Drosophila melanogaster to investigate how lack of Ih current affects dopamine levels and the behavioral consequences in the sleep∶activity pattern. Unlike mammals, in Drosophila there is only one gene encoding HCN channels. We generated a deficiency of the DmIh core gene region and measured, by HPLC, levels of dopamine. Our data demonstrate daily variations of dopamine in wild-type fly heads. Lack of Ih current dramatically alters dopamine pattern, but different mechanisms seem to operate during light and dark conditions. Behaviorally, DmIh mutant flies display alterations in the rest∶activity pattern, and altered circadian rhythms. Our data strongly suggest that Ih current is necessary to prevent dopamine overproduction at dark, while light input allows cycling of dopamine in an Ih current dependent manner. Moreover, lack of Ih current results in behavioral defects that are consistent with altered dopamine levels.HCN channels are becoming pharmacological targets mainly in cardiac diseases. But apart from their well-known role in heart pacemaking, these channels are widely expressed in the nervous system where they contribute to the neuron firing pattern. Consequently, abolishing Ih current might have detrimental consequences in a big repertoire of behavioral traits. Several studies in mammals have identified the Ih current as an important determinant of the firing activity of dopaminergic neurons, and recent evidences link alterations in this current to various dopamine-related disorders. We used the model organism  Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are responsible for the Ih current, which regulates rhythmic electrical activity, contributes to the resting membrane potential, and shapes the input-output curve in excitable cells. The Ih current was originally described as the inward cation current activated at hyperpolarized membrane potentials which substantially contributes to the spontaneous pacemaker activity of the heart sinoatrial cells. Indeed, attempts to ameliorate cardiac rhythm disorders have established HCN channels as promising drug targets Drosophila there is only one HCN channel encoding gene, DmIhIn mammals, HCN channels are encoded by four genes, HCN1–4, that show distinctive but partially overlapping expression in different tissues and brain regions in vivo output have not been reported. Therefore, we analyzed how impairment of Ih current in DmIh mutant flies may alter dopamine outcome in vivo. DmIh gene is broadly expressed in the Drosophila brain Drosophila brain dopaminergic cells express DmIh. Moreover, we provide the first demonstration of significant circadian variation in levels of dopamine in Drosophila head extracts, and show that the daily cycling of dopamine is drastically modified when Ih current is eliminated. Therefore, our data associate Ih current with dopamine signaling also in flies, suggesting a role for DmIh channels in influencing dopamine neurotransmission either in a dopaminergic cell-autonomous manner or at different steps in neuronal communication.In rodents, dopaminergic neurons display characteristic rhythmic spontaneous firing activity, which is dynamically modulated by multiple afferent inputs. Several studies have identified Ih current as an important determinant of this spontaneous firing rate DmIh mutant flies display alterations in the rest∶activity pattern which correlate with the aberrant dopamine levels. Finally, flies lacking Ih current show altered circadian rhythms, which translate into an arrhythmic behavior or a shorter period in constant darkness conditions.Several experiments and observations demonstrate a central role for dopamine in regulating sleep–wake states. For instance, mice with varying degrees of dopamine depletion show different levels of REM sleep Drosophila, HCN channels are encoded by a single gene  that suffers alternative splicing, potentially giving rise to at least 12 isoforms DmIh by FLP-FRT based recombination RB01599 is inserted upstream of exon 4 and pBacWH01485 is inserted downstream of exon 12. Upon heatshock, recombination occurs between these two piggyBac elements, deleting the intervening region and regenerating a complete piggyBac element from half of each of the original piggyBacs (DmIh deletion line was backcrossed into a Canton-S (CS) background for five generations for further analysis, and one control (wt) and one mutant (DmIh) isogenic line was established and used in all experiments. Care was taken to completely eliminate the 1118w mutation to avoid an effect on dopamine levels In iggyBacs . The delDrosophila, both TH Drosophila heads every four hours, with some additional measures when considered necessary, under 12 h light∶dark (LD) conditions . Although previous indirect cues suggested cycling of dopamine levels in DrosophilaDopamine amount shows a rhythmic behavior, peaking at the beginning and the end of the light period, with lowest levels in the middle of the day. At nighttime dopamine is maintained to a low level, slowly increasing towards the end of the night . CoincidDrosophila heads is influenced by light or under circadian control, we measured dopamine in flies kept in constant darkness (DD).In rat, the release of dopamine in the striatum also shows daily oscillations in LD conditions 6,21 = 3.670; p = 0.012), although post hoc analysis failed to identify significant differences between any of the dopamine data points.Flies were entrained for two days in LD prior to being released to DD, and dopamine was determined after three days in this condition. As shown in 13,42 = 4.760; p<0.001; for details see 6 = −3.18; p<0.5 for ZT/CT0 and t6 = −3.57; p<0.5 for ZT/CT7), which corroborates a role for light in controlling dopamine levels.The different pattern of dopamine oscillation observed in LD and DD suggests that light affects dopamine levels also in fly heads. In addition, the similar pattern of dopamine oscillations during the night period in LD and the subjective night in DD indicates that dopamine levels could be under circadian control during dark periods. To test this hypothesis, we analyzed the effect of circadian time (ZT or CT) and light conditions (LD or DD) on dopamine levels by a two-way ANOVA. The results show that both factors, as well as their interaction, have a significant effect  were rhythmic . In contrast, at nighttime mutant flies show a dramatic increase of dopamine . The different behavior of dopamine oscillation in control and mutant flies was confirmed by a two-way ANOVA, which showed that both genotype and ZT, and the interaction between these two factors, significantly affect dopamine levels  of the flies. This result suggests an association between the pattern of dopamine oscillation and the pattern of locomotor activity, both being affected by light.When analyzing activity behavior, the mean overall activity of mutant flies is lower (see below), and peak of activity at dawn is blunted . As meanactivity , resemblactivity . To get DmIh mutant flies. Therefore, we measured dopamine levels in DmIh mutants in DD conditions to verify if the increase observed at nighttime in LD correlates with a lack of light input whatever the circadian conditions. In constant dark, the proportion of rhythmic DmIh mutants drops to 24.5% . Two-way ANOVA  showed that in this condition too, genotype and CT significantly affect dopamine levels. However, the pattern of circadian changes is not different between control and mutant flies, because the interaction between these factors was not significant , leads to a severe reduction of sleep DmIh mutants we expected to see a change in the rest-activity pattern of these flies. Activity was monitored in individual flies in a 12 h light∶dark cycle and rest∶activity parameters were analyzed , a competitive antagonist of tyrosine hydroxylase (TH) which has been shown to reduce dopamine in flies without producing significant effects on basic behavior and viability DmIh mutant flies, which is basically an increase in sleep fragmentation, is dopamine-dependent, a reduction on dopamine levels would at least partially correct this fragmentation. Indeed, when analyzing the rest∶activity parameters of 3IY-fed flies, it is clear that sleep becomes more consolidated in both control and mutant flies  but only 39% in controls (p<0.05), while episode number is reduced by 33% (p<0.001) and 30% (p<0.05) respectively. Nevertheless, drug treatment increased total sleep only in control flies, suggesting that the effect of dopamine on total sleep time may occur, to some extent, through the modulation of Ih current.Altering dopamine signaling affects total sleep time and sleep consolidation nt flies . MoreoveDrosophila flies live more than two months and the effect of age in sleep fragmentation is not perceptible until the fourth week. Our sleep∶activity recordings were always performed in 6 to 8 days old flies, precluding an effect of chronological age in our results. In order to see if DmIh mutant flies might be considered physiologically elderly, we performed a test of longevity. The survival curves of DmIh mutant male flies reared at 25°C did not show a reduction in the maximum life-span and only a slight reduction in the median (10%), but the death rate during the exponential phase was increased , have been shown to cycle circadianly in whole heads but not in bodies Ih in adult heads has been detected in neural tissues Even though our data on dopamine levels was obtained from whole heads, various reasons make us confident that dopamine oscillation precisely reflects the behavior of neural dopamine. First, contribution of hypodermic dopamine to whole head dopamine levels has been reported to be no more