Researchers in evolutionary genetics recently have recognized an exciting opportunity in decomposing beneficial mutations into their proximal, mechanistic determinants. The application of methods and concepts from molecular biology and life history theory to studies of lytic bacteriophages (phages) has allowed them to understand how natural selection sees mutations influencing life history. This work motivated the research presented here, in which we explored whether, under consistent experimental conditions, small differences in the genome of bacteriophage φX174 could lead to altered life history phenotypes among a panel of eight genetically distinct clones. We assessed the clones’ phenotypes by applying a novel statistical framework to the results of a serially sampled parallel infection assay, in which we simultaneously inoculated each of a large number of replicate host volumes with ∼1 phage particle. We sequentially plated the volumes over the course of infection and counted the plaques that formed after incubation. These counts served as a proxy for the number of phage particles in a single volume as a function of time. From repeated assays, we inferred significant, genetically determined heterogeneity in lysis time and burst size, including lysis time variance. These findings are interesting in light of the genetic and phenotypic constraints on the single-protein lysis mechanism of φX174. We speculate briefly on the mechanisms underlying our results, and we discuss the potential importance of lysis time variance in viral evolution. We overcame this challenge by developing a novel inferential apparatus that complemented the experimental framework with a maximum likelihood-based approach.From this assay, we obtained data that could help determine mean and variance in both lysis time and burst size . HoweverIn this article, we describe our finding that, under the tested conditions, genetic heterogeneity could give rise to significant heterogeneity in φX174 lysis time and burst size, including lysis time variance. These findings are interesting in light of the genetic and phenotypic constraints on the single-protein lysis mechanism of φX174, and they point to promising avenues for future theoretical and experimental work.2 was used throughout. We studied the performance of φX174 on E. coli C , the phage’s natural host. A stock of E. coli C was stored in 40% glycerol at –80°. We used this primary stock to seed bacterial colonies on agar plates stored at 4°. Colony plates were created anew every 2–4 wk. To generate experimental bacterial cultures, 10 mL LB was inoculated with a single colony and shaken in a 50-mL flask at 200 rpm and 37° for about 16 hr. The resulting stationary-phase cells were then diluted somewhere between 33- and 80-fold into 40 mL LB in a 250-mL flask. The diluted culture was shaken at 37° for about 3 hr until it reached an optical density Difco LB Lenox Broth , pH 7.50, and supplemented with 2 mM CaClDiscussion for the cell cycle’s relevance.) First, we transferred 1 mL cells to a 1.5-mL centrifuge tube and pelleted them by spinning in an Eppendorf centrifuge (5417R) for 2 min at 20,800 rcf. After aspirating the supernatant, we resuspended the cells in 1 mL HFB-1 solution [0.06 M NH4Cl/0.09 M NaCl/0.1 M KCl/0.1 M Tris-HCl (pH 7.4) /1.0 mM MgSO4/1.0 mM CaCl2] by vortexing for 8 sec. Then, we centrifuged the solution, removed the supernatant, and resuspended in HFB-1 twice more. After removing the supernatant from the third and final HFB-1 rinse, we resuspended cells in 1 ml HFB-2 solution (HFB-1/10 mM MgCl2/5 mM CaCl2). This served as our culture of starved bacterial cells, ready for phage adsorption.To synchronize phage infections, we treated cells using a starvation protocol adapted from Epos mutants carrying point mutations in the E gene, whose product is the chief player in φX174 lysis  at five sites: G833A (nonsynonymous), G1650A (nonsynonymous), T2811C (synonymous), A4518G (nonsynonymous), and T4784C (synonymous) (E (but not the D) gene, though it fell well outside the region coding for the E protein’s essential transmembrane region , and plating in top agar held at 42°, consisting of 7.0 g/L Difco BactoAgar  added to LB medium (as described above). After several minutes on the bench at room temperature, we incubated plates agar-side-up at 37°. Preliminary results (not shown) demonstrated that accurate plaque counts required at least 4 hr of incubation. Occasionally, for logistical reasons, we interrupted incubation with overnight refrigeration at 4°.We began by adding 90 μL starved cells to each well of a TempPlate nonskirted 96-well PCR plate (USA Scientific item no. 1402-9598) at 15°. The PCR plate was mounted on a thermocycler to control its temperature precisely. Using a 12-channel pipettor, we then added 10 μl 30-PFUs/mL phage dilution to each well in each row of 12 wells, allowing for a 30-sec interval between rows. PCR plates were held at 15° for at least 20 min, during which time phages could adsorb to the starved cells. We estimate that 10–20% of phage particles adsorbed to hosts during this incubation period  to each well in a row with a 12-channel pipettor, again allowing 30-sec intervals between rows. Consequently, the infective process in each row was staggered by half a minute, allowing ample time for the sampling procedure that followed. Note that the temperature increase alone did not trigger infection. The addition of rich medium, such as LB, was required for infection to advance . Based oD-promoter mutants and other clones in coat protein F. While a change in the F protein could theoretically have affected adsorption . We assumed that each phage particle infected and then lysed a cell T minutes after the addition of warm media, where T could not be less than a biologically imposed minimum (the latent period), and lysis was equally probable every moment thereafter . We assumed that burst size grew linearly with lysis time, a relationship generally assumed and supported by past work  that had occurred, we did not know when they occurred, except that they must have occurred prior to the moment of sampling. From a statistical perspective, this suite of hidden variables made the problem both interesting and challenging.Within the wells, we assumed phage particles adsorbed instantaneously to starved cells upon the initiation of infection. . Then, we noted that each sampled well fell into one of three categories: samples with zero phage particles, samples with phage particles but no lysis events, and samples showing lysis events. The probability of a well belonging to each category was, respectively, Our first task was to estimate β is less than or equal to one, counts greater than seven are highly improbable in wells where lysis has not occurred . In addition, certain experimental manipulations  could feasibly have induced very premature lysis of some cells, resulting in plates with extremely low plaque counts that otherwise would have had just one plaque.There was a nonzero chance of slight inaccuracies in some plaque counts. Rare errors could have occurred in the counting of plaques that would have increased counts on average .We defined wells not showing lysis events as those having plaque counts greater than zero, and less than or equal to seven. When the Poisson parameter a be p, set each equal to zero, and did a considerable amount of algebra (see Appendix 1 for details). This resulted in the following estimator:X is the mean among observed plaque counts, applied only to wells sampled before we witnessed any lysis events). The results (which we will not cover in detail) revealed that Equation 2 had a very slightly positive bias , we binned the P-values into four categories  and conducted an exact multinomial test against expected frequencies in each bin under the null. The null bin frequencies were equal across the four categories .To test whether the distribution of T minutes had elapsed since i.e., the slope parameter), and ε captured the normally distributed variation from expectation  could, like burst size, be expressed as a linear function:r sampled at time t from assay a, r (assay a) that had lysed their hosts by time t, We modeled the potential for burst size to change with time as a linear function, terating . More spi.e., the central 95%) to define the confidence intervals. To keep our analysis simpler, we did not conduct formal likelihood ratio tests for significant differences between clones for these parameters. Instead, we compared confidence intervals and applied the conservative criteria that when confidence intervals were nonoverlapping, the two clones were significantly different.To obtain confidence intervals and test for differences between clones in the regression parameters, we used a parametric bootstrap approach. For each genetically distinct clone, we simulated 1000 datasets using the estimated parameters and, each time, estimated α, μ and σ. We then sorted the bootstrap set of parameter values individually, using the 25th and the 975th values  and D-promoter ancestor are given by GenBank accession numbers J02482.1 and AF176034, respectively. Regions of the mutants' genomes were sequenced, and these sequences also were archived in GenBank (accession numbers KU646482 to KU646588).Strains are available from D.M.W. upon request. File S1.) The estimated Poisson parameter Statistical methods).Thirty-seven assays were conducted for the eight clones (four to six each).  to 14.5 min . Estimates of pos6, and pos4B infections, though about 22min for pos5) and D-promoter mutants and wild type).Across the clones, the earliest times at which lysis was observed ranged from 11.0 min (mut321), meaning that the cumulative probability of lysis after The smallest observed value of λ among the clones was 4.8 min  wild type and pos4B, and (3) the remaining clones. This clustering can be observed clearly when the parameters composing those functions, λ and ch other .P-values associated with the null hypothesis that the clones being compared came from the same function , we would expect the P-values of the 28 pairwise tests to follow a uniform distribution, and we would expect just one or two false positives .  