O-methyl transferase; monoamine oxygenase), transport  and receptors (DRD1-D5)] would be changed by testosterone or its metabolites, dihydrotestosterone and 17β-estradiol, in the nigrostriatal pathway of adolescent male rats. We found that testosterone and dihydrotestosterone increased DAT and VMAT mRNAs in the substantia nigra and that testosterone increased DAT protein at the region of the cell bodies, but not in target regions in the striatum. Dopamine receptor D2 mRNA was increased and D3 mRNA was decreased in substantia nigra and/or striatum by androgens. These data suggest that increased testosterone at adolescence may change dopamine responsivity of the nigrostriatal pathway by modulating, at a molecular level, the capacity of neurons to transport and respond to dopamine. Further, dopamine turnover was increased in the dorsal striatum following gonadectomy and this was prevented by testosterone replacement. Gene expression changes in the dopaminergic cell body region may serve to modulate both dendritic dopamine feedback inhibition and reuptake in the dopaminergic somatodendritic field as well as dopamine release and re-uptake dynamics at the presynaptic terminals in the striatum. These testosterone