than 15% Drosophila based on the circadian oscillations of TH and dopamine receptor responsiveness, but a detailed study of dopamine levels at different time points was lacking. Our results show rhythmicity in the daily levels of dopamine in Drosophila, with a pronounced bimodal pattern at daylight and a smooth increment towards the end of the night. When analyzing the contribution of the light signal versus the circadian control in this rhythmic behavior, through a comparison of LD and DD dopamine values, it turns out that the bimodal pattern during the day is driven by light while dopamine levels at night are under circadian control. A link between light and dopamine is supported by studies in vertebrates showing that dopamine plays critical roles in the light-induced resetting of the peripheral retinal circadian clock Drosophila demonstrating that dopamine is necessary for low light circadian entrainment Drosophila dopamine receptor dDA1 responds homeostatically by downregulating its expression when dopamine signaling is increased Daily fluctuations of dopamine have already been reported in mammals, and also suggested in DmIh is expressed in photoreceptors How light affects the dopamine outcome is not known, but our results clearly demonstrate the implication of Ih current in maintaining its bimodal pattern. DmIh mutant flies lack Ih current not only in dopaminergic neurons, but in the entire nervous system, possibly affecting input signals to these cells. Finally, dopamine autoreceptors provide important feedback control during dopamine signaling by governing firing rate, synthesis, and release. Therefore, by acting at different cellular and subcellular locations, Ih current could be critical for proper functioning of the dopaminergic negative feedback loop to prevent excessive release of neurotransmitter.Ih current is also involved in maintaining physiological levels of dopamine, because abolishing this current strongly increases dopamine amount in dark conditions. A number of studies have implicated the hyperpolarization-induced, nonselective cation conductance Ih in the firing activity of mammalian dopaminergic neurons, emerging as one determinant of their spontaneous firing rate DmIh deficient flies show a dramatic fragmentation of sleep at nighttime, which is consistent with their increased dopamine levels. This trend towards sleep fragmentation is also observed in mutant flies at daytime, suggesting that disrupting dopamine cycling during the light period can also affect sleep consolidation. Results showing that pharmacologically decreasing the amount of dopamine restores sleep consolidation in mutant flies are consistent with this phenotype being dopamine-dependent , more than 50% of the flies also lack the bimodal activity pattern  and 1118;pBac{RB}Ihe01599w, and 1118;pBac{WH}Ihf01485w. DmIh mutant flies were obtained by deleting the S1–S3 core domain upon recombination of the two pBac elements. Recombination was induced following described protocols 1 w1118 hspFLPy; {RB}Ihe01599/SM6apBac×1 w1118 hspFLP;pBac{WH}Ihf01485/SM6ay, starting at the second day after the cross was established. Heat-shocked males of the genotype hspFLP; {RB}Ihe01599/pBac{WH}Ihf01485pBac were crossed to 1118;snaSco/SM6aw females, and individual lines were established from male descendants and checked by PCR to determine if recombination had occurred. Two DmIh-deficient lines were identified, and one of them was backcrossed into a Canton-S (CS) background for five generations to establish one isogenic w+ control (wt) and one isogenic w+ mutant (DmIh) line, which were used in all experiments.Flies were grown at 25°C under a 12 h∶12 h light cycle in standard AACGATGATCTGAGCACACG and CAGCGTTGTCTTGTTGCATAA were used for amplification of the DmIh core region. A single primer TGCATTTGCCTTTCGCCTTAT was used as both forward and reverse primer for amplification of the newly formed pBac element with the Taq Long Extent (Roche). PCR products were analyzed in 0.8% agarose gels.Genomic DNA was isolated from 10 control and recombinant flies with High Pure PCR Template Preparation Kit (Roche), and used for PCR amplification. Primers For HPLC analysis, 10 heads of 6 to 8 day-old males, entrained for 3 to 4 days in LD or DD conditions, were homogenized in 100 µl of ice-cold 0.1 M perchloric acid. The samples were centrifuged at 13.000 rpm for 10 min and supernatant filtered through a 0.45 µm PVDF centrifuge filter (Teknokroma). Dopamine levels were measured with a Varian 1200L HPLC with triple quadrupole LC/MS equipped with a Zorbax SB C18, 5 µm, 4.6 mm×150 mm column (Agilent).t tests.Each ZT and CT point represents the mean value of two independent experiments, with duplicated HPLC measures. HPLC measurements coefficient of variation were 8.04% and the inter-experiment coefficient of variation was 7.97%. All statistical evaluations were performed with SPSS13 . Because dopamine measurements fulfilled the assumptions of the normal distribution and the homogeneity of variance, data were compared with one or two way ANOVA followed by the Bonferroni's post hoc test. Significant interactions in the ANOVAs were followed up with DmIh gene, we performed RT-PCR assays in isolated ple (TH) expressing cells by Fluorescence Activated Cell Sorting (FACS). 50 THG4/UAS:GFP adult brains were dissected and mechanically dissociated in Trypsin 5′-AACGATGATCTGAGCACACG-3′ and 5′-CAGCGTTGTCTTGTTGCATAA-3′ for Ih amplification, and 5′-CGCAGCAAGGCAAATGAT-3′ and 5′-AGGAGATGCCCTCCTTGAG-3′ for the neural isoform of the ple gene.In order to determine whether dopamine cells express the Drosophila food. Locomotor activity was collected with Drosophila Activity Monitoring Systems  in 1 min bins, and circadian data were analyzed using Clocklab (Actimetrics Software). For LD analysis, flies were kept in 12 h∶12 h light-dark cycles for at least 6 days. For DD analysis, flies were first entrained in 12 h∶12 h LD cycles during 3 days, and activity data were analyzed for 6 days. To assess rhythmicity, data were analyzed by chi-square periodogram. Flies that showed clear rhythms through actogram analysis and a well-defined peak close to 24 h in the periodogram were classified as rhythmic and included in the calculation of average period. Flies that failed to yield a significant period in the periodogram analysis and showed random distribution of activity in actograms were classified as arrhythmic. Those that showed recognizable, but weak rhythms in actograms, and resulted in periodograms that displayed insignificant or barely significant periods, were considered weakly rhythmic. To analyze the bimodal vs. unimodal pattern of locomotor activity in LD, data from 6 consecutive days were analyzed by Fourier transformation to yield the predominant frequency. In all cases, at least two independent experiments were analyzed.Two to three day-old males were placed in 65 mm×3.5 mm glass tubes  containing standard For rest∶activity analysis, flies were acclimated in behavior tubes at 25°C in 12 h light∶12 h dark (LD) conditions for 3 to 4 days, and data were collected for the following 2 consecutive days. Active Sleep was defined as any period of uninterrupted behavioral immobility lasting more than 5 min. Waking state was any non-sleeping state, and an active minute was defined when ≥1 count/min was recorded. DAMS data were analyzed with FlySiesta custom-designed MATLAB software Pharmacological treatment with 3-iodotyrosine (3IY) (Sigma) was carried out as described 2 anesthesia and housed in groups of 25. They were raised at 25°C under a 12 h∶12 h light cycle and transferred to fresh food vials every 2–3 days. Dead flies were scored daily.Males of genotypes under study were collected within 24 hr of eclosion under brief CO2O2 for the first 24 h. In subsequent days 5% H2O2 was used. Dead flies were scored every 8 h.Three day-old males were transferred to vials with filter papers soaked with 1 ml of a 10% sucrose solution with or without 1% HDrosophila adults were entrained for three days in LD and were dissected in the third day in DD at CT2 and CT14. Brains were fixed in 4% Formaldehyde solution. The primary antibody used was mouse anti-PDF  and was detected with anti-mouse antibody conjugated with Rhodamine-RedX . Images were acquired under the same parameters using a Leica Wild MPS52 microscope and analyzed with ImageJ software. Eight hemispheres were measured for each genotype and condition.Table S113,42 = 4.760; p<0.001). Both factors have a significant effect, and also their interaction, meaning that the dopamine cycling along the 24 h period is different in LD and DD.Comparison of Dopamine cycling in LD and DD conditions in control flies. Two way ANOVA on dopamine datapoints was performed to assess the effect of circadian time (ZT/CT) and light condition (LD or DD). A two-way ANOVA including both factors and their interaction is significant (F(DOC)Click here for additional data file.Table S2DmIh mutation on Dopamine cycling in LD and DD conditions. Two way ANOVA on dopamine datapoints was performed for both conditions (LD and DD) to assess the effect of genotype (control or DmIh mutant) and circadian time (ZT/CT). In L∶D, both genotype and ZT significantly affect dopamine levels. Dopamine levels vary with ZT in both genotypes, but the changes along the day are different in control and DmIh mutant flies, given that the interaction between Genotype and ZT is significant. In D∶D, genotype and CT significantly affect dopamine levels. However, the daily changes are not different between control and DmIh mutant flies, because the interaction between these factors is not significant.Effect of (DOC)Click here for additional data file.