had little effect on these findings. By ignoring adsorption time, we treated the time to lysis as a single exponential process, when in reality it involves the sum of two exponential waiting times  pos4B and pos5, (3) wild type and mut319, and (4) mut321, mut323, and mut324. Formal testing of parameter differences was not conducted. However, where confidence intervals were nonoverlapping, there was good evidence for a significant difference. For slope, the confidence intervals within the four clusters were generally overlapping while intervals between clusters were not.The regression analysis of burst size by time revealed that burst size increased with time for all clones . The pare.g., lysis time variance), could be subject to the phage’s genetic control.In recent years, it has been found that the probability that a beneficial mutation will fix in a lytic phage population depends sensitively on the life history parameter it affects , 2009. FD promoter that have been reported to downregulate the transcription of several genes, including the lysis gene (E); three Epos clones with mutations in E that have been found to upregulate its expression; and the wild-type ancestor of the Epos mutants. To this end, we used a serially sampled parallel infection assay to interrogate the lysis time and burst size phenotypes of a panel of eight genetically distinct φX174 clones. Among these clones were four with mutations in the Using a novel statistical approach that allowed us to overcome the challenge of hidden variables, we found significant variation in both lysis time and burst size among these clones, including evidence that φX174 exerts genetic control over lysis time variance under our experimental conditions.D promoter  By contrast, burst size increased more quickly than the wild type in the three Epos mutants. This may be related to the findings of E within the D gene  .The strength of the experimental and statistical approach taken in this study lies in its power and applicability. It is powerful in that, more effectively than most other methods, it simultaneously captures information related to lysis time variance and burst size as a function of time. The approach also has potential application to a range of microbiological questions. The principles and tools underlying the assay and inferential apparatus could be applied usefully to other cases in which one seeks to make inferences about dynamic, Poisson-distributed processes that are sampled destructively over a time course , they limit the generality of some conclusions drawn from this work. The unique conditions of the assay complicate comparisons to previous estimates of the clones’ burst size and lysis time phenotypes, and they make it impractical to validate our methods against traditional single-step growth experiments with much confidence.However, we concede our approach has limitations. There is increasing evidence that host cells’ physiology plays a vital role in determining phage infection dynamics  differed significantly. Furthermore, it is reasonable to assume that the degree of genetic variability among the clones is comparable to that which is the substrate for natural selection acting in the wild.Our finding that variance in the lysis time of φX174 can be subject to at least some genetic control inspired us to ask, “What, then, could be the evolutionary implications of mutations affecting variance in phage lysis time?” Trivially, if burst size grows more slowly than exponentially once a cell has been infected, then a mutation increasing lysis time variance will be selectively favored, because early lysis events contribute more to population growth than late lysis events detract . EmpiricIn order to control for the influence of this mode of selection, we recently have explored a model in which burst size increases exponentially in time since infection . This has allowed us to infer the intrinsic selective consequences of mutations that influence lysis time variance. As expected, in phage populations growing at their stable age-of-infection distribution in unlimited host cells , we foune.g., as employed in lab conditions, but also surely in nature), we observed that mutations increasing lysis time variance can enjoy a dramatic increase in population frequency, despite their neutrality when they finally reach their stable age-of-infection distribution . We therefore suggest a second mode of selection acting on mutations affecting variance in lysis time.However, the moment a mutation appears, the mutant subpopulation is by definition not at stable age distribution, and indeed we have found in simulations that mutations increasing variance in lysis time are transiently enriched in the population . This effect follows from the fact that the frequency of phages with high lysis time variance exceeds (sometimes considerably) the frequency of their low-variance brethren early in each lysis cycle. While the reverse is true late in the cycle, the effect is of smaller magnitude and much shorter lived. Finally, overlaying periodic demographic bottlenecks (The findings reported here suggest two complementary avenues for future investigation. One is microbiological, and it involves exploring φX174 lysis in relation to its genetic determinants and its host’s biology. It would be interesting to explore the mutants’ performance in a range of assays, such as those involving thermally regulated microscopy , single-Theoretical evolutionary problems present another opportunity for future research. Does natural selection act on lysis time variance in nature? What is the fixation probability of a mutation that affects lysis time variance, and how do populations carrying such mutations evolve in the face of periodic bottlenecks? Existing computational models could be strengthened by more thoroughly exploring the models’ parameter sensitivity and investigating how mutations that pleiotropically influence adsorption, lysis time, and burst size affect predicted population dynamics. In addition, the models must be integrated with other theoretical work exploring the fixation probabilities of mechanistically distinct beneficial mutations in lytic phages , 2009. O


Evaluate the role of NT-proBNP levels in Preterm neonates suffering from PDA and used as a screening tool for predicting HsPDA and guiding physicians to consider early echocardiographic evaluation.This is a monocentric prospective blind study which was conducted at Arar Central Hospital, Ar’ar, Saudi Arabia, during the period between Jan 2014 to June 2014. Thirty-three (33) preterm infants born at less than 31 weeks of gestation or weighing less than 1200 g at birth infants were initially enrolled during a 6-month period. Blood samples were collected along with routine blood tests on days 1, 2, 3, and 7 of life for NT-proBNP analysis. Two echocardiographies were systematically performed on day two of life to ascertain about the status of Ductus Arteriosus.The Plasma NT-proBNP levels were high on day one of life and decline from day three to day seven of life except in those infants with significant hsPDA. Plasma NT–proNBP levels on day 2 of infants in the HsPDA group were significantly higher (<0.001) than those in non-HsPDA group. Echocardiogram parameters indicates the significant difference (p<0.002) in Left Atrial and Aortic ratio (LA/AO), Interventricular septum thickness (P<0.03), Left ventricular posterior wall thickness (p<0.05), diastole PDA gradient (p<0.005) between HsPDA and non-HsPDA.Plasma NT-proBNP level peaked during the first few days after birth and declined rapidly within a week. Therefore, its level may have a role as a screening tool to predict HsPDA and provide more information regarding its spontaneous closure or otherwise. Patent ductus arteriosus (PDA) is a common problem in premature infants born before 32 weeks.In HsPDA there is significant shunting of blood from the systemic to the pulmonary circuit resulting in increased pulmonary blood flow with reduction in effective systemic blood flow.15Therefore a biomarker like NT-proBNP is a useful tool in diagnosis of PDA in premature neonates, especially in cases where Doppler echocardiography is not easily available21The objective of this study was to evaluate the role of NT-proBNP levels in Preterm neonates suffering from PDA and to see whether it may be used as a screening tool for predicting HsPDA and guiding physicians to consider early echocardiographic evaluation.This monocentric prospective blind study was conducted at Arar Central Hospital, Ar’ar, Saudi Arabia, during the period between Jan 2014 to June 2014.Thirty-five (35) preterm infants were initially enrolled during a 6-month period (from Jan 2014 to June 2014). The data of two infants were excluded from analysis because the data were incomplete. Thirty one 33 infants  were eligible for evaluation. Their mean GA was 28.6 (±2.6) weeks and the mean birth weight was 991 (±294) g.The Preterm infants born at less than 31 weeks of gestation or weighing less than 1200 g at birth. The infants were enrolled within 24 hours of birth.The Preterm infants with the presence of cardiac congenital anomaly other than PDA, life-threatening congenital malformation, severe asphyxia at birth, persistent pulmonary hypertension, and death within the first week of life.We prospectively evaluated the infants for PDA within the first 24 h of life determine whether the patient is suffering from hsPDA or non-HsPDA diagnosis based on a large ductal flow with left to right shunt on color Doppler echocardiography.Blood samples were collected along with routine blood tests on days 1, 2, 3, and 7 of life for NT-proBNP analysis.Two echocardiographies were systematically performed on day two of life.Blood collected for routine sampling in lithium–heparin tubes was transported to the laboratory at room temperature where it was centrifuged at 20009g for 6 minutes. Supernatant plasma was analyzed for NT-proBNP using the Roche Elecsys 2010 electrochemiluminescence immunoassay, following the manufacturer’s recommendations. In most referral hospitals in Saudi Arabia, these tests are frequently available and are carried out free of cost in these hospitals whenever the physicians feel, they send a request and tests are done.Echocardiographies were performed using an 8–MHz probe (Philips CX 50). Conventional echocardiographic measurements were obtained in accordance with the guidelines of the American Society of Echocardiography.The definition of Echocardiographic hsPDA was based on the evidence from the previous report, and the diagnosis was made if at least two of the following criteria were met: (i) LA⁄Ao ratio ≥1.4, (ii) ductal diameter ≥1.5 mm and (iii) the percentage of the ratio of the TVI of the diastolic retrograde flow to the TVI of the systolic anterograde flow along the descending aorta ≥30%.23According to Nuntnarumit et al; the Clinical hsPDA was defined as the presence of the ductal flow with predominant left to right shunt on color Doppler which measured at least 1.5mm on two-dimensional echocardiography plus at least two of the following signs, including heart murmur, persistent tachycardia (heart rate >160 beats/min), hyperactive precordium, bounding pulse, pulse pressure >25 mmHg, hepatomegaly, pulmonary hemorrhage , increasing respiratory support by 20% increase in oxygen supplementation or in pressure support and chest radiographic evidence of cardiomegaly or pulmonary congestion.24Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) version 15.0. Continuous variables are presented as mean values with standard deviation. P<0.05 is considered significant.Demographic and clinical parameters are listed in this table showing; mean±SD, Gestational age in weeks (wks), Birth weight in grams (gm), etc. Mean (± SD) of NT-proBNP levels in pmol ⁄ L on day1, 2, 3 and 7 of PDA are shown in Echocardiogram parameters in the PDA infants, which indicates the significant difference (p<0.002) in Left Atrial and Aortic ratio (LA/AO), Interventricular septum thickness (P<0.03), Left ventricular posterior wall thickness (p<0.05), diastole PDA gradient (p<0.005) between HsPDA and non-HsPDA are shown in In this study we observed that the Plasma NT-proBNP levels were high on Day-1 of life and showed a decline from Day-3 to Day-7 of life except in those infants with significant hsPDA. Plasma NT–proNBP levels on day 2 in the HsPDA group (n=18) were significantly higher than in non-HsPDA group (n=15). In non-HsPDA group, the plasma level of NT–proBNP was found to peak in the first two days of life (1286 pmol/L) and then declined during day 3–7 after birth which is highly predictive of a ductus that will close spontaneously.26El-Khuffash et al. also reported a significantly high level of NT-proBNP at 2 day of life, not at Day-1, in preterm infants with HsPDA and a rapid decrease after successful PDA closure. 29In Farombi-Oghuvbu et al study the high levels of NT-proBNP on day 10 were also found in infants with sepsis.Another study of El-Khuffash et al. has also shown that NT-proBNP on day 3 significantly correlated with LA/Ao ratio, and PDA diameter.Plasma NT-proBNP level peaked during the first few days after birth and declined rapidly within a week. Significantly high levels of plasma NT-proBNP on Day-2 were observed in infants who developed HsPDA. Therefore, its level may have a role as a screening tool to predict HsPDA and provide more information regarding its spontaneous closure or otherwise.


