Male Sprague Dawley rats were infused with isotonic saline solution during 120 minutes and randomized in nine different groups: control, pargyline plus tolcapone (P+T), ANP, dopamine (DA), D-22, DA+D-22, ANP+D-22, ANP+DA and ANP+DA+D-22. Renal functional parameters were determined and urinary dopamine concentration was quantified by HPLC. Expression of OCTs and D1-receptor in membrane preparations from renal cortex tissues were determined by western blot and Na+, K+-ATPase activity was determined using in vitro enzyme assay. 3H-DA renal uptake was determined in vitro. Compared to P+T group, ANP and dopamine infusion increased diuresis, urinary sodium and dopamine excretion significantly. These effects were more pronounced in ANP+DA group and reversed by OCTs blockade by D-22, demonstrating that OCTs are implied in ANP stimulated-DA uptake and transport in renal tissues. The activity of Na+, K+-ATPase exhibited a similar fashion when it was measured in the same experimental groups. Although OCTs and D1-receptor protein expression were not modified by ANP, OCTs-dependent-dopamine tubular uptake was increased by ANP through activation of NPR-A receptor and protein kinase G as signaling pathway. This effect was reflected by an increase in urinary dopamine excretion, natriuresis, diuresis and decreased Na+, K+-ATPase activity. OCTs represent a novel target that links the activity of ANP and dopamine together in a common mechanism to enhance their natriuretic and diuretic effects.The aim of this study was to demonstrate the effects of atrial natriuretic peptide (ANP) on organic cation transporters (OCTs) expression and activity, and its consequences on dopamine urinary levels, Na The renal dopaminergic system is a local independent natriuretic system that contributes to preserving the normal balance of sodium and water, blood pressure levels and renal redox steady state . Renal d+, K+-ATPase, in almost the entire nephron [As a major regulator of proximal tubule salt and water reabsorption, renal dopamine exerts its physiological actions through two families of receptors located at the tubular cell surface: D1-like receptors (D1R and D5R) and D2-like receptors  . The imp nephron .+, K+-ATPase [in vivo and in vitro the effects of ANP infusion on OCTs expression and activity, respectively, and its consequence on dopamine urinary levels, Na+, K+-ATPase activity and renal function.Atrial natriuretic peptide (ANP), discovered by de Bold, is a 28-amino-acid peptide synthesized and stored in the atrial myocytes and released in response to cardiac wall stretching or after endothelin and α-adrenergic stimulation . Natriur+-ATPase ,13,14. TMale Sprague Dawley rats weighing 300–350 g  were used following international guiding principles and local regulations regarding the care and use of laboratory animals for biomedical research as well as the “International Ethical Guiding Principles for Biomedical Research on Animals” established by the CIOMS . The protocol was approved by the Institutional Committee for Care and Use of Laboratory Animals of the School of Pharmacy and Biochemistry of University of Buenos Aires . Animals were housed in cages with a 12-hour light/dark cycle under conditions of controlled temperature and humidity. All animals had free access to water and standard chow  until the day of the experiment. All surgery was performed under ethyl urethane anesthesia and all efforts were made to minimize suffering.3H-dopamine, 28.0 Ci/mmol specific activity ; nomifensine; decynium 22 (D-22); benserazide; pargyline; tolcapone; dopamine; imidazol; ATP (adenosine 5′ triphosphate); bovine seroalbumin fraction V of Cohn; ouabain ; Folin Ciocalteus phenol reagent  and EcoLite, a liquid scintillation . Standard Krebs bicarbonate (SKB) solution of the following composition (mM) was employed as incubation medium: 118 NaCl; 4.7 KCl; 1.2 MgSO4.7H2O; 1.0 NaH2PO4; 2.4 CaCl2; 0.004 EDTA; 11.1 glucose; 0.11 ascorbic acid and 26.0 NaHCO3.The following drugs were used in the experiments: rat atrial natriuretic peptide 1–28 (ANP) ; In order to minimize the confounding effects of endogenous dopamine, benserazide (an inhibitor of dopamine synthesis) was administrated to rats 24 and 2 hours before treatment . Rats were anesthetized with 10% w/v ethyl urethane  and placed on a thermostatically controlled heating pad to keep body temperature at 37°C. A tracheotomy was performed and a PE-90 tube (3 cm long) was inserted into the trachea to maintain an open airway. The left femoral vein was cannulated with a Silastic cannula (0.12 mm i.d.) for continuous infusion. The right carotid artery was catheterized with a T4 tube for blood sampling and arterial pressure monitoring, using a Statham GOULD P23ID transducer coupled to a Grass Polygraph 79D. The bladder was cannulated with a PE-75 cannula for urine collection. During a stabilization period of 60 minutes, the animals were infused with 0.15 M NaCl isotonic saline solution (ISS) at a rate of 3.0 mL/h  to reach a steady state in diuresis and allow urine collection (baseline period). After this period, the animals were infused at the same rate with ISS plus different drugs (which are stated below) for 2 hours . To determine renal functional parameters, urine and blood samples were collected every 30 and 60 minutes, respectively. Mean arterial pressure (MAP) was recorded every 30 minutes .The animals were randomized into the following experimental groups:Control group (C) (n = 8): infused with ISS for 120 minutes .With the exception of the control group, all groups received an i.v. bolus of pargyline (20 mg/kg) and tolcapone (300 μg/kg) dissolved in 0.1 ml of ISS, inhibitors of the dopamine catabolic enzymes monoaminoxidase (MAO) and cathecol-O-methyl-transferase (COMT) respectively, 30 minutes before the beginning of the experimental period in order to prevent the catabolism of the exogenous dopamine infused.Pargyline + tolcapone (P+T) (n = 6): animals were infused with ISS for 120 minutes.ANP (n = 7): animals received ANP  in i.v. bolus at the beginning of the experimental period. Thereafter, rats were infused with ISS plus ANP (10 μg/kg/h) for 120 minutes.Dopamine (n = 7): animals were infused with ISS plus dopamine (100 μg/kg/h) for 120 minutes.D-22 (n = 6): D-22, a specific inhibitor of OCTs, was infused one hour before the experimental period at 10 μg/kg/h. Thereafter, animals were infused with ISS plus D-22 (10 μg/kg/h) for 120 minutes.Dopamine + D-22 (n = 6): D-22 was infused one hour before the experimental period at 10 μg/kg/h. Thereafter, animals were infused with ISS plus dopamine (100 μg/kg/h) and D-22 (10 μg/kg/h) for 120 minutes.ANP + D-22 (n = 6): D-22 was infused one hour before the experimental period at 10 μg/kg/h. An i.v. bolus of ANP (5 μg/kg) at the beginning of the experimental period was followed by ISS infusion plus ANP (10 μg/kg/h) and D-22 (10 μg/kg/h) for 120 minutes.ANP + Dopamine (n = 6): animals received ANP (5 μg/kg) in i.v. bolus at the beginning of the experimental period, followed by ISS infusion plus ANP (10 μg/kg/h) and dopamine (100 μg/kg/h) for 120 minutes.ANP + Dopamine + D-22 (n = 6): D-22 was infused one hour before the experimental period at 10 μg/kg/h. An i.v. bolus of ANP (5 μg/kg) at the beginning of the experimental period was followed by ISS infusion plus ANP (10 μg/kg/h), dopamine (100 μg/kg/h) and D-22 (10 μg/kg/h) for 120 minutes.To evaluate renal functionality, the following parameters were determined: urine and plasma sodium and creatinine , glomerular filtration rate (GFR) estimated by creatinine clearance (CrCl), fractional and urinary sodium excretion  and urine flow rate (UV). UNa, PLNa, UCr and PLCr were measured by standard methods using an autoanalyzer. FENa, UNa.UV and CrCl were calculated according to standard formula. UV is expressed as μL/min, UNa and PLNa as mEq/L, FENa as the percentage (%) of filtered sodium, UNa.UV as μEq/min and CrCl as mL/min. To determine urinary dopamine concentration by HPLC, a fraction of urine was collected at the end of the experimental period into polyethylene tubes containing 100 μl of 6 N HCl.Western blot analysis was performed to evaluate ANP effects on total amount of OCTs and D1 receptor, by using membrane preparations. Right kidney from each animal was removed and renal cortex was immediately dissected and separated under refrigeration. In order to isolate cellular membranes, additional sets of tissue samples were homogenized on ice with a Tissue Tearor (Biospec Products Inc), 1:9 weight/volume, in a buffer mixture: 50 mmol/L Tris and 300 mmol/L sucrose, pH 7.40, and centrifuged at 4000 rpm and 4°C during 15 minutes. Supernatants were centrifuged at 17000 rpm and 4°C during 45 minutes. Pellets were suspended in buffer containing 50 mmol/L Tris, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1% Triton-X-100, 1 mmol/L PMSF, 1 μmol/L pepstatin, and 2 μmol/L leupeptin, 1x protease inhibitor cocktail (Roche Diagnostics), and afterwards, samples were centrifuged at 12000 rpm during 20 minutes. Protein concentration in the Triton-soluble supernatant was determined by Lowry technique [+, K+-ATPase activity was assayed in the supernatant using Fiske-Subbarrow method [+, K+-ATPase activity [+, K+-ATPase activity, considering control values as 100%.Sample tissues from renal cortex weighing 50 mg were homogenized (1:10 weight/volume) in 25mM imidazole/1mM EDTA/0.25 M sucrose solution and centrifuged at 5.000 rpm and 4°C for 15 minutes. Naw method . ATPase activity . Protein50-μl aliquots of urine were partially purified by batch alumina extraction, and then separated by reverse-phase high-pressure liquid chromatography (RP-HPLC) using a 4.6×250 mm Zorbax RxC18 column  and quantified amperometrically by the current produced upon exposure of the column effluent to oxidizing and then reducing potentials in series using a triple-electrode system . Recovery through the alumina extraction step averaged 80–90%. Dopamine concentrations in each sample were corrected for recovery of an internal standard. Levels of dopamine were further corrected for differences in recovery of the internal standard in a mixture of external standards. The limit of detection was about 20 pg/volume assayed.3H-Dopamine uptake was measured as previously described [2 and 5% CO2 for 15 min; nomifensine (50 μM) was added in the medium to avoid neuronal dopamine uptake. After preincubation, tissues were transferred to fresh SKB and incubated, in similar conditions, with 0.625 μCi/mL 3H-dopamine (22.32 nM), 17 μM nomifensine and the different tested drugs. To determine ANP effects and signaling pathway involved in the regulation of dopamine uptake, 3H-dopamine uptake was measured in the absence or in the presence of D-22, anantin and KT-5823. The following groups were studied: (a) control; (b) 100 nM ANP; (c) 1 μM D-22; (d) 100 nM ANP plus 1 μM D-22; (e) 100 nM anantin; (f) 100 nM ANP plus 100 nM anantin; (g) 1 μM KT-5823; (h) 100 nM ANP plus 1 μM KT-5823.Another set of rats (300–350 g) were anesthetized with 10% w/v ethyl urethane . Both kidneys were excised and washed with fresh Saline Krebs Buffer (SKB) to remove the residual blood. The renal cortex was dissected from the medulla by using a small scalpel. After then, slices of renal cortex were cut, minced and weighed (approximately 50 mg). escribed . BrieflyAll values are expressed as means ± SEM and normalized respect body weight. Data was processed using Graph Pad InStat Software . Statistical analysis was performed using Student’s t-test and one-way ANOVA. A value of p<0.05 was considered statistically significant.The involvement of OCTs transporters in renal dopamine and ANP diuretic and natriuretic effects was investigated. To verify this objective, we determined the effects of MAO and COMT inhibition, the effects of the administration of dopamine and ANP alone, and the co-administration of ANP plus dopamine together, on renal functionality  and on tversus control and P+T groups  did not alter any renal functional or hemodynamic parameter compared to control rats Tables . Under MT groups . The addT groups . HoweverT groups . HoweverT groups . MAP andT groups .