Because of its strongly theoretical nature, evolutionary biology is a field full of controversy. For example, in the late 1970s, UCLA hosted a forum in which two famous biologists, Motoo Kimura and Francisco Ayala, engaged in a heated debate. Kimura had posited in the late 1960s that molecular evolution is dominated by neutral processes, i.e., genetic drift and random mutation. Ayala argued for the more traditional view, i.e., Darwinian natural selection as the primary force driving evolution of genes and proteins. Ideological camps formed around this controversy, and the debate continues to this day. Nevertheless, these discussions have been a positive driver of research in the field—our view of molecular evolution has benefited and been shaped by the continuing development of the concepts surrounding this debate.mBio series explores a new topic of controversy in the field of evolutionary biology—the hologenome concept. This view advances the idea that evolutionary forces, both neutrality and selection, act on the host and its symbiotic microbes as a conjoined “holobiont,” and the genomic complement of all partners equals the “hologenome.” The opposing view adheres to the traditional concept that evolution is acting at the level of the individual species that comprise the whole functional biological unit and that this set of closely interacting organisms is most clearly conceptualized and studied as an ecological community. As in the debate between the neutral and selectionist theories, there may be no one answer that applies to all systems under all circumstances, but the exploration of these ideas promises to drive the field forward in exciting ways.This entire biosphere. The application of molecular features to study microbial evolution, particularly in the last 10 years, continues to have profound effects on all of evolutionary biology. For example, the Modern Synthesis, developed in the 1930s and ’40s, is the most widely held view of evolutionary biology; it couples Darwin’s theory of natural selection with Mendel’s genetics and provides a mechanistic explanation for evolutionary processes in animals and plants. However, because most microbes can acquire genes in a Lamarckian fashion, by lateral gene transfer, much of the biosphere does not adhere to the precepts of the Modern Synthesis. Thus, Eugene Koonin has suggested that we need a “postmodern synthesis” to capture mechanisms of evolution beyond the strictly vertical inheritance of traits. Relevant here is that genome sequencing of the microbiota has demonstrated tight relationships between hosts and their microbial partners. Such relationships span from mitochondria through obligate, vertically transmitted intracellular symbionts, to the extracellular consortia of gut microbes acquired each generation by animal hosts. The host and its microbial partners constitute a complex amalgamation of genomes that is driven by both Darwinian and Lamarckian processes. The stability, connectivity and predictability of symbiotic associations have precipitated a reconsideration of the most fundamental principles of macroevolution. Not only classically trained evolutionary biologists but also others in the field of symbiosis are entering the debate around the hologenome concept.From where does the hologenome controversy arise? Although microbiologists have been strong contributors to the neutralist/selectionist debate of molecular evolution, until recently there has been less focus on macroevolution, i.e., the evolution of separate gene pools, occurring at the species level and above. Through the lens of macroevolutionary concepts, animal and plant biologists had recognized patterns of relatedness among organisms, from species to the higher taxonomic levels, and gained insight into how they arose and diversified across geologic time. It was the pioneering work of Carl Woese, beginning in the late 1970s, that brought macroevolutionary approaches to microbiology. Using the sequences of slowly evolving genes to produce molecular phylogenies, Woese could characterize the relationships of organisms across the As the contributions in this series illustrate, evolutionary biology joins a growing set of subdisciplines of biology that have been revolutionized by an emerging realization: that the microbial world is fundamental to all processes of the biosphere across the hierarchy of life, from ecology to cell and molecular biology. For example, with phylo- and metagenomics enabling characterization of the forms and functions of microbial communities, for the last two decades the field of ecology has debated the extent to which the well-developed concepts describing the ecology of macroorganisms can be applied to microbial ecology. Similarly, at the cellular and molecular levels, fields such as immunology, developmental biology, and neuroscience have been transformed by the increasing awareness of our own dependence on the microbial world for growth and health. Evolutionary biology is similarly experiencing the need to question and affirm existing ideas as the field integrates new microbiology-inspired visions. One such vision is the hologenome concept.mBio series with an open mind: consider the arguments and embrace the controversy, and think about how such debate will shape our concepts of the biological world. As with the neutralism/selectionist debate, it is likely that the hologenome concept will be a focus of contention for many years to come. Right now, for those of us who are not evolutionary biologists, it is enough to recognize that the very foundations of biology are being shaken by both the integration of microbiology into concepts of macroevolution and the recognition that host-microbe symbioses are a major theme in biological systems.When patterns do not fit an existing construct, controversy arises, and … that’s when we learn. The reader would be advised to go into this


A key feature of precision medicine is that it takes individual variability at the genetic or molecular level into account in determining the best treatment for patients diagnosed with diseases detected by recently developed novel biotechnologies. The enrichment design is an efficient design that enrolls only the patients testing positive for specific molecular targets and randomly assigns them for the targeted treatment or the concurrent control. However there is no diagnostic device with perfect accuracy and precision for detecting molecular targets. In particular, the positive predictive value (PPV) can be quite low for rare diseases with low prevalence. Under the enrichment design, some patients testing positive for specific molecular targets may not have the molecular targets. The efficacy of the targeted therapy may be underestimated in the patients that actually do have the molecular targets. To address the loss of efficiency due to misclassification error, we apply the discrete mixture modeling for time-to-event data proposed by Eng and Hanlon  to devel On February 20, 2015, in his State of the Union Address, US President Obama announced the launching of a new Precision Medicine Initiative (PMI). As pointed out by Collins and Varmus . The enrThe enrichment design may be an efficient design for evaluation of targeted therapies. However, very few diagnostic devices have a perfect accuracy with 100% positive predictive value (PPV). In particular, the PPV can be very low for some rare diseases with low prevalence. Consequently, a sizable proportion of the patients tested positive during the enrichment phase may not actually have the molecular targets of interest. On the other hand, the targeted therapy is only designed to be efficacious for the patients actually having the molecular targets. The targeted therapy may not only be ineffective but also cause harmful adverse reactions in those patients who do not have the molecular targets despite testing positive for them. Liu, et al.  showed tMost of clinical trials for the near-term goal in the PMI are for evaluation of targeted therapies in treatment of various cancers with progression-free survival (PFS) or overall survival (OS) as the primary or secondary efficacy endpoints. The Cox proportional hazard (PH) model is the most commonly employed statistical method for inference of treatment effects which takes individual patient characteristics into account. Consequently, we propose an application of the discrete mixture modeling for time-to-event data proposed by Eng and Hanlon  to develIn the next section, our proposed method for taking into consideration the diagnostic inaccuracy and uncertainty, based on discrete mixture modeling and the EM algorithm for the Cox PH model, is introduced with inferential procedures. The results of numerical studies, including numerical examples and simulation studies, are provided in the third section. Numerical examples illustrate the application of our proposed method in practical scenarios. Simulation results provide empirical performance of our proposed methods in terms of size, power and coverage probability of confidence intervals. Final remarks and discussion are presented in the last section.We developed the proposed procedure for the following situations. First, a specified molecular target has been identified in the pathway of pathogenesis of the disease. Secondly, a validated diagnostic device is available for detection of the specified molecular target with a known estimated positive predicted value from validation studies. Third, this device is only for detection of the molecular target and is not prognostic for clinical outcomes of patients. A targeted therapy is currently being developed for true-positive patients with the molecular target. Finally, the enrichment design is employed to evaluate the treatment effect of the targeted therapy for the patients tested positive for the molecular target by the diagnostic device. Our objective is to develop statistical inference for the treatment effect of the targeted therapy for the true-positive patients with the specified molecular target.Although under the enrichment design, all randomize patients have tested positive for the molecular target, due to inaccuracy of the diagnostic device, some positive patients still may not have the specified molecular target. Therefore, there are two latent classes of patients: the patients with the molecular target (true-positive patients) and the patients without the molecular target . (Pepe ) By naïvyi+, δi+, zi+), i = 1,…,n+ be an independent right-censored sample with right-censored survival time, yi+, covariates zi+, and censored indicator δi+ .  