Pyoderma gangrenosum is a rare clinical entity of poorly understood pathogenesis, characterized by rapidly progressing skin necrosis. In around half of patients pyoderma gangrenosum is a manifestation of underlying systemic diseases, such as rheumatoid arthritis, inflammatory bowel disease or myeloproliferative disorders. There have been very few reports on the association of pyoderma gangrenosum with solid malignancies.We report a case of a 68-year-old Caucasian woman in whom pyoderma gangrenosum first appeared around 30 years earlier, at the time of exacerbation of rheumatoid arthritis, and recurred as a manifestation of locally advanced breast cancer. The causative role of the neoplastic process was partly confirmed by the healing of the skin ulceration only following effective endocrine cancer therapy, whereas earlier attempts with standard anti-inflammatory therapy were unsuccessful.Pyoderma gangrenosum has a recurrent nature and may be reactivated by various causes within several years. Therefore, a prompt and thorough diagnosis accompanied by treatment of the underlying disease is necessary. Pyoderma gangrenosum is a rare clinical condition of poorly understood pathogenesis, characterized by rapidly progressing skin necrosis. In around half of patients, this entity is a manifestation of underlying systemic diseases, such as rheumatoid arthritis, inflammatory bowel disease or myeloproliferative disorders. There have been very few reports on the association of pyoderma gangrenosum with solid malignancies. We present a case of a 68-year-old woman in whom pyoderma gangrenosum first appeared around 30 years earlier, at the time of exacerbation of rheumatoid arthritis, and recurred as a manifestation of locally advanced breast cancer.A 68-year-old Caucasian woman presented with an ulceration of the anterior surface of her left leg. Thirty years earlier, at exacerbation of rheumatoid arthritis, our patient underwent therapy for a similar lesion located on her left forearm, diagnosed as pyoderma gangrenosum. Since then, the rheumatoid arthritis had been quiescent and had not required any therapy, and at admission there were no signs of recurrence. A physical examination showed an ulcerating tumor involving the lower quadrants of her left breast. The breast lump had appeared four years earlier, progressed rapidly within the last month and at diagnosis was clinically staged at T4aN1M0. A histopathology examination showed invasive ductal breast carcinoma, with estrogen receptor expression in 90 percent of the tumor cells, and no expression of progesterone receptor and epidermal growth factor receptor type 2 (HER2). A biopsy specimen from the edge of the leg ulceration showed pyoderma gangrenosum: an intensive mixed inflammatory infiltrate including mainly neutrophils, with accompanying vessel destruction and necrosis of the epithelium Figure  and aftePyoderma gangrenosum is a rare dermatosis characterized by rapidly progressive skin ulceration. The etiology of this entity is poorly understood but neutrophil and monocyte dysfunctions (defects in phagocytosis and chemotaxis) have been reported -6. In arIn our patient, pyoderma gangrenosum was most likely induced by progressing breast cancer. The causative role of the neoplastic process was partly confirmed by the healing of the skin ulceration only following effective endocrine cancer therapy, whereas earlier attempts with corticosteroids were unsuccessful. The lack of improvement with corticosteroids has been atypical, as this therapy usually induces dramatic response within the first two weeks . AnotherDue to association with serious underlying conditions, rapid development and potentially serious consequences, pyoderma gangrenosum necessitates prompt and thorough diagnosis, including the search for coexisting disease ,16. If tWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.HER2: epidermal growth factor receptor type 2.The authors declare that they have no competing interests.RD treated the patient and drafted the manuscript. EZ treated the patient and drafted the manuscript. AG treated the patient and drafted the manuscript. BG carried out the immunoassays and drafted the manuscript. All authors read and approved the final manuscript.


Cattle risk factors  were more strongly associated with herd risk than area-level measures of badger social group density, habitat suitability or persecution (sett disturbance). Highest risks were in areas of high badger social group density and high rates of persecution, potentially representing both responsive persecution of badgers in high cattle risk areas and effects of persecution on cattle bTB risk through badger social group disruption. Average badger persecution was associated with reduced cattle bTB risk (compared with high persecution areas), so persecution may contribute towards sustaining bTB hotspots; findings with important implications for existing and planned disease control programmes.Bovine TB (bTB) is endemic in Irish cattle and has eluded eradication despite considerable expenditure, amid debate over the relative roles of badgers and cattle in disease transmission. Using a comprehensive dataset from Northern Ireland (>10,000 km Mycobacterium bovis, has proven difficult to control and eradicate in Irish cattle, despite most EU countries being officially disease free. Intensive ongoing efforts to control the disease are based on a Government sponsored regime of tuberculin testing and slaughter of infected cattle, an expensive programme for both farmers and the government234Bovine tuberculosis (bTB), caused by the bacterium Meles meles) has been identified as a major factor contributing towards the difficulty of bTB eradication in Ireland781012The presence of a wildlife reservoir of infection . The perturbation hypothesis states that when a badger sett is disturbed or some resident badgers killed, surviving social group members are more likely to disperse, increasing contact rates with badgers in neighbouring social groups and potentially spreading bTBWe combined data on the cattle and badger populations in Northern Ireland to produce a comprehensive list of risk factors concentrating on aspects of bTB risk potentially influenced by interactions between the two host populations. We also addressed several other important issues including the impact of international, cross-border cattle imports. By identifying herd-level risk factors of bTB infection, we aim to aid the eradication programme by informing farmers, veterinarians and policy-makers on the strategies most likely to reduce disease incidence. We specifically address the following questions: i) What are the cattle-related risk factors for herd-level bTB detection (herd breakdowns)? ii) Is cattle bTB risk associated with badger social group density and/or habitat suitability? iii) Is badger persecution  associated with increased bTB risk?Anonymised tuberculosis test results (tuberculin skin test) and animal movement data for all active cattle herds  in Northern Ireland during the period 2004 to 2011 were made available from the Animal and Public Health Information System APHIS curated n = 212, 2  and an index of habitat suitability derived from a spatially explicit Species Distribution Model (SDM) associating badger main sett presence with landscape features (for full methodology of sett classification seeData describing the badger population were provided courtesy of DARD from the ‘Badger Survey of Northern Ireland 2007/08’22M. bovis) within a given year and 0 otherwise . Herds with unconfirmed or inconclusive skin test results scored 0. The aim was to identify potential risk factors associated with new herd breakdowns so herds undergoing a continuing breakdown were excluded from the risk-set  until the calendar year following that in which the breakdown had been declared over, which occurs after two subsequent negative follow-up tests occurring at least 60 days apart (i.e. a breakdown extending over two years would only be counted in the first year). A summary of the number of herds in each category is given in P < 0.1 in univariable models were retained , so changes in incidence cannot be unambiguously associated with persecution at a particular time point. The study period was split into two year intervals  and the selected cattle-badger model was fitted to data from each interval separately. Herd-level predictions of bTB risk were extracted and used to calculate expected bTB incidence by badger main sett density. Preliminary analysis showed persecution risk was low but had a strongly right-skewed distribution. Herds in areas with persecution indices in the upper quartile of the distribution (probability of sett disturbance >0.35) were perceived as high persecution areas representing 29%  of the land area with active cattle herds compared with herds in other areas .To further investigate relationships among cattle and badger variables, we conducted a descriptive analysis comparing temporal trends in bTB incidence in cattle in areas with high and low badger persecution, both before and after the badger population data were collected (autumn-winter 2007/08) in order to infer the directionality of any identified relationships between persecution and bTB incidence. High incidence in areas subsequently found to have relatively high persecution would be consistent with persecution being largely a response to high local bTB risk. We also reasoned that if persecution reduced risk we might expect a disproportionately large decrease in incidence immediately after the badger survey in areas with high persecution compared with low persecution areas, over and above any longer term incidence trends. However, such an effect might be obscured if patterns of persecution changed dramatically in the period immediately after the badger survey . Areas with the highest social group density and highest rates of persecution were in the south-east of the region i.e. Co. Down  and high rates of persecution remained at elevated risk of breakdown (bTB hotspots) regardless of temporal trends in bTB incidence in the rest of the country. In low persecution areas the weak positive association between social group density and incidence was similar before (2004–2007) and after (2008–2011) collection of badger population data. Breakdown risks before the badger survey were greater in high compared with low persecution areas, especially at higher badger social group densities  and a low rate of persecution and so the predicted bTB incidence in areas of low persecution tracked the overall decreasing trend in cattle bTB incidence better than the high persecution profile . Areas wensities . High peIn our population scale comparison of bTB risk factors for new herd breakdowns, cattle-related factors were a far better predictor of bTB risk than badger-related factors in terms of model fit. Herd size was a key predictor and high bTB risks have been previously associated with large herds in the UK and Ireland1011293031323436373538394042Variation in incidence of bTB in cattle associated with the badger population was small in comparison with cattle variables but high badger social group density, habitat suitability and badger persecution were associated with elevated cattle bTB risk. The highest risks were found in areas of high badger social group density coupled with high rates of persecution through sett interference, with two potentially non-exclusive explanations for such an association. Firstly, badger persecution may be initiated in response to high local cattle bTB prevalence in an effort to protect herds from (re)infection, especially where badgers are common and, hence, a more visible perceived threat and accessible target47An important consideration is whether these findings, based on data from Northern Ireland can be extrapolated to other parts of the British Isles. The relative influence of badgers on bTB dynamics in Great Britain and the Republic of Ireland has been widely debated, with large scale badger culling trials in each country yielding dramatically different results and policy recommendationsBadger population density in Ireland (in both Northern Ireland and the Republic of Ireland) is thought to be low, having remained relatively stable whilst the population in Great Britain has increased122212M. bovis between the two host populations occurs relatively frequentlyDespite the population-scale dataset used, our models had relatively modest predictive power and considerable unexplained variation, indicating that the selected variables did not capture all of the dynamics of the system and that, in common with many observational studies, unquantified covariates were likely to have had an influence on outcomes. Most of the herd-level predictors identified in a global review of bTB risk factors2505110How to cite this article: Wright, D. M. et al. Herd-level bovine tuberculosis risk factors: assessing the role of low-level badger population disturbance. Sci. Rep.5, 13062; doi: 10.1038/srep13062 (2015).