Both molecules displayed picomolar and nanomolar binding association with albumin across multiple species and circulating half lives of 16 and 11 hours, respectively, post a single IV dose in rats. Unlike exenatide, both molecules elicited robust glucose lowering when injected 1 day prior to an oral glucose tolerance test, indicative of their extended duration of action. [Leu14]exenatide-ABD was compared to exenatide in a ob/obLep  mouse model of diabetes. Twice-weekly subcutaneously dosed [Leu14]exenatide-ABD displayed superior glucose lowering and weight loss in diabetic mice when compared to continuously infused exenatide at the same total weekly dose. A single oral administration of each molecule via an enteric coated capsule to cynomolgus monkeys showed superior pharmacokinetics for [Leu14]exenatide-ABD as compared to [Leu14]exenatide-ABP with detectable exposure longer than 14 days. These studies support the potential use of these novel long acting exenatide analogs with different routes of administration for the treatment of type 2 diabetes.The design, synthesis and pharmacology of novel long-acting exenatide analogs for the treatment of metabolic diseases are described. These molecules display enhanced pharmacokinetic profile and potent glucoregulatory and weight lowering actions compared to native exenatide. [Leu Peptide hormone-based therapeutics deliver significant therapeutic benefits while displaying exquisite receptor selectivity. While insulins have long been a treatment mainstay for diabetes, the development of peptide drugs from somatostatin Long-acting injectable peptide drugs, given by injection, afford greater convenience and promote increased compliance due to less frequent administration. The design of such drugs needs to address proteolytic degradation and renal filtration which plays a significant role in their clearance from the bloodstream. Efforts to reduce renal clearance have focused on increasing molecular size beyond the 60 kDa renal threshold via non-covalent association of the fatty acid alkyl moiety with albumin, and is exemplified by the once-daily anti-diabetic drugs Insulin detemir Alternate strategies involve modification of peptide and protein drugs to allow a reversible non-covalent association to serum albumin, resulting in half life extension commensurate with their albumin binding affinity et alOur half-life extension strategy focused on the fusion of bio-active peptides with small peptidic motifs that display high affinity for albumins across species. Albumin Binding Peptides (ABP) and Albumin Binding Domains (ABD) have been reported in the literature. Dennis 14]exenatide to generate potent, metabolically stable and long acting exenatide analogs with prolonged glucoregulatory action. The ABP and the ABD components are 1.4 and 5 kDa’s, respectively, and confer extended circulation half-lives to the conjugates through tight, reversible binding to plasma albumin. Our fusion strategy is amenable to chemical or recombinant manufacturing processes, and results in significantly smaller conjugates compared to those with molecular scaffolds such as Fc, XTEN and PEG. They display comparable half-life extension with preservation of biological potency, afford the ability to screen the molecules in animal models from different species and offer flexibility to deliver the fusion molecules by alternate routes of administration.In our studies, we utilized a previously reported ABP and an ABD licensed from Affibody AB (ABD035) as fusion partners for [Leu14]exenatide-ABD and [Leu14]exenatide ABP was administered to cynomolgus monkeys. The preliminary pharmacokinetics results highlight the potential to provide novel exenatide analogs for daily and potentially weekly oral administration.As a proof of concept study, an oral formulation of exenatide  using standard solid phase peptide synthetic protocols on a Prelude automated peptide synthesizer ). Fmoc amino acid residues were activated using 4.0 eq of 0.5 M O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 8.0 eq of 1 M diisopropylethylamine in dimethylformamide (DMF) and double coupled for 1 hour. Fmoc group was removed by treatment with 20%(v/v) piperidine in DMF. Cleavage and deprotection was performed by treatment of the resin with reagent B (93% trifluoroacetic acid (TFA), 3% phenol, 3% water and 1% triisopropylsilane) for 3 hours. The cleaved peptide was precipitated using t-butyl methyl ether, pelleted by centrifugation and lyophilized. The pellet was re-dissolved in 10% acetonitrile/water (10–15 min), filtered and purified via reverse phase HPLC using C5 column and an acetonitrile/water gradient containing 0.1% TFA. The compound was re-purified via reverse phase HPLC C18 column using a shallower gradient. The purified compound was analyzed by ESI-MS . The pure peptide obtained as a TFA salt was used for all biological evaluations.[Leu14]exenatide-ABP was assembled on Fmoc-Trp(Boc)-Novasyn TGT resin using standard solid-phase peptide synthetic protocols on a Prelude ™ peptide synthesizer on a 0.2 mmole scale. After cleavage of the peptide from the resin, the lyophilized material was re-dissolved in 20% acetic acid (7–10 mg/ml) and oxidized with an iodine solution (0.125 M in acetic acid) for 10–15 minutes, filtered and purified via reverse-phase HPLC using C5 column and an acetonitrile/water gradient containing 0.1% TFA. The purified product was analyzed by ESI-MS and re-purified via reverse phase HPLC C18 column using a shallower gradient. Final peptide was analyzed by ESI-MS . The pure peptides thus obtained as a TFA salt were used for all biological evaluations.Similarly, exenatide-ABD and 3.1 µM, 1.03 µM and 0.344 µM for [Leu14]exenatide-ABP. The highest concentration was allowed to disassociate for 3 hours. The running buffer contained 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20 and 0.2 mg/ml ovalbumin. A 12-second pulse of 1/200 dilution of phosphoric acid was used to completely regenerate the surfaces between samples. After subtraction of reference surface and buffer injection, curves were fitted to a 1∶1 (Langmuir) binding model using BIA evaluation software (BioSensor Tools) with correction for mass transfer and with RU max set as a local parameter.Binding of the engineered peptides was tested as follows using 3 concentrations in duplicate: 100 µM, 33.3 µM and 11.1 µM for [Leu14]exenatide-ABD’s in-vitro activity was affected by albumins, we also performed this assay in the presence of 1% serum albumin of different species .Peptides were diluted in assay buffer  and incubated with 6–23 clone 6 cells endogenously expressing the rat GLP-1 receptor, in the presence of 250 µM IBMX . Following 30 minutes incubation, the cAMP was measured using the cAMP Dynamic 2 assay  as per the manufacturer’s instructions. cAMP was detected by a decrease in time-resolved fluorescence energy transfer using a GeniousPro plate reader . To test whether [Leu14]exenatide and GLP-1 were used as positive and negative controls. The final results were normalized to the positive control and expressed as percent peptide remaining.Standard solutions of the peptides (200 µM in PBS) were prepared and diluted in human plasma  to a final concentration of 50 µM at 37°C. At 0, 1, 2, 3, 4, and 5 h, aliquots of 20 µl were removed, quenched with 5% phosphoric acid (100 µl ) and the samples were analyzed as triplicates by using analytical HPLC coupled with a mass detector. exenatide-ABD or [Leu14]exenatide-ABP in phosphate buffer (PBS) as a bolus into the tail vein.In all studies, blood samples were collected into tubes containing EDTA and were kept at approximately 5°C until and during centrifugation. Plasma, collected from the centrifuged blood samples, was stored at −70°C until analyzed for peptide concentration. Male drug-naïve Sprague Dawley rats received 2 nmol/kg of exenatide or [Leu14]exenatide-ABD, 2.4 nmol/kg in pH 4 acetate buffer or [Leu14]exenatide-ABP, 2.4 nmol/kg in pH 7.4 phosphate buffer administered through an indwelling catheter in the cephalic vein. The test articles were supplied in ready to use IV solutions. All animals were dosed in the morning (09∶09–09∶33) and blood samples collected while in a restraint chair. The animals were housed in their standard cages between sample collections. Blood samples were collected from the cephalic or saphenous veins pre-dose and at various time points up to 14 days post-dose. The study consisted of 4 experimental groups containing 3 male nonhuman primates each dosed in a parallel design. The animals were selected based on their availability with no randomization being performed. The animals used came from the Xenometrics non-naïve colony. No animals were sacrificed in the course of this study and all animals were returned to the non–naïve stock colony following completion of the study.Monkeys were fasted overnight prior to dosing, and through approximately 2 hours post-dose, and received exenatide-ABD at 100 nmol/kg/dose in PBS was administered IP twice a week (BIW). To match the [Leu14]exenatide-ABD total weekly dose, exenatide was administered at 30 nmol/kg/d in 30 mM Na-acetate buffer (pH 4.5) via continuous SC infusion using osmotic mini-pumps . To assure the same animal handling across all groups, [Leu14]exenatide-ABD-treated mice received mini-pumps with infusion vehicle, the exenatide-treated group was injected BIW with PBS, and vehicle control mice were dosed BIW with PBS and implanted with mini-pumps delivering infusion vehicle. After 2 weeks, all mice were re-implanted with new mini-pumps with the same treatment administered for the next 2 week. HbA1c and body weight were assessed periodically. Following the study, all mice were sacrificed with an overdose of isoflurane.Mice were singly housed and randomized into three treatment groups based on glycated hemoglobin A1c (HbA1c) prior to the 4-week study. exenatide-ABP or [Leu14]exenatide-ABD with a single enteric coated capsule , followed by up to a 5 ml flush of water to ensure delivery completion. Blood samples were collected from the cephalic or saphenous veins predose and at 0.25, 0.5, 1, 2, 3, 4, 8, 24 and 48 h post-dose and on days 5, 7, 10, 12, and 14. A half banana was given following the 2 h PK sample collection, ½ daily ration was given ∼4 h post-dose and the remaining daily rations were fed ∼6 h post-dose. Absolute bioavailability was calculated by dividing the dose-adjusted AUC of the PO PK curve by the AUC of the IV PK curve.Cynomolgus monkeys were dosed PO with either exenatide-ABP and [Leu14]exenatide-ABD to albumin from different species are presented in a of 1.51±0.03×106 M−1s−1 and a dissociation rate constant kd of 7.47±0.06×10−6 s−1 (based on a 3 h measurement), resulted in an overall affinity KD of 4.94±0.09 pM for [Leu14]exenatide-ABD. This value corresponds closely to that of the unconjugated ABD035 (KD 16.1±0.4 pM).Binding constants . The 100-fold reduction of potency in the receptor functional assay for [Leu14]exenatide-ABD when compared to [Leu14]exenatide (0.006 nM) is possibly due to steric hindrance caused by the ABD fused to the C-terminus of the peptide. Similar to the parent peptides, [Leu14]exenatide-ABD was stable in human plasma over 5 hours. However some degradation of [Leu14]exenatide-ABP was noted. Significant degradation of all peptides occurred at pH 6.8 when exposed to a mixture of pancreatic enzymes. The stability in the pancreatin mix improved with the decrease of pH and degradation was negligible in the most acidic milieu (pH 4.0) for all peptides.Results from y medium . [Leu14]14]exenatide-ABP and [Leu14]exenatide-ABD were 11 and 16 hours, respectively  profiles of the compounds intravenously dosed in rats were markedly extended versus the PK profile of exenatide. In rats, the half-life of the un-conjugated exenatide was 30 minutes, whereas the absolute calculated half-lives for [Leuectively . Furtherectively .14]exenatide-ABP and [Leu14]exenatide-ABD blunted the rise in plasma glucose relative to vehicle-treated mice with efficacy comparable to exenatide or [Leu14]exenatide .When dosed 5 min prior to an oral glucose challenge at 25 nmol/kg, exenatide-ABD for 4 week resulted in a significant vehicle-corrected reduction in HbA1c (−1.78±0.16%) vs. continuous infusion of exenatide at a similar total weekly dose of 210 nmol/kg/week (−0.55±0.27%) or vehicle (p<0.05)  . [Leu14]infusion . This reinfusion  with [Le14]exenatide-ABP and [Leu14]exenatide-ABD was <1%, detectable plasma exposure of [Leu14]exenatide-ABP was <50 pM at all time points, whereas exposure of [Leu14]exenatide-ABD was >50 pM for 7 days and detectable for 14 days  and mouse (253 pM) albumins. Based on its picomolar affinities for albumins from various species and the presence of high concentration of serum albumin in blood, it is expected that [Leu14]exenatide-ABD will exist essentially in the bound state in vivo. Despite its tight binding to albumin, [Leu14]exenatide-ABD retained potent GLP-1 receptor functional activity in the presence of high concentrations of serum albumin (1%) of different species. This is surprising in part because liraglutide, a GLP-1 analog with much weaker albumin binding properties 14]exenatide-ABD also functions as a spacer to minimize undesirable steric interactions between exenatide and the bound albumin, thereby permitting the exenatide component to interact with the GLP-1 receptor. The calculated half-lives of [Leu14]exenatide-ABD and [Leu14]exenatide-ABP in rats was determined to be 16 hours and 11 hours respectively, while the half lives of the non-conjugated exenatide molecules were less than 30 minutes. The terminal half-lives of liraglutide ranging from 4–8 hrs has been reported in mice, rats, rabbits and monkeys  and is approximately 4 hrs in rats 14]exenatide-ABP and [Leu14]exenatide-ABD, which are in line with the stronger affinity of the peptides for albumin.Both molecules displayed extremely high affinity for serum albumin from various species. [Leu14]exenatide-ABP and [Leu14]exenatide-ABD in monkeys produced exposure of 2 days and greater than 14 days, respectively. In other studies, [Leu14]exenatide-ABD exposure after a single intravenous dose persisted up to four days in dogs (data not shown). The turnover of albumin in the cynomolgus monkey is about 11–13 days, and about 19 days in humans. Thus, allometric scaling combined with the observed strong affinity of [Leu14]exenatide-ABD for human albumin suggest that a duration of exposure of at least 14 days could be expected in human subjects. The shorter duration of exposure of [Leu14]exenatide-ABP in monkeys, coupled with its susceptibility to degradation in human plasma, led us to focus on [Leu14]exenatide-ABD for additional investigation in diabetic mice.We then evaluated the pharmacokinetic profile of the engineered peptides in higher mammal species. Intravenous dosing of [Leu14]exenatide-ABP and [Leu14]exenatide-ABD were both full GLP-1 receptor agonists, albeit with somewhat reduced potency compared to native exenatide. A 100 fold loss of in vitro functional activity of [Leu14]exenatide-ABD compared to exenatide may be attributed to steric hindrance associated with the larger peptide chain. This finding is consistent with the reduced receptor potencies reported for liraglutide  were achieved and were sustained over 7 days. It remains to be seen whether the flat pharmacokinetic profile and a relatively low Cmax noted in our study will enhance patient tolerability in future studies. The absolute oral bioavailability of [Leu14]exenatide-ABD calculated in monkeys was <1%. Despite this relatively poor bioavailability, therapeutic concentrations of [Leu14]exenatide-ABD were attained in plasma, and its extended plasma residence support the potential for weekly oral dosing.In the study, cynomolgus monkeys were dosed orally with a single enteric coated capsule containing [Leu14]exenatide-ABP and [Leu14]exenatide-ABD that display potent glycemic control and significantly prolonged duration of action compared to their parent hormone. These molecules bind albumin with high affinity while in circulation, leading to their extended duration of action. The analogs are significantly smaller in molecular size compared to long-acting GLP-1 clinical candidates that rely on Fc, albumin or XTEN conjugation for half life extension, are amenable to production by cost-effective recombinant processes, and hence offer the promise of superior manufacturing cost of peptide per therapeutic dose. In addition, their albumin binding affinities can be tuned for daily or even weekly delivery via different administration routes. Long exposure of [Leu14]exenatide-ABD obtained in non-human primates after oral delivery warrant further studies for optimization of the oral delivery formulations.In summary, we have shown that fusion of an exenatide analog to albumin binding motifs of various origin provide novel long acting analogs [Leu


This prejudiced approach usually does not allow for a reasonable debate”.In my article on a Jewish perspective of the theory of evolutionUnfortunately, Dr Jacob in a response to my article entitled “Reflections on Darwinian Evolution – Is there a Jewish Perspective?”some of Dr Jacob’s arguments in a factual manner.By contrast, I fully respect opposing views even if I disagree with them, such as those which are proposed in Dr Jacob’s article, and hence I shall relate briefly to may begin with theories and concepts, but he is obviously wrong by stating that all experimentation begins in such a way. In fact, science can – legitimately – start from facts and build up toward theories and vice versa. My point, however, was apparently missed or misunderstood by Dr Jacob: in either way theories remain only theories until proven experimentally. In my article I phrased it in the following way: “The fundamental aspect of modern science is the experimentally proven data under controlled conditions which confirm or reject the theoretical hypotheses about how phenomena work”. The theory of the ancient scientists about the sun turning around the earth as well as the theory about the earth being flat with four corners were accepted theories by all scientists. I assume that if Dr Jacob was living then he would have adopted these theories as “facts” and fought against whoever would have said differently, since those facts were “confirmed then to such a degree that it would have been perverse to withhold assent”. However, these theories were experimentally unproven and with time were proven to be totally wrong. The fact that ancient scientists had a theory about the world and the universe did not prove it to be true because it was not validated by experimental proofs. The example of the apple and gravitation given by Dr Jacob is obviously ridiculous, because the fact that apples fall by gravitation has been proven time and again, and hence gravitation is a scientifically proven theory. Nonetheless, even this theory underwent significant modification by the theory of relativism, pointing again to the fact that scientific theories are constantly changing. Indeed, this universally accepted notion that scientific theories come and go seems in the eyes of scientists like Dr Jacob not to apply to the theory of evolution. This theory, even though not scientifically proven in some of its major components, is considered by many current scientists such as Dr Jacob as immortal. Some of the scientists believe in the absolute truth of this theory, and they do not want to be confused by the lack of experimentally proven major components of the theory. Such an approach, however, is contrary to basic scientific thinking and methodology, and in fact undermines the very foundation of modern science.Dr Jacob argues that “science does not begin with facts; rather, all experimentation begins with the premise ‘Let us assume that ...’. In short, science starts with theories and concepts about the physical world”. Dr Jacob is right that experimentation fact that apples fall from trees, and so can Dr Jacob and millions of human beings today and in the past. This fact can be explained either by Newton’s or by Einstein’s theory. However, neither I, nor Dr Jacob, nor anyone ever in history has ever seen the transition of an apelike creature to a human being. Moreover, scientists have never been able to use evolutionary mechanisms to transform one species into another. Also, the time-line for generating all of the necessary mutations to get to our point in time is several fold longer than what science claims is the age of life. In response, science must invent “periods of accelerated mutations” of which there is little proof. Hence, this was, and stays, a theory, not a fact, based on very problematic assumptions, which I discussed at length in my article, and which Dr Jacob, for reasons unknown to me, fails to address at all. The fact is that there are human beings, equivalent to the fact that apples fall from trees – both are seen clearly by every observer. The falling apples can be explained either by Newton’s or by Einstein’s theory; the existence of human beings can be explained either by Darwin’s theory or by direct creation by God. Neither is experimentally proven. However, Darwin’s theory, being within the domain of science, ought to be experimentally proven, otherwise it remains a speculative academic exercise; creation by God cannot – and need not – be experimentally proven, being part of a religious belief, and not negated by any valid scientific fact.Dr Jacob further argues that evolution is a fact as well as it is a theory. He claims that “Humans evolved from apelike ancestors whether they did so by Darwin’s proposed mechanism or by some other yet to be discovered”. He further compares this “fact” to the fact of falling apples, whether it happens by Newton’s or by Einstein’s theories. In my view, this comparison of facts is an excellent example of a distorted scientific approach: I can see for myself every day the Dr Jacob admits that the origin of life or of a common ancestor is beyond evolution theory. I certainly agree with this statement. However, it ought to lead any serious thinker – scientist or otherwise – to ask the questions: Then how did it all start? How did life start? How did the common ancestor start? How did the Big Bang happen? There are various approaches to answer these questions. One way is to say: Science, through the theory of evolution, deals with what has happened after the common ancestor was – mysteriously – created; however, in the future, science, in some as yet unknown way, will be able to discover how it all started. Another way is to say: Science does not deal with this question; therefore, the question does not exist. These obviously are very simplistic and problematic responses to a very serious question. Yet another approach is to say: Since the origin of life is beyond the sphere of science, one can believe that it was – or at least might have been – created by a power beyond any scientific comprehension. Such an answer is based upon systems of religion and faith, which can neither be proven or negated by scientific methods. Accepting this approach actually puts one in the category of a “fundamentalist creationist”, which Dr Jacobs apparently finds to be repulsive. In any event, Dr Jacob chose to avoid this difficult problem by a meaningless statement: “To put it mildly, this is a rather odd statement for a biologist”. It might be odd to a biologist, but it certainly is not odd to any sincere and serious thinker.On the Origin of Species, published in 1869 and on. Whatever his understanding of the term might have been, it was widely used in the strictest sense of survival of the fittest not only by natural scientists but also by sociologists, psychologists, and even politicians. Neo-Darwinism has indeed modified the concept, and perhaps even eradicated it altogether, but it again demonstrates the volatility of scientifically unproven concepts and theories.Dr Jacob further argues against the concept of the survival of the fittest. Assuming that many or even most of the current scientists agree with Dr Jacob’s views on this matter, it once again proves the fact that accepted theories and concepts are changing with time. Darwin himself used this term from the fifth edition of religion – not the scientific quotes – are absolute truths, whereas for a serious and sincere scientist it is obvious that scientific theories and principles are objective, being