model of β-amyloid neuropathology We used a 3 day-long experimental design with continuous video-EEG . After tThe muscarinic antagonist atropine suppressed IIS in the first 4 hours after administration compared to the first 4 hours after saline injection . However36The nicotinic antagonist mecamylamine induced a slight but significant increase in IIS frequency during the first 4 hours after drug injection . In contInterestingly, the cholinesterase inhibitor donepezil did not significantly affect IIS . These dThroughout the pharmacological experiments we noticed that some Tg2576 mice had particularly high IIS frequencies before drug administration. These animals appeared to be the mice that had the greatest reduction in IIS by atropine. To quantify this relationship, we asked if there was a positive correlation between the effect of drug and the IIS frequency prior to drug administration. Indeed, there was a significant positive correlation . In cont2039By investigating neuronal hyperexcitability in young Tg2576 mice prior to β-amyloid deposition, we found that IIS occur at just 5 weeks of age, confirming a prior studyin vivo hyperexcitability that has been identified in these mice. However, there are many abnormalities that have been reported in Tg2576 mice, although most studies do not examine mice at 5 weeks of age. At 2 months of age, there is a decline in spines of the principal cells of the dentate gyrus and decreased long-term potentiation (LTP) of the major afferent pathwayA receptor antagonists or convulsantsin vivo in the current study are not reproduced in slices. Instead, IIS may require behavioral states (e.g. sleep) or projections that span long distances (e.g. cholinergic).Our data show that large amplitude fast transients similar to IIS in animals with epilepsy occurred early in life in Tg2576 mice, and corroborate a recent studyOther early changes that have been reported in young Tg2576 mice include impaired olfaction at 3–4 months of ageConsidering the evidence that β-amyloid deposition begins at approximately 6 months of age in Tg2576 mice40434445In this context it is interesting to consider that one of the regions where Aβ40 and Aβ42 were detected in our earlier study of 2 month-old Tg2576 mice was the brainstem505153Notably, IIS were recorded synchronously from both hemispheres using cortical and hippocampal recording sites. Therefore, IIS reflect synchronized activity in large areas of the neocortex and hippocampus. Cholinergic pathways are good candidates to entrain widespread areas of the cortex and hippocampus because of their diffuse projections. One that is relevant because of its role in sleep is the cholinergic projection of the lateral dorsal tegmentum and pedunculopontine nuclei in the brainstem which innervate areas of the midbrain reticular formation, thalamus, and the forebrain. Another cholinergic projection - one that has been extensively implicated in AD - is from the forebrain cholinergic nuclei (including the MS), with neurons that also have widespread projections to cortical regions. Our data support the idea that these cholinergic projections are important because increased ChAT-ir was evident in the areas corresponding to the axon terminals at only 4 months of age and there was an increase in c-fos-ir in an area corresponding to the cell bodies, the MS. Our findings are consistent with a prior study showing greater vesicular acetylcholine transporter (VAChT)-labeled cholinergic boutons in frontal cortex of Tg2576 mice at 8 months of ageBased on these considerations, we suggest that IIS occur early in life and are rare initially. As IIS become significant in the first months of life, an increase in cholinergic tone contributes to IIS, possibly caused by initial elevations in Aβ. IIS are initially specific to REM, presumably because of the increased release of ACh during this sleep stage. By divergent projections to hippocampus and cortical regions, ACh could facilitate synchronized discharges in these locations – manifested as an IIS. Muscarinic receptors appeared to be important to IIS, but additional mechanisms are likely to contribute based on pharmacology and data from APP51 mice. Later in life as Aβ levels and other abnormalities emerge, the data suggest that IIS become variable rather than continuing to escalate .In the preceding discussion we consider IIS to reflect abnormal excitability, consistent with the underlying cellular correlate of IIS, a sudden and synchronous depolarization in principal neurons . In contrast to the idea that IIS are a reflection of hyperexcitability, interictal IIS in epilepsy may abort or inhibit seizuresRegardless of the consequences of IIS on spontaneous seizures, there is good reason to believe that IIS adversely affected cognition, because their frequency became most robust at the age when behavioral impairments have been shown to emerge,  mutations driven by the hamster prion protein promoter751; C57BL6 background) were generously provided by Dr. Paul Mathews. These mice overexpress the 751 isoform of human WT-APP without familial mutations of AD driven by the Thy-1.2 promoterad libitum.Experimental procedures were approved by the Institutional Animal Care and Use Committee at The Nathan Kline Institute and experiments were conducted in accordance with approved guidelines. Mice which express human APPSurgical and implantation procedures were performed as previously describedMice were housed individually for at least a week after surgery in the room where EEG was conducted so that they would acclimate to the recording environment. Recordings were performed in a 21 cm × 19 cm cage similar to a standard mouse cage with a multichannel commutator (Pinnacle Technologies) to allow freedom of movement.Signals were acquired at 2000 Hz sampling rate and bandpass filtered at 0.5–200 Hz (Pinnacle). Simultaneous video was recorded continuously at 10 frames per second (synchronized with the EEG record) during both light and dark periods using an infrared LED camera with Sirenia Video Acquisition software (Pinnacle).Data analysis was performed using Spike2 (Cambridge Electronic Design) and MATLAB (MathWorks) with the FieldTrip toolboxIIS were defined as transient (≤75 milliseconds), large (7 zscores above and below the mean of the baseline of each channel) amplitude events occurring in all 4 recording channels. An amplitude threshold of 7 zscores was selected because this criterion most closely approximated IIS detection performed by manual review . SelectiPETHs were constructed using standard methods with custom-written scripts in MATLAB using the peak of the IIS and the onset of the behavioral state . Raw histograms were smoothed with a Gaussian kernel (defined by a 3 second bandwidth) and averaged across animals of the same genotype/age.Ratio of theta to delta power Wideband power (0.5–200 Hz) of the frontal cortical recording.Movement, detected by positional tracking and confirmed by simultaneous manual review of video.Video-EEG was analyzed in 10 second-long periods to characterize behavioral statesAnimal position during video-EEG recordings was tracked using the Kalman filter, image segmentation, and blob detection technique . The raw velocity vector was smoothed with a Gaussian kernel (bandwidth = 500 milliseconds).REM sleep was defined by a high ratio of theta/delta power . All spectral thresholds were verified manually for each recording. After manual staging of behavioral states, we implemented a Random forest supervised learning algorithmA Sleep Fragmentation Index (SFI)Seizures were detected offline based on the wideband power in the EEG, using a threshold of 4 zscores for a minimum of 5 seconds in any single recording channel . These criteria were chosen because they easily discriminated seizures from other behaviors not only in Tg2576 mice but also other mice with seizures using the same recording systemData were downsampled to 1000 Hz and cut into 10 second-long segments. Segments that were contaminated by mechanical noise or movement/cable artifacts were discarded. Power spectra were computed using a single Hann taper Fast Fourier Transform (FFT) approach. Raw FFT power spectra were then standardized by zscore in the frequency domain using the mean and standard deviation of the EEG during periods of quiet wakefulness within a given recording session. For analysis of peak theta frequency, power spectra between 5 and10 Hz  were computed during REM periods. Distributions of the peak theta frequency were calculated from the local maxima estimation of each power spectrum in all REM sleep epochs during a 24 hour-long recording for every animal.Data were downsampled to 1000 Hz and the time-frequency representation was computed using a wavelet approach. We convolved the data with a complex Morlet wavelet (7-cycle density per frequency). Raw wavelet power spectra were then standardized by zscore in the frequency domain using the mean and standard deviation of wavelet power from periods of quiet wakefulness within a given recording session.20) followed by 40 ml of 4% paraformaldehyde in 0.1 M phosphate buffer . The brains were removed immediately and post-fixed for 24 hours in 4% paraformaldehyde in 0.1 M PB at 4 oC. After post-fixation, brains were hemisected and one hemisphere was cut in the horizontal plane into 50 μm-thick sections using a vibratome (Vibratome Co.). Sections were collected sequentially so that when choosing sections to process it was possible to compare equivalent dorso-ventral levels across animals and the sections were equidistant (300 μm apart).Mice were deeply anesthetized by isoflurane inhalation followed by urethane (2.5 g/kg i.p.) and transcardially perfused with a peristaltic pump (Minipuls 1) using 10 ml of 0.9% NaCl in double-distilled water -glucose and 2% NiCl2 in 0.1 M Tris buffer.Sections from WT and Tg2576 were processed together. Methods similar to those used previously were used for washing and preparation of the tissue16Sections were examined using a brightfield microscope  and photographed using a digital camera . For the hippocampal ChAT analyses shown in All drugs were made on the day of use, dissolved in 0.9% NaCl, and injected i.p. The dose of atropine sulfate  was based on its ability to reduce theta in rodents7719p criterion was 0.05. Statistical comparisons were performed in MATLAB. For comparison of two means, either paired t-tests or two sample t-tests were used. For comparison of more than 2 groups, we used one-way, two-way, or three-way ANOVA with Tukey-Kramer’s post hoc test. Repeated measures ANOVA followed by the Tukey-Kramer post hoc test was used to analyze sequential EEG recordings. For data sets with zero values or a failed Lillefors test of normality (p < 0.05), non-parametric tests were used .All data are reported as mean ± S.E.M and the How to cite this article: Kam, K. et al. Interictal spikes during sleep are an early defect in the Tg2576 mouse model of β-amyloid neuropathology. Sci. Rep.6, 20119; doi: 10.1038/srep20119 (2016).