This perspective aims at underlining the usefulness of event-related potentials (ERP) to better understand the brain correlates of Korsakoff’s syndrome (KS), a neuropsychiatric disease characterized by severe memory impairment and most frequently resulting as a neurological complication of alcohol-dependence (AD). While ERP have been broadly used in AD, it has up to now been very little applied in KS or in the comparison of KS and AD. Within the framework of dual-process models, an influential theory postulating that addictive states result from an imbalance between under-activated reflective system and over-activated automatic-affective one, this paper proposes: (1) a brief synthesis of the main results of ERP studies in AD and KS, and (2) new research avenues using ERP to identify the electrophysiological correlates of cognitive and emotional dysfunction in KS. These experimental perspectives aim at exploring the continuity hypothesis, which postulates a gradient of impairments from AD to KS. We conclude on the possibility of developing neuropsychological strategies with electrophysiological follow-up to ensure KS diagnosis and test the efficacy of patient’s neurocognitive rehabilitation. Korsakoff’s syndrome (KS), a frequent neurological complication of alcohol-dependence (AD), is mainly caused by the combined effect of thiamine deficiency and alcohol neurotoxicity. KS is classically associated with disorientation, confabulation, and amnesia , severe Figure 1 illustrating the sequence of main ERP components during  information processing; An interesting way to conduct these cognitive-emotional explorations in KS is the event-related potentials (ERP) technique. Indeed, this non-invasive tool, which allows measuring brain electrical activity associated with cognitive functioning, constitutes a method of choice to assess cognitive deficits in pathological populations and has notably proven its usefulness among several psychiatric disorders . In partAccordingly, the aim of the present paper is twofold. First, we report the results of the few ERP studies in KS. We also report the most robust findings from studies in AD that investigated both systems postulated by dual-process models . This kind of modified Flanker paradigm, which allows measuring the classical stimulus-locked and response-locked ERP , would enable determining whether there is a graduated effect of: (1) deficits in inhibition abilities from AD to KS for conflict detection (indexed by reduced NoGo N2) and/or response inhibition (indexed by reduced NoGo P3), both stages being affected in AD; (2) greater allocation of resources to performance monitoring as previously shown by response-locked ERP modifications in AD .A gradual decline from AD to KS has been recently hypothesized for inhibition , given tHow the increased “salience” attribution to alcohol-related cues and associated biases in AD e.g.,  evolve w(1) What is the real nature of the biases? Are they perceptual , attentional  or decisional ? Visual oddball paradigms e.g.,  should b(2) Do alcohol-related stimuli capture attentional resources of KS individuals? ERP recording during a dot-probe task e.g.,  would he(3) Is the putative hyperactivity for alcohol cues conversely associated with a decreased reactivity for non-alcohol related natural rewards ? Future ERP studies in KS should investigate whether the possible hyperactivity of the automatic subsystem for alcohol-related cues is accompanied by hypoactivity of this subsystem for another type of rewards, as indexed by lower P3 amplitude to incentive stimuli in AD .Figure 2), and are more severe in KS. Importantly, further ERP studies should investigate more deeply the putative deficits of visual pathways in KS and the interaction between vision and emotion as recently proposed for AD . ERP components related to inhibition  and response monitoring could be therefore studied in the specific context of affective subsystem  or automatic subsystem .Future ERP should directly investigate reflective and automatic-affective systems interactions in AD and KS  to deterThe main aim of this article was to stress the need for developing studies employing ERP, which have been up to now under-used in the domain, to obtain a clearer picture of KS impairments thanks to the high temporal resolution of this technique. We proposed new research avenues within the framework of dual-process models to better understand KS deficits regarding the reflective and automatic-affective systems, whose unbalanced interactions are supposed to be at the heart of AD.According to the continuity hypothesis from AD to KS, and on the basis of ERP findings showing that activity of both systems is affected in AD, we can suppose: (1) a linear hypo-activation of both reflective system and affective subsystem; and (2) a graduated over-activation of the automatic subsystem. From a cognitive point of view, this assumption would lead to the deterioration from AD to KS of both executive functions and emotional decoding abilities and to a stronger bias toward alcohol-related stimuli. Alternatively, a linear worsening of brain impairments from AD to KS could also lead to a decreased activity in prefrontal and limbic areas and, therefore, a hypo-activation of both reflective and automatic-affective systems. This latter assumption calls into question the continuity hypothesis as it implies that deficits in KS do not result from a mere strengthening of deficits already present in AD. Instead, modifications in the activity of the automatic subsystem would be variable over the course of the disease, with an over-activation at the early stages  and then an under-activation at later stages . At this time, maintenance of the AD could be predominantly caused by an accentuation of the reflective system deficits. ERP investigations should help to examine the evolution of the two systems and their interactions during and between AD and KS. Moreover, further ERP studies should also investigate the possible role of a third system involving the insula as recently proposed by Figure 2) is that the massive cognitive deficits associated with KS would limit the ability of KS individuals to understand tasks instructions, to keep them in memory during the tasks, and to focus their attention during a sufficiently long period to complete the tasks. This explains why we tried to propose experimental procedures as simple as possible However, there are several lines of evidence in favor of the feasibility of our proposal: previous studies have been carried out with KS individuals using complex tasks ; there test see ; there aAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, Geraldine Petit, and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


As predicted by the temporal difference learning models, dopamine neurons respond during the early stages of classical and operant conditioning with a burst of action potentials (a phasic-like response) after reward presentation   , phasic DA stimulation enhanced the effects of food-reward seeking . Interestingly, they also found that after a series of extinction sessions during which no food reward or phasic DA stimulation occurred, preferential lever pressing  could be reestablished by DA stimulation in the absence of both external cues and, critically, food reward. Finally, the authors used a reversal learning session where the relationship between the active  and inactive  levers were switched, and demonstrated that ChR2 mice switched their lever pressing to the previously inactive lever compared to control mice. This finding is particularly important because it suggests that not only is the phasic DA signal driving and enhancing simple stimulus-reward associations but it is also causally involved in flexible behavioral adaptations that occur as a result of changes in stimulus-reward contingencies.With regards to the causal role of DA in behavioral flexibility, in a recent study  dopamine D1 and D2 function in the striatum is another important component to take into account when trying to establish a causal link between neural activity and behavior. Dopamine depletion, for example, in the dorsomedial striatum results in reversal learning impairments (O'Neill and Brown, Studies that have looked at increasing dopamine concentration have demonstrated that DA stimulation by injection of amphetamine in the NAc core or shell increased instrumental responding to a conditioned stimulus predictive of reward (Pecina and Berridge, In conclusion, increasing evidence from optogenetic, voltammetry, and pharmacological studies over the recent years have added a new dimension to the established but mostly correlation role between the midbrain DA neurons and reward learning. This evidence suggests that this phasic response may have a causal role not only in reward prediction error signaling, but also in driving flexible behavioral adaptations to changes in stimulus-reward contingencies.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Today’s psychiatry is progressively entering the realm of science. Significant advances in structural neuroimaging and insight in functional as well as dysfunctional connectivity permit us to see the world from a patient’s perspective and generate hypotheses driven by objective brain-based data . In addiThe first domain is related to psychotropic drug metabolism: If we know how well a patient’s liver enzymes deactivate and eliminate medications, we can choose a medication whose metabolism is neither too quick nor too slow. We must clearly understand both metabolism scenarios before prescribing any psychotropic medication because if the patient metabolizes a medication quickly, the medication may never achieve sufficient plasma levels to influence the brain. On the other hand, when an individual has deficient enzymatic machinery for a particular medication, they may develop significantly elevated drug concentrations that result in side effects and/or toxicity. In both cases, the medication may be deemed ineffective without truly assessing its efficacy, and the patient would have no benefit from the therapy . MoreoveCOMT Val158Met) , which renders COMT hyper-efficient and may result in dopamine deficiency. In contrast, genotype Met/Met results in a hypo-active enzyme and causes excessive dopamine in the synaptic cleft. There are also heterozygotes Val/Met, carrying both variants of the enzyme. They are associated with intermediate function and often considered within the normal spectrum.Second, different genetically controlled biochemical targets influence therapeutic outcomes. Among these are the pre- and postsynaptic receptors, neurotransmitter transporter molecules, and voltage-gated ion channels. Of recent interest are enzymes involved in the degradation of released neurotransmitters. One such enzyme is catechol-O-methyltransferase (COMT), an enzyme that degrades dopamine and other catecholamines. This enzyme is present in two variants due to the single nucleotide polymorphism, resulting in a valine-to-methionine substitution at codon 158  took greater sexual risks than individuals with Val/Val genotype. Perhaps, excessive prefrontal dopaminergic activity causes cognitive dysfunction that, in turn, causes inappropriate decisions (Val/Val individuals, who had significantly more postsynaptic dopamine receptors, had lower dopamine levels as a consequence of pre-synaptic deprivation. The study confirmed the up-regulation of the cortical dopamine-1 receptor type in patients who were homozygous Val/Val compared to those who were homozygous