+, K+-ATPase specific activity was investigated. To quantify this, Na+, K+-ATPase specific activity was determined in samples of renal cortex obtained at the end of the experimental period.The involvement of OCTs transporters in the inhibitory effects of renal dopamine and ANP on Na+, K+-ATPase activity by approximately 56% and 53% respectively  of Na+, K+-ATPase activity elicited by ANP or dopamine alone. On the other hand, the addition of D-22 partially reversed the inhibition produced by the co-administration of ANP plus dopamine, reaching values very similar to those registered with the infusion of ANP or dopamine alone.In accordance with previous studies , the infectively . To evalThe possibility that the inhibition of OCTs may affect the stimulatory effects of ANP on dopamine tubular transport or may alter dopamine urinary excretion was evaluated. To quantify this, dopamine excretion was determined in urine during the last experimental period. Because of the administration of benserazide, almost all urinary dopamine was considered to be from the infusion of exogenous dopamine. As expected, the exogenous infusion of dopamine significantly increased the excretion of the amine when compared with the control group. This increase was inhibited by the administration of D-22. The co-infusion of ANP and dopamine together further increased dopamine urinary excretion even compared with dopamine alone group. This increase was also inhibited by D-22, reaching very similar urinary dopamine levels to those observed in control group .Based on the above results, we tested the possibility that ANP might stimulate the protein expression of OCTs and/or dopamine D1R at membrane level, and consequently, the fact that the natriuretic peptide could increase the urinary excretion of dopamine and enhance its natriuretic effects. To assess this possibility, western blot analysis of OCTs and D1R were performed on membrane preparations from renal cortex tissues. Samples were probed with anti-OCT-1, anti-OCT-2, anti-OCT-3 and anti- D1R antibodies and quantified using GAPDH as an internal standard. Although difference in expression did not reach statistical significance, a trend towards to increase OCT-3 expression can be observed in the ANP group. In 3H-dopamine. 3H-dopamine uptake (approximately 35%) in renal cortex slices compared with control group. In presence of D-22 (1μM), 3H-dopamine uptake was significantly reduced by approximately 60%. As demonstrated in 3H-dopamine uptake by itself, but when added together with ANP, it was able to abolish the increased dopamine uptake elicited by the natriuretic peptide. Moreover, the specific inhibitor of PKG, KT-5823 (1 μM) reversed ANP stimulatory effects on dopamine uptake. On the other hand, KT-5823 by itself did not alter 3H-dopamine uptake.In order to evaluate whether the increased urinary excretion of dopamine elicited by ANP administration is caused by stimulation of dopamine tubular transport by OCTs, we carried out an in vitro assay in renal cortex slices using We have previously reported that ANP stimulates dopamine uptake in renal cortex, this process being characterized as a typical extraneuronal uptake . In our The identification of a dopamine specific tubular transporter is of great interest for basic and clinical research since it would allow insight into the mechanisms that affect the exogenous dopamine availability in the tubular lumen and therefore its natriuretic and diuretic efficacy. The clinical efficacy of dopamine infusion to prevent renal failure  has been questioned by a number of clinical studies –23. Seve+, K+-ATPase specific activity. As expected, either ANP or exogenous dopamine inhibited renal specific activity of Na+, K+-ATPase, an enzyme closely linked to water and sodium retention. Moreover the co-infusion of ANP plus dopamine increased further pump inhibition, showing an enhanced response. On the other hand, the inhibition of OCTs by D-22, prevented the inhibition of Na+, K+-ATPase activity caused by dopamine alone and also reduced the inhibition caused by dopamine plus ANP.Our next step was to test in vivo the possibility that the inhibition of OCTs by D-22 could reduce the inhibitory action of exogenous ANP and dopamine on Na+, K+-ATPase inhibition, as well as water and sodium excretion, both stimulated by dopamine. Moreover, the co-infusion of ANP and dopamine further increased urinary dopamine levels when compared with dopamine group, and this increase was also blocked by inhibition of OCTs by D-22. These results clearly suggest that ANP promotes the uptake of exogenous dopamine by the renal tubules into the tubular lumen through stimulation of OCTs synthesis and/or their specific activity. These results also correlate with our findings that the co-infusion of ANP and dopamine further increase water and sodium excretion by the kidney as well as inhibiting Na+, K+-ATPase activity. We have previously demonstrated that ANP and urodilatin stimulate dopamine uptake in renal cortex, while angiotensin II exerts the opposite effect [In order to confirm the stimulatory effect of ANP on dopamine tubular transport, mediated by OCTs, we proceeded to test whether the urinary excretion level of dopamine could be altered by the presence of ANP and/or D-22. This experiment allowed us to discard a simple addition of independent effects of both natriuretic agents. Our results indicate that the inhibition of OCTs by D-22 can decrease urinary dopamine excretion observed after the infusion of exogenous dopamine. This supports the fact that the administration of D-22 reduced Nae effect ,31,32. Oe effect . In linee effect ,35.The effects of ANP on OCTs may be the result of an increase in their protein expression and/or activity. The protein expression of both, OCT-1 and OCT-2, is reduced in experimental model of diabetes induced by streptozotocin in rats, thereby affecting the transport of a large number of exogenous and endogenous compounds . On the 3H-dopamine. 3H-dopamine uptake (by approximately 35%) over a short time period, in renal cortex as compared with the control group. OCTs inhibition by D-22 reduced 3H-dopamine uptake compared with control group by approximately 60%. Once again, this result demonstrates that OCTs are specific transporters for dopamine, and their activity is critical in the process of dopamine uptake by the kidney. On the other hand, even in the presence of ANP, D-22 decreased 3H-dopamine uptake by 50% compared with control group, reinforcing the idea that ANP stimulates dopamine tubular uptake by increasing OCTs activity. The enhancing effects of ANP on dopamine uptake are mediated by stimulation of NPR-A receptors and PKG activation, since anantin (specific blocker of NPR-A receptor) and KT-5823 (specific inhibitor of PKG) reverse ANP stimulatory effects on dopamine uptake in kidney slices. Moreover, ANP promotes the urinary excretion of dopamine by stimulating OCTs activity. The analysis of all these results as a whole suggests that the secretion of dopamine into the tubular lumen would allow D1R activation and Na+, K+-ATPase activity inhibition, leading to decreased sodium reabsorption and increased natriuresis. By these mechanisms, ANP and dopamine may regulate and enhance natriuresis and diuresis through a common pathway.As it was previously mentioned, even if ANP did not alter OCTs expression, the fact that ANP enhances OCTs activity may provide another way to temporarily regulate the dopamine tubular transport by OCTs. To test whether the increase in urinary dopamine excretion observed after ANP infusion could be related to the stimulation of OCTs activity by ANP, we performed an in vitro experiment in renal cortex slices incubated with +, K+-ATPase activity are due to the stimulatory effects of ANP on exogenous dopamine OCTs-dependent transport into the tubular lumen.Under physiological conditions, the natriuretic and diuretic effects elicited by dopamine are exerted mainly by D1-like receptors . Aperia This work provides evidence of a novel mechanism by which ANP acts together with dopamine to enhance sodium and water excretion by increasing dopamine tubular bioavailability .A failure in the mechanism of dopamine transport in renal tubule cells, associated to changes in OCTs activation or expression, could be postulated as a cause of sodium and water retention and contribute to the development of hypertension. Moreover, this mechanism could explain the lack of effects observed in clinical studies in which exogenous dopamine infusion is not capable of preventing the impairment of renal function in patients with acute renal injury ,24. In mTogether, these results highlight the role of OCTs as transporters of dopamine in the kidney, the relationship between ANP and dopamine to enhance each other´s natriuretic and diuretic properties, and the importance of OCTs as an essential factor for dopamine to exhibit its full natriuretic and diuretic effects. Therefore it may be hypothesized that the lack of clinical benefits reported after dopamine infusions could be related to an alteration in its transport to the lumen side by changes in OCTs activity. Further experimental and clinical studies must be done to confirm this hypothesis.In summary, this study demonstrates a novel mechanism for how ANP increases dopamine levels at the tubular lumen by stimulation of dopamine uptake by OCTs located at the basolateral side of tubular proximal cells. This effect increases the availability of dopamine to interact with D1R located at the luminal side of tubular cells. Through this mechanism, both natriuretic agents enhance their effects to promote sodium and water excretion by the kidneys.