is similarly defined for the patients without the molecular target (-), i = 1,…,n−. Denote the collection of right-censored times, censored indicator and covariate for all patients asFor simplicity, we only consider one covariate Z for treatment identification in the Cox PH model. Z is 1 for the targeted therapy and 0 for the concurrent control. For the patients with the molecular target (+), let (hh0(t) and λh are the baseline hazard and the treatment effect (regression coefficient: log hazard ratio) with treatment indicator zhi, respectively, for patient i with true target status h, for i = 1,…,nh;h = +,−.We further assume a Cox PH model separately for the patients with and without the molecular target. Hence, under the PH assumption, the hazard function for true target status h is given asThe hypothesis of interest for the true-positive patients with the molecular target is given asHh0(yhi) is the cumulative baseline hazard function for patient i with molecular status h,i = 1,…,nh;h = +,−.The density function of the PH model for right censored survival times for true target status h is given asXi for the true target status of patient i which is independently and identically distributed (i.i.d.) as a Bernoulli distribution with probability γ, where γ is the PPV of the diagnostic device, i = 1,…,N; N = n+ + n_. In other words, Xi = 1 if patient i has the molecular target; = 0 if patient i lacks the molecular target and P(Xi = 1) = γ = 1−P(Xi = 0), i = 1,…,N. The density function of  given latent variable Xi is given asTo accommodate the inaccuracy of the diagnostic device and the variability of its estimate, we introduce a latent binary variable yi+, yi−, xi) isHence, the joint density function of  denote the vector of unknown parameters, where h = {h0+(yi+), h0−(yi−)} indicates the set of baseline hazard functions. It follows that the complete-data log-likelihood function for ψ is given asLet Xi given Yi+, Y−i, δi+, δi−, Zi+, Zi−; γ, h, λ+, λ−. Denoting the current estimates of the parameters after k iterations as To obtain the maximum likelihood estimates of the treatment effect by the expectation-maximization (EM) algorithm, in the E-step, we need to find the conditional expectation of γ is given asIn the M-step, the updated positive predicted value (PPV) Following Eng and Hanlon , the estThe estimation procedure of the EM-based approach is summarized as follows and then explained more fully step-by-step:γ(0),Set initial values for Eψ(K){Xi|Yi+, Yi−, δi+, δi−, Zi+, Zi−;γ,h,λ+,λ−} using h0+, h0−, λ+, λ− by Calculate Repeat Step 2 until convergence.γ(0). For the initial values, the estimated PPV from the validation studies can be used as the initial value of ocedures .Step 1. Choose a large bootstrap sample size B. We would suggest B ≥ 1000. For 1 ≤ b ≤ B, generate the bootstrap samples Step 2. Estimates Step 3. An estimator of the variance of whereα/2 is the α/2 upper percentile of a standard normal distribution, and + can be constructed as The null hypothesis is rejected and the efficacy of the targeted therapy is found to be different from that of the control group for the true-positive patient population at the α significance level if+, λ− according to the status of the molecular target. It is presumed that the placebo control is not efficacious in the patients either with or without the molecular target. In addition, the targeted therapy is presumed ineffective in the false-positive patients without the target. The pair of survival times and censored indicators  was generated according to the methods described in Chen, et al. [We conducted extensive simulation studies to empirically investigate and compare performance of our proposed method with the current procedure in terms of relative bias, coverage probability of confidence intervals, size and power. A modified R function by Eng and Hanlon  was empl, et al. .The following specifications of parameters were considered in the simulation studies. The PPV was set to be 0.5, 0.6, 0.7, 0.8, and 0.9, which reflect a range of low to high positive predicted value. The censored proportions considered in the simulation studies were 0, 0.1, 0.2, 0.3 and 0.4. To investigate the finite sample properties, the sample sizes were set as 300, 600, and 900 per group. The size was evaluated at the hazard ratio of 1. The power of the proposed procedure was investigated at hazard ratios of 0.70, 0.75, 0.80 and 0.85. For each of 300 combinations, 1000 random samples were generated and the number of the bootstrap samples was also set to be 1000.0: λ+ = 0 vs. Ha: λ+ ≠ 0) was rejected for the two-sided test at the 5% significance level. For a 95% confidence level, a simulation study with 1000 simulated random samples implies that 95% of the empirical coverage probabilities of all combinations would be within 0.9365 and 0.9635 if the proposed method provides sufficient coverage probability. In addition, for the 5% nominal significance level, a simulation study with 1000 random samples implies that 95% of the empirical sizes would be within 0.0365 and 0.0635 if the proposed method can adequately control the size at the nominal level of 0.05.For estimation, we investigated the bias of the estimators and the coverage probability of the 95% confidence interval. For hypothesis testing, the performance measures included empirical size and power. The bias was estimated as the average of the differences between the estimates and the true value of the hazard ratio over 1000 simulated samples. The coverage probability was calculated as the proportion of the 1000 95% confidence intervals that contain the true value of the hazard ratio. The size and power were computed as the proportion of the 1000 samples for which the null hypothesis  in the package insert of Herceptin®. A targe®. The basWhen the PPV was 0.75, the naive approach without consideration of inaccuracy of diagnostic device and the variability of its estimate yields the estimate of hazard ratio for mortality of 0.8318 with a 95% CI from 0.6637 to 1.0425. Because the 95% CI does contain 1, the observed hazard ratio of death is not statistically significantly different from 1 at the 5% significance level. The targeted therapy therefore fails to prove its superior efficacy over chemotherapy alone. On the other hand, our proposed EM method resulted in an estimated hazard ratio of 0.7026. The 95% CI for the hazard ratio is , which does not contain 1. As a result, the efficacy of the targeted therapy plus chemotherapy is concluded superior to chemotherapy alone for the true-positive patients with the molecular target at the 5% significance level. Because 25% of the patients do not have the specified molecular target, failure to take into consideration inaccuracy of the diagnostic device leads to underestimation of the hazard ratio based on the naive method by a magnitude of 18.39%.The simulation studies provide empirical results of performance of our proposed method compared with the current approach with respect to relative bias, coverage probability, size, and power. Due to the large volume of results generated in the simulation studies, we only present the results concerning relative bias, coverage probability and empirical power for the combinations with sample size of 300. They are provided in The simulation results show that the empirical estimates of the positive predicted values are close to the specified values for all the different combinations, with a maximal absolute difference of only 0.008. The simulation results on relative bias and coverage probabilities are given in The results concerning the empirical coverage probabilities of the 95% confidence intervals for the hazard ratios by the current method and the EM method are described below. Only 20 of the 300 coverage probabilities (6.67%) of the 95% confidence intervals by the naive method exceed 0.9365. The coverage probability by the naive method are as low as 0.546. On the other hand, there are 245 of 300 coverage probabilities (81.7%) of the 95% confidence intervals by the EM method exceeding 0.9365. However, 280 of the 300 coverage probabilities (93.3%) of 95% confidence intervals constructed by the EM algorithm are above 0.93. No coverage probability of the EM method is below 0.91. The coverage probability is an increasing function of the PPV and a decreasing function of the hazard ratio and sample size. In summary, the proposed EM procedure not only gives nearly unbiased estimated treatment effect for the true-positive patients with the molecular target but also provides sufficient coverage probability.The simulation results on the sizes are given in Although all patients enrolled into the enrichment design are tested positive for the specified molecular target, some of them may not actually have the target due to inaccuracy of the diagnostic device. The proportion of false-positive patients may be sizable for rare diseases with low prevalence rates. Consequently, the treatment effect of the targeted therapy may be severely underestimated for the true-positive patients with the molecular target. In addition, the magnitude of underestimation is a decreasing function of the PPV. The Cox PH model is the most frequently employed method for evaluation of targeted therapies for cancer trials with progression-free survival (PFS) or overall survival (OS) as the primary efficacy endpoint. We applied the method of discrete mixture modeling proposed by Eng and Hanlon , based oλ+−λ− is given byBecause of the discrete mixture modeling, the PH assumption is relaxed such that the hazards are only assumed to be proportional within each group of patients either with or without the molecular target. Since the PPV is not 100%, the log hazard ratio for the patients without the molecular target can be similarly estimated by the EM algorithm. Its estimated standard error can be also obtained by the bootstrap method. Inference regarding the efficacy of the target therapy can similarly be made to the patients without the molecular target. In addition, the patients with the molecular target are independent of the patients without the target. We can also make inferences about the target-by-treatment interaction, i.e., the difference in the treatment effect between the groups of patients with and without the molecular target. Denote λ+−λ− not only can be used to make inferences about the existence of target-by-treatment interaction but also can test whether the efficacy of the targeted therapy of the patients without the target is either equivalent or non-inferior to that of the patients with the molecular target.A confidence interval for λ+ − λ− is  which includes 0. The target-by-treatment interaction is, therefore, not statistically significant at the 0.05 significance level. However, the target-by-treatment interaction is significant at the 0.10 level because the 90% asymptotic confidence interval is , which does not include 0. Therefore, this analysis of the target-by-treatment interaction further demonstrates that even under the enrichment design our proposed procedure can evaluate whether the new targeted therapy is efficacious for false-positive patients if the PPV is not too high.Using the same dataset used in the Numerical Example, the estimated log hazard ratio for the patients without the molecular target is 0.259 with an estimated standard error of 0.3049. The corresponding estimated hazard ratio is 1.2956 with a 95% asymptotic confidence interval of . It follows that a 95% asymptotic confidence for j and βj are the other covariates and their corresponding regression coefficients.Our proposed method only considers one covariate in the PH model, which is the treatment indicator. However, we can also incorporate other covariates such as gender, age, or disease status in the model. The full likelihood with more than one covariate is given asSimilarly, the regression coefficients can be estimated by the EM algorithm and their estimated standard errors can be obtained via the bootstrap technique.Since no diagnostic tool is perfect with 100% PPV, our proposed EM procedure is based on the Cox PH model given two latent class memberships. However, we would be interested in the impact of violation of the two latent classes’ assumption. An additional simulation study was conducted to assess the performance for the special case of PPV = 1.00. The simulation results on the relative bias of the estimator for hazard ratio by both EM and naïve methods are provided in p>0.We conducted another simulation study to investigate the robustness of our proposed EM procedure when the true-positive data is generated from the Weibull distribution with a shape parameter of 2 and the false-positive data is from an exponential distribution. S1 FigBlack line: naive; red line: proposed EM.(PDF)Click here for additional data file.S2 FigBlack line: naive; red line: proposed EM.(PDF)Click here for additional data file.S3 FigBlack line: naive; red line: proposed EM.(PDF)Click here for additional data file.S4 FigBlack line: naive; red line: proposed EM.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