Met/Met. The study evaluated the brain’s decision-making regions related to immediate and delayed gratification in COMT genotype patients. This study showed that individuals who carried the less active COMT Met variant exhibited a decreased immediate reward bias, while Val/Val individuals showed greater potential for impulsivity (Val/Val have significant processing insufficiency in frontal lobe circuits. Nevertheless, for most of these correlational studies, the hypotheses await additional studies to see if they will endure the test of time.For now, let us focus on studies that investigate the consequences of hyper- and hypoactive COMT and ask whether individuals with more efficient COMT differ from the heterozygous population and from patients with less enzyme efficiency. For example, one study showed that Cannabis use may have significant negative effects in some vulnerable individuals and lead to psychosis and/or other chronic psychotic symptoms. It seemed that individuals who were homozygotes with isorders . This asisorders . It alsoisorders . Individecisions . A recenulsivity . TherefoVal/Val gentoype have decreased frontal dopaminergic activity and potential executive function impairment. As such, agents such as stimulants may be considered for augmenting dopamine. Also, these individuals may respond to non-pharmacological prefrontal stimulations such as transcranial magnetic stimulation that seemingly increases dopamine activity (COMT Met/Met genotype have increased prefrontal dopamine and, thus, well-developed executive function that would respond negatively to stimulant therapy. How exciting to think that, until very recently, these genetic tools were simply fantasy. Now, these genetic developments have brought the world of mental health closer to the reality of individualized person-centered therapy.Finally, for psychopharmacology, genetic differences may have significant implications for how we choose medication for patients with specific genetic characterizations: Individuals with activity ,12. On t


The method considers that the functional molecule provides a spatial distribution of effective potential field for the electronic transport. The electrons are injected from one electrode by bias voltage, then transmit through the potential field around the functional molecule, at last are poured into the other electrode with a specific transmission probability which is calculated from one-dimension Schrödinger equation combined with three-dimension correction. The electron-transport properties of alkane diamines and 4, 4′-bipyridine molecular junctions are studied by applying OTCTCA method. The numerical results show that the conductance obviously exponentially decays with the increase of molecular length. When stretching molecular junctions, steps with a certain width are presented in conductance traces. Especially, in stretching process of 4, 4′-bipyridine molecular junction, if the terminal N atom is broken from flat part of electrode tip and exactly there is a surface Au atom on the tip nearby the N atom, the molecule generally turns to absorb on the surface Au atom, which further results in another lower conductance step in the traces as the experimental probing.Based on the  G) decreases exponentially with a decay constant 2 group262931233435363741424446It is noticeable that, due to the rapid progress of single-molecule technologies123567810111213141516171819202112172627As one knows, in the lower bias voltage regime, resonant transmissions contributed by delocalized transmission channels are often very weak. Hence, nonresonant transmissions, which attribute to the quantum scattering and quantum transmission effects of effective potential fields4950n = 2, 4, 6, 8, denoted as C2, C4, C6 and C8, respectively) molecular junctions experimentally probed by different groups29D) between the two electrodes. For each distance, the molecule and some Au atoms which close to the molecule are relaxed by performing geometric optimization, and the other Au atoms are fixed. Then the electrodes’ distance is elongated by adjusting the fixed Au atoms a little, and the relaxed part of the system that is just optimized is taken as the initial geometry to perform further geometric optimization. By this way, the stretching process is implemented at B3LYP level with Lanl2DZ basis set in Gaussian03 packagesThe electronic transport properties of alkane diamines D = 0.95, 1.21, 1.47 and 1.73 nm for C2, C4, C6 and C8 molecular junctions, which correspond to the equilibrium electrode distances . The second probable process is that, the end of the molecule shifts to close the surface Au atom, simultaneously, the surface Au atom glides on the gold tip from one hollow position to the neighbor hollow position to close the end of the molecule, and further elongation also induces the other end of the molecule gliding on the other gold tip until the molecule is approximately perpendicular to the surface of gold tip. Finally, at 1.63 nm, the molecule is broken down from the gold tip without surface Au atom . However, if the molecule is broken from the electrode without surface Au atom on the tip, the molecular junction is straight ruptured.4, 4′-bipyridine molecular junction is also investigated by OTCTCA method. In order to simulate the stretching process, considering existence of single surface Au atom on the tip of breakpoint, we construct a 4, 4′-bipyridine molecular junction as In order to understand why the end of 4, 4′-bipyridine molecule is prone to adsorbing on the surface Au atom, we show the evolution of single-point energy when elongate 4, 4′-bipyridine molecular junction in et al. very well495049When the two electrodes both possess single surface Au atom, the conductance trace still shows two conductance steps . From Fii.e., inexpensive computational cost, intelligible electronic transport picture, and well consistent results. The disadvantage of OTCTCA method is difficult to deal with longer molecular chain with large bending angle. For the strong coupling molecular junctions such as molecular device with dithiol linker groups, the strong coupling may enhance the conductivity of the molecular junction for some extent, which is not included in the present OTCTCA method and will be considered in future.It is not an occasional event to successfully simulate the dependence of conductance on molecular length and the conductance steps in the stretching processes of the molecular junctions with OTCTCA method. In fact, these results are inevitable due to the wave character of electrons. As one knows, for the electronic transport in the molecular junction, the electronic waves are injected into the functional molecule from electrode and forced to transfer along the negative bias direction in the molecule by external bias voltage. Simultaneously, the atoms in the molecule can be considered as barriers or scattering centers in the path of transmission waves, which result in the diffracting and reflecting of electronic waves. One can find that the OTCTCA method exactly matches with this physical picture. Additionally, the OTCTCA calculations show that the shape of the effective potential and the number of scattering centers are the key factors to influence nonresonant transmission, which not only matches the essential characteristic of nonresonant transmission, but also matches the original predicted mechanism of exponential length dependence of the conductance562 unit in the alkane diamines molecular chains with decay constant of about 0.85~0.95 per CH2 unit. Conductance trances are calculated by elongating molecular junctions successively and steps are found in the conductance traces. Especially, for 4, 4′-bipyridine molecular junction, the conductance trances show bi-stable conductive states as the experimental probes, which is induced by single surface Au atom that lies on the tip of gold electrode. The exponential decaying of length dependence and the existence of conductance steps both indicate that, in the lower bias regime, nonresonant electronic transmission dominates electronic transport of molecular device.In summary, based on hybrid density functional theory, a method of one-dimension transmission combined with three-dimension correction approximation is developed and the electron-transport properties of alkane diamines and 4, 4′-bipyridine molecular junctions are investigated. The conductance of alkane diamines decays exponentially with the increase of CHx axis) and the state density of reservoirs are two key factors when calculate current and conductance of molecular device.A molecular device consists of a functional molecule sandwiched between two metal electrodes. In electronic transport process, by driving of bias voltage, the electrons are injected into functional molecule from one electrode (defined as source reservoir) and scattered by the molecule, at last poured into the other electrode (defined as drain reservoir). It is obvious that the spatial distribution of the effective potential that around the molecule dominates electrons transmit through the molecule. The transmission probability in the direction of bias voltage , then the current that flows through molecular device isIf the incident probability current density is S is the effective inject area. Here we apply plane wave to approximately simulate incident wave, thus the probability current density58where E is the energy of incidence electron relative to the Fermi energy of drain reservoir.and T(E), we plot spatial distribution of effective potential of a molecule in x components of probability current density have contribution to the current, we developed one-dimension transmission combined with three-dimension correction method to approximately calculated transmission probability of molecular device. In this method, we calculate a series of transmission probability τis of electrons with given energy along a corresponding series lines that parallel x axis  and (9), we haveFrom equation (10) and with the help of Schrödinger equation (3), we can obtainLet Additionally, and with the help of recurrence relations we further derive Then we obtain the reflected coefficientBased on the particle conservation law we know that i.e., the atoms, thus for lower incident energy, only the s partial wave has obvious contribution to the scattering. Considering that s partial wave is isotropic, one can easily understand that, when electrons pass through each scattering center, only a half of electrons are scattered forward. Hence the transmission probability should be reduced by half when the electrons pass each scattering center, i.e., K is the number of scattering centers in the transmission path. In addition, the coupling between different lines will cause the electrons pass through more scattering centers compared with those only transporting along the line parallel x axis, which will further reduce the transmission probability to about 0.9 ± 0.1, and the exact proportion of reducing is related to the geometries of molecules.However, considering three-dimension effect, especially the diffraction effect of wave function in three-dimension space, the transmission probability obtained by one-dimension approximation is generally much larger than the exact value. In fact, the molecule consists of series scattering centers, How to cite this article: Liu, R. et al. A method to study electronic transport properties of molecular junction: one-dimension transmission combined with three-dimension correction approximation (OTCTCA). Sci. Rep.6, 21946; doi: 10.1038/srep21946 (2016).