In a subgroup, we explored the effects of raised NEFA enriched with SFA, with or without LC n-3 PUFA, on whole body insulin sensitivity (SI) and responsiveness of the endothelium to insulin infusion.We have reported that adverse effects on flow-mediated dilation of an acute elevation of non-esterified fatty acids rich in saturated fat (SFA) are reversed following addition of long-chain (LC) 2) consumed oral fat loads on separate occasions with continuous heparin infusion to elevate NEFA between 60 and 390 min. For the final 150 min, a hyperinsulinaemic-euglycaemic clamp was performed, whilst FMD and circulating markers of endothelial function were measured at baseline, pre-clamp (240 min) and post-clamp (390 min). NEFA elevation during the SFA-rich drinks was associated with impaired FMD (P = 0.027) whilst SFA + LC n-3 PUFA improved FMD at 240 min (P = 0.003). In males, insulin infusion attenuated the increase in FMD with SFA + LC n-3 PUFA (P = 0.049), with SI 10% greater with SFA + LC n-3 PUFA than SFA (P = 0.041).Thirty adults (mean age 27.8 y, BMI 23.2 kg/mThis study provides evidence that NEFA composition during acute elevation influences both FMD and SI, with some indication of a difference by gender. However our findings are not consistent with the hypothesis that the effects of fatty acids on endothelial function and SI operate through a common pathway.NCT01351324 on 6th May 2011.This trial was registered at clinical trials.gov as  •The impact of raised NEFA on endothelial function and insulin sensitivity was studied.•NEFA elevation during the SFA drink reduced FMD while SFA + LC n-3 PUFA improved FMD.•n-3 PUFA than SFA, with SI similar in women.Men had a 10% higher SI with SFA + LC •Changes in FMD were not mirrored by changes in circulating NOx.•Gender mediated the effect of NEFA composition on both endothelial function and SI. In vitro studies report more adverse effects of saturated (SFA) than unsaturated fatty acids on the endothelial PI3K insulin signalling pathway and NO production n-3 polyunsaturated fatty acids (LC n-3 PUFA) found in fish oil has been consistently shown to improve endothelial function in a variety of populations n-3 PUFA Non-esterified fatty acids (NEFA) have been proposed to be a mediator of insulin signalling defects in both skeletal muscle and endothelial tissue 2). All subjects were healthy non-smokers who were not taking greater than 1 g eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) per day, or any medication known to influence blood clotting, lipids or blood pressure. The subjects were screened for fasting cholesterol (mean ± SD 4.62 ± 0.76 mmol/L), triglyceride (TG) (1.06 ± 0.28 mmol/L) and glucose (5.15 ± 0.64 mmol/L). Subjects were recruited between March 2009 and January 2010.From a larger cohort genotyped for a common polymorphism in the eNOS gene  This was a single-blind crossover study; subjects attended the Hugh Sinclair Unit of Human Nutrition on two occasions separated by four weeks for females  or at least one week for males. Subjects were randomly assigned to one of the fat loads on each day using an online number generator. Investigators responsible for performing and analysing the FMD and insulin clamp measures were blinded to the allocation and were not involved in the preparation or serving of the fat loads.The study protocol has been described elsewhere The procedures followed in the current study were in accordance with the ethical standards of the University of Reading Research and Ethics Committee. Written informed consent was obtained from all subjects.Oral fat loads were prepared according to bodyweight  using paFMD of the brachial artery was measured by trained researchers using an ATL Ultrasound HDI5000 broadband ultrasound system  and a procedure based on standard guidelines, as previously described +, HemoCue AB, Ängelholm, Sweden). The steady state glucose infusion rate over the final 30 min of the clamp provided an index of whole body SI and was expressed as mg · min−1 · kg−1 of fat-free mass (FFM). FFM was measured using a bioimpedance device .Venous blood glucose was sampled immediately prior to the commencement of the hyperinsulinaemic-euglycaemic clamp at 240 min in vitro lipolysis, the EDTA samples were placed immediately on ice and processed within 30 min Venous blood samples were collected every 30 min into K3 EDTA  or serum tubes . To limit Plasma C-peptide and insulin were quantified using a multiplex assay system  with a Milliplex Endocrine Panel . NEFA composition analysis was performed by extracting lipids from 800 μl of plasma collected at baseline (the two baseline samples were pooled) and 240 min At 95% power and 5% significance, the minimum number of subjects required to detect a difference of 1.5% in FMD response between the two oral fat loads was calculated to be 22. Additional subjects were recruited (n = 30) to allow for possible dropouts.SPSS version 17.0  was used for all statistical analyses. Summary measures calculated for the time-course data included area under the curve (AUC) and incremental AUC (iAUC). Data were tested for normality; it was necessary to log transform NEFA and TG values and use non-parametric tests for the fatty acid composition of NEFA. Independent and paired t-tests  were used to compare baseline and summary measures between genders and fat loads, respectively. For postprandial time-course data, repeated measures ANOVA were performed using a mixed model approach. Bonferroni correction was applied to control for multiple pair wise comparisons. P ≤ 0.05 was considered significant.The fat loads were well tolerated by the subjects. Initial analysis of the results revealed some differences by gender; therefore data are also presented separately for males and females where appropriate.n-3 PUFA compared to SFA regime (P = 0.041) whereas SI was similar in females between the two fat loads (P = 0.420).There were no differences in fasting values or metabolic responses as measured by iAUC for insulin, C-peptide, or C-peptide: insulin ratio (a marker of insulin clearance) by fat load or gender . Males hFMD response and circulating markers of endothelial function are shown in n-3 PUFA improved (P = 0.003) the FMD response at 240 min. The mean absolute difference in change from baseline between the two fat loads was 1.35 ± 0.22% (P < 0.001). There were some indications of differential effects by gender for the change in FMD following the fat loads  but not males (P = 0.179); conversely the impairment of FMD associated with the SFA load was evident in males (P = 0.017) but not in females (P = 0.387). Serum NOx declined to a similar extent during both fat loads (P < 0.001) and did not differ by gender. Plasma ET-1 did not change during either fat load in males or females.For the group as a whole, compared with baseline, the SFA load resulted in an impairment (P = 0.027) whilst SFA + LC at loads ; the benn-3 PUFA regime  during the SFA + LC A regime a. The deA regime b; this wA regime a and b. A regime c, with sIn the group as a whole, there were no significant differences in FMD or circulating markers of endothelial function after the insulin infusion (390 min) for either fat load.0–390min was 70% greater in males than females (110.3 ± 14.2 mmol/L × 390 min vs. 64.6 ± 11.3 mmol/L × 390 min), P = 0.015.Baseline NEFA and TG did not differ by fat load or gender. The oral fat-heparin protocol resulted in a two-fold elevation of serum NEFA at 240 min as compared to baseline a. Concenn-3 PUFA regime (P = 0.016). TG iAUC0–390min revealed a greater reduction in TG over the study day in males (−64.4 ± 14.3 mmol/L × 390 min) than females (−20.5 ± 13.2 mmol/L × 390 min) (P = 0.029), with no difference by fat load.The TG response remained within a narrow range b but wasn-3 PUFA; 43.7% (42.0–45.9); both P < 0·001). A significant increase in the proportion of LC n-3 PUFA during the SFA + LC n-3 PUFA load (from 1.3% (1.0–1.8) to 6.8% (5.8–7.2)) was observed at 240 min, consistent with a three-fold increase in EPA and a five and a half-fold increase in DHA .There was a significant increase in the percentage weight of SFA in the NEFA fraction of plasma from baseline  to 240 min during both fat loads ; SFA + LC n-3 PUFA to reverse impairment in FMD observed with SFA alone n-3 PUFA on insulin sensitivity. Conversely, there were indications that females were more responsive to the beneficial effects of LC n-3 PUFA on FMD. Differences in lipid metabolism during the protocol were also observed, with males having significantly higher NEFA concentrations than females.We have previously shown acute ingestion of SFA with LC n-3 PUFA fat load the effects observed were complex as in males, insulin infusion following this fat load was actually associated with a decrease in FMD response. As FMD has been shown to be dependent on NO bioavailability n-3 PUFA, a reduction in circulating NOx does not result in a decrease in FMD. However, the lack of association between these two measures may be attributed to the plasma NOx measurement which represents not only NO production but also its degradation and excretion. We propose that LC n-3 PUFA enhance endothelial function through a number of mechanisms, some of which may be independent of NO production and/or insulin signalling. For example, in vitro studies show that cytochrome P450 epoxygenases (CYP450) present in endothelial cells can metabolise LC n-3 PUFA such as DHA to fatty epoxides, which promote vasodilation through activation of calcium-activated potassium channels present in smooth muscle cells n-3 PUFA on FMD observed in females in this study remains to be determined but is worthy of further investigation given the almost two fold greater response we have seen in our female subjects.Contrary to our hypothesis of a positive effect of insulin infusion on endothelial function, attenuation of the FMD response following exposure to NEFA rich in SFA did not improve after insulin infusion in the group as a whole, or in either gender. For the SFA + LC n-3 PUFA in subjects with type II diabetes n-3 PUFA than SFA alone.Whilst experimental elevation of NEFA has been consistently shown to impair whole-body insulin sensitivity The sampling of venous rather than arterial or arterialised blood during the insulin clamp is a limitation of this study. With the current protocol, it was not possible to measure the primary outcome measure FMD whilst heating the hand as this has been shown to induce changes in systemic vasodilation n-3 PUFA on FMD and on SI during acute NEFA elevation, with some indication of a difference in response by gender. We had postulated that the effects of elevated NEFA composition on impairment of FMD might reflect the differential effects of dietary fatty acids on the PI3K/Akt pathway that have been reported from in vitro studies n-3 PUFA may improve endothelial function and to speculate on differences in insulin dependent endothelial pathways in males and females. The clinical and public health relevance of the beneficial effects we have observed of LC n-3 PUFA on FMD and SI are potentially significant and need to be substantiated by clarification of the underlying mechanisms involved.In conclusion, our study provides evidence for differential effects of SFA and LC