The vascular effects exerted by GLP-1 are mediated by several synergistic mechanisms such as involvement of nitric oxide and natriuresis. Recently, it was demonstrated that atrial natriuretic peptide (ANP) is essential for the glucagon-like peptide-1 (GLP-1)-stimulated vascular smooth muscle relaxation that mediates anti-hypertensive action in rodents. Therefore a GLP-1–ANP axis has been suggested. The aim of this study was to investigate whether this effect can be demonstrated in patients with type 2 diabetes and congestive heart failure.The study was a post hoc analysis of a randomized double-blinded, placebo-controlled trial. Twenty male patients with type 2 diabetes and congestive heart failure were randomized to receive a 6-h infusion of exenatide or placebo. Cardiac filling pressures were measured by right heart catheterization, and plasma levels of ANP, N-terminal pro-brain natriuretic peptide, and exenatide were measured at baseline and at the end of the exenatide infusion.Exenatide infusion resulted in a significant decrease of circulating ANP levels compared with placebo, concomitant with a decrease in pulmonary capillary wedge pressure (PCWP), pulmonary artery pressure (PAP) and right arterial pressure (RAP), and increased cardiac output. There was no correlation between plasma ANP levels and exenatide levels. A negative correlation between ANP levels and PCWP, PAP, and RAP, which remained significant after adjustment for plasma exenatide levels, was demonstrated during exenatide infusion.Exenatide infusion decreases cardiac filling pressure and ANP levels. The reduction of ANP levels was primarily because of the reduction in cardiac filling pressure, independent of exenatide levels. It seems unlikely that this was mediated via ANP.http://www.isrctn.org/ISRCTN47533126Trial registration:  Glucagon-like peptide-1 (GLP-1) is a gut-derived hormone released after meal ingestion , 3 and hThe vascular effects exerted by GLP-1 are mediated by several synergistic mechanisms as earlier studies have linked GLP-1 to both nitric oxide (NO)-dependent, and NO-independent vasorelaxation , 8, 9. AANP is synthesized and stored in secretory granules of atrial myocytes , 15. In The current study is a post hoc analysis of a recently randomized controlled clinical trial, where the aim was to determine whether exenatide improves hemodynamic function in patients with type 2 diabetes and CHF . Since aThe study population comprised 20 participants with type 2 diabetes hospitalized for CHF. Inclusion and exclusion criteria have been described elsewhere . Brieflyhttp://www.isrctn.org/ISRCTN47533126.The protocol was approved by the Swedish Central Ethical Review Board and the Medical Products Agency and conducted according to the principles of the Declaration of Helsinki, 1975. Written informed consent was obtained from all participants. The trial was registered at The trial used a randomized, crossover, double-blind design . In summHemodynamic measures were determined by right-heart catheterization (method described elsewhere ). ThermoA catheter with an arterial line primed with NaCl (0.9 %) was positioned in the radial artery in the right wrist of all patients. Calculation of HR was based on the average R–R interval over the final 10 s (Siemens Sirecust SC 9000XL).ANP was determined by radioimmunoassay specific for h-ANP . Exenatide was measured by sandwich immunoassay . Two monoclonal antibodies  were used to immobilize and detect exenatide.NT-proBNP was determined by a sandwich immunoassay (Roche Diagnostics Scandinavia AB). Two monoclonal antibodies in a two-step procedure (capture antibody and detection antibody) were used to immobilize and detect brain natriuretic peptide (BNP). NEFA levels were determined using a NEFA-HR kit  on a Thermo T20xti instrument .P value <0.05 indicated statistical significance. Statistical analyses were performed using the statistical software package SPSS 22.0 .Data are presented as mean (±standard error of the mean) or percentages. Normal distribution of the variables was tested with the Shapiro–Wilk test. Differences in paired data were evaluated using the Wilcoxon signed-rank test. Correlations between hemodynamic variables and ANP were tested by Spearman’s correlation coefficients. To detect potential carry-over effects, Wilcoxon signed-rank tests between baseline levels for days 1 and 2 (for each variable studied) in Group A (the group that received active treatment on day 1) were performed. A general linear mixed model with participant and time as repeated factors and treatment as a fixed factor was used to test the effect of treatment on continuous parameters with data for more than two time points. To test whether the associations between ANP and NT-proBNP, and the hemodynamic variables were confounded by exenatide concentrations, we created multivariable linear regression models with ANP and NT-proBNP, respectively, as the dependent variable, and exenatide concentrations as a covariate. The assumptions for the linear regression models were tested as the standardized residuals were plotted against standardized predicted values to test statistical independence and variance of the errors. All tests were two-tailed, and a Baseline characteristics of the study population are shown in Table Table We found no significant carry-over effects in any of the primary endpoint variables.2) as a result of increased heart rate (8 ± 3 bpm), but with a concomitant decrease in PCWP (−2.2 ± 0.9 mmHg) and RAP (−0.85 ± 0.7 mmHg) [The results of the original study including all hemodynamic data have been published elsewhere . Hemodyn.7 mmHg) . Stroke .7 mmHg) .Table 2Hp < 0.001. Exenatide infusion significantly decreased circulating ANP levels by 21.9 ± 9.2 pmol/mL (Table p = 0.02). Exenatide did not change NT-proBNP levels , or NT-proBNP levels   compared with the placebo infusion (0.6 ± 0.5 to 1.0 ± 0.5 pmol/L), r = 0.50, p = 0.029), PAP , and PCWP  during exenatide infusion, with no such correlations during placebo infusion  have natriuretic effects. In the current study, the decrease in filling pressure, evoked by exenatide, might simply be the result of increased natriuresis , 21, or Our study demonstrates that short-term exenatide infusion resulted in a significant decrease in circulating ANP levels. There was no correlation between ANP level and exenatide level. During exenatide infusion there was a significant positive correlation between ANP levels and cardiac filling pressure, independent of exenatide levels.


The drug-loaded composite electrospun nanofiber has attracted more attention in biomedical field, especially in cancer therapy. In this study, a composite nanofiber was fabricated by electrospinning for cancer treatment. Firstly, the carbon nanotubes (CNTs) were selected as carriers to load the anticancer drug—doxorubicin (DOX) hydrochloride. Secondly, the DOX-loaded CNTs (DOX@CNTs) were incorporated into the poly(lactic-co-glycolic acid) (PLGA) nanofibers via electrospinning. Finally, a new drug-loaded nanofibrous scaffold (PLGA/DOX@CNTs) was formed. The properties of the prepared composite nanofibrous mats were characterized by various techniques. The release profiles of the different DOX-loaded nanofibers were measured, and the in vitro antitumor efficacy against HeLa cells was also evaluated. The results showed that DOX-loaded CNTs can be readily incorporated into the nanofibers with relatively uniform distribution within the nanofibers. More importantly, the drug from the composite nanofibers can be released in a sustained and prolonged manner, and thereby, a significant antitumor efficacy in vitro is obtained. Thus, the prepared composite nanofibrous mats are a promising alternative for cancer treatment. Up to date, cancer is still one of the deadliest killers to human lives, because both the incidence and mortality rates of cancers  are continuously rising , 2. UnfoNanotechnology as an emerging technology may be a reasonable choice and an effective strategy for solving the above problem. According to the report from the National Cancer Institute (NCI) in USA, nanotechnology seeks to exploit distinct technological advances towards cancer prevention and treatment . ElectroIn this present work, we have successfully fabricated DOX-loaded PLGA/CNTs composite nanofibrous mats by using electrospinning technique and then have assessed its antitumor efficacy against HeLa cells. The morphology and structure of electrospun nanofibers were characterized. After the evaluation of the drug loading capacity and efficiency, the in vitro release characteristics of DOX from the composite nanofibers were also measured. And further cytotoxicity experiments demonstrate that the drug-loaded nanofibers exhibit obviously therapeutic effect for cancer cell, implying that it may become a novel therapy strategy for preventing local cancer recurrence in future cancer therapy.PLGA with lactic acid/glycolic acid ratio of 75:25 (Mw = 110 kDa) was purchased from Daigang Biomaterials Inc. . Carbon nanotube (CNTs) was purchased from Chengdu Organic Chemicals Co., Ltd. Doxorubicin (DOX) hydrochloride was purchased from Beijing Huafeng United Technology Co., Ltd. HeLa cells were supplied by Institute of Biochemistry and Cell Biology . Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), 3--2,5-diphenyltetrazoliumbromide (MTT), trypsin, penicillin (100 U/mL), and streptomycin (100 mg/mL) were all purchased from Shanghai Yuanxiang Medical Equipment Co., Ltd. All other chemicals were obtained from Sino-pharm Chemical Reagents Co., Ltd. .The DOX-loaded CNTs (DOX@CNTs) were fabricated by the previously described method with some modifications . BrieflyLoading percentage (%) = /weight of DOX-loaded CNTs × 100 %.w/v %, and then stirred thoroughly to form a homogenous spinning solution. The solution was placed into a 5-mL plastic syringe with an 18-gauge blunt-ended needle. The electrospinning parameters were set as follows: the applied voltage of 20 kV, the collection distance of 15 cm, and the solution flow rate of 1.0 mL/h controlled by a syringe pump . The collected nanofibers were vacuum-dried at least 72 h to remove the residual solvent before further use.The PLGA/DOX@CNTs composite nanofibers were fabricated by using a blend electrospinning. Firstly, the prepared DOX@CNTs was completely dispersed in hexauoroisopropanol (HFIP) and the PLGA was added into the above solution at 20 The CNTs content in the nanofibers were 0.5, 1, and 2 wt. % (CNTs relative to PLGA), and the corresponding DOX contents in nanofibers can be calculated from the loading efficiency of CNTs. For comparison, the PLGA/DOX composite nanofibers were also prepared by electrospinning with the same DOX contents.The morphology of the composite nanofibers was observed by a scanning electron microscope , and the distribution of DOX@CNTs in the nanofibers was characterized by a transmission electron microscope  at an operating voltage of 200 kV. At least 40 nanofibers were selected from different SEM image, and the nanofiber diameter was measured using Image J 1.40 G software . The fluorescent images of the different composite nanofibers were observed by a fluorescence microscope . The thermogravimetric analysis (TGA) was conducted on a thermal analyzer  from the room temperature to 600 °C at a heating rate of 10 °C/min. The tensile testing of the composite nanofibers with a planar area of 50 × 10 mm was measured using a universal material tester  with a cross-head speed of 10 mm/min.The DOX release behaviors from the DOX-loaded samples including PLGA/1.5 % DOX and PLGA/1.5 % DOX@2 % CNTs electrospun nanofibers were carried out in PBS at pH 7.4. Briefly, a certain amount of the different DOX-loaded samples with the same content of DOX were put in a dialysis bag (cutoff molecular weight 7000 Da), and the bag was immersed in 15 mL of PBS (pH 7.4) at 37 °C in a thermostatted shaker with shaking at a rate of 100 rpm. At selected time intervals, the release medium (5 mL) was taken out and supplied with the same volume of fresh PBS. The concentration of DOX in the release media was determined by recording the absorbance of DOX at 480 nm using UV-vis spectrophotometer.2. MTT assay was employed to evaluate the viability of the HeLa cells cultured with different samples. For all experiments, cells were harvested by using trypsin solution and resuspended in the fresh DMEM medium. Before cell seeding, the samples were sterilized under UV light for 3 h and washed with PBS for three times.HeLa cells were cultured in the DMEM medium supplemented with 10 % FBS 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were cultured at 37 °C in a humidified incubator containing 5 % CO4 cells/well were seeded in 24-well plates and cultured for 12 h to allow cells to attach. Then, the medium was replaced with a fresh medium (negative control) and the medium containing the PLGA/2 % CNTs, free DOX (positive control), PLGA/1.5 % DOX, and PLGA/1.5 % DOX@2 % CNTs composite nanofibers, and the total DOX concentrations was designed 10 mg/mL, 25 mg/mL, and 50 mg/mL. After the cells were cultured for 24 h, the cells were washed twice with PBS and the 360-μL fresh culture medium and 40-μL MTT solution (5 mg/mL in PBS) were then added to each well. The cells were incubated for another 4 h. Thereafter, the 400 μL suspension was discarded, and 400 μL of DMSO was added to each well to completely dissolve the precipitate by stirring for 15 min. Then, 100 μL of the supernatant was transferred to 96-well microplates, and the OD value was measured with a microplate reader  at 492 nm. The relative cell viability was calculated by dividing the mean OD value of the control group, and the average value was obtained from five parallel samples.For MTT assay, HeLa cells at a density of 1 × 104 cells/well and incubated for 24 h. Then, the culture medium was removed, and the cells were incubated with PLGA/2 % CNTs, free DOX, PLGA/1.5 % DOX, and PLGA/1.5 % DOX@2 % CNT composite nanofibers (DOX concentrations at 25 μg/mL) for another 24 h. After washing with PBS twice, the cells were fixed with 4 % paraformaldehyde for 10 min. The cells were then permeabilized in 0.1 % Triton X-100 in PBS for 5 min, followed by blocking with 1 % BSA for 20 min. The F-actin of cells was stained by using Alexa Fluor 488® phalloidin solution for 10 min. Finally, all samples were washed several times with PBS and observed by confocal laser scanning microscope (CLSM).For confocal microscopy observation, HeLa cells were seeded into 24-well plates at a density of 1 × 10Figure It is known that the morphology of electrospun nanofibers is influenced by various parameters, such as the concentration of polymers, applied voltage, and flow rate of solution. For the organic/inorganic composite nanofibers, the morphology and the diameter can be dramatically affected by the content of inorganic component in polymers , 26. TheFigure Tonset) of the PLGA nanofibers was 247 °C, whereas the Tonset value (270 °C) of PLGA/1.5 % DOX@2% CNTs was prominently increased, indicating that the addition of CNTs can improve the thermal stability of the composites, which was similar to the results obtained by Qiu et al. [TGA was used to examine the thermal properties of obtained electrospun nanofibers and confirm the successful incorporation of drug-loaded CNTs within the composite nanofibers. As seen from Fig. u et al. .Fig. 4TG2-hybridized carbon surfaces and a large surface area of CNTs [To investigate the DOX loading and releasing behavior of composite nanofibers, DOX-loaded CNTs were firstly prepared. The high loading percentage 81.5 %) was attributed to the special sp of CNTs . Then, t % was atThe DOX release behavior was investigated under stimulated physiological environment (pH = 7.4) at 37 °C. For comparison, the release profiles of PLGA/1.5 % DOX and PLGA/1.5 % DOX@2 % CNTs electrospun nanofibers containing the equivalent DOX contents were studied. As shown in Fig. P < 0.01). However, PLGA/1.5 % DOX@2 % CNTs composite nanofibers showed a statistically significant lower inhibition effect than PLGA/1.5 % DOX and free DOX when the DOX concentration was less than 50 μg/mL (P < 0.01), which was due to the slow release rate of DOX from the composite nanofibers. Additionally, PLGA/1.5 % DOX and PLGA/1.5 % DOX@2 % CNTs composite nanofibers started to have apparent cytotoxicity only at DOX concentration of 25 μg/mL or above. It is noted that the inhibition effect of PLGA/1.5 % DOX@2 % CNTs composite nanofibers was equal to PLGA/1.5 % DOX when the DOX concentration was 50 μg/mL but lower than the free DOX. Although PLGA/1.5 % DOX@2 % CNTs composite nanofibers have not shown the relatively prominent anticancer efficacy compared with the PLGA/1.5 % DOX and free DOX group from the MTT assay, but we expected that a long-term growth inhibition in cancer cells could be achieved with the sustained DOX release from this matrix.In antitumor application of drug-containing materials, a high antitumor effect was needed. Therefore, we next investigated the antitumor activity of the DOX upon release from DOX-loaded composite nanofibers by MTT assay. Figure CLSM imaging technology was further employed to gain more insights into the therapeutic effect caused by PLGA/DOX@CNTs. The Alexa Fluor® 488 phalloidin was used to label cytoskeleton and the red fluorescent nature of DOX facilitated the in situ observation. As shown in Fig. In summary, we have reported a very effective and reproducible route to prepare a drug delivery system based on PLGA/CNTs composite nanofibers. The highly uniform and smooth nanofiber is successfully designed and developed. By combining the CNTs, as a proof-of-concept, we demonstrated that the PLGA/CNTs composite nanofibers could be used for the sustained release of the anticancer drug molecule DOX, which is important for biomedical applications requiring the drug molecule to maintain long-term anticancer efficacy. And the release profiles clearly indicated that the DOX can be loaded in the inner cavities or on the outside surface of the CNTs weakening the initial burst release of DOX. In addition, our results indicated that the prepared PLGA/DOX@CNTs nanofibers platform could effectively inhibit the cell viability of HeLa cells in vitro. Our results shed light on a promising use of the PLGA/DOX@CNTs composite nanofibers as a long-term drug release nanoplatform for chemotherapy in clinical cancer treatment.


The mycobacterial ESX-1 virulence locus accelerates macrophage recruitment to the forming tuberculous granuloma. Newly recruited macrophages phagocytose previously infected apoptotic macrophages to become new bacterial growth niches. Granuloma macrophages can then necrose, releasing mycobacteria into the extracellular milieu, which potentiates their growth even further. Using zebrafish with genetic or pharmacologically induced macrophage deficiencies, we find that global macrophage deficits increase susceptibility to mycobacterial infection by accelerating granuloma necrosis. This is because reduction in the macrophage supply below a critical threshold decreases granuloma macrophage replenishment to the point where apoptotic infected macrophages, failing to get engulfed, necrose. Reducing macrophage demand by removing bacterial ESX-1 offsets the susceptibility of macrophage deficits. Conversely, increasing macrophage supply in wild-type fish by overexpressing myeloid growth factors induces resistance by curtailing necrosis. These findings may explain the susceptibility of humans with mononuclear cytopenias to mycobacterial infections and highlight the therapeutic potential of myeloid growth factors in tuberculosis.  •Myeloid deficiencies increase innate immune susceptibility to mycobacterial infection•Depletion of macrophage supply below a critical threshold hastens granuloma necrosis•Increasing macrophage supply to the granuloma delays granuloma necrosis The early tuberculous granuloma promotes intracellular bacterial growth by co-opting host macrophages to serve as bacterial growth niches. Pagán et al. show that, conversely, an adequate macrophage supply to the granuloma is required to prevent necrosis of infected macrophages and subsequent extracellular bacterial growth. Mycobacterium marinum-zebrafish model of TB find that despite the ability of participating macrophages to partially restrict mycobacterial growth, the bacteria can actually co-opt the tuberculous granuloma to expand intracellularly  is the granuloma, a cellular aggregate consisting of macrophages and other immune cells . Studiesllularly . MycobacThe mycobacterial ESX-1/RD1 virulence locus promotes both apoptosis of infected macrophages and recruitment of new macrophages to the granuloma . Accordicsf1 in wild-type animals curtailed granuloma necrosis. This work highlights the role of an adequate macrophage supply in increasing host resistance by limiting infected macrophage necrosis and extracellular bacterial expansion.In active human TB, granulomas can progress to central necrosis caused by lysis of the infected macrophages that releases viable mycobacteria into a growth-permissive extracellular milieu . Thus grM. tuberculosis (csf1raj4blue homozygous mutant (csf1r−/−) zebrafish   A and S2Bon (dpf) , similaron (dpf) .csf1r mutants had 14% fewer macrophages in the CHT, a deficit that became more pronounced by 6 dpf (49% reduction) and extended to other tissues normally populated by these macrophages   by 32 hpf before it transitions into the adult kidney through developmental pathways analogous to the transition of hematopoiesis from mouse fetal liver to adult bone marrow . At 3 dpctively) C and S2Dctively) D (Herbomctively) . Neutrop mutants E.csf1r mutant macrophages were more spherical and moved more slowly than wild-type macrophages  knockdown had higher bacterial burdens in their macrophages . Mutant macrophages had similar bacterial burdens to wild-type at this time point   Figure A. In conrophages A. csf1r capacity  , 67%, and 84%  zebrafish larvae have a near complete lack of macrophages without any obvious developmental abnormalities   at the two temperatures  . Csf-1r eratures . We usedgh 7 dpf A. By 4 dnd 7 dpf B. In con− larvae B. Thus, csf1rts/− larvae was higher than in wild-type animals at 33°C but not at 24°C , the corded extracellular bacteria therein can no longer be phagocytosed by new macrophages . This caM. marinum M strain (ATCC #BAA-535) and its mutant derivatives ΔESX-1 (ΔRD1) and Δerp expressing tdTomato or tdKatushka2 under control of the msp12 promoter (promoter  were gropromoter .mpeg1:Brainbow)w201 line was generated by cloning the Brainbow 1.0L cassette  as previously described  gl25 × Tg(UAS:E1bKaede)s1999t (mpeg1:YFP)w200 (mpeg1:Brainbow)w201, and Tg(lysC:EGFP)nz117 s1999t , Tg(mpegYFP)w200 , Tg(mpegFP)nz117  larvae wFP)nz117 . Adults FP)nz117 . Homozyg deficit . irf8st9analysis  of PCR pDanio rerio csf1a was cloned into a Gateway middle entry vector encoding nuclear-localized Venus and a viral 2A peptide cleavage sequence (pCMV:nlsVenus-V2A-csf1a (gift from D. Parichy). In vitro transcription was performed with mMessage/mMachine SP6 kit (Life Technologies).sequence  and subsirf8 splice-blocking morpholino (5′-AATGTTTCGCTTACTTTGAAAATGG-3′) (csf1a mRNA were diluted in a 1× Buffer Tango (Thermo Scientific) containing 2% phenol red sodium salt solution (Sigma) and injected into the yolk of one- to two-cell-stage embryos in ∼2 nl (http://clodronateliposomes.org) (ATGG-3′)  (Gene Toin ∼2 nl . Lipo-PBmes.org)  were dilbactin for ΔΔCt analysis.Quantitative RT-PCR was performed as described . Total RSudan Black staining was performed as described . EmbryosHistology was performed as described . EuthaniN-phenylthiourea (PTU)-supplemented fish water containing 0.025% Tricaine and embedded in 1.5% low-melting-point agarose on optical bottom plates or dishes (MatTek Corporation). A Nikon A1 confocal microscope with a 20× Plan Apo 0.75 NA objective was used to generate 40 μm z stacks consisting of 1.3–2 μm optical sections. The galvano scanner was used for all static imaging and for time-lapse imaging of the CHT. Time-lapse images were taken at 5 min intervals for 8 hr. Data were acquired with NIS Elements (Nikon). Macrophage tracks were generated using the 3D tracking feature of Imaris (Bitplane Scientific Software).Fluorescence microscopy was performed as described . QuantifSplenocyte single-cell suspensions of 4- to 5-month-old zebrafish were prepared by dissociating the tissue in flow cytometry buffer  and filtering it through a 70 μm cell strainer (BD Biosciences). Liver samples were digested in 1× PBS supplemented with DNase I and type I/II collagenases for 15 min at 32°C. Cells were then stained with Alexa Fluor 594-conjugated Peanut Agglutinin (PNA) (Life Technologies) for 30 min at 4°C to enrich for myeloid cells, as previously described . Samples∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.Statistical analyses were performed on Prism (GraphPad). Not significant, p ≥ 0.05; mpeg1:Gal4FF Tol2 plasmid and mpeg1:Gal4FF; UAS:E1bKaede fish prior to their publication. A.J.P. and C-T.Y. prepared the figures, and A.J.P. and L.R. wrote the paper with input from C-T.Y.A.J.P., C-T.Y., and L.R. designed experiments and analyzed the data. A.J.P. and C-T.Y. performed experiments. J.C. performed adult fish experiments in


Locusta migratoria, shows remarkable phenotypic plasticity at behavioral, physiological, and morphological levels in response to fluctuation in population density. Our previous studies demonstrated that dopamine (DA) and the genes in the dopamine metabolic pathway mediate phase change in Locusta. However, the functions of different dopamine receptors in modulating locust phase change have not been fully explored. In the present study, DA concentration in the brain increased during crowding and decreased during isolation. The expression level of dopamine receptor 1 (Dop1) increased from 1 to 4 h of crowding, but remained unchanged during isolation. Injection of Dop1 agonist SKF38393 into the brains of solitary locusts promoted gregarization, induced conspecific attraction-response and increased locomotion. RNAi knockdown of Dop1 and injection of antagonist SCH23390 in gregarious locusts induced solitary behavior, promoted the shift to repulsion-response and reduced locomotion. By contrast, the expression level of dopamine receptor 2 (Dop2) gradually increased during isolation, but remained stable during crowding. During the isolation of gregarious locusts, injection of Dop2 antagonist S(–)-sulpiride or RNAi knockdown of Dop2 inhibited solitarization, maintained conspecific attraction-response and increased locomotion; by comparison, the isolated controls displayed conspecific repulsion-response and weaker motility. Activation of Dop2 in solitary locusts through injection of agonist, R(-)-TNPA, did not affect their behavioral state. Thus, DA-Dop1 signaling in the brain of Locusta induced the gregariousness, whereas DA-Dop2 signaling mediated the solitariness. Our study demonstrated that Dop1 and Dop2 modulated locust phase change in two different directions. Further investigation of Locusta Dop1 and Dop2 functions in modulating phase change will improve our understanding of the molecular mechanism underlying phenotypic plasticity in locusts.The migratory locust,  Locusta migratoria, one of the important economic pest species, shows density-dependent polyphenism, gregarious phase and solitary phase  in dopamine pathway in gregarious locusts results in their behavioral changes toward solitary phase  is applied to the thoracic ganglia of this locust  from the solitary and gregarious colonies maintained in the Institute of Zoology, Chinese Academy of Sciences, Beijing, China. Gregarious locusts were cultured in large boxes (40 × 40 × 40 cm3) at a density of 500–1000 locusts per container. Solitary locusts were obtained from the gregarious colony and cultured individually in separate white metal boxes (10 × 10 × 25 cm3). These boxes were supplied with charcoal-filtered compressed air. Gregarious and solitary locusts were maintained for at least three generations before the experiments were conducted. Gregarious and solitary colonies were maintained in a 14 h light/10 h dark cycle at 30 ± 2°C and fed with fresh wheat seedlings and bran  and electrochemical detection (ECD), as previously described  of Locusta . The other sequences for phylogenetic analysis were downloaded from NCBI databases. Multiple sequence alignments of 300 amino acids (including TM2 to TM7) in these insect dopamine receptors were performed in Clustal W and curated in MEGA 5.34  and allowed to live with 20 gregarious locusts at the same stadium. After these solitary locusts were allowed to stay with the gregarious locusts for 1, 4, 16, or 32 h, the brains of the crowded locusts were dissected and immediately placed in RNAlater solution for subsequent qRT-PCR analysis. The brains of solitary locusts were sampled as the control group for crowding treatment. All of the locusts were sampled at the same time point for eight biological replicates, and equal numbers of male and female locusts were sampled per replicate.Fourth-stadium gregarious locusts were isolated and individually reared as solitary locusts. After 1, 4, 16, or 32 h of isolation, the brains of isolated locusts were dissected and immediately placed in RNAlater solution  for quantitative real-time PCR (qRT-PCR) analysis. The brains of gregarious locusts were sampled as the control group for isolation treatment. Meanwhile, 10 solitary locusts at the fourth-stadium were introduced to an optic perplex-made box . DNase was applied to eliminate DNA contamination in RNA samples. To analyze the expression levels of the target genes, we reversely transcribed 2 μg of total RNA in each sample by using MMLV reverse transcriptase  in accordance with the manufacturer's instructions. PCR amplification was conducted in Roche Light Cycler 480 using RealMaster-Mix (SYBR Green) kit . Amplification was initiated by incubation at 95°C for 5 min, followed by 40 cycles at 95°C for 20 s, 58°C for 20 s, and 68°C for 20 s. Melting curve was detected to confirm the amplification specificity of target genes. Table Locusta Dop1 in modulating behavioral transition from solitary to gregarious phase. All injection procedures were performed under a stereo-dissection microscope using a NANOLITER injector 2000  with a glass micropipette tip  for 4 h before behavioral assay. The control group received the same volume of saline before behavioral assay was conducted.We injected SKF38393, a specific agonist of D1-like dopamine receptor -TNPA] , a D2-like receptor selective agonist -2,10,11-trihydroxy-We injected SCH23390 , a specific antagonist of D1-like receptor -sulpiride , a specific antagonist of Dop2  according to the manufacturer's instructions. We then selected the fragment without homologies with other genes in the genome database to avoid non-specificity in RNAi knockdown. Table DA1-injected gregarious locusts were directly assayed. The dsDA2-injected gregarious locusts were directly assayed or lived individually in solitary-rearing cages for 15, 30, or 60 min before behavioral assay was conducted. The effects of RNAi on the relative mRNA level were detected through qRT-PCR after dsRNA was injected for 72 h.Double-stranded RNA (dsRNA) of green fluorescent protein (GFP), 3). The wall of the arena is opaque plastic, and the top is clear. One of the separated chambers (7.5 × 30 × 10 cm3) contained 20 fourth-stadium gregarious locusts as the stimulus group, and the other end of the chamber with the same dimensions was left empty. Both ends of the chamber were equally illuminated to prevent formation of locust shadows. The floor of the open arena was covered with filter paper to avoid contamination during the behavior assay. The locusts were gently transferred through a tunnel to the arena. Each locust was recorded for 6 min and examined only once  was used for video recording and data extraction. The arena behavior assay was performed in a rectangular arena (40 × 30 × 10 