valid as long as they are accepted by the current experts in the particular area of science, but they are constantly changing and often rejected with advancements in scientific and technological comprehension. Indeed, Dr Jacob describes at length the change in the very definition of “Science” itself, from what was understood and accepted beyond doubt by all scientists in the Middle Ages to what is understood by current scientists. Was science in the Middle Ages an absolute truth? Is current science an absolute truth? Can Dr Jacob be sure that what happened to the science of the Middle Ages in its most fundamental comprehension will not happen to current science? Dr Jacob is mistaken by assuming that my statement is intended to attempt to protect the truth of the Torah by casting doubt on the certainty of science. Rather, it defines the boundaries of the two sets of human thought – religion and science – which are fundamentally different in their aims and purposes, in their methods of operation, in their scope of interest and issues, and in their origin and ramifications. Hence, “Whenever science surpasses its limits, or religion exceeds its boundaries, it actually is a form of an abuse of both. This has happened to the theory of evolution in a more powerful mode than any other interaction between science and religion”.1In the section on “The ‘Jewish faith’ and the theory of evolution” Dr Jacob challenges my approach that religion believes in absolute truths whereas science is an objective method with inherent limits and with constant changes. I know of no serious philosophers of religion and science that would argue with my general statement. To a true religious believer the fundamentals of the Dr Jacob goes on to discuss fundamental theological differences between his comprehension of Jewish faith and mine. Dr Jacob’s religion as described in this article is definitely not my way of understanding our religion. I certainly hope that Dr Jacob does not propose to impose his understanding of Judaism upon others. Such a paternalist approach would be absolutely intolerable. It is beyond the scope of this article to argue and reject Dr Jacob’s theological statements, which in my view are mistaken. In my article I discussed the question whether or not the accepted Orthodox perception of the Jewish faith and the theory of evolution can coexist. Dr Jacob’s way to reconcile between Jewish faith and the theory of evolution is to blindly accept the theory of evolution and completely ignore the very significant scientific difficulties with major parts of this theory on the one hand, and significantly modify Jewish religion to the degree that nothing in the theory of evolution would contradict it. This approach is serving both science and Judaism in a misleading and dishonest way.facts of the theory of evolution”. Indeed, already the Talmudic sages and subsequent commentators of the Bible understood the verses of the beginning of Genesis not in their literal meaning. In my article I gave numerous examples to this fact. This happened hundreds of years before the theory of evolution came into existence and hence, obviously, unrelated to it. However, Maimonides, as quoted by Dr Jacob, states very clearly: “For if the Creation had been demonstrated by proof ... if on the other hand Aristotle had a proof ...” Maimonides understood very clearly that scientific theories are far from sufficient to disprove religious beliefs; only scientific proof can do so. This is exactly my argument in my article: Those parts of the theory of evolution that have been scientifically proven should be – and are – accepted by Jewish theology, even if it requires some adaptations of the Biblical and Talmudic statements: “No scientifically proven facts negate these statements. Moreover, all experimentally proven facts of the theory of evolution, as well as some of its assumptions and interpretations, are compatible with and accepted by Judaism”. However, major parts of the theory of evolution are unproven, speculative assumptions and therefore do not fulfill Maimonides’ criteria for theological revision.Dr Jacob cites Maimonides as stating that if science would demonstrate evidence that directly contradicts Jewish theology we would have to change our beliefs accordingly. Indeed, in my article I quoted another, earlier, Jewish philosopher stating that the Torah can never contradict scientifically proven matters –1140(?)).Dr Jacob challenges my Talmudic examples on changes in nature. However, in my article I referred to the concept used by Jewish scholars, “nature has changed”, only to point out that the basic concept of intraspecies changes was well accepted in Jewish thought and law. Also, in my article I referred to extinct species described in ancient Jewish sources to point out the fact that the findings of fossils of extinct creatures were well known and accepted in ancient Jewish sources. It was beyond the scope and the purpose of my article to discuss the specific examples.believe in the entire theory of evolution as such and to disregard any serious scientific criticism of major parts of it, but that he can do as a private person, not as a scientist.Dr Jacob concludes his article by stating the following: “After 150 years of the most intense analysis, debate, and critical testing, the theory of evolution stands as strong as ever with thousands of facts as its empirical base ... Whether we like it or not, biology simply means evolution”. In my view, such bombastic statements do not make them right or true. In fact, such a statement indeed conceals and covers up many fundamental and unresolved difficulties with the theory. Firstly, I assume that even Dr Jacob would admit that the theory of evolution itself underwent a significant evolution within the 150 years of its existence. I assume that Dr Jacob agrees with some of the observations I cited in my article: “Various fundamental scientific principles that are today accepted without dispute were rejected for many years by the scientific community because of the ‘danger’ that accepting them might cast doubts on the whole theory. Furthermore, in the early years, various ‘scientific’ theories were offered to strengthen Darwin’s theory, and even scientific forgeries were given to justify the theory of evolution”; “In the beginning of the 20th century various basic assumptions of the original Darwinian theory were found to be scientifically invalid, and the theory was in disarray. In the 1940s the evolutionists recruited scientists from different disciplines in order to revive and modernize Darwin’s theory. They proposed fundamental changes and developed the modern synthetic theory of evolution, currently known as the neo-Darwinian theory”. How then can Dr Jacob be so sure that this revised theory will withstand history without further major changes? Secondly, in my article I reviewed numerous scientific problems casting doubt on major parts of the theory of evolution, but Dr Jacob chose to ignore them completely. It is obviously legitimate for Dr Jacob to biological theory of evolution, which portrays the natural evolution, and the extrapolation of this theory to the spheres of beliefs, human behavior, values, and ethics. Some scientists have expanded the biological theory of evolution into a type of a ‘religion’, explaining the universe and the psycho-ethical and political behavior of Man on the basis of beliefs and speculations which are not experimentally proven and indeed cannot be proven by scientific methods, and hence are beyond the scope of science”. Such troublesome extension of parts of the theory of evolution is based on the acceptance of the supposition that the evolution of Man from apelike creatures was accidental without purpose or intent; hence, leading to the conclusion that Man’s creation was without a goal and without a plan. “Therefore, it may lead to the conclusion that people have a right to ignore the ethical and moral foundations of humanity. This attitude is the basis for the theory of the stronger races having dominion over lower ones in accordance with the randomness of natural selection and survival of the fittest over the weakest in society. Judaism totally rejects all the extensions of the theory of evolution beyond natural sciences.” In my view it would be wrong to hide behind an argument that biological scientists have no responsibility toward other scientific fields that use – or rather abuse – unproven natural theories that are blindly supported by biological scientists.Finally, Dr Jacob refrains from dealing with the argument in my article that “there is a difference between the


Comprehensive elucidation of genomic, transcriptomic, proteomic, and metabolomic networks that define normal and pathological states, in combination with reprogrammed patient cells are thus poised to become high value resources in modern diagnosis and prognosis of patient disease.Development of innovative high throughput technologies has enabled a variety of molecular landscapes to be interrogated with an unprecedented degree of detail. Emergence of next generation nucleotide sequencing methods, advanced proteomic techniques, and metabolic profiling approaches continue to produce a wealth of biological data that captures molecular frameworks underlying phenotype. The advent of these novel technologies has significant translational applications, as investigators can now explore molecular underpinnings of developmental states with a high degree of resolution. Application of these leading-edge techniques to patient samples has been successfully used to unmask nuanced molecular details of disease vs healthy tissue, which may provide novel targets for palliative intervention. To enhance such approaches, concomitant development of algorithms to reprogram differentiated cells in order to recapitulate pluripotent capacity offers a distinct advantage to advancing diagnostic methodology. Bioinformatic deconvolution of several “-omic” layers extracted from reprogrammed patient cells, could, in principle, provide a means by which the evolution of individual pathology can be developmentally monitored. Significant logistic challenges face current implementation of this novel paradigm of patient treatment and care, however, several of these limitations have been successfully addressed through continuous development of cutting edge  Disease anticipation prior to symptomatic presentation provides multiple pre-emptive opportunities for clinical management. Palliative or curative effectiveness is enhanced by early detection of disease-promoting factors, as early stage treatment can preclude catastrophic pathological progression. Translationally relevant risk factors can be identified through techniques that interrogate and assess biological marker status , where mDistinctions among high throughput methodologies confer specific advantages and disadvantages that determine efficiency and application of genomic, proteomic, or metabolomic approaches. For example, genes validated from tissue biopsies and measurement of glucose uptake with the glucose analogue tracer 18F-fluoro-2-deoxyglucose are among the tools that oncologists rely upon to diagnose and manage tumors . ExperimDifferential gene expression provides direct indicators of genetic background and transcriptome reprogramming as a consequence of evolving disease, but cannot provide information regarding dynamic functional changes. Peptide screening, a hallmark of proteomic analyses, can be employed to obtain this type of information ,8. For eAdvantages of genomic/transcriptomic and proteomic screens provide relevant data for clinical management of disease, though the techniques themselves require several days to weeks for quality-controlled processing and data analyses. Metabolic screening offers a distinct advantage in that samples can be assayed to provide feedback on disease states with greater celerity than the “-omic” approaches described previously. Though the prototype for metabolite analysis is glucose monitoring used in diabetes management , other eThese techniques allow deep interrogation of the molecular complement that comprises the functional background of a cell or tissue sample. Clinical