Knowledge of phylogeny is of fundamental importance for understanding evolution. It has become an indispensable tool in modern genomics as a framework for interpreting genomes and metagenomes, for understanding evolution of genes, proteins, and noncoding RNAs as well as different types of regulations including secondary RNA and protein structures, and for reconstructing ancestral genomes . The eraAn important avenue of modern phylogenetics is the study of coevolution. Here a major focus is the developing of effective algorithms to infer common history of genes, proteins, molecular machines , signal transductions, metabolic pathways, chromosomal structures and gene synteny, and so forth. An important direction is to define and reconstruct the evolutionary scenario on the basis of polytomous trees, also in application to study fast evolving bacteria and viruses with high medical impact, and in shaping the primary genomes. Dating of the phylogeny immediately depends on progress in detecting zones of active horizontal gene transfers or other genomic events, defining time slices to describe tree-like phylogenies, and so forth.20 vertices, 109 parts, and the same average vertex degree can be processed on a multiprocessor machine in reasonable time. A high priority for testing modern algorithms is to obtain primary data with known predefined solutions. The call is for realistic computational models that allow simulating large-scale phylogenies and can serve for future studies, as well as real data with known phylogenies or at least few data sets where everybody would agree what the correct phylogeny is.Mathematics of phylogenetics will grow to foster alternative ways to describe evolution: generalization of trees into nets and developing models based on stochastic and partial differential equations. An important focus is to develop low polynomial complexity algorithms for exact models that are usually solved heuristically. Contemporary methods of clustering and discrete optimization have already proved very effective. Thus, graphs with 10The contents of the special issue of 2015 cover research of various groups that use the toolkit of phylogenetics to tackle a spectrum of evolutionary questions. Apart from classic molecular systematic applications to infer taxon phylogenies, the trend is obvious to approach molecular and biodiversity assessment at different levels in various communities, at intraspecific level and in environmental samples, including systematic studies of bacterial and viral pathogenic agents. Molecular markers such as mobile elements are being developed and exploited in studies of population polymorphisms, and RNA secondary structures are used to detect signatures of selection.A historical profile of molecular phylogenetics with some extrapolations into the future, as well as a brief outline of hot spots in this field, can be found in this special issue . We trulVassily LyubetskyWilliam H. PielPeter F. Stadler


Since biomarker imaging is traditionally understood as imaging of molecular probes, we highly recommend to avoid any confusion with the previously defined term “imaging biomarkers” and, therefore, only use “molecular probe imaging (MPI)” in that context. Molecular probes (MPs) comprise all kinds of molecules administered to an organism which inherently carry a signalling moiety. This review highlights the basic concepts and differences of molecular probe imaging using specific biomarkers. In particular, PET radiopharmaceuticals are discussed in more detail. Specific radiochemical and radiopharmacological aspects as well as some legal issues are presented. The terms “imaging biomarker” and biomarker imaging” are currently used in a variety of different contexts. Therefore, a clear definition of these terms is pivotal. “Biomarker” has already been defined by the National Institutes of Health  director’s initiative on biomarkers and surrogate endpoints to be “a characteristic, that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” . But thiimago meaning “picture”. Hence, imaging is a tool enabling the operator to obtain a picture of the organism. It can enable the assessment of the biological basis of the body, including changes due to disease, response to therapeutic intervention. Nowadays, imaging is mostly used as a synonym for visualisation (derived from the Latin word video meaning “to see”), although visualising processes and conditions are only part of imaging these things. The aspect that distinguishes imaging from mere visualisation is quantitation. This brings us back to the definition of biomarker being a measurable signal. For that reason, a visualisation biomarker could not exist! Imaging (and therefore imaging biomarkers) can be characterised as structural, functional and molecular, respectively. Hence, one should distinguish truly molecular information from rather functional or even structural measurements and restrain from thoughtlessly terming everything “molecular imaging”. So what makes imaging become molecular? The answer is simple: the visualisation and quantification of a molecular interaction between a molecular probe and a molecular target.Moreover, “imaging” is also a term with widespread use demanding for closer characterisation; coming from the Latin word Recently, Lucignani strengthened the term “imaging biomarkers” and pointed out that they allow identifying measurable variables that are otherwise undetectable . FurtherNow, that we have defined imaging biomarkers can we simply rearrange these terms and consequently derive a definition of biomarker imaging as well? Unfortunately, it is not that simple and thus requires further reflection. Since biomarker imaging traditionally is understood as imaging of molecular probes, we highly recommend to avoid any confusion with the previously defined term “imaging biomarkers” and, therefore, only use “molecular probe imaging (MPI)” in that context. Molecular probes (MPs) comprise all kinds of molecules administered to an organism which inherently carry a signalling moiety. This moiety can for example be a radionuclide, a fluorescent chromophore, a paramagnetic core or a gas-filled or gas-producing nanostructure. The resulting signal has to be visualised and quantified by dedicated imaging procedures and technologies ,5,6,7,8.The blood-oxygen-level dependent (BOLD) signal is an indirect measure for the local oxygenation level in tissue . It is qInorganic gadolinium (Gd) compounds as contrast enhancers for MRI are MPs . Dependi18F]FMISO ([18F]fluoromisonidazole) is an example of an MP used in positron emission tomography (PET) for the assessment of hypoxia [18F]FMISO. Comparing these complementary techniques to assess information on oxygen levels, it is evident that the quality and depth of the information is different.FDG (2-deoxy-2-[18F]fluoro-d-glucose) is a radiolabelled analogue of glucose. Like its parent compound it is transported through the blood stream and taken up by cells through specific glucose transporters (GLUT). GLUT is expressed by cells in correlation to its energy demand. Therefore, tumour cells, inflammatory tissue or myocytes as well as neurons exhibit up-regulated GLUT. After being taken up by the cell, the fate of [18F]FDG and native glucose is different: due to the substitution of a hydroxyl moiety by the 18F-label in the molecule the biochemical catabolism is altered. In the first step of glycolysis, both glucose and [18F]FDG are phosphorylated by hexokinase enzyme. Subsequently, the glucose molecule is rearranged by an isomerase enzyme which is not able to use [18F]FDG as substrate. The 18F-substituent is hindering this transformation. Since hexokinase activity is the “driving force” in the cell to gain energy out of sugar molecules, it is—more or less—unidirectional allowing for almost no back-reaction. Hence, [18F]FDG is trapped within the cell and its uptake correlates to the energy demand of the tissue [18F]FDG; and the IB is a combination of hexokinase activity and GLUT expression, both representing a surrogate for energy demand.acetyl]-d-phenylalanyl-l-cysteinyl-3--l-alanyl-d-tryptophyl-l-lysyl-l-threonyl-N-[-2-hydroxy-1-(hydroxymethyl)propyl]-,cyclic(2-7)-disulfide), a radiolabelled peptide from the octreotide family, selectively targets somatostatin receptors FDG vs.19F-FDG, [11C]methionine vs.12C-methionine. It should be of note that [18F]FDG and glucose are not strictly the same chemical compound and therefore a distinct difference in their biochemical behaviour is observed! Since radioactive molecular probes are chemically indistinguishable from their stable  physics and technology, radiopharmaceutical sciences (radiochemistry and biomarker development) and  molecular imaging see . All expBinding and/or uptake characteristics in terms of affinity, selectivity and unspecific binding;Pharmacokinetics yielding information on input function, elimination rate and principal fate of the compound;Metabolic issues with respect to formation of metabolites potentially interfering with the original signal and stability considerations;Involvement in different (patho-)physiological processes such as parallel representation of both vital tumour tissue and inflammation or combined information on perfusion and hypoxia; andnota bene: pharmacodynamic effects are in general of no interest in molecular imaging using radiopharmaceuticals because of the minuscule mass of compounds that is administered. Therefore, pharmacodynamics are not considered herein).Pharmaceutical issues referring to interactions of molecular probes and co-medication as well as influence of the pharmaceutical formulation of the molecular probe chemical properties of the molecular vehicle, especially for molecular modelling and dosimetry. Of particular interest is the deep understanding of:Even a basic understanding of radiochemical issues and preparation techniques is required for the application of PET/MRI in a scientific or clinical setting .Receptor systems and their subtypes;Transporter proteins;Antigens;Specific intra- or extracellular enzymes;Alterations in gene and protein expression;Changes in physiology and metabolism, such as hypoxia or differences in vascularisation and perfusion;Energy turnover.The molecular probe—and especially the targeting component—has to display a high degree of specificity and selectivity towards the target site. Important targets are:The molecular probes either interact directly with the aforementioned targets and processes or enable an indirect assessment of imaging biomarkers.In a pathophysiological state, these targets and/or processes may be altered distinctively in comparison to their baseline status. Hence, the quantifiable signal resulting from the interaction of the MP with these molecular targets may also be changed considerably. For example, in many tumours the expression rate of some receptors or transporters as well as enzymatic activity and representation of antigens on the cell surface is modified and these changes may serve as a suitable predictor for stage and allocation of these tumours (IB).18F]fluoride delivered from the target is received as an aqueous solution that is chemically inactivated due to its shell of water molecules and therefore has to be removed from its aqueous environment. This is achieved by azeotropic drying with acetonitrile in presence of an aminopolyether as phase transfer catalyst. The next step is a coupling reaction directly with or incorporation into the predesigned precursor compound. This substitution reaction generally demands elevated temperature or other ways of chemical activation . This means that, in contrast to conventional organic chemistry, the main aim is not a quantitative conversion but the maximum achievable total yield of radioactivity (in GBq). It is therefore sometimes the better choice to sacrifice chemical conversion output (in %). This is of particular interest for Carbon-11: if for example in a 11C-methylation reaction the conversion yield is 15% after 1 min at room temperature  and increases to 50% after 35 min under heating at 80 °C (which means that for heating up and cooling down an other 5 min are required) the total achievable yield is lower for the latter conditions! Other differences to conventional chemistry are:i.e., the radionuclide and the precursor molecule)! A huge excess of the precursor is present in the reaction solution compared to the amount of radionuclide. An illustrative extrapolation reflecting this imbalance would be to react a whole shipload of precursor material with as little as one single sugar cube of radionuclide!There is no stoichiometry between the reaction partners , the use of special miniature equipment is demanded.Working with high amounts of radioactivity  requires powerful radiation protection devices. All radiolabelling procedures have to take place in a fully lead shielded environment (dedicated hot cells) under remote control using special equipment for manipulation like tele-tongs or robotic arms.For the routine delivery of radiopharmaceuticals with maximum quality, the radiosyntheses are performed