cmP−sol = eη/(1 + eη), where η = β0 + β1·X1 + β2·X2 + … + βk·Xk, X1, X2, …, where Xk is the behavioral covariates. P-sol is the probability that the locusts should be regarded as a member of the solitary phase population. The probability value ranges from 1 to 0, where 1 indicates that individuals display solitary behavior and 0 indicates that individuals display gregarious behavior. The most robust indicators  of the phase state were retained in the model. P-sol was calculated according to the following equation: P-sol = 2.361 − 0.016 × TDM − 0.172 × FOM − 0.005 × AI , latency of first occurrence in the stimulus area (LFOISA), total duration in the area close to the arena wall (TDCW), entry frequency in the area close to the arena wall (EFCW), entry frequency in the region opposite the stimulus area , latency of first occurrence opposite the stimulus area (LFOIOSA), mean distance to the stimulus group (MDTSG), total distance moved (TDM), total duration of movement (TDMV), frequency of movement (FOM), and attraction index . The behavioral parameters of this model were adjusted until the regression model discriminated the two phases at the optimum level according to the following equation: post-hoc Tukey test for multiple comparisons. The data for behavioral phase change were analyzed by Mann–Whitney U (MWU) test because of their non-normal distribution characteristics. Student's t-test was conducted to analyze the changes of specific parameters, including TDM and FOM, between saline- and agonist-injected, saline- and antagonist-injected, dsGFP- and dsDop1-injected groups. The changes of TDM and FOM during isolation were analyzed by One-Way ANOVA, followed by post-hoc Tukey test for multiple comparisons. The change of parameter AI was analyzed by MWU test. The data were expressed as mean ± standard error of the mean. P < 0.05 was considered statistically significant. The probabilistic metric of solitariness (P-sol) is presented as median values. All statistical data were analyzed with SPSS 15.0 .DA content and expression levels of dopamine receptors over a time course of crowding and isolation were analyzed by One-Way ANOVA, followed by Locusta during crowding and isolation. During the crowding of solitary locusts (CS), the DA level in the brains increased after 4 h of crowding and decreased thereafter    Figure . During ) Figure . These rLocusta. A phylogenetic analysis was performed using MEGA5.34    during crowding   Figure . However) Figure . By contg Figure , signifi) Figure . These rLocusta Dop1 expression in the brains of solitary locusts after 4 h of crowding leads us to explore the roles of Locusta Dop1 in regulating locust gregarization. Thus, we injected SKF38393, the specific agonist of D1-like receptor, into the brains of the migratory locust and detected its effects on behavioral changes after 4 h. The behavioral assay showed that solitary locusts changed their behaviors toward gregarious phase       Figure . We also) Figure . In addi) Figure . These rt-test, t = 1.795, P = 0.078 for TDM; t = 1.925, P = 0.062 for FOM; MWU, U = 265, P = 0.039 for AI)   , frequency of movement (FOM), and attraction index (AI) in phase change. Injection of SKF38393 into the brains of solitary locusts did not increase their TDM and FOM, but induced the shift from avoidance to approaching the stimulus locusts (the gregarious locusts in one of the end chambers in the assay arena)  Figure . Thus, DLocusta Dop1 mRNA in the brains of gregarious locusts significantly reduced their TDM and FOM, as well as induced the shift from approaching to avoiding the stimulus locusts   -sulpiride, the antagonist of Dop2, in the brains of gregarious locusts and detected its effects on their behavioral state. The injection of S(-)-sulpiride into the brains of gregarious locusts (IG 0 min) did not change their behavioral phase  , but significant difference was not observed between saline-injected gregarious controls and S(-)-sulpiride-injected gregarious locusts  , despite that the gregarious controls injected with saline exhibited the solitary-like behavior compared with gregarious locusts   Figure . After 1) Figure . After 3 Figures .Dop2 and designed the dsRNA to specifically knockdown the expression of Locusta Dop2 mRNA for functional validation. The injection of Locusta Dop2 dsRNA in the brains of solitary locusts resulted in approximately 50% reduction of Locusta Dop2 mRNA level   , whereas the deficiency of Locusta Dop2 inhibited the behavioral change of gregarious locusts toward solitary phase, as compared with the corresponding dsGFP-injected controls   Figure . As abov Figures .Locusta Dop2 in solitary locusts through injection of Dop2 specific agonist, and detected their behavioral state after 60 min. However, the reported agonists in insects will activate both Dop1 and Dop2  , whereas changes were not observed after Dop2 antagonist was injected  , whereas the antagonist-injected groups tended to approach the stimulus locusts   Figure .F = 2.246, P = 0.086) compared with the dsGFP-injected groups, which displayed a significant decrease in TDM during isolation  . However, the deficiency of Dop2 signaling caused by RNAi-knockdown inhibited the decrease in FOM, and the treated gregarious locusts showed significant decrease in FOM after 60 min of isolation  , whereas the dsDop2-injected groups tended to approach the stimulus locusts    Figure . The iso) Figure . After 6) Figure . In soliLocusta. The increased DA level and up-regulation of Locusta Dop1 expression in crowding suggested their correlations with the gregarization of Locusta, and we verified that dopamine-Dop1 signaling mediated behavioral gregariousness. Moreover, the increased expression of Locusta Dop2 in isolation suggested its correlations with solitarization, and we confirmed that dopamine-Dop2 signaling mediated the behavioral solitariness in Locusta. This study clarified the different functions of dopamine receptors underlying behavioral phase change in Locusta.In this study, we found two dopamine receptors playing different roles in phase change of Locusta Dop1 mediated the gregariousness of Locusta by inducing the attraction-response and increasing motility, and Locusta Dop2 regulated the solitariness of Locusta by inducing the repulsion-response and decreasing the motility, suggesting that DA-Dop1 and DA-Dop2 signaling mediate sensory interaction between conspecifics during aggregation. A number of studies have suggested that dopamine mediates the sensory perception of vertebrates and invertebrates, such as olfaction , AmDOP3 receptors regulate their attraction-response to the queen mandibular pheromone (QMP) , the fluctuation of DA levels in brains and ovaries are respectively associated with ritualized combat and the formation of a reproductive hierarchy  is critical in the acquisition of the gregarious behavior (Ott et al., Schistocerca (Ott et al., Locusta, cAMP-PKA signaling may be greatly activated by the increasing of DA level and Dop1 mRNA expression in crowding, and the up-regulation of cAMP-PKA signaling promotes the locust gregarization. During isolation, the tendency of dopamine level in isolation is contray to the expression of Dop2 mRNA. The decrease in dopamine concentration in the brain will reduce the cAMP-PKA level, and the increasing of Dop2 expression may regulate other downstream signaling and finally result in quick solitarization in Locusta. Thus, the tendency of DA concentration and the expression and functional properties of Locusta Dop1 and Dop2 may result in the divergence of these two receptors underlying locust phase change.The level of DA, the expression, and the functional properties of Schistocerca, injection of DA into the thoracic cavities of gregarious locusts induces solitarization and avoidance behavior (Alessi et al., Schistocerca did not point out that dopamine mediates solitarization through Dop1 or Dop2, or other receptor subtypes simultaneously. Meanwhile, the non-selective antagonist fluphenazine (Degen et al., Schistocerca has been employed for administrations to confirm the function of dopamine (Alessi et al., Schistocerca is contrary to the function of Locusta Dop1 in the gregarization of Locusta. The applications of 5-HT also lead to distinct effects on phase change of Schistocerca from different laboratories and Locusta (Anstey et al., Locusta and Schistocerca belong to different Acrididae subfamilies, Cyrtacanthacridinae and Oedipodinae, respectively.In Locusta, the fluctuations and functions of DA and its receptors were analyzed and verified in the brain, whereas in Schistocerca, the investigation of dopamine function in phase change mainly focused on the thoracic ganglia. In Schistocerca, the application of DA induced a significant reduction in the amplitude of the CS-FETi EPSP in the metathoracic ganglia, and the systematic injection of dopamine in gregarious locust induced more behavioral avoidance (Alessi et al., Locusta suggests that the functions of dopamine in gregarization and solitarization are closely related to the receptor subtypes to which dopamine binds. By contrast, in Schistocerca, the expression patterns and functions of different receptor subtypes were unclear; the injection of dopamine and non-selective antagonist in thoracic cavity may merge the functions of multi receptor subtypes in behavioral plasticity.The function of dopamine in regulating locust phase change may be closely correlate with the target tissues where dopamine is located and where its function is apparent. In Locusta were confirmed via pharmacological intervention and RNAi-knockdown. Pharmacological intervention by injecting agonists and antagonists of dopamine receptors may introduce off-target effects and therefore non-specifically activate or inhibit other biogenic amine receptors. Meanwhile, RNA interference, through exogenous introduction of dsRNA with sequences complementary to the targeted genes, provides specific manipulation of gene expression and is widely used to investigate gene functions (Fire et al., Locusta Dop1 and Dop2 during phase change of Locusta. By contrast, in Schistocerca, pharmacological injection was applied to confirm the function of dopamine in solitarization (Alessi et al., Schistocerca. Therefore, genetic information and functional verification of dopamine signaling involved in modulating phase change from Schistocerca are very essential, because it will be beneficial for the clarification of the divergence and convergence of the functions of dopamine in phase change of the two locust species.The functions of dopamine-Dop1 and Dop2 signaling in phase change of In conclusion, different neurochemicals and molecular mechanisms are likely to perform on the same behavioral paradigm of transition in different species. With the development of next generation sequencing, dissection of genome sequences in closely related locust species and deep exploration of molecular mechanism of phase change will be helpful in understanding the divergence and convergence of mechanisms underlying the classical phenotypic plasticity in different locust species.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.