application of these approaches gains significant leverage when combined with a pluripotent cell platform that can provide readouts of the molecular origin of disease phenotype.in silico methods. Indeed, bioinformatic approaches that employ systems biology principles to parse multidimensional biodata extracts multiple levels of integrated information, and application of such techniques to targeted stem cell populations may offer novel modalities of advanced pathology diagnosis.Current generation high throughput technology, originally developed to address rapid high volume sequencing needs , has galStem cells possess a unique potential to anticipate disease phenotypes as they harbor the fundamental molecular baseline that gives rise to genomic content and its derived transcriptomic, proteomic, and metabolomic strata. Interrogation of these “-omic” layers provides rich data to deconvolute systems biology of developmental programs, elucidation of which is critical to understanding clinical pathology etiology. Molecular cartography of pluripotent disease-prone backgrounds would facilitate pre-emptive diagnosis through comparison of healthy wild type templates with diseased states ,15,16, aEmbryonic stem (ES) cells can be harvested and profiled to establish a molecular baseline for comparison to diseased genomes. Recent data indicate that in addition to patient-donated material, patient-specific ES cells can be successfully cloned for potential therapeutic applications . Partly Full equivalency of induced compared to developmentally derived ES cells must be demonstrated to employ reprogrammed cells as proxies for a naturally occurring primordial baseline . Indeed,Systems-wide biodiagnostics, in which entire molecular complements can be assayed, provides an opportunity to comprehensively quantify molecular elements that underlie normal as well as clinically dysfunctional development. Current advances in cellular reprogramming methodology, along with the advent of high throughput technology and advanced bioinformatic computational approaches, presents a unique confluence of molecular tools and techniques by which diseased cells can be reprogrammed to a primordial state , then scThe genomic, transcriptomic, proteomic, and metabolomic elements of systems biology approaches provide biological information unique to each layer of the molecular network, and a variety of specialized techniques exist to interrogate each. From a molecular network ontogeny perspective, the genome is the origin of subsequent transcriptomes, proteomes, and metabolomes, and is a useful starting point for a discussion regarding roles the genome has played in translational diagnosis.The genome is the molecular blueprint composed of genic and non-genic sequences that ultimately determine organismal phenotype , and theImplementation of genome-wide analyses to stem cell (re)programming in the context of therapeutic application has yielded a rich body of novel data that provides details on changes in genomic regulatory elements, epigenomic landscape transitions, and three-dimensional chromatin shifts critical to the process of phenotype (re)acquisition ,33. ThesThe transcriptome is the full complement of RNA produced in response to signaling cues processed by, and transcribed from, the underlying genome, and technology and methods employed for genomic deconstruction are applicable to transcriptome resolution. Comprehensive transcript analysis is an attractive option for biomarker identification, as panels of differentially expressed genes (DEGs) are used to establish indices of disease progression . Prioritin silico approaches to properly resolve transcriptome dynamics of fate commitment/reversion  and function, including their physical and functional interactions . Modern Proteomic studies have increased our understanding of protein complexes and dynamics contributing to cell fate determination, of cell state transitions during development and reprogramming cells to pluripotency, and of the extensive molecular impact mediated by disease-targeted stem cell-based therapy. An expanded pluripotency network described by proteomic assessment of protein-protein interactions between known transcription regulatory factors required for maintenance of ES cells identified combinatorial effects of transcriptional activator complexes required for pluripotency, together with repressor complexes necessary to prevent expression of proteins associated with differentiated cells -49.Stoichiometric correlation between proteins and transcripts can vary , and proProteomic studies are also being used to define functional consequences of stem cell-based therapy. For example, extensive proteomic remodeling underlying structural and functional changes associated with onset of dilated cardiomyopathy was reversed by ES cell therapy, with derived protein networks exhibiting a pro-cardiogenic developmental response with concomitant demotion of dysfunctional disease-associated categories . UltimatThe metabolome consists of small molecular weight compounds that undergo chemical transformation within the cell. Metabolomics captures the complexity of global metabolism in the context of (patho)physiology, and a multitude of analytical tools have been developed to detect metabolite levels, such as enzymatic analysis, flame ionization, and Raman/Fourier transformed infrared and UV-VIS spectroscopy. Nuclear magnetic resonance (NMR) and mass spectrometry have become methods of choice due to their ability to resolve a wide range of chemical moieties in a high throughput manner . The majTo bridge the genotype-phenotype continuum, metabolite screening offers a minimally invasive diagnostic approach associated with high patient value and can provide a wealth of information as metabolite profiles serve as functional signatures of enzymatic activity . MetabolA growing number of studies have utilized metabolomic techniques to examine stem cell biology, critical for defining the baseline stem cell metabolic landscape and its perturbation in the diseased state, as well as identifying principal roles of energy metabolism in controlling stem cell fate -67. ThisGenomic, transcriptomic, proteomic, and metabolomic networks possess unique traits that endow these strata with qualities suitable for use as clinical tools, yet an added level of diagnostic sophistication may be accomplished by leveraging the integrated molecular architecture of these networks against complex disease phenotypes. Network biology, as part of a clinical management strategy, can potentially be employed to identify molecular candidates for pharmacological intervention ,72. DissBiological networks possess a discriminant set of characteristics that can be quantified ,75. ThisSystems biology integrates multiple disciplines to create a novel area for translational application, yet faces a plethora of challenges that constrain full implementation. High throughput techniques are expensive and availability to clinical populations at present is financially prohibitive, but as technology improves and assay costs diminish, application of these techniques to the greater clinical population becomes feasible . For exaAnother parameter associated with high throughput approaches is generation of massive amounts of biodata . LogistiTechnology development in the post-genomic area has given rise to advanced high throughput methodologies and modern, integrative bioinformatic tools that permit an unprecedented level of molecular resolution. Dissection of these discrete, yet interrelated molecular strata, in combination with the ability to reprogram phenotypically committed cells to a pluripotent state that provides a unique and patient-specific embryonic pool of cells, enables feasible dynamic and individualized diagnostic strategies for translational application -93. For ad hoc diagnosis and real-time molecular tracking of patient pathology and disease progression.Resetting diseased cells to a pluripotent state provides opportunities to track patient-specific changes at primary (genomic), secondary (transcriptomic), tertiary (proteomic), and quaternary (metabolomics) molecular network strata. Construction of an accessible electronic archive, to house large volumes of biodata produced from high grade bioinformatics analyses, is critical for establishing a dynamic clinical resource essential to fully realize comprehensive


Matched molecular pairs (MMPs), i.e., pairs of compounds that are related to each other by a specific molecular transformation, have become an integral tool of drug discovery ,2. Gener


Aim. Cavernous hemangioma-like Kaposi sarcoma is a rare morphologic type of Kaposi sarcoma. So far there are no cases in the literature defining the histological features of this morphologic spectrum in detail. In this study we presented two classical-type cutaneous Kaposi sarcoma cases with histologic findings resembling cavernous hemangioma in company with clinical and histopathological data. Cases. One hundred and eighty-five classical-type cutaneous Kaposi sarcoma lesions in 79 patients were assessed retrospectively in terms of histopathological features. Findings of two cases showing features of cavernous hemangioma-like Kaposi sarcoma whose clinical data could be accessed were presented in accompany with the literature data. Both cases were detected to have bluish-purple, protruded, irregularly bordered cutaneous lesions. Histopathological examination revealed a lesion formed by cavernous hemangioma-like vascular structures organized in a lobular pattern that became dilated and filled with blood. Typical histological findings of early-stage KS, consisting of mononuclear inflammation, extravasated erythrocytes, and a few immature vascular structures in superficial dermis, were observed. All cases were serologically HIV-1 negative. A positive reaction with HHV-8, CD31, CD34, and D2-40 monoclonal antibodies was identified at both cavernous hemangioma-like areas and in immature vascular structures. Results. Cavernous hemangioma-like Kaposi sarcoma is a rare Kaposi sarcoma variant presenting with diagnostic challenges, that may be confused with hemangioma. As characteristic morphological features may not be observed in every case, it is important for diagnostic purposes to show immunohistochemical HHV-8 positivity in this variant. Kaposi sarcoma (KS) is a rare vascular lesion with four different epidemiological forms , iatrogenic, and AIDS related) caused by human herpes virus-8 (HHV-8) . It may Since they are considerably rare, we discussed two cases of classical-type cutaneous KS exhibiting cavernous hemangioma-like features in company with clinical and histopathological findings and stressed the importance of HHV-8 immunoreactivity in differential diagnosis of these lesions.This study enrolled 185 cases of classical-type cutaneous KS lesions of 79 patients who were diagnosed at Bülent Ecevit University, Faculty of Medicine, Department of Medical Pathology, and Dr. Lütfi Kırdar Kartal Training and Research Hospital between 2001 and 2012 and whose data could be retrieved. Clinical information of the cases was obtained by screening medical files and reassessing materials of pathology archive for histopathological and immunohistochemical data. No patient had a history of immunosuppressive drug use or transplantation. All patients were human immunodeficiency virus-1 (HIV-1) negative. Diagnosis of KS was confirmed by reviewing of Hematoxyline&Eosin (H&E)-stained sections. Histopathological progression of the lesions were grouped into early (patchy), intermediate (plaque), and late (nodule/tumor)-stage KS according to previously specified criteria , 2, 8. AAmong retrospectively examined biopsy samples, lesions, the dominant histopathological component which is composed by cavernous hemangioma-like vascular structures and in which typical histological features of KS are observed at focal areas, were classified as cavernous hemangioma-like Kaposi sarcoma (CHLKS) see . VasculaRetrospective assessment of archive materials of 185 classical-type cutaneous KS lesions belonging to 79 patients revealed that histopathological features of lesions of 2 cases were consistent with CHLKS. Clinical, histopathological, and