in automated synthesis modules which can be remotely controlled from outside the shielded hot cells . The production sequences are programmed in advance based on optimization procedures and performed automatically with constant and reproducible quality.12C-CO2 always present in air may contribute significantly to a reduction in specific activity. In case of fluorine-18, the isotopically enriched target material  also contains traces of non-radioactive fluorine-19 as dissolved fluoride anions. Typically, the content of 19F-fluoride is specified as <0.1 mg/L (=100 ppb) fluoride equal only 30.0 pg (=1.6 pmol) Therefore, up to a 16-fold excess of non-radioactive fluoride may be present in terms of amount of substance!Any carrier (stable isotope of the radionuclide taking part in the reactions) influences the radiochemical conversion leading to a significant amount of indistinguishable, non-radioactive product that “dilutes” the final radiopharmaceutical. This is followed by a significant change in stoichiometry. This carrier also determines the maximum achievable specific radioactivity which is a measure for the radioactivity per mass unit  ,36. Such100 ppb) ,39,40 an18F]FDG and [18F]fluoride for labelling) concentrations up to 35 GBq/mL (700 GBq in 20 mL) can be reached [When working with solutions with high radioactivity concentrations, one has to consider radiolysis as a major factor reducing product purity leading to formation of unwanted by-products. Especially when dealing with batches that are to be shipped  is normally not predisposed for direct labelling reactions, the first step in the labelling sequence is an activation reaction. For instance, [sistance ,32, micrsistance ,34, highsistance . As a maunder vacuo for product purification [Radiolysis may also occur during the production process , particularly when applying excessive heating and evaporation to dryness fication . Hence, fication . Overallmax) determine the maximum achievable signal. Specific radioactivity  therefore allows predicting the amount of detectable signal, reflecting the ratio of signal-evincing to signal-erasing binding. Consequently, the required specific radioactivity has to be higher when the density of the target sites is lower. Nota bene, the specific radioactivity should be kept constant throughout a (quantified) clinical study and should always be included as a co-variable in statistical analysis of the data .As already mentioned earlier in this review, the specific radioactivity is an essential parameter for the quantification and visualisation of the MP’s signal—the imaging biomarker. In many (patho-)physiological imaging procedures the actual number of target sites is limited and the process of binding between the MP and the specific target  is saturable. Then, the maximum available binding sites  is cleaved from the intact parent molecule through active metabolism. Subsequently, the measured signal would be wrongly assigned and quantification would be systematically biased. Therefore, knowledge on and quantification of the metabolic stability of the MP (both in target tissue and blood stream) are pivotal.in vivo behaviour of the MP due to the insertion of this radionuclide unfortunately point into opposite directions. . Although the structural change may appear small it can not be excluded that this will lead to pronounced changes in the MP’s characteristics—both, in vitro and in vivo. This could be in both directions: it might lead to reduced affinity, stability or selectivity but could also lead to ameliorated in vivo behaviour [The availability of the desired radionuclide and the potential alterations in the ons. cf. For examc-TRODAT ,45. Moreehaviour .With respect to the short-lived nature of the used radionuclides, also some distinct differences in the (radio-)pharmacological behaviour of the MPs occur that have to be taken into consideration when dealing with radiopharmaceuticals.The choice of the used radionuclide (and MP) will therefore always be a compromise between availability and economical considerations on the one hand, and physical properties, radiolabelling possibilities and radiopharmacological issues on the other hand.By law in most countries, PET-radiopharmaceuticals are drugs. Hence, a manifold of regulatory aspects have to be addressed prior to release for human. Since the majority of radiopharmaceuticals are administered intravenously, these parenteral drugs have to follow even stricter regulations. A specific monograph in the European Pharmacopoiea has been established to generally cover radiopharmaceutical preparations . FurtherFinally, the time for multi-modal molecular imaging probes has finally come and the simultaneous gathering of molecular information on the basis of imaging biomarkers and hybrid systems has already started, paving its way into personalized medical diagnosis and treatment.


Survival after diagnosis is a fundamental concern in cancer epidemiology. In resource-rich settings, ambient clinical databases, municipal data and cancer registries make survival estimation in real-world populations relatively straightforward. In resource-poor settings, given the deficiencies in a variety of health-related data systems, it is less clear how well we can determine cancer survival from ambient data.We addressed this issue in sub-Saharan Africa for Kaposi’s sarcoma (KS), a cancer for which incidence has exploded with the HIV epidemic but for which survival in the region may be changing with the recent advent of antiretroviral therapy (ART). From 33 primary care HIV Clinics in Kenya, Uganda, Malawi, Nigeria and Cameroon participating in the International Epidemiologic Databases to Evaluate AIDS (IeDEA) Consortia in 2009–2012, we identified 1328 adults with newly diagnosed KS. Patients were evaluated from KS diagnosis until death, transfer to another facility or database closure.Nominally, 22 % of patients were estimated to be dead by 2 years, but this estimate was clouded by 45 % cumulative lost to follow-up with unknown vital status by 2 years. After adjustment for site and CD4 count, age <30 years and male sex were independently associated with becoming lost.In this community-based sample of patients diagnosed with KS in sub-Saharan Africa, almost half became lost to follow-up by 2 years. This precluded accurate estimation of survival. Until we either generally strengthen data systems or implement cancer-specific enhancements  in the region, insights from cancer epidemiology will be limited. Survival after diagnosis is one of the most fundamental parameters in cancer epidemiology. Survival encompasses both the natural biologic history of a malignancy as well as the effects of therapeutic interventions. Monitoring survival over time, for example, can be a powerful tool to measure the cumulative impact of medical advancements. In resource-rich settings, ambient clinical systems diagnose cancers as they occur in the community, municipal registries record all deaths, and well-established cancer registries and epidemiologic platforms  program in the U.S. ) combineKaposi’s sarcoma (KS) in sub-Saharan Africa is an example of a malignancy in a resource-limited setting which would benefit from knowledge about current survival. KS was among the more common cancers in Africa even before HIV , 6, explTo address whether ambient data can answer a fundamental question of cancer survival in sub-Saharan Africa, we examined the feasibility of estimating survival after a KS diagnosis in the current era of burgeoning ART use. We took advantage of an epidemiologic network of clinical data from throughout sub Saharan Africa  to identAmong HIV-infected adults diagnosed with KS, we performed a cohort analysis of time to death using ambient clinical and administrative data. We retrospectively identified a consecutive sample of HIV-infected adults (≥18 years old) diagnosed with KS from January 2009 to July 2012 who were receiving their primary care at one of thirty-three HIV clinics in Kenya, Uganda, Malawi, Nigeria and Cameroon. The sites included 26 clinics in a network in western Kenya ), one clinic in Uganda (Immune Suppression Syndrome Clinic (ISS) in Mbarara), two clinics in Nigeria , three clinics in Cameroon , and one clinic from Malawi (Lighthouse Trust in Lilongwe). Each site administers ART in accordance with their national guidelines, and each participates in the International Epidemiologic Databases to Evaluate AIDS (IeDEA) Consortium, which, in Africa, consists of East, West, Central and Southern Africa regions . IeDEA wAt the participating clinics, KS diagnosis was made during the course of routine clinical care, either by clinical examination alone or with biopsy confirmation. Both individuals who were diagnosed at their initial clinic visit and during the course of their care were included. Ascertainment of death was through review of clinic charts, clinic databases and municipal death registries, where available. Clinics are informed of a patient’s death in a variety of ways, including from reports from adjacent hospital units or from family members. All of the participating clinics had some form of a tracking program in place to follow-up on lost patients. Demographic characteristics, including age and sex, were routinely collected by the clinics and extracted from the respective clinic databases. CD4+ T cell counts were performed at clinical laboratories associated with each of the clinics. The CD4 count proximal to KS diagnosis, when available, was defined as CD4 count closest to date of diagnosis, within 180 days prior to KS diagnosis or up to 14 days after.Time zero for the analysis was date of KS diagnosis. Patients were followed until death, and for those not known to be dead, the last known date known to be alive either from a clinic visit or transfer to another facility. Cumulative survival following KS diagnosis was estimated via the Kaplan-Meier technique, with censoring at transfer or last clinic visit. This approach assumes that the rate of death among those lost to follow-up is the same as those whose disposition is known. Loss to follow-up was defined as absence from clinic during the 3 months prior to database closure with no evidence of death or transfer. Incidence of loss to follow-up was calculated using the Aalen-Johansen estimator with death as a competing event , 25. Pro3 (IQR: 59–299) at time of KS diagnosis : 30–41), and the median proximal CD4 + T cell count was 159/mmPatients were followed for a median of 8.9 months (IQR: 4.0–20) and a collective 1473 person-years in which 191 patients died. Cumulative mortality, as estimated by the Kaplan-Meier technique, was 13 % at 6 months following KS diagnosis, 18 % at 1 year, and 22 % at 2 years. These estimates of mortality were clouded, however, by a cumulative loss to follow-up of 23 % : 21–26 %) at 6 months, 36 % (95 % CI: 33–38 %) at 1 year, and 45 % (95 % CI: 42–48 %) at 2 years , although this did not meet conventional levels of statistical significance. When the analysis was restricted to those with biopsy-proven KS (n = 378), the results were largely unchanged, with the exception that CD4 count ≤50 cells/mm3 was significantly associated with loss to follow-up .Given the high frequency of lost to follow-up, we also explored its determinants. After adjustment for geographic site in proportional hazards regression, age <30 years (hazard ratio (HR) 1.41, 95 % CI: 1.11–1.79) and male sex  were independently associated with loss to follow-up , we actively sought after those who were lost by searching for them in the community . We founA limitation of this work is that many of the KS diagnoses are based on clinical suspicion only. The high frequency of clinical diagnosis of KS has been documented by others  and is lIn summary, this work demonstrates on a large scale the challenges of accurately estimating cancer survival in sub-Saharan Africa. This issue is gaining significance as interventions (such as chemotherapy for KS) become more readily available, such that monitoring survival over time is increasingly important. Until we either generally strengthen data systems or implement cancer-specific enhancements to derive more accurate survival estimates  in the region, insights from cancer epidemiology will be severely limited. Strengthening data systems across this entire region may not be possible in the short term, but sentinel regional sites could be selected for enhanced monitoring and tracking of the lost. Additionally, the recent expanding efforts in cancer registries in sub-Saharan Africa –40 will