Noninvasive imaging of atherosclerosis could potentially move patient management towards individualized triage, treatment, and followup. The newly introduced combined positron emission tomography (PET) and magnetic resonance imaging (MRI) system could emerge as a key player in this context. Both PET and MRI have previously been used for imaging plaque morphology and function: however, the combination of the two methods may offer new synergistic opportunities. Here, we will give a short summary of current relevant clinical applications of PET and MRI in the setting of atherosclerosis. Additionally, our initial experiences with simultaneous PET/MRI for atherosclerosis imaging are presented. Finally, future potential vascular applications exploiting the unique combination of PET and MRI will be discussed. Molecular imaging can be defined as noninvasive visualization and quantification of distinct molecular pathways. Important features of molecular imaging are the ability to target a molecular process in living organisms without perturbing them. Both positron emission tomography (PET) and magnetic resonance imaging (MRI) are classic methods used for molecular imaging.Atherosclerosis is traditionally assessed using luminal stenosis by anatomical imaging such as angiography or ultrasound. However, atherosclerosis is now recognized as a systemic degenerative inflammatory vascular disease that develops over decades, with a long subclinical period. Postmortem analyses have shown that most fatal vascular events originate from nonstenotic atherosclerotic lesions , and aboThe concept of the vulnerable plaque is a hallmark in atherosclerosis. The vulnerable atherosclerotic plaques are those with a high short-term risk of rupture and thrombosis. The vulnerability of a plaque is characterized by a number of factors like a thin, collagen-poor fibrous cap, a large necrotic core, and abundant macrophages in the cap, whereas the luminal protrusion is not a marker of vulnerability .The current goal in noninvasive imaging is to identify vulnerable atherosclerotic plaques that may subsequently lead to myocardial infarction or stroke. This identification could lead to more optimal and individualized risk stratification and thereby enabling personalized therapy.The aim of this review is to give a summary of current relevant clinical applications of PET and MRI in the setting of atherosclerosis and to discuss potential future uses of the newly introduced combined PET/MRI system.A state-of-the-art MRI scanner offers the ability to perform both anatomical and functional examinations. The atherosclerotic plaque components can be differentiated using dedicated imaging sequences . T1-, T2MRI for plaque characterization in the coronary arteries is technically more challenging than carotid plaques due to cardiac and respiratory motion of the often small tortuous vessels. Several methods are under development to deal with these challenges. Some studies have in fact shown that positive remodeling and intracoronary thrombus detection is feasible in the coronary arteries , 12.Molecular imaging with MRI is also possible using specific contrast agents that allow for visualization of processes in the atherosclerotic plaque at the molecular level. Target specific MRI contrast agents are typically based on paramagnetic gadolinium or iron oxide. Clinical studies have demonstrated uptake of small iron oxide (USPIO) particles in carotid plaques and the uptake was found to correspond to areas of macrophage infiltration . One stuIt is relevant in this context to consider what PET can add to the “MRI-one-stop-shop.” Clinical PET has a spatial resolution around 3 mm at best and can thus in no way compete with the spatial resolution of MRI. PET is based on photon emission from positron emitting radioactive tracers and does not contain anatomical information. Thus, PET cannot be used to image morphological components of the plaque. PET, however, is a sublime technique for molecular imaging. Molecular imaging with MRI has a limited sensitivity typically in the micromolar range, whereas a typical clinical PET scanner can detect concentrations in nano- to picomoles per liter. This sensitivity, however, is lower in plaque imaging since the plaque size is near the spatial resolution of the system and thus subject to signal loss by partial volume effects.PET is based on the tracer principle for imaging. The radioactive tracer utilized in PET is a compound where an atom is replaced by a radioisotope or a radioisotope is added. Only traces of the substance are applied; therefore, it has no pharmacologic effect in vivo. Typically, the tracer is a biomolecule that reflects a particular body function or metabolism. PET tracers can be nonspecific following a biochemical pathway or allowing for measurement of tissue extraction or metabolism. These radiotracers include the glucose analogue fluorine-18-fluorodeoxyglucose (FDG) which is taken up by high-glucose-utilizing cells, where FDG is trapped intracellularly by phosphorylation to allow tissue glucose metabolism assessment. PET tracers can also be specific radioligands involved in an interaction with receptors. PET tracer distribution can be quantified in absolute terms and with dynamic measurement of PET tracer uptake and distribution kinetic analysis is possible.In summary, we find that molecular imaging with PET in most cases will be far superior to molecular imaging with MRI. In our opinion, optimal utilization of the complementary information from hybrid PET/MR system will require a better understanding and foremost a better use of the pathophysiologic information that can be acquired by MRI. Simply using the anatomical information in conjunction with the metabolic information from the PET will be a suboptimal utilization of the potential of the system.As compared to preclinical animal scanners, human hybrid PET/MRI systems can physically contain larger animals. This opens the opportunity to study atherosclerosis in human-like atherosclerosis models such as rabbits and porcines. So far, few feasibility data on this topic has been published; Dregely et al.  presenteOur institution recently performed a study aiming at evaluating the feasibility of integrated PET/MR imaging of the carotid arteries in humans . Six HIVPET imaging of atherosclerosis has so far focused primarily on FDG. The first report on FDG accumulation in the large arteries emerged in 2001 –23. The One very interesting trial is the ongoing prospective observational PESA study . A subgrPET imaging of atherosclerosis has thus far focused primarily on FDG. A major drawback of imaging atherosclerosis with FDG PET, however, is the lack of specificity of the tracer. Another limitation is the high uptake of FDG in the myocardium, which produces a suboptimal signal-to-noise ratio when coronary arteries are imaged. As an alternative, some researchers have suggested 18F-deoxy-mannose as a more atherosclerosis specific tracer than FDG , but thi68Ga-DOTATATE is utilized in diagnosis and staging of neuroendocrine tumors and has high affinity for somatostatin receptors and a few studies have suggested a future role in plaque imaging [A hunt for new and more specific tracers has started. The tracers should specifically target cell-mediated key molecular processes associated with the vulnerable atherosclerotic plaque. The most prominent of these targets include macrophage infiltration, apoptosis, hypoxia, and neoangiogenesis of the intima/media. Activated macrophages express the somatostatin receptor subtype 2, and this could be a target for PET imaging.  imaging –34. PET  imaging –37.18F-sodium fluoride (NaF). This tracer is deposited by chemisorptions onto hydroxyapatite and is used in oncology to identify bone metastasis. Recent evidence suggests that NaF uptake is not equivalent to calcification as identified by CT imaging [Another promising PET tracer for plaque imaging is  imaging  but that imaging ; however imaging . Joshi e imaging  recentlyIn summary, we find that the combination of morphologic and functional information from the MRI with molecular imaging from PET may lead to optimal characterization of the plaque and thus improved individualized counseling and therapy.1H-MRS) of atherosclerotic plaques in carotid arteries using clinical 3-T MR systems is feasible [Image-guided proton magnetic resonance spectroscopy  like pyruvate can be enriched with 13C and injected into humans or animals following ex vivo hyperpolarization. The increased signal from the 13C allows both imaging and spectroscopy of the tracer within a limited time window. Hyperpolarized MRI for atherosclerosis has only been reported in few preclinical reports [13C-pyruvate and plaque hypoxia can be assessed with a specific radiolabelled PET tracer like 64Cu-ATSM simultaneously. In this way, HyperPET can combine two molecular imaging techniques with a possible synergistic effect in atherosclerosis. HyperPET, however, is a very demanding and expensive solution that will most likely be restricted to experimental use in few selected cases. Nevertheless, the distinct and small volume of interest, the plaque, makes HyperPET more realistic to be applied than in diseases where whole body evaluation is needed.Our institution has recently installed a hyperpolarizer with our PET/MRI system allowing us to do simultaneous hyperpolarized MR and PET (HyperPET). Hyperpolarization of nuclear spins like  reports . The HypThe acquisition of a PET/MRI system is a huge task that requires widespread knowhow by technicians, physicists, and physicians in addition to extensive funding both initially and for running costs. It is our expectation that this will limit the technique to mainly experimental use in a limited number of major university centers for some time. At our institution, we have set up an atherosclerosis research workflow in collaboration with the vascular surgeons . This woOne apparent area for simultaneous PET/MRI is cross validation of new imaging modalities when similar molecular imaging probes exist in PET and MRI. The simultaneous acquisition allows for experiments to be performed under one and the same physiologic condition. One very elegant example is the use of a reporter gene approach. Higuchi et al.  transducBoth PET and MRI have been used for followup after clinical intervention. The dal-PLAQUE study randomly assigned 130 patients with, or with high risk of, coronary heart disease to placebo or dalcetrapib . CoprimaTo date, no interventional studies of human atherosclerosis using hybrid PET/MRI have been published.We have presented our initial experiences with simultaneous PET/MRI in the field of atherosclerosis. It is our belief that the synergy between PET and MRI will justify its use in atherosclerotic imaging despite its higher cost and more complex management. At the same time though, we do not see a translation from experimental applications to clinical routine in the very near future.We expect a continuous development of the integration of molecular, functional, and anatomical imaging as well as the clinical indications within atherosclerosis. This novel imaging methods may lead to early detection of high-risk vulnerable plaques, enabling clinicians to improve risk stratification and thus paving the way for individualized therapy.


C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na6[TeW6O24] within the liquid–liquid phase-separation zone.Latent and active aurone synthase purified from petals of  cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P212121 and P1211 and diffracted to ∼1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3121. The crystals of latent cgAUS1 belonged to space group P1211 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na6[TeW6O24] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI).Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active  Aurone synthase (AUS) is a homologue of PPOs that exclusively possesses diphenolase activity and catalyzes the oxidative conversion of chalcones to aurones  and carnations (Dianthus caryophyllus)  and catechol oxidases (EC 1.10.3.1). Tyrosinases catalyze the cgAUS1 (A0A075DN54) from Coreopsis grandiflora has been reported by Molitor et al. (2015cgAUS1 is a member of the novel group 2 PPOs. An insertion (V237ANG240 in the cgAUS1 sequence) in a loop region near to the dinuclear copper centre is characteristic of this subgroup and might be involved in substrate docking . The homogeneity of the protein samples was verified by mass determination by means of ESI-Q-TOF MS  has been described in Kaintz, Molitor et al. (2014cgAUS1-a1 and cgAUS1-a2) were stored at a concentration of 6 mg ml−1, the latent form originating from the natural source (cgAUS1-ln) at a concentration of 10 mg ml−1 and the recombinantly expressed latent cgAUS1 (cgAUS1-lr) at a concentration of 14 mg ml−1 in 10 mM sodium acetate buffer pH 5.0 at 4°C for crystallization experiments.The preparation of active  al. 2015. Another al. 2014. The two2.2.cgAUS1-a2 was performed on a nanoESI-QTOF mass spectrometer  using 20 µl protein solution with a concentration of approximately 1 µg µl−1. Buffer exchange to 10 mM ammonium acetate pH 5.0 was performed by ultrafiltration. Just prior to the measurements, acetonitrile was added to a final concentration of 25%(v/v) and formic acid was added to a final concentration of 0.05%(v/v).Electrospray ionization mass spectrometry (ESI-MS) of purified active 2.3.et al., 1986et al.  was synthesized according to a modified procedure  and the recombinantly expressed latent proenzyme (cgAUS1-lr).Four different cgAUS1-a1 were performed with the Crystallization Basic Kit for Proteins from Sigma–Aldrich at 20°C by the hanging-drop vapour-diffusion technique in 15-well EasyXtal plates  at 293 K. Screen condition No. 9  resulted in a hit. Owing to the low quality of the obtained crystals, an intensive screen for additives was necessary. The final crystallization condition . To investigate the reaction