immunohistochemical findings of these cases were summarized below.A 69-year-old man presented with purple spots at both feet. The lesions had begun at the lateral aspects of the feet and their number and size increased gradually. He had a 10-year hypertension history for which he had been taking medications. Dermatologic examination revealed multiple purple-colored plaques and papules of 0.5–1.5 cm, some of which were covered with crust on the dorsum of feet and around the ankles. He had no pathology in laboratory and serologic examinations. Physical examination revealed no signs suggestive of systemic involvement of KS, including suggestive symptoms, lymphadenopathy, and visceral involvement. He was HIV-1 negative in serological examination. Biopsies were taken from the lesions with an initial diagnosis of KS. The patient underwent cryotherapy for the Kaposi sarcoma lesions in lower extremity. The patient was followed at 6-month intervals for 22 months, and new cutaneous lesions of Kaposi sarcoma in lower extremities were treated with cryotherapy. No systemic involvement or an aggressive course was present.H&E sections of the lesion demonstrated a dilated, blood-filled vascular proliferation lined by flattened endothelium stretching from papillary dermis under hyperkeratotic squamous epithelium to reticular dermis, forming lobular patterns. A few thick-walled vascular structures were observed in that lesion that was composed of thin-walled vessels. A hemosiderin accumulation was noted at the vessel wall and surrounding stroma. Chronic inflammatory cells and histological findings of early-stage KS such as vascular structures forming clefts, hemosiderin pigment, and extravasated erythrocytes were observed around the cavernous hemangioma-like vascular structures, and these findings were more prominent at papillary dermis. At these areas a prominence of nucleolus and mild atypia was notable in endothelial cells lining vascular structures. The lesion did not show spindle cell proliferation, a finding observed at advanced stages of KS. Cavernous hemangioma-like vascular structures formed about 80% of the lesion, and typical histopathological findings of early-stage KS were observable at focal sites inside the lesion.A 70-year-old female patient presented with the complaint of purple discolorations on the dorsum of the right foot, which had been present for 2 years and growing in number. Her past history was remarkable for an operation against colon carcinoma 10 years ago. She had not taken chemotherapy and radiotherapy. She also had goiter for 5 years. Dermatologic examination revealed multiple purple-colored macular lesions with diameters ranging from 0.5 to 1 cm on the dorsum of the right foot. Her serologic examination was negative for HIV-1. Systemic examination and laboratory tests were negative for a finding suggestive of systemic involvement of KS. Biopsies were taken from the lesions with an initial diagnosis of KS. The patient was treated with local excision and cryotherapy for cutaneous Kaposi sarcoma lesions. The patient was followed at six month periods for a total of 11 months. During follow-up, local recurrences were detected in lower and upper extremities and treated with cryotherapy. No systemic involvement or an aggressive course was present during follow-up.H&E sections of the lesion revealed a vascular proliferation forming more than 90% of the lesion at the dermis, which had similar features with the first case. The lesion was composed of vascular structures filled by blood, which were lined by flattened endothelium and organized as lobular patterns, some of which included fibrous septa. Some vascular structures were closely spaced from skin adnexae that were surrounded by hemosiderin pigment. At the papillary dermis adjacent to the lesion were immature vascular structures intersecting collagen and extravasated erythrocytes, findings of the early-stage KS. No spindle cell proliferation was evident in the lesion.Immunohistochemical examinations of lesions from both cases showed a strong reaction with endothelial markers  and HHV-8 (LNA-1) both in endothelia of the cavernous hemangioma-like vascular structures and at the areas of early-stage KS. Endothelia of the mature vascular structures adjacent to the lesions had a positive reaction with Factor VIIIra whereas neoplastic vascular structures were negative. A focal positive reaction was present with actin and desmin at the walls of the cavernous hemangioma-like vascular structures. In the light of clinical, histopathological, and immunohistochemical findings, both lesions were interpreted as classical-type early-stage cutaneous KS, that showed features similar to cavernous hemangioma.KS is a vascular neoplasm with a low malignancy potential, which is caused by HHV-8, has four different epidemiological forms, and has considerably different clinical and histopathological features , 8, 12. A wide spectrum of clinical and histopathological morphologies of KS poses a difficulty in differential diagnosis and a delay in the diagnosis and treatment of the disease , 16. In Studies investigating histological subtypes of KS have been mostly case reports including a low number of cases. There are a few studies studying histological subtypes of KS in large samples , 16, 17.In our study both cases considered as having CHLKS were elderly patients having multiple cutaneous lesions of blue-purple color located in lower extremity. These lesions include typical clinical findings of early-stage KS and do not contain distinguishing features. No history of drug use with attending immunosuppression, transplantation, or HIV infection was notable after clinical and laboratory examinations in the patients.The common histopathological features of the lesions considered as CHLKS were formation of nearly all lesions by cavernous hemangioma-like vascular structures and the presence of typical histological features of early-stage KS at focal areas. The congested vascular structures forming the main component of the lesions are lined by flattened endothelium, and an intimal thickening is notable at the walls of some vascular structures. Although there are no evident atypia in endothelial layer, some endothelial cells became prominent to form protrusions into the lumen. Hemosiderin pigment accumulation is observed at the vessel walls and the surrounding stroma see . In the Differential diagnosis of CHLKS includes many reactive and benign vascular lesions , 15, 16.The most important lesion in the differential diagnosis of CHLKS is cavernous hemangioma. Cavernous hemangioma is a vascular lesion that is seen particularly in childhood period and more commonly involves the upper part of the trunk , 19, 20.Another lesion in the differential diagnosis of CHLKS is spindle cell hemangioma. Clinically, they present with purple-red, painless, nodular at the acral regions. Histologically, they are formed by spindle cell proliferation forming clefts intertwined with thin-walled cavernous hemangioma-like vascular structures. In some cases, similar to hemangioendotheliomas, an intracytoplasmic lumen or a vacuole may be seen in neoplastic cells. The component consisting of spindle cells does not react with endothelial cell markers and it is positive for vimentin/actin/desmin. Differential diagnosis with KS is possible with histological findings as well as immunohistochemical features supporting the pericytic origin of the spindle cells and an absence of HHV-8 positivity , 19–22. Presence of cavernous hemangioma-like features in only 2 (1%) cases of 185 lesions present in our study suggests that this histological finding is a rare morphological spectrum of KS. Macroscopic appearance of the lesions and the clinical data of the cases are consistent with classical KS. Whether this different morphological appearance may be considered as a histological subtype of KS will be elucidated by future studies with larger sample size.CHLKS has a histological appearance very closely resembling a hemangioma, in which typical morphological findings of KS are not prominent. Thus, a careful morphological examination as well as demonstration of HHV-8 immunoreactivity is important for the diagnosis.


Toxoplasma gondii manipulates its host's behavior. In infected rodents, the behavioral changes increase the likelihood that the parasite will be transmitted back to its definitive cat host, an essential step in completion of the parasite's life cycle. The mechanism(s) responsible for behavioral changes in the host is unknown but two lines of published evidence suggest that the parasite alters neurotransmitter signal transduction: the disruption of the parasite-induced behavioral changes with medications used to treat psychiatric disease  and identification of a tyrosine hydroxylase encoded in the parasite genome. In this study, infection of mammalian dopaminergic cells with T. gondii enhanced the levels of K+-induced release of dopamine several-fold, with a direct correlation between the number of infected cells and the quantity of dopamine released. Immunostaining brain sections of infected mice with dopamine antibody showed intense staining of encysted parasites. Based on these analyses, T. gondii orchestrates a significant increase in dopamine metabolism in neural cells. Tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, was also found in intracellular tissue cysts in brain tissue with antibodies specific for the parasite-encoded tyrosine hydroxylase. These observations provide a mechanism for parasite-induced behavioral changes. The observed effects on dopamine metabolism could also be relevant in interpreting reports of psychobehavioral changes in toxoplasmosis-infected humans.The highly prevalent parasite  Acanthocephala spp. Toxoplasma gondiiT. gondii infection and altered aversive behavior. The underlying mechanism(s) responsible for this behavior change are presently unknown. The aim of our study was to identify a possible explanation for this phenomenon.A complex range of interactions exist between a pathogen with its host, which may include manipulation of the host for the pathogen's own advantage. There are several examples of viruses, such as rabies virus T. gondii is a common, global protozoan parasite, which requires both a definitive host and an intermediate host to complete its life cycle. Although felines are the only definitive host of T. gondii, any warm-blooded animal, including humans, can be infected T. gondii infection . Prevalence in some areas can be as high as 95% in older populations. Latent, chronic infection, which is characterized by parasite encystment in the host muscle and brain cells , persists following the resolution of acute infection and continues with seropositivity throughout the host's lifetime T. gondii infection in the brain.T. gondii, exhibit a distinct repertoire of specific behavioral changes, including a loss of aversion to cat odors Toxoplasma genome whose expression is induced during differentiation to tissue cyst stages T. gondii infection in humans can have serious neurological effects T. gondii seroprevalence and schizophrenia T. gondii infection have been found to be greater than the risk factors associated with an individual's genes and with other environmental factors During the chronic stage of infection, infected rodents, which are a key intermediate host for T. gondii infection, particularly based on evidence of a tyrosine hydroxylase encoded by T. gondii. To address these questions, dopamine metabolism was monitored in vivo in the brains of chronically infected mammals and monitored in vitro during infection of neural cells.Thus, it is crucial to examine whether dopamine metabolism is affected by All animal work was performed according to national and international guidelines following approved animal procedures by the Beltsville Area Animal Care Committee, United States Department of Agriculture . This protocol is reviewed annually, and any amendments are approved separately.T. gondii strains were maintained in human foreskin fibroblasts (HFFs) as previously described T. gondii VEG strain were used for histology.Female Swiss Webster mice infected with T. gondii VEG strain oocysts 6–8 weeks prior to processing. Tissues were collected, formalin-fixed and paraffin-embedded using standard protocols and following approved guidelines. Slides were deparaffinized and rehydrated with an alcohol descending row, which was then followed by epitope retrieval in 10 mM sodium citrate buffer (pH 6.0) overnight at 60°C following sectioning. Slides were blocked with 2% normal goat sera for 1 h at room temperature. TRITC-conjugated lectin from Dolichos biflorus  was introduced to the slides for 4 h at room temperature, diluted 1∶200 in primary staining solution . Next, samples were washed (3×10 min) in wash buffer (TBS pH 8.4 with 0.1% Triton X-100 and 1% fish skin gelatin) and blocked using a biotin-streptavidin blocking kit  according to the manufacturer's protocol. Samples were incubated with primary antibody (raised in rabbit) against dopamine  (diluted 1∶200) or tyrosine hydroxylase  (diluted 1∶500) overnight at 4°C. Samples were rinsed with wash buffer and incubated for 1 h with biotinylated anti-rabbit IgG secondary antibody  diluted 1∶500 in secondary antibody solution (0.05% Tween in 0.1 M PBS pH 7.2). Sections were rinsed and incubated with FITC-conjugated streptavidin  diluted 1∶100 according to the manufacturer's guidelines in secondary antibody buffer at room temperature. Finally, slides were rinsed in wash buffer containing DAPI and double-distilled water prior to mounting in Fluoromount G . All incubation and blocking steps were carried out in a wet chamber. All slides were kept at 4°C in the dark before imaging using a Zeiss LSM510 META laser scanning inverted AxioVert 200M confocal microscope with DIC optics. 