The Brain Reward Cascade (BRC) is an interaction of neurotransmitters and their respective genes to control the amount of dopamine released within the brain. Any variations within this pathway, whether genetic or environmental (epigenetic), may result in addictive behaviors or RDS, which was coined to define addictive behaviors and their genetic components.To carry out this review we searched a number of important databases including: Filtered: Cochrane Systematic reviews; DARE; Pubmed Central Clinical Quaries; National Guideline Clearinghouse and unfiltered resources: PsychINFO; ACP PIER; PsychSage; Pubmed/Medline. The major search terms included: dopamine agonist therapy for Addiction; dopamine agonist therapy for Reward dependence; dopamine antagonistic therapy for addiction; dopamine antagonistic therapy for reward dependence and neurogenetics of RDS.While there are many studies claiming a genetic association with RDS behavior, not all are scientifically accurate.PREDX™), the first test to accurately predict one’s genetic risk for RDS.Albeit our bias, this Clinical Pearl discusses the facts and fictions behind molecular genetic testing in RDS and the significance behind the development of the Genetic Addiction Risk Score (GARS In 1990 Blum’s laboratory at the University of Texas along with Ernest Nobles’ group at UCLA discovered the first genetic association with severe alcoholism, the Dopamine D2 receptor gene located on chromosome 11 q22-q23 .To carry out this review we searched a number of important databases including: Filtered: Cochrane Systematic reviews; DARE; Pubmed Central Clinical Quaries; National Guideline Clearinghouse and unfiltered resources: PsychINFO; ACP PIER; PsychSage; Pubmed/Medline. The major search terms included: dopamine agonist therapy for Addiction; dopamine agonist therapy for Reward dependence; dopamine antagonistic therapy for addiction; dopamine antagonistic therapy for reward dependence. Our results produced the following: dopamine agonistic therapy for addiction-Cochrane Systematic reviews-o; DARE-0; Pubmed Central Clinical Quaries-9, National Guideline Clearinghouse-0; PsychINFO-0; ACP PIER-83; PsychSage-15; Pubmed/Medline-501; dopamine agonist for addiction-Cochrane Systematic reviews-3; DARE-3; Pubmed Central Clinical Quaries-10; National Guideline Clearinghouse-0; ACP PIER-0; Psychsage-15; Pubmed/Medline-13; dopamine agonistic therapy for reward dependence-Cochrane Systematic reviews-0; DARE-0; Pubmed Central Clinical Quaries-1; National Guideline Clearinghouse-0; PsychINFO-0; ACP PIER-0, PsychSage-0; Pubmed/Medline-62; dopamine agonist for reward dependence- Cochrane Systematic reviews-0; DARE-0; Pubmed Central Clinical Quaries-337; National Guideline Clearinghouse-0; PsychINFO-1; ACP PIER-0; PsychSage-0; Pubmed/Medline-120; dopamine antagonistic therapy for addiction-Cochrane Systematic reviews-0; DARE-0; Pubmed Central Clinical Quaries-0; National Guideline Clearinghouse-0; PsychINFO-0; ACP PIER-0; PsychSage-0; Pubmed/Medline-633. Clearly we utilized a combination of Pubmed Central Clinical Quaries and Pubmed/Medline for our reliable review search as well as author searches based on personal knowledge of the field. In terms of neurogenetics we utilized PUBMED primarily.Clark et al.  analyzedWe believe these previous studies –3 laid dAdopting a Bayesian approach, earlier studies from Blum’s laboratory determined a Positive Predictive Value (PPV) for the DRD2 A1 variant (low number of D2 receptors) of 74%, indicating that if a child is born with this polymorphism they have a very high risk of becoming addicted to either drugs, food, or aberrant behaviors at some point in their future , 15.Over the many years to come since the 1990 finding, laboratories all across the globe including NIDA and NIAAA not only confirmed this early work  but exteImportantly, Blum’s laboratory proposed  that anyWhile there is a plethora of very positive experiments involving thousands of studies for many candidate gene associations with all RDS behaviors, there are also negative results . CurrentOtherwise utilization will lead to spurious and false results . AnotherPREDX™) in conjunction with the Institute of Behavioral Genetics, University of Colorado, Boulder.While we the authors may have a personal bias because over the many years that Blum’s laboratory has dedicated work to develop an accurate genetic test to predict true liability/risk for RDS and associated behaviors, we will attempt to explain why our current laboratory has successfully developed the first Genetic Addiction Risk Score , a test mandated in 13 states, for both alcohol and drug severity risk scores [To develop GARSk scores .PREDX™ panel. All the data was genotyped and analyzed at the Institute for Behavioral Genetics (IBG) at the University of Colorado Boulder. Without going into specifics we found a significant association between a summed score of all GARS panel risk alleles (variant forms) and both the ASI-MV alcohol and drug severity indices in a total of 273 subjects.We contacted eight very diverse treatment centers across the United States resulting in a total of 393 subjects that were genotyped using the selected GARSPREDX™ panel is and any deviation will produce false results that may occur with other commercial tests that have no research to validate their tests. A full length paper on these results will be published elsewhere whereby all gene polymorphisms will be displayed.In fact, the higher the number of risk alleles the stronger the prediction of alcohol or drug use severity. It was also found that family problems, psychological issues and medicalization significantly correlated as well. One important caveat was that if we changed any specific SNP the significance was lost. This strongly suggests how important the selected GARSIn summary, based on this seminal research and unlike other existing genetic tests we are poised to launch the first proven and validated Genetic Addiction Risk Score that will have important clinical benefits including personalized medicine and assessment of RDS risk. The test will also include the P450 system of genetic variants that influence how individuals metabolize opioids . Clinica


I-V) measurements. Contrary to conventional conductance studies, this I-V approach provides a correlated statistical description of both, the degree of electronic coupling across the metal-molecule interface, and the energy alignment between the conduction orbital and the Fermi level of the electrode. This exhaustive statistical approach was employed to study single-molecule junctions of 1,4-benzenediamine (BDA), 1,4-butanediamine (C4DA), and 1,4-benzenedithiol (BDT). A single interfacial configuration was observed for both BDA and C4DA junctions, while three different interfacial arrangements were resolved for BDT. This multiplicity is due to different molecular adsorption sites on the Au surface namely on-top, hollow, and bridge. Furthermore, C4DA junctions present a fluctuating I-V curve arising from the greater conformational freedom of the saturated alkyl chain, in sharp contrast with the rigid aromatic backbone of both BDA and BDT.Electronic and structural detail at the electrode-molecule interface have a significant influence on charge transport across molecular junctions. Despite the decisive role of the metal-molecule interface, a complete electronic and structural characterization of the interface remains a challenge. This is in no small part due to current experimental limitations. Here, we present a comprehensive approach to obtain a detailed description of the metal-molecule interface in single-molecule junctions, based on current-voltage ( For the majority of single-molecule junction studies, structural identification of junctions have been performed by combining measured conductance at a fixed bias voltage and theoretical support of transport calculations at the low bias limit. This is because in BJ experiments at room temperature, the lifetime of a single molecule trapped between two electrodes is relatively short. The short life time of single-molecule junctions (<100 ms20I-V) characteristics422232425I-V characteristics of single molecular junctions provide useful information on the molecule-metal contact configurations, such as the electronic coupling between the metal and the molecule (Γ ) and the energy difference between the Fermi level energy and the conduction-orbital (ε0)272829I-V characteristics of single molecular junctions, these measurements still remain a challenge from both, the experimental point of view, and in terms of analysis and interpretation. For example, the influence of the voltage scan rate remains a matter of debate. Previous studies have typically employed I-V acquisition times of approximately 100 ms22I-V measurements will certainly benefit from faster voltage scan rates. Furthermore, reducing the I-V acquisition times will reduce the structural instability caused by current-induced Joule heating effectsUnderstanding charge transport through single molecules is a fundamental issue in molecular electronics123456781011121315s <100 ms19 makes I-V measurements based on the STM-BJ method , statistical analysis of the I-V curve provides Γ and ε0, essential parameters needed to understand the metal-molecule contact configurations in molecular junctions. Combined analysis in G and both Γ and ε0 enabled us to resolve the structural details of the metal-molecule interfaces at the molecular junctions. We applied this approach to the single-molecule junctions of 1,4-benzenediamine (BDA), 1,4-butanediamine (C4DA), and 1,4-benzenedithiol (BDT)  . BDA andI-V measurements of the single-molecule junction at slow scan rates due to significant current fluctuation, most probably arising from variation in the metal-molecule contact configuration structure and molecular conformation. We checked bias-voltage-scan-rate dependence of the current fluctuation within the range of 4 to 400 Hz, where the current fluctuation displayed considerable scan-rate dependence for BDA molecular junctions  I-V histograms  conductance states, appear as current peaks at 290 and 450 nA  at 0.3 V in the current histogram  are the energy of the conduction channel  and the electronic coupling energy between the molecule and the left (right) electrode, respectively2333EF, to zero. The current through the molecular junction is written bywhere n is the number of bridging molecules and f is the Fermi distribution function. When electronic temperature, T, is set to 0 K, Eq. 2 can be analytically evaluated aswhere Γ = ΓL + ΓR and α = ΓL/ΓR. Note that the temperature effect of the Fermi-Dirac distribution is several percent of I(V) at 300 Kwhere i.e., the two averaged I-V curves in n . For the BDA molecular junctions, the “single” conductance state and the corresponding set of Γ and ε0 values were obtained by fitting statistically averaged I-V characteristics within a reasonable choice of n .The “Au–NH2” contact configuration originates from a simple delocalization of the lone pair of electrons from the amine-nitrogen to the Au atoms, and the bonding is not strongly directionalCurve fitting the bias of Eq. 3 for the two preferential conductance states . We extended our I-V measurement technique to C4DA molecular junctions. I-V curve of the C4DA single-molecule junction, in which the significant current fluctuation, most probably due to the flexibility of the methylene backbone, is apparentI-V histogram constructed from 1,000 I-V curves for the C4DA molecular junctions. The histograms exhibit several faint band structures, which can be explained by formation of preferential conformers with trans and gauche conformations at the C4DA molecular junctions. Here, we focus on the one of the bands, whose current ranged between 15 