mechanism of PPOs, active cgAUS1-a2 was co-crystallized with 1,4-resorcinol, an inhibitor of PPOs and a suicide substrate for tyrosinase .Initial crystallization screens for active M ammonium acetate, 0.1 M sodium citrate pH 5.6, 30% PEG 4000) of the Crystallization Basic Kit for Proteins (Sigma–Aldrich) resulted in fully precipitated latent cgAUS1-ln. However, redissolving the precipitate in six equivalents of water and subsequent re-equilibration produced needles and bunches of needle-like crystals resembling sea urchins. Stepwise optimizations led to rod-like crystals . Notably, the protein started to precipitate several hours after crystallization setup and subsequently redissolved with the formation of a liquid–liquid phase-separation (LLPS) zone . The crystallization of the recombinantly expressed enzyme (cgAUS1-lr) using crystallization condition D (Table 1e). Optimization of the crystallization conditions led to the substitution of 100 mM magnesium chloride by 1 mM hexatungstotellurate(VI) (Table 1E). Crystals appeared within 2 d and the crystals grew to final dimensions of up to 500 × 50 × 50 µm within several days . Lowering the pH of the crystallization buffer to 5.0 (Table 1F) improved the stability of the crystals significantly and also enhanced crystal growth. Crystals were soaked for 30–45 min in cryoprotectant solution containing 5 mM hydrogen peroxide to generate the oxy-form of the dinuclear copper centre.Condition No. 9  possessed an intensity ratio between species A (unmodified) and B (phosphorylated/sulfated) of ∼0.14:1, indicating a significantly higher level of phosphorylation or sulfation . As the petal tissue (harvested from June to September 2011) was stored in 0.5 kg packages and no enrichment of phosphorylated/sulfated forms had been observed within the different cleaved active cgAUS1 forms  (Na6[TeW6O24]) drastically improved the nucleation and crystal growth of cgAUS1-lr within the LLPS. Crystals diffracting to 2.08 Å resolution were obtained within several days . The crystal structure of latent cgAUS1-ln was solved by molecular replacement followed by automated model building. Refinement of the obtained models is in progress.Only two crystals of latent s Table 2 improved


Evidence-based decision making (EBDM) is an effective strategy for addressing population health needs. Assessing and reducing barriers to using EBDM in local health departments may improve practice and provide insight into disseminating EBDM principles among public health practitioners.Administrative leaders at the Pueblo City–County Health Department, Pueblo, Colorado, used a systematic approach for implementing EBDM. Research partners engaged staff to understand factors that increase or deter its use.A survey was distributed to staff members at baseline to identify gaps in administrative and individual practice of EBDM. In-depth interviews were also conducted with 11 randomly selected staff members. Results were shared with staff and administration, after which activities were implemented to improve application of EBDM. A follow up survey was administered 1 year after the initial assessment.Survey data showed evidence of progress in engaging and educating staff members, and data showed improved attitudes toward EBDM . For example, staff members reported having the necessary skills to develop evidence-based interventions (73.9%), the ability to effectively communicate information on evidence-based strategies to policy makers (63.0%), access to current information on improving EBDM processes (65.2%), and a belief that evidence-based interventions are designed to be self-sustaining (43.5%).Within a local health department in which leaders have made EBDM a priority, addressing the culture and climate of the department may build EBDM. Future research may provide insight into tailoring EBDM within and across local health departments. Evidence-based decision making (EBDM) provides a framework to address many critical challenges  facing the public health system . EBDM isAlthough the key tenets of EBDM are now well established ,4,5, infOrganizational structures and activities associated with EBDM performance, called administrative evidence-based practices (AEBPs) , fall inThe Pueblo City–County Health Department (PCCHD), located in southeastern Colorado, was selected for this case study because of the public health director’s interest in establishing EBDM as standard practice throughout the department. Initial contact was made between the director and the research team through an EBDM training. PCCHD is a mid-sized health department governed by a local board of health. It serves a population of 159,000 that is 54% non-Hispanic white and 42% Hispanic. Approximately 18% of residents in PCCHD’s jurisdiction have incomes below the federal poverty limit, and 72% have completed high school . PCCHD administrators encouraged the staff to complete the surveys through face-to-face interaction or email. No incentives were given. The follow-up survey contained the same questions as the initial one with an additional 4 questions to assess the impact of activities that occurred during the time between surveys. The follow-up survey was administered to all staff members who received the initial survey and were still employed by PCCHD in April 2014 (n = 46).In February 2013 a baseline survey was sent to 74 PCCHD staff members chosen by administrators on the basis of how pertinent EBDM was to the type of work the staff members performed. Email addresses were acquired from PCCHD, and surveys were distributed via individualized links generated using the Qualtrics online research suite  for items related to organizational and individual skills. To describe the magnitude and direction of any significant differences, we calculated effect sizes. Cohen’s d values were calculated for mean differences observed in t tests. Cohen suggests the following effect ranges for d: 0.2, small; 0.5, medium; and 0.8, large , and χ8, large . In addiect size . All anaAll responses were recorded and were confidential and anonymous. The interviewer transcribed the qualitative interviews. Open coding was completed for each transcript to ascertain general themes. The interviewer next used focused charting to develop a codebook that was distributed to a team of researchers. The team made changes to more adequately capture codes and subcodes. The final codebook was then used by the interviewer to analyze each script. In addition, 1 research assistant analyzed 3 scripts for interrater reliability. Discrepancies between reviewers were minimal. A nonparticipant transcribed the content of the focus group, which was then analyzed by verbally verifying content for accuracy among the group, and then capturing major themes and concluding remarks by using open coding.Of the 74 staff members who received the baseline survey, 59 completed it, an 80% response rate. The majority of respondents were technical experts  or other (23.7%), which included nurses, a registered dietician, administrative assistants, accountants, and the vital statistics staff. On average, participants had spent 5.7 years in their current position , and 9.5 years in public health . Two AEBP domains ranked lowest in the surveys, leadership and organizational climate and culture . The perAmong 11 staff members interviewed, the length of employment at PCCHD ranged from 1.5 to 21 years. Nine interviewees were line staff, and 2 held manager or supervisory roles. Five of 8 departments were represented.Qualitative findings were grouped into benefits and barriers to EBDM use and action steps to promote EBDM at PCCHD . The benStaff recommended several action steps for improving the use of EBDM at PCCHD. First, health department leaders and managers should clearly communicate EBDM messages to various departments and model EBDM as a process, in a manner that crosses categorical programs. A second suggestion was to listen to and follow up on staff suggestions. Third, staff members requested positive feedback from leadership to encourage use of EBDM. Finally, staff members wanted to understand the relevance of EBDM to their work. As for training in EBDM, and consistent with adult education theories , staff rFocus group findings were also clustered into 3 general topics related to EBDM: benefits, barriers, and action steps. Respondents said that benefits to using EBDM in a local health department were “smart business”: EBDM can improve programs, help government agencies stay accountable, determine program effectiveness, and improve efficiency. The barriers were internal and external politics, funding and time limitations, personal agendas, differences in recognizing the value of EBDM across departments within local health departments, and limited knowledge and self-efficacy for using the process. When asked, “If you were the administrator and wanted to make EBDM a priority, what would you do to bridge the gap between benefits and barriers to EBDM?,” the overwhelming majority agreed that securing sustainable funding, rather than grant-focused funding, would be the most beneficial. Other responses included explaining how EBDM is pertinent to each department, demonstrating and explaining specific steps in EBDM, and improving the public’s understanding of the purpose of public health.Baseline survey findings were presented to key administrators at PCCHD, the local board of health, and to all PCCHD staff in several meetings. Presentations were a collaborative effort between the research team and PCCHD administration and staff. The results were shared in order to engage the PCCHD staff in developing policies and activities aimed at reducing the barriers to EBDM.The following is a summary of the recommendations given at the conclusion of the research assistant’s (A.H.’s) time with PCCHD:Clearly define and differentiate workforce improvement initiatives.Pay attention to EBDM messaging, modeling, and follow-up activities, because they are vital for the success of EBDM.Ensure every person and division understands the value of EBDM and how each staff member can use the process to improve service.Ensure that administration and management regularly discusses EBDM ideas and progress in meetings.Create an EBDM promotion team from among staff members trained in EBDM.Two-thirds of the staff agreed or strongly agreed that these recommendations accurately captured staff behaviors and feelings about EBDM.Following the assessment period, the research team maintained communication with the PCCHD community via conference calls and email to learn about, and track progress on, their plans for addressing improvement in the uptake of EBDM. Notably, many strategies were operationalized through collaboration between staff members and external partners. For example, PCCHD partnered with a state university to tailor a 3-day EBDM training for PCCHD staff that was based on an established training model . Agency The follow-up survey was sent to the 46 staff members who were respondents to the initial survey and who were still employed at PCCHD; the survey had a 100% response rate. Like the initial survey, the majority of respondents were technical experts (50.0%) or other (23.9%). Participants had an average of 6.9 years in their current position , and 10.3 years in public health . Between surveys, 55.9% of the respondents participated in an all-staff meeting where information on EBDM was presented, and 52.5% attended the tailored 2-day training.Significant changes were made in the AEBPs in the follow-up survey for access to training in EBDM and quality improvement processes, access to current information on EBDM, and knowing PCCHD allocated resources for quality improvement . SignifiChanges between pre- and post-survey data on activities that would most encourage staff to use EBDM  are noteThe success of PCCHD staff engagement was largely due to administrative support. Administrators were dedicated to delivering messages about EBDM in ways their staff members found helpful. They combined EBDM and quality improvement processes in order to make using both less confusing. The partnership between a state university and the PCCHD was crucial to delivering the 2-day EBDM training, which was tailored to address the needs identified by staff during the assessment phase.This case study provides evidence that a local health department can improve attitudes toward EBDM among its staff in a relatively short time. First, through qualitative and quantitative evaluation, we were able to understand the barriers a local health department faced in using EBDM. Second, we showed evidence of staff engagement in improving attitudes and perceptions of EBDM principles as shown by two-thirds of the staff reporting that the team accurately captured their attitudes toward EBDM and significant improvement in several quantitative outcomes.This study had several limitations. The main limitation relates to community engagement in that not all staff members were included in the survey. Because the pool of respondents was chosen by PCCHD administrators on the basis of job descriptions, results may be skewed in favor of EBDM practice. Another limitation is that 17% (10/59) of the original sample was lost to follow-up because they were no longer employed at PCCHD. In addition, organizational activities occurred during the year from baseline to follow-up, and activities occurring close in time to follow-up data collection may have been recalled more clearly, allowing the potential for bias. Finally, the context-specific nature of a case study limits the generalizability of any findings to other local health departments.The activities conducted by PCCHD show evidence of improvement in attitudes and perceptions toward EBDM over 1 year. Previous studies suggest that strong leadership is a key ingredient in creating a work climate conducive to EBDM in a local health department . At baseThis case study of a local health department community builds on previous work ,15 by pr