3D reconstructions of serial sections were generated with the same equipment using the LSM imaging software for the 3D deconvolution. To assess the specificity of dopamine staining, sections were incubated either without primary antibody or with primary anti-dopamine antibody in the presence of freshly prepared dopamine or serotonin for 30 min prior to and for overnight following addition to the sections.Immunofluorescence against multiple targets was performed on paraformaldehyde-fixed, paraffin-embedded mouse brain sections. Female Swiss Webster mice were infected with T. gondii tyrosine hydroxylase antibody custom antibody  was developed to assess the parasite enzyme in animals. The affinity purified antibody is directed against a unique sequence (CIRSSPDPLDLRKMT) in the amino terminal domain that is not found in mammalian tyrosine hydroxylase and has no significant similarity to any protein in the predicted mammalian proteome or other proteins of the T. gondii proteome. The specificity of the antibody for T. gondii tyrosine hydroxylase was confirmed by Western analysis. Total protein from half mouse brains was isolated in 20 volumes (wt/vol)lysis buffer ) and quantified using Bradford reagent (Sigma) as per manufacturer's instructions. Expression and purification of T. gondii tyrosine hydroxylase was as previously described T. gondii tyrosine hydroxylase antibody was used for immunofluorescence (diluted 1∶500) following a similar protocol as described above.A T. gondii stably expressing RFP-conjugated GRASP protein  in human foreskin fibroblasts grown on polylysine-coated coverslips were alkaline induced for differentiation as published and differentiation monitored by counting the number of parasites in the vacuoles in the normal and pH shifted cultures. These conditions yielded bradyzoite forms as shown by RT-qPCR with the bradyzoite markers BAG1 and SAG4 Immunofluorescence of tyrosine hydroxylase in cultured parasites was performed with paraformaldehyde-fixed cell cultures. Cultures of 2O2 (in 0.1 M PBS) to quench endogen peroxidases. Following secondary antibody treatment, 5 µg/ml HRP-conjugated streptavidin  was applied, and next, the peroxidase substrate kit  was used according to the manufacture's protocol. Prior to mounting, sections were stained with haematoxylin to visualize cell nuclei. Imaging of slides was performed using a Zeiss Axioplan microscope equipped with DIC optics. Photomicrographs were collected with a Photometrics CoolSNAP camera and Improvision Openlab software.For immunohistochemical assays, sections were treated as described above, except for the following steps: for washing and dilution buffers, 0.1 M PBS supplemented with 0.1% Tween was used. After antigen retrieval, slides were incubated in 0.3% HA cytochemical method was used to assess the dopamine staining of tissue cyst-containing neural cells in infected mice brain. Glyoxylic acid reacts with catecholamines in a gaseous reaction to form fluorescent products. Dopamine reacts with glyoxylic acid to form a product that specifically emits at 478–480 nm T. gondii tachyzoites that had been alkaline shocked to induce bradyzoite differentiation. Liberated tachyzoites were incubated in RPMI media at pH 8 with 1% FCS at 37°C and ambient CO2 for 16–18 h, then rinsed with DMEM and returned to standard PC12 cell culturing conditions. PC12 cultures were infected 2.5×105–7.5×105 parasites and cultured for five days prior to assay. The cultures infected with higher numbers of parasites had parasitemia of 40–50%. Prior to assay, samples were normalized to equivalent numbers of cells (2.5×106) per assay. One set of cultures was harvested by centrifugation and lysed by sonication in 0.1 M perchlorate for total dopamine measurement by HPLC with electrochemical detection. A parallel set of cultures were equilibrated with wash buffer with low KCl containing buffer  for 30 min followed by incubation with two volumes high KCl containing buffer  for 2 min to induce dopamine release as previously described The dopaminergic cell line PC12 (ECACC) was infected with Prugniard strain of Following centrifugation, cell homogenates and media were assayed by HPLC-ED. Reverse phase chromatography, combined with electrochemical detection, was performed with a Dionex HPLC system consisting of a P580 Pump (Dionex) and Ultimate 3000 Autosampler Column Compartment with a C18 Acclaim 120 column  and an ESA Coulochem III cell, equipped with a glassy carbon electrode used at 700 mV versus Ag/AgCl reference electrode for detection of monoamines. The mobile phase consisted of degassed 57 mM anhydrous citric acid , 43 mM sodium acetate  buffer containing 0.1 mM EDTA (Sigma Aldrich), 1 mM sodium octanesulphonate monohydrate, and 10% methanol. The pH was adjusted to 4. The mobile phase was delivered at a flow rate of 1.5 ml/min, and the column temperature was set at 40°C. Applied standards  were dissolved in 0.1 M perchlorate for chromatography. The concentration of compounds was determined using Chromeleon software.T. gondii was increased by 14% (114% of uninfected (P<0.01)), whereas other neurotransmitters were unchanged T. gondii infection on dopamine metabolism in tissue cysts have not been examined. T. gondii forms intracellular tissue cysts in neurons with each tissue cyst containing hundreds of bradyzoites (slowly dividing stage) that may remain in situ through the host's lifetime T. gondii were probed with dopamine antibody (Abcam). Dopamine antibody staining was readily apparent in infected cells  into the tissue cyst stages (ie. bradyzoites) with alkaline pH and decreased CO2 content as described by others To assess whether the dopamine detected in T. gondii and total dopamine was quantitated by HPLC-ED following washes with low KCl buffer. We found that infected cultures accumulated significantly greater levels of dopamine and the increase correlated with infection rate , and hence could potentially import neuronal tyrosine hydroxylase into the tissue cyst It is possible that the tyrosine hydroxylase expression observed within the tissue cyst could be either the T. gondii could provide an enzyme with tyrosine hydroxylase activity. We previously described a T. gondii encoded tyrosine hydroxylase that could be expressed in the brain tissue cysts T. gondii has two copies of the tyrosine hydroxylase gene encoding nearly identical proteins (97.5%) with one gene induced in bradyzoite-stage parasites. The parasite tyrosine hydroxylase has a high degree of homology (53% identity) with mammalian tyrosine hydroxylases. Unique for tyrosine hydroxylases, the parasite orthologue enzyme contains a putative signal sequence that could permit the enzyme to be trafficked to an organelle or secreted by T. gondii. Additionally, it was observed that the commercial tyrosine hydroxylase antibody used in these studies recognizes the T. gondii encoded orthologue, as well as the mammalian tyrosine hydroxylases (unpublished observations). Indeed, in vitro cultivated parasites under alkaline conditions that induce formation of bradyzoites bind commercial tyrosine hydroxylase antibody  was developed. The target sequence of this custom antibody is located in the amino-terminal domain of TgTH, which is unique and divergent from mammalian tyrosine hydroxylases. T. gondii tyrosine hydroxylase was localized within tissue cysts in neural cells in chronically-infected mouse brains  from the host cell cytosol into the parasitophorous vacuole could allow passage of small compounds from the vacuole into the host cytosol T. gondii mutants that will provide a conclusive dissection of the role of the parasite's tyrosine hydroxylase in the dopamine synthesis and release are in progress, but with either site of dopa decarboxylase action, the increased dopamine metabolism has important implications on the host neurochemistry.The observed intense dopamine staining within the dopamine . The parT. gondii-induced behavioral changes. Prior studies have proposed that the host immune response to T. gondii infection may lead to altered neurotransmitter levels T. gondii infection with a T-lymphocyte–driven defense T. gondii elicits a dominant Th1 response involving interferon-gamma (IFN-γ), interleukin-12 (IL-12), IL-18, and tumor necrosis factor alpha (TNF-α). TNF-α induction has a serious impact on T. gondii induced pathology at early stages of infection. Th2-associated cytokines, such as IL-4 and IL-10, appear relatively late after infection and may limit immune pathology. To resolve acute infection, IFN-γ induces indoleamine 2,3-dioxygenase (IDO) release, resulting in tryptophan degradation and kynurenic acid accumulation T. gondii chronic infections although there may be localized undetected changes T. gondii infection could contribute to alterations in neurotransmitter levels that could affect behaviour in conjunction with the increased dopamine mediated by the parasite. Further studies are essential to investigate these possibilities.In addition to dopamine, neurotransmitters such as serotonin and glutamate need to be considered in T. gondii infection may contribute to serious neurological disorders in humans. Several studies have observed an association between T. gondii seroprevalence with schizophrenia T. gondii infection has been found to last throughout the lifetime of the host, seroprevalence is likely to reflect chronic infection T. gondii infected rodents. It is possible that the increased dopamine accumulation and release observed during T. gondii infection may contribute to T. gondii associated schizophrenia. Dopamine metabolite concentrations have been inversely correlated with gray matter volume in schizophrenia patients, and recent MRI evidence found that the majority of volume reduction is in those patients seropositive for T. gondii, suggesting that T. gondii infection leads to an increase in dopamine metabolite concentrations T. gondii.Behavioral changes associated with T. gondii's ability to directly alter dopamine levels will not only help to better understand the relationship between schizophrenia and T. gondii seroprevalence, but these findings may be critical for understanding the mechanism(s) involved in a variety of pathogen-associated neurological disorders T. gondii infection to other dopamine-related diseases Malfunctions of dopamine metabolism have a serious impact on human behavior. Dopamine dysfunction has been associated with a variety of neurological disorders including schizophrenia, attention deficit hyperactivity disorder, tic disorders, Tourette's syndrome, and dyskinesias. The novel findings of this study, that demonstrate