and 25 nA at 0.3 V, covering the previously reported conductance values of single C4DA molecule junctions measured by STM-BJI-V measurement, the curves within the current window have been averaged, and these are indicated by the black line shown in I-V curve of the C4DA molecular junctions is remarkably dissimilar to the nonlinear curves of the BDA molecular junctions. To qualitatively discriminate the I-V characteristics and related electronic structures between the molecular junctions, we fitted the averaged C4DA-I-V curves with eq. 3 under a constraint condition of n = 1. A set of Γ and ε0 values was determined to be Γ = 48 meV and ε0 = 1.7 eV. The obtained ε0 of 1.7 eV is substantially larger than the ε0 of 0.7 V obtained for the BDA molecular junction. This remarkably large energy of 1.7 eV is caused by intrinsic molecular nature in the junctions, which is a wide HOMO-LUMO gap of the insulating alkane moiety in the C4DA junction. Statistical analysis of the I-V curves enabled us to capture molecular dependent I-V characteristics in a qualitative manner, which was used to assess the applicability of the present method. It is interesting to note that, despite both of the C4DA and BDA junctions have the same Au-N binding group, much lower electronic coupling strength was found for C4DA (48 meV) than BDA (75~85 meV). The electronic coupling strength depends on not only the local structure of the binding group but also electronic details in the molecular backbones . The terminal N atom in BDA binds to a sp2-hybridezed carbon atom and the lone pair in the N atom is partly delocalized into the π-electron network in the molecular backbone. The resulting electronic interaction between the terminal binding group of N and the molecular backbone results in the larger coupling strength. On the other hand, the N atom in C4DA binds to a sp3-hybridized C atom and electronic interaction through the lone-pair in the N atom and the molecular backbone cannot be expected for C4DA. Thus, the electronic coupling of the molecular junction becomes smaller for C4DA. Such electronic interaction between the binding group and the molecular backbone has been demonstrated in rupture force measurements of BDA and C4DA molecular junctions sandwiched by Au electrodes, in which the BDA and C4DA junctions with the same Au-N binding group exhibited distinct rupture forces of the molecular junctionsTo clarify the effect of molecular backbone on the molecular I-V analysis to BDT molecular junctions. Since the pioneering MCBJ-I-V experiments of Reed et al., BDT is well-known as a prototypical molecule for electronic transport study of single-molecule junctions in the field of molecular electronics. Over the last decade, much research has been carried out, and a wealth of information on the variable transport properties of single molecule junctions has been accumulated, allowing a better understanding of the atomistic details at the junctions. The current understanding is that different “S-Au” bonding patterns, such as on-top, bridge and hollow adsorption-modes, and BDT-metal contact configurations at the molecular junctions, which have been demonstrated to display a variety of configurations, lead to differences in molecular conductance. One of the most challenging tasks is to identify the contact configurations and reliably characterize the corresponding charge transport properties and electronic structures at the molecular junctions. I-V curves and 2D I-V histograms in two different current regimes. In contrast to the BDA junctions with a single “N-Au” bonding mode, the BDT junctions with multiple “S-Au” bonding modes features a variety of statistically significant I-V characteristics, as shown in I-V characteristics, we developed an automated algorithm to fit each I-V curve with eq. 3 and extract the values of Γ and ε0. Γ and ε0 values constructed from 1,000 individual I-V curves measured for the BDT molecular junctions; in these, three preferential distributions, H, M, and L, are noticeable. The histograms of each Γ and ε0 . The current histograms at the bias voltage of 0.3 V revealed one current-peak at 7 nA in the small conductance regime  5~20, (M1) 20~40, (M2) 40~100 nA, and (H) 440~2200 nA at 0.3 V are divided into four groups to obtain statistically significant I-V characteristics. Averaged I-V curves within the windows are indicated by black dotted curves in I-Vs of each using eq. 2, Γ, ε0, and α were determined for the H, M, and L states  for several model systems using nonequilibrium Green’s function combined with DFT (NEGF-DFT)3940i.e., hollow, bridge, and on-top, to the Au electrodes and determined the conformations of each junction model by standard DFT geometry optimization. The conductance values of hollow and on-top conformations were 0.024 G0 and 0.009 G0, respectively. On the bridge type configurations, we found slightly different three stable structures, which are termed as (i) bridge, (ii) bridge-top, and (iii) tilted bridge. In the case of bridge-top, one anchoring point is the bridge site and the other site shifted slightly to the on-top position from the bridge site. The tilted bridge configuration is that the BDT molecule tilts by anchoring nonequivalent bridge sites of left and right electrodes. The schematic figures of these structures are given in e.g., (i) bridge, 0.22 G0; (ii) bridge-top, 0.32 G0; and (ii) bridge tilt, 0.27 G0, respectively  by diagonalizing the molecular projected Hamiltonian (MPSH) and identify the conductive MO, whose energy εa should be close to EF and whose coupling strength to electronic state of the electrodes, γ, is sufficiently large42γ is the imaginary part of the normalized self-energy to MPSH and was obtained for each PMO. Generally, the conductive MO is not the conduction channel stateεa, γ) is not equal to , as defined by eq. 2. However, identifying conducting MO’s is useful to check validity of our analysis via eq. 1. In addition,  is a good approximation that allows discussion of the tendency of γ as far as we can select suitable conductive MOs. When we rewrite  of the conductive MO’s of the H state as  etc., the calculated values εH, εM, and εL are −0.75, −1.09, and −0.47 eV, respectively. Since the conductive MO energies of the three states are of the same order, analysis of the relationship between the contact configuration and Γ, as evaluated by I-V curves using eq. 1, is reasonable. The relative coupling strength i.e., the correlation of conductance and Γ agree reasonably with that of conductance and γ.Next, we discuss the relationship of the assigned sites and I-V measurements and analysis of single-molecule junctions measured by STM-BJ method under ambient conditions. For BDA, C4DA, and BDT molecules that are commonly used in the break junction-based-conductance measurements at a fixed bias voltage, we applied our statistical I-V-approach to determine the molecule dependent properties Γ and ε0 in a qualitative manner within the single channel transport model. For BDA and C4DA, the molecule dependent ε0 of 0.7 and 1.7 eV were obtained, which assessed applicability of the statistical I-V-approach. For BDT with a variety of metal-molecule contact configuration, three sets of statistically significant I-V characteristics with remarkable difference in the Γ values were captured and, by combining the result of first principle charge transport calculations, we identified the BDT junction-structures with on-top, hollow, and bridge sites-adsorption-configurations in the order of the molecular conductance.Towards the characterization of metal-molecule interface of single-molecule junctions, we developed a statistical approach for treatment of I-V measurement was prepared by dipping the Au substrate into a 1 mM ethanol solution containing the target molecules. After evaporation of the solution, the substrate surface was washed with ethanol. We used a commercially available STM  operating at ambient conditions. Two current amplifiers, 1 μA/V and 10 nA/V, were used to access wide molecular conductance ranges from 10–5 to 101G0. STM tips were prepared by mechanically cutting an Au wire . The I-V curves of the single-molecule junction were obtained by the following procedure (G0) was made between the STM tip and the sample surface. Secondly, the tip was withdrawn by 10 nm at a speed of 38 nm/s to break the Au contact and to make a nanogap between the Au electrodes, forming the molecular junction during current monitoring at a fixed bias voltage of 20 mV. Thirdly, the tip position was fixed and one I-V curve was recorded by scanning the bias voltage from 20 to 1000, –1000 mV, and back to 20 mV within a time period of 2.5~25 ms at constant tip-sample separation. Finally, the junction was broken by pulling the STM tip away from the substrate. To capture possible structural variation of the junction structures, we cycled the molecular junction making and breaking process and reformed the junction-structure after obtaining each I-V curve. This I-V measurement-scheme was performed though a signal access module III  using an external piezo driver  and a data-acquisition-device with LabVIEW2014 . More than 1,000 I-V curves for the molecular junctions were collected for each molecule. The I-V curves of molecular junction were obtained by automatically removing I-V curves corresponding to Au-metallic junctions and vacuum gap formation. For the I-V measurement using the 1 μA/V (10 nA/V) current amplifier, I-V curves with <100 nA (<5 nA) current at the bias of 1.0 V was classified as vacuum tunneling, while I-V curves with <10,000 nA (<100 nA) current at the bias of 0.2 V was classified as charge transport through an Au-metallic contact.BDA, C4DA, and BDT were purchased from TCI Japan  and wererocedure . Firstlyi.e., the alignment of S-S axis of BDT molecule tilts to the surface. We used density functional theory (DFT) to carry out the calculations and the B3LYP exchange-correlation (XC) functional and LanL2DZP basis set for cluster model calculations. For the DFT calculations, Gaussian09 was used. To carry out transport calculations, we replaced the clipped bulk part of the cluster model with a c(5 × 5) bulk model by adding shortage Au atoms and then adding three more Au atomic layers to set up a scattering region. A periodic boundary condition was used, thus the unit cell was defined by a c(5 × 5) structure. We used the Perdew–Burke–Ernzerhof (PBE) XC functional for NEGF-DFT calculations. For the NEGF-DFT calculations, double-zeta plus polarization function basis set for all atoms in the molecule and a single-zeta plus polarization function basis set for the Au atoms were used. To check the validity of the evaluated conductance values and analysis, we also examined the XC using the local density approximation self-interaction correction (LDA-SIC). We confirmed that the PBE functional provides sufficient results for qualitative analysis in the present purpose, i.e., evaluation of the electronic coupling strength and identification of each conductance state . All the NEGF-DFT calculations were performed using the HiRUNE subroutine40Γ and ε0, were evaluated directly by calculating projected MO’s (PMO) and renormalizing the self-energy of each PMO. The details of the method, which is called the effective molecular projected state Hamiltonian (MPSH) approach, is given in ref. [In this section, we survey the computational details used in the present first-principles calculations. The adsorption structures of BDT on Au electrodes were determined using a cluster model, where each side of the junction consists of 19 Au atoms and the cluster structure is taken to model the apex and the (111) surface of bulk electrodes. We fixed the two outermost Au layers with the structure of the bulk (clipped bulk), and the other atomic positions and the distance between the distance of the left and right electrodes were allowed to relax. We took hollow, bridge, and top adsorption sites as initial geometries and examined the tilt adsorption structure,  in ref. .How to cite this article: Komoto, Y. et al. Resolving metal-molecule interfaces at single-molecule junctions. Sci. Rep.6, 26606; doi: 10.1038/srep26606 (2016).


An overwhelming segment of the world’s population possesses certain genetic variations that increase risk for genetic predispositions to substance seeking and even behavioral addictions  that preclude them from reaching their optimum health potential, contribute to impaired health, and/or can cause involuntary indulgence in detrimental and self-destructive behaviors. This is especially true when this genetic reward deficiency (RDS)  problem It is our notion that the real genesis of all behavior, whether so-called normal or abnormal behavior, derives from an individual’s genetic makeup at birth involving gene variations that make up about 3% of the human genome. While people are not doomed because of carrying these so called variations  this risk predisposition, due to multiple gene combinations and polymorphisms (gene variations), is expressed differently based on numerous environmental (epigenetic) elements. So carrying these known gene variants especially in those genes known to control what is called the “Brain Reward Cascade (BRC) influencing how the brain programs feelings of well-being, mediate the actual expression of these important genes [It is well-established that psychiatric disorders are complex multifactorial illnesses involving chronic alterations in the brain reward circuitry. While it is agreed that genetic factors are important in the etiology of disorders such as RDS, relatively high rates of discordance among identical twins suggest the importance of other mechanisms. Certainly, environmental factors such as stress or prior drug exposure play a role in the onset of these illnesses. There is evidence that sustained abnormalities are maintained by epigenetic modifications in specific brain regions. Addictive behavior can be modeled in animals by inducing disease-like states through environmental manipulations  .As David Smith the founder of the Haight-Ashbury Clinic stated “Love Needs Care” and that is what positive caring has in common with powerful epigenetic effects. We firmly embrace this “love and care” concept as part of any treatment goal with the knowledge that through epigenetic effects either methylation and or deacetylation gene expression will lead to positive dopamine release at the reward centers of the brain . UnderstIn fact, we promote the concept that the core of predisposition to these behaviors is a set of genes , which mediate a feeling of well-being via chemical messenger (i.e. neurotransmitters) interaction at the “reward site” of the brain (N. Accumbens), leading to normal dopamine release and influencing dopamine receptor density . A low number of Dopamine receptors suggest a hypodopaminergic function as manifested in all addictive disorders. When there are a low number of dopamine receptors, the person will be more prone to seek any substance or behavior that stimulates the dopaminergic system (a sort of “dopamine fix”). In fact, Nora Volkow the current director of the National Institutes of Drug Abuse (NIDA) stated that “all roads lead to dopamine” further described by Blum et al. .To understand generalized craving behavior, due to hypodopaminergic function  attempts to alleviate or compensate for the low dopaminergic brain activity via drug-receptor activation  . This wiIn order to help explain this so called pseudo self-healing process, we are cognizant of known dysfunctional diagnosis known as “Legacy Cases.” These cases are a major problem within the Workers Compensation insurance carriers system. They are very similar to another dysfunctional diagnose such as Fibromyalgia . Legacy One of the major underlying problems relating to these Legacy Cases is the inappropriate misdiagnosis and non-productive treatments these patients experience because of utilization of paper-pencil tests rather than sound candidate genetic testing . We stroRx™) especially at the portal of entry of patients presenting with either acute or chronic pain will identify patients with high RDS risk and as such will flag these patients’ with genetic knowledge and attenuation of over-prescribed powerful pain reliving opiates at inception of their treatment and physician promotion of subsequent alternatives for pain control  [Our goal in terms of assisting insurance carriers is to reduce iatrogenic induced opioid addiction by reducing subsequent dependence on these powerful opiates such as even Suboxone thereby promoting “Legacy Case” closure . Closurere etc.) . MoreoveTo reiterate utilizing a new paradigm shift in both genetic testing and enhancing functional connectivity of the brain with DAM should improve the health and well-being of the “Legacy Claim” patients because the major underlying root causes of opiate dependency will be reduced. It will additionally curb abuse within the Workers’ Compensation Program and provide a significant monthly cost savings to the carrier and employer. In fact utilizing our approach in a hypodopaminergic genetically tested patient with GARS, having a workers Comp payment for medications over $50,000 per month has been successfully treated with DAM as well as other natural modalities, being opioid free for over two years . Our mai


To illustrate the clinical and radiological features of idiopathic interstitial pneumonias (IIPs), according to the American Thoracic Society (ATS)/European Respiratory Society (ERS) classification updated in 2013.IIPs include a subset of diffuse and restrictive lung diseases, resulting from damage to the parenchyma characterised by inflammation and fibrosis of the interstitium. Classification into major and rare IIPs is based on the 2013 ATS/ERS committee.The diagnosis of idiopathic pulmonary fibrosis (IPF) needs to exclude other well-known causes of interstitial lung diseases. According to the 2011 evidence-based guidelines, usual interstitial pneumonia (UIP) can be diagnosed by HRCT when all criteria are fulfilled. Non-specific interstitial pneumonia (NSIP) is characterised by patchy ground-glass opacities and irregular linear/reticular opacities. The imaging patterns of respiratory bronchiolitis associated-interstitial lung disease (RB-ILD) and desquamative interstitial pneumonia (DIP) show centrolobular nodules and ground-glass opacities. Cryptogenic organising pneumonia (COP) consists of patchy peripheral or peribronchial consolidations, while ground-glass opacities are typically associated with diffuse lung consolidation, evolving to fibrosis, in acute interstitial pneumonia (AIP). Rare IIPs include lymphoid interstitial pneumonia and idiopathic pleuro-parenchymal fibroelastosis (IPPFE).The knowledge of IIP imaging features on HRCT images help radiologists in diagnosis. Moreover, the overlap of imaging features needs a multidisciplinary approach.UIP findings are reticulations, bronchiectasis, honeycombing and absence of inconsistent features.• Bilateral patchy ground-glass areas represent the most encountered features in NSIP.• Poorly defined centrilobular nodules are typical of RB-ILD, whereas a ground-glass appearance is typical of DIP.• HRCT features of COP include characteristic peripheral or peribronchial patchy consolidations.• Rare IIPs include idiopathic LIP and idiopathic pleuro-parenchymal fibroelastosis (PPFE).•  The idiopathic interstitial pneumonias (IIPs) are a group of acute and chronic diffuse parenchymal lung diseases with many important features in common. Although all IIPs are characterised by an unknown aetiology , 2, a stIn 2002 The American Thoracic Society (ATS) and the European Respiratory Society (ERS) published a classification of IIPs . This clThe ATS/ERS Classification has been recently updated, distinguishing major IIPs from rare IIPs Table . Major IIIPs are generally distinguished from other diffuse parenchymal lung diseases on the basis of clinical properties, laboratory tests, physical examination, imaging and histopathological features , 2, 7, 8The role of the radiologist in the diagnosis of IIP has being continuously emphasised in recent years because of the significant contribution of HRCT in the identification and diagnosis of diseases. In fact, according to the 2011 evidence-based guidelines, UIP can be diagnosed by HRCT when all criteria are fulfilled , 9474, 9The association of perivascular cysts and ground-glass opacities is highly suggestive of LIP.Centrilobular and subpleural nodules Fig.  with thiPneumocystis carinii pneumonia may be considered because of cystic abnormalities. A very important differential diagnosis is from Hodgkin’s lymphoma [Regarding the differential diagnosis, the HRCT pattern of LIP should be differentiated from hypersensitivity pneumonitis, sarcoidosis and lymphangitic spread of tumour. lymphoma .Idiopathic PPFE has been recently included in the ATS/ERS IIPs classification  as a rarIt is a characterised by the development of fibrosis along pleural surfaces and subpleral parenchymal lung, with predominantly upper lobe involvement.Some clinical data report an association between recurrent pulmonary infection and development of PPFE. Many authors have recently reported PPFE as a pulmonary reaction associated with chronic graft-versus-host disease following bone marrow transplantation . Some hiIn a PPFE series reported by Reddy et al. a male/female ratio of 1/1.4 was found, whereas in the literature the tendency is a male/female ratio of 2/1 .Histologically, the hallmarks consist of pleural thickening and subpleural fibrosis.Chest X-ray shows aspecific pleural thickening of apical regions, with small subpleural pulmonary consolidations. This imaging pattern is indistinguishable from many other chronic pulmonary infections (tuberculosis). Superior hilar retraction has been described in all five cases reported by Kusagaya et al. .HRCT Fig.  shows plIIPs diagnosis is currently based on a multidisciplinary approach, and HRCT offers a special contribution because some patterns have been associated with specific IIPs. Consequently, radiologists should be more able to detect ILDs on HRCT images.According to the 2011 evidence-based guidelines [The overlap of imaging features among interstitial pneumonia limits the diagnosis. NSIP's diagnosis can be difficult because of the heterogeneity of radiological appearances. Smoking-related IIP patients could improve their course with smoking cessation. Clinical presentation could help radiologists in their approach to acute/subacute IIDs (COP and AIP).


Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus.The cattle tick  Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world . The. TheCaenR. microplus. To our knowledge, this is the first report of a GlyR from ticks. Electrophysiological studies are underway to determine GlyR regulation by the agonist glycine and the acaricide IVM. Finally, the identification of molecular targets for IVM helps develop the foundation for the study of molecular mechanisms that lead to acaricide resistance and for the development of novel acaricides.In summary, we report the molecular characterization of a glycine-like receptor from