CT), and transluminal attenuation gradient (TAG), CCTA has emerged as a noninvasive method for the assessment of both anatomy of coronary lesions and its physiological consequences during a single study. This review provides an overview of the current status of new CT techniques for the physiologic assessments of CAD.Coronary CT angiography (CCTA) has become an important noninvasive imaging modality in the diagnosis of coronary artery disease (CAD). CCTA enables accurate evaluation of coronary artery stenosis. However, CCTA provides limited information on the physiological significance of stenotic lesions. A noninvasive “one-stop-shop” diagnostic test that can provide both anatomical significance and functional significance of stenotic lesions would be beneficial in the diagnosis and management of CAD. Recently, with the introduction of novel techniques, such as myocardial CT perfusion, CT-derived fractional flow reserve (FFR Coronary CT angiography (CCTA) has been widely used as an effective noninvasive imaging modality in the diagnosis of coronary artery disease (CAD) . MultiplCT, and transluminal attenuation gradient, CCTA has emerged as a noninvasive method for the assessment of both anatomy of coronary lesions and its physiological consequences during a single study. This review provides an overview of the current status of new CT techniques for the physiologic assessments of CAD.Detection of the physiological severity of intermediate coronary lesions has significant implications for the diagnosis, prognosis, and optimal treatment , 19. It Nuclear medicine for a long time has played an important role in the noninvasive evaluation of known or suspected coronary artery disease. Over the past decades, myocardial perfusion imaging (MPI) using single photon emission computed tomography (SPECT) or positron emission tomography (PET) has been well established as the reference standard for the diagnosis and decision making in patients with known coronary artery disease , 26. A mMyocardial CT perfusion imaging (CTP) can be performed in two ways: first-pass static perfusion imaging and dynamic CTP. First-pass CCTA imaging has the potential to assess the physiologic significance of coronary artery lesions . In thisBy using resting CCTA images, coronary artery stenosis and myocardial perfusion may be evaluated simultaneously using the same raw data without the need for further scans, or additional radiation and contrast. However, the value of rest myocardial CTP for the evaluation of myocardial ischemia is uncertain. There have been some reports investigating the detection of myocardial ischemia at rest using multidetector CT (MDCT). Iwasaki and Matsumoto  studied Stress CTP is performed under pharmacological administration of stress agents, such as adenosine, dipyridamole, or regadenoson, similar to nuclear medicine MPI . In the Studies have been conducted to investigate the diagnostic performance of stress CTP for the evaluation of CAD. Blankstein et al.  studied CORE320 is a multicenter multinational diagnostic study which was designed to evaluate the diagnostic performance of 320-MDCT for detecting coronary stenosis and myocardial perfusion deficits in patients with suspected CAD compared with conventional coronary angiography and SPECT MPI . In thisThe use of adenosine during stress CTP may increase the heart rate. CTP is prone to motion artifacts particularly in patients with high heart rate, which may compromise the evaluation of myocardial perfusion. Combined CCTA/CTP has been proven to have better diagnostic performance than CCTA alone. Clinical outcome studies are still needed to determine the effectiveness of stress CTP in evaluating patients with known or suspected CAD compared with other modalities.P = 0.004). The results indicate that combined CCTA and CTP may provide incremental diagnostic value compared with CCTA alone for the detection of significant coronary stenoses in patients with CAD, especially in patients with heavily calcified plaques or implanted stents. To evaluate the feasibility and diagnostic accuracy of stress DECT for detecting hemodynamically significant stenosis causing reversible myocardial perfusion defect, the same group studied 41 patients with known CAD compared with stress MR perfusion and conventional coronary angiography. Stress DECT had sensitivity, specificity, and accuracy of 89%, 78%, and 82%, respectively, for detecting segments with reversible perfusion defects compared with MR perfusion. For the detection of vascular territories with reversible perfusion defect that had hemodynamically relevant CAD, stress DECT had 89% sensitivity, 76% specificity, and 83% accuracy compared with conventional coronary angiography. The results demonstrate that stress DECT has the potential to identify stress-induced myocardial perfusion defect in patients with CAD. It should be noted that DE-CTP is more susceptible to motion artifacts during adenosine infusion and beam-hardening artifacts which mimics perfusion defects and led to false positives [Recently, CTP has been performed by using the dual-energy mode. Dual-energy CT (DECT) is based on the principle that body tissues and iodinated contrast have specific spectral characteristics at different energy of X-rays , 52. DECositives . Recent ositives , 61. AltSingle-phase first-pass CTP is highly dependent on accurate bolus timing and thus the peak attenuation may be missed because of the acquisition of only one sample of the data. A dynamic CT perfusion scan can be performed while the scanning table is stationary or in shuttle mode , 63. Durr = 0.639). The combination of CTP with CCTA improves the diagnostic accuracy for identifying flow-limiting stenosis compared with CCTA alone.The feasibility of dynamic CTP for the detection of hemodynamically significant coronary stenosis was investigated by Bamberg et al.  with invThe comparison of dynamic with single-phase CT acquisition for MPI has been recently studied. One study demonstrated similar performance of single-phase peak enhancement to the perfusion parameter MBF in the detection of ischemic myocardium . AnotherDelayed enhancement CT was found to be feasible for the assessment myocardial infarction and viability. Hyperenhanced myocardial areas on delayed CT images may represent scar tissue caused by myocardial infarction. However, the contrast-to-noise ratio between the infarcted region and the normal myocardium is limited compared with CMR delayed enhancement imaging , 71. OneThe main limitations of CTP currently are artifacts, radiation exposure, and contrast load. Artifacts can alter myocardial attenuation and interfere with myocardial perfusion analysis, which may result in misinterpretation of myocardial blood flow and perfusion defects . It has The increasing radiation exposure to the total population due to CT scans has raised serious concerns . The maiBesides the radiation, concerns about iodine contrast associated with CTP remain to be addressed, especially in patients with abnormal renal function. Combined CCTA/CTP requires doubling the iodinated contrast dose compared with CCTA alone. The repeated scanning in stress testing requires additional iodine contrast. Recent studies have demonstrated 74% reduction in radiation and 28% reduction in contrast dose without significantly compromising the ability to detect stress-induced myocardial ischemia by using lower tube voltage . TherefoMyocardial CTP has the potential to noninvasively assess both coronary artery stenosis and its functional significance at a single examination. Current evidence indicates that the combination of CCTA with CTP imaging may improve diagnostic accuracy compared with CCTA alone for the evaluation of suspected CAD. Future advances of CT technology with improved spatial and temporal resolution and detector widths, as well as beam-hardening correction algorithms, will enhance the diagnostic accuracy of CTP. CTP would be performed with very low radiation dose and free of beam-hardening artifacts. More studies will be required to further define the diagnostic value of combined CCTA and CTP. Longer term follow-up studies are needed to validate the prognostic value of CTP, as well as the patient outcomes of CTP-guided clinical decision making. Although CTP has shown the potential to evaluate the hemodynamic significance of stenotic lesions, there is still no strong evidence to support the routine clinical use of this novel technique.FFR is widely accepted as the gold standard invasive physiologic test for the assessment of lesion-specific ischemia , 84. FFRCT) with the advances in computational fluid dynamics and image-based modeling. FFRCT is a novel noninvasive technique that can be used to determine the physiologic significance of coronary stenosis without any modification of CCTA protocols, additional image acquisition, or administration of medications [CT is calculated by computational fluid dynamic (CFD) modeling after semiautomated segmentation of coronary tree and left ventricular mass. Three-dimensional blood flow and pressure of the coronary arteries can be simulated, with blood modeled as a Newtonian fluid with incompressible Navier-Stokes equations and solved subject to appropriate initial and boundary conditions with a finite element method on parallel supercomputer [CT is modeled for conditions of adenosine-induced hyperemia without adenosine infusion. This process is computationally complex and time-consuming and may require several hours. Similar to invasive FFR, FFRCT was obtained by dividing the mean pressure distal to the coronary stenosis by the mean aortic pressure. An FFRCT < 0.80 was considered diagnostic of lesion-specific ischemia [CT studies have been performed. In the DISCOVER-FLOW  trial [CT and CCTA stenosis for the detection of ischemia-causing lesions was assessed with invasive FFR as the reference standard. The results demonstrate that the per-vessel accuracy, sensitivity, specificity, PPV, and NPV of FFRCT were 84.3%, 87.9%, 82.2%, 73.9%, 92.2%, respectively, and were 58.5%, 91.4%, 39.6%, 46.5%, 88.9%, respectively, for CCTA stenosis. The area under the ROC was 0.90 for FFRCT and 0.75 for CCTA (P < 0.001). The specificity and PPV were remarkably improved by FFRCT compared with CCTA stenosis. The DeFACTO  trial [CT for the diagnosis of ischemia with invasive FFR as a reference standard. On a per-patient basis, diagnostic accuracy, sensitivity, specificity, PPV, and NPV of FFRCT plus CCTA were 73%, 90%, 54%, 67%, and 84%, respectively. When comparing FFRCT with CCTA stenosis for diagnosing obstructive CAD, FFRCT demonstrated improved discrimination with area under the ROC 0.81 for FFRCT and 0.68 for CCTA (P < 0.001). The study results demonstrated improved diagnostic accuracy of FFRCT plus CCTA versus CCTA alone for the diagnosis of ischemia, although the study did not achieve its prespecified primary outcome goal of diagnostic accuracy of greater than 70% of the lower bound of the sided 95% confidence interval . The study suggests the potential of FFRCT as a promising noninvasive method for the identification of individuals with ischemia. More recently, the multicenter study NXT  trial [CT for the diagnosis of ischemia was determined with invasive FFR as the reference standard. As compared to previous studies [CT technology was used with an emphasis on CCTA image quality. The area under the ROC curve for FFRCT was 0.90 versus 0.81 for CCTA (P = 0.0008). On per-patient basis, sensitivity and specificity to identify ischemia were 86% and 79% for FFRCT versus 94% and 34% for CCTA, respectively. When compared to CCTA, the improved diagnostic performance of FFRCT is in particular with regard to specificity. The study demonstrates that FFRCT has relatively high diagnostic accuracy compared to invasive FFR for identifying hemodynamically significant CAD.Recently, The FFR can be computed from standard CCTA scans  trial , FFRCT wy) trial , a multi studies , 89, theCT technology enables the “virtual stenting” modeling and may be served to predict hemodynamic effect of coronary stenting on ischemia-causing stenoses [CT in 44 patients (48 lesions) to predict FFR value changes after stenting with invasive FFR as the reference standard. Before intervention, invasive FFR was 0.70 ± 0.14 and increased to 0.90 ± 0.05 after stenting. FFRCT was 0.70 ± 0.15 before intervention and increased to 0.88 ± 0.05 after virtual coronary stenting. There was a good correlation between FFR and FFRCT before  and after stenting . This study demonstrates that virtual stenting by FFRCT is feasible and may be helpful for revascularization planning before invasive procedures.FFRstenoses . Kim et stenoses  studied CT. First, FFRCT is calculated by computational simulation of adenosine mediated hyperemia rather than by actual administration of adenosine. Second, the value of FFRCT is influenced not only by stenosis severity but also by the presence of viable or scarred myocardium [CT values may be lower than those of measured FFR in patients with microvascular disease, for the modeling of adenosine-induced hyperemia may overestimate the degree of vasodilation [CT requires accurate anatomic models, numerous artifacts on CCTA may affect FFRCT calculation, including the presence of heavy calcification, motion artifacts, lower SNR, and misregistration. Therefore, CCTA data with good image quality is essential for the accuracy of FFRCT interpretation.There are several limitations for physiologic assessment of coronary stenosis by FFRocardium . Third, dilation . Fourth,CT is feasible for noninvasive determination of lesion-specific ischemia. Although the accuracy of FFRCT was reported as modest in several multicenter trials, this novel technique shows promise not only for diagnosing lesion-specific ischemia but also for predicting revascularization benefit. The present studies support the potential of CCTA with FFRCT as a gatekeeper to invasive coronary angiography and revascularization. Further improvements in the technology or new algorithms for a reliable estimation of FFR will be a prerequisite for wide acceptance of FFRCT, and further studies are needed to determine its clinical utility compared with other noninvasive testing. Combination of CCTA and FFRCT might permit evaluation of CAD in a “one-stop-shop” approach in the future.The results of the limited studies indicate that FFRAssessment of the change in intraluminal contrast attenuation across a coronary stenosis on CCTA may allow prediction of functional significance of coronary stenosis . A methoHowever, the measurement of gradients across coronary stenoses is inherently more robust than evaluation of opacification difference. Transluminal attenuation gradient (TAG) is defined as the linear regression coefficient between luminal attenuation (Hu) and axial distance along the vessel from the coronary ostium. TAG is reflected by the kinetics of the iodinated contrast media within the coronary arteries. Study confirmed that opacification gradients exist in patients with normal coronary arteries. The TAG along the course of a coronary artery can be reproducibly evaluated with CCTA . The hypIt has been reported that TAG has the potential to improve the classification of stenosis severity on 64-slice CCTA compared with coronary angiography as a reference standard, especially in calcified lesions, which is particularly useful because CCTA is limited in accuracy when lesions are calcified . The limCT. However, given the limited evidence so far, larger studies with current acquisition and reconstruction protocols as well as new analysis software are required to validate the diagnostic and prognostic value of this approach.Combined TAG and CCTA assessment may have incremental value over CCTA alone for detecting functionally significant coronary stenoses, especially in severely calcified lesions. TAG depends on the luminal attenuation values, and the accuracy of quantification may be influenced by various artifacts on CCTA. So, maintaining image quality of CCTA is important for TAG analysis. TAG assessment has particular attraction, as it does not require additional scan or complex computation compared with CT perfusion or FFRCT, which simulate FFR from CT data using CFD method and TAG derived from the gradient in contrast opacification along a coronary artery. The current studies have demonstrated that these methods are feasible for noninvasive assessment of CAD and have the potential to provide incremental value in detecting functionally significant coronary stenosis over CCTA alone. Although the currently available data are promising, these approaches are still in its early stage, and their diagnostic values still need further validation. Further research is required to identify the prognostic value and clinical outcomes of decision making based on these techniques.CCTA has become an important noninvasive imaging modality in the diagnosis of CAD. CCTA enables accurate evaluation of coronary artery stenosis. However, CCTA provides limited information on the physiological significance of stenotic lesions. There is a great interest in evaluating both coronary anatomy and its hemodynamic significance during a single examination. Combination of anatomic and physiologic information would be beneficial for clinical decision making, particularly in lesions with moderate stenoses. Recently, novel CT techniques which can provide both anatomical and functional assessments of CAD were developed, including myocardial CTP and noninvasive FFR


Consistent with a role of Shh in the regulation of neuronal proliferation in this region, we find that the number of tyrosine hydroxylase (TH)-positive neurons is increased in the VTA of −/−Cdon mice at birth and that this effect endures into adulthood. In contrast, the number of TH-positive neurons in the SNc is not altered in −/−Cdon mice at either age. Moreover, adult −/−Cdon mice have a greater number of medial prefrontal cortex (mPFC) dopamine presynaptic sites, and increased baseline concentrations of dopamine and dopamine metabolites selectively in this region. Finally, consistent with increased dopamine function in the mPFC, we find that adult −/−Cdon mice fail to exhibit behavioral plasticity upon repeated amphetamine treatment. Based on these data, we suggest that Cdon plays an important role encoding the diversity of dopamine neurons in the midbrain, influencing both the development of the mesocortical dopamine pathway and behavioral outputs that involve this neural circuitry.Motivated behaviors and many psychopathologies typically involve changes in dopamine release from the projections of the ventral tegmental area (VTA) and/or the substantia nigra pars compacta (SNc). The morphogen Sonic Hedgehog (Shh) specifies fates of midbrain dopamine neurons, but VTA-specific effects of Shh signaling are also being uncovered. In this study, we assessed the role of the Shh receptor Cdon in the development of VTA and SNc dopamine neurons. We find that Cdon is expressed in the proliferating progenitor zone of the embryonic ventral midbrain and that the number of proliferating cells in this region is increased in mouse  Sonic hedgehog signaling is involved in the specification and development of dopamine neurons in the ventral midbrain. Here we demonstrate that the Shh receptor Cdon plays a role in the development of dopamine neurons in the ventral tegmental area. Moreover, this effect of Cdon is selective to the dopamine neurons that project to the medial prefrontal cortex. Adult mice that lack Cdon also fail to show amphetamine-induced behavioral plasticity. Our findings show that the Cdon receptor is important in encoding the diversity of dopamine neurons in the midbrain, influencing both the development of the mesocortical dopamine pathway as well as behavioral outputs that involve this neural circuitry.Midbrain dopamine neurons are involved in diverse behavioral and psychological processes, and alterations in their development can have implications that range from motor deficits to psychopathology . DopaminOne example is the sonic hedgehog (Shh) signaling pathway, which is involved in the specification of dopamine cell fate  and acts−/−Cdon embryos at embryonic day 12.5 (E12.5), and identified a potential role for Cdon in the regulation of proliferation in the midbrain dopaminergic progenitors. Consistent with a putative increase in the proliferation of dopamine progenitors in −/−Cdon mice, we observed an increase in the number of dopamine neurons in −/−Cdon mice immediately after birth and in adult life. Importantly, this increase was specifically observed in the VTA. Next, we examined dopamine concentrations in forebrain regions that receive dopamine projections from the VTA or the SNc and found increased levels of dopamine and dopamine metabolites in the medial prefrontal cortex (mPFC), but not in the nucleus accumbens (NAcc) and dorsal striatum (DS), of adult −/−Cdon mice. Furthermore, we found that adult −/−Cdon mice have an increased number of mPFC dopamine presynaptic sites. To determine potential behavioral consequences of these neuroanatomical and neurochemical changes, we evaluated amphetamine-induced behavioral plasticity in adult −/−Cdon mice and found important deficits. These findings show that Cdon is important in the development of VTA dopamine neurons, particularly those projecting to the mPFC, and in turn influences adult behaviors that are dependent on these pathways.Cell adhesion molecule-related/downregulated by oncogenes (Cdon) is a Ptch1 coreceptor that binds Shh  and moduhttp://www.ccac.ca). −/−Cdon mice  studies, as well as in the quantification of tyrosine hydroxylase (TH)-positive varicosities in the mPFC. All other experiments used only male mice.All animal housing, experiments, and procedures were approved by the Animal Care Committee at the Douglas Mental Health University Institute, McGill University  and at the Institut de Recherches Cliniques de Montréal, and were all performed in accordance with the guidelines set out by the Canadian Council of Animal Care , cryoprotected in a sucrose solution , then snap frozen in optimal cutting temperature medium  and stored at −80°C until slicing. Embryos (14 μm sections) and P0 (35 μm sections) pups were sliced on a cryostat , sections were collected on charged Superfrost Slides (Fisherbrand), and stored at −80°C until use. Adult male mice  were deeply anesthetized with sodium pentobarbital , perfused transcardially with ∼50 ml of 0.9% saline followed by ∼50 ml of 4% paraformaldehyde. Brains were dissected, postfixed overnight (4°C), and sliced on a Vibratome . Serial coronal sections were stored free-floating in Watson’s cryoprotectant at −20°C until processing .Immunohistochemistry and immunofluorescent staining was performed  with ant−/−Cdon mice included in the study had weights that were similar to those of the WT littermates.Serial coronal sections of embryos and adult brains were examined with Leica DM4000 and DM6000 microscopes with an Orca ER CCD camera (Hamamatsu) using Volocity (PerkinElmer) or Stereo Investigator (MBF Bioscience) software. In order to avoid including mice with signs of holoprosencephaly (HPE), we inspected for HPE on the live/intact mouse or embryo and performed a careful and systematic morphological analysis under the microscope. Specifically, all embryos and mice were inspected for any signs of cebocephaly and incomplete forebrain clefting . At P0, Genotype×Level. Stereology was performed to quantify the number of TH-positive cell bodies in the VTA and SNc, and the number of TH-positive varicosities in the mPFC , and the optical fractionator probe was then used to count TH-positive varicosities. The Gundersen coefficient of error was <0.15 for all regions of interest in all sampled brains.To obtain a measure of the presynaptic density of dopamine synapses in the pregenual mPFC, TH-positive varicosities were quantified in this structure. TH-positive varicosities are sites of putative synapses with a dendritic spine or shaft , and areAs described previously , mice wem phosphate buffer, centrifuged, the supernatant was then removed and filtered for HPLC testing, and the pellet was resuspended for quantification of the protein content . The HPLC assay for dopamine, DOPAC, and HVA was performed with an EZChrom Chromatography System (Scientific Software Inc). Dopamine and metabolites were detected and quantified with a Coulochem III detector, and concentrations were calculated from peak height comparisons with known amounts of injected pure standards (Sigma-Aldrich). Significance levels used to evaluate statistical differences were adjusted using the Holm-Bonferroni’s sequentially rejective procedure  and homovanillic acid (HVA) in the DS, NAcc, and mPFC were assessed using high-performance liquid chromatography HPLC; . Brieflyrocedure .As described previously , locomotAs described previously , prepulst tests, analyses of variance, and Bonferroni’s post hoc tests were performed using Prism 5 (GraphPad Software). For each figure and statistical test, F and t values are reported in t test was used, and in Genotype×Level was used. In t test. In Genotype×Region was used. In Genotype×Time was used to compare groups over the duration of the test, and in Genotype×Time was used with Bonferroni’s post hoc comparisons. In Genotype×ppvolume was used. All graphs illustrate the mean ± standard error.All Student’s Cdon gene by homologous recombination  of the midbrain dopamine somatodendritic region.We next assessed whether this increase in progenitor proliferation leads to an increase in the numbers of dopamine neurons later in brain development and in adulthood. Stereological counts of TH-positive neurons in the VTA and SNc at P0 revealed a significant increase in the number of TH-positive cells in the VTA of −/−Cdon mice are significantly elevated compared to WT littermates  or a stimulant drug of abuse .To examine the possible consequences of the neuroanatomical changes that we observed in the VTA and mPFC of ironment  and in rironment . Cdon−/−post hoc test on WT, p = 0.0018; −/−Cdon mice when pre- and postsensitization levels were compared  and then were left undisturbed in their home cage for 8 d , and only mice that did not show any obvious signs of HPE and that remained healthy into adulthood were included in our study. This variability between studies is in part attributed to the fact that the expression of HPE in −/−Cdon mice depends strongly on the genetic background and genetic modifiers of this receptor (Cdon mutant mice (A previous report described a reduction in the number of TH-positive cells in E13.5  embryos . One posdulthood . In contreceptor . Importaant mice . TherefoThe increased numbers of Ki67-positive cells that we observe at E12.5 coincides with the second stage of Shh signaling. At this stage in development, it would appear that Cdon modulates the proliferation rate of dopamine neuron progenitors. Indeed, it has been shown that once dopamine neuron progenitors begin to express Shh, the duration and timing of Shh expression contributes to fate decisions made by these cells . Therefo−/−Cdon mice exhibit a greater number of presynaptic dopamine sites in the mPFC without showing increases in the expanse that dopamine fibers occupy in this regions indicates that Cdon plays a role in the proliferation of mesocortical dopamine neurons, but not in their guidance toward forebrain targets.An increasing number of reports show that dopamine neurons in the VTA are a heterogeneous population, and that the neuroanatomical, electrophysiological, and developmental properties of these neurons are dictated by the targets they innervate. It is therefore possible that in the midbrain, only a subset of the medial portion of the ventral midbrain dopamine neuron progenitors coexpress Cdon and Shh, namely those dopamine progenitors that are fated to innervate the mPFC. Increased numbers of mesocortical dopamine neurons would presumably lead to increased dopamine input to and dopamine concentrations in the mPFC. Because the dopamine innervation to the mPFC is a protracted event, which extends into early adulthood, we would also predict that these effects will only manifest fully in adulthood. The fact that Dcc exhibit increased baseline concentrations of dopamine and dopamine metabolites in mPFC, which in turn causes blunted amphetamine-induced dopamine release in the NAcc (Dcc haploinsufficient mice also fail to show sensitization to the locomotor effects of amphetamine upon repeated exposure (−/−Cdon mice could result from blunted responsiveness of NAcc-projecting dopamine neurons, which are associated with increased mPFC dopamine function (Locomotor responses to amphetamine depend mainly on drug-induced dopamine release in the NAcc , which ithe NAcc . Interesexposure . Thus, ifunction .−/−Cdon mice (The selective effects of Cdon on the mesocortical dopamine projections are particularly interesting in light of another recent study that also highlighted the sensitivity of this circuit to changes in Shh signaling . Gli2 is−/− mice .−/−Cdon mice is surprising, and at this point we cannot provide a conclusive mechanistic explanation of this finding. Deficits in baseline prepulse inhibition have been shown to result from reduced mesocortical dopamine function (−/−Cdon and WT mice. It is possible that impaired sensorimotor gating function in −/−Cdon mice results from alterations in mPFC and/or nucleus accumbens dopamine release that could only be captured via in vivo microdialysis or voltammetry. Moreover, it is also possible that either insufficient or excessive extracellular dopamine concentration in the mPFC lead to deficits in PPI as has been shown for the effects of mPFC dopamine function on cognitive processing (The relationship between increased dopamine concentrations in the mPFC and impaired PPI in function . Furtherfunction , it has function . Howeverocessing . Future ocessing .In conclusion, it is increasingly becoming clear that the diversity of midbrain dopamine neurons results from developmental processes that determine the heterogeneity of these cells, potentially long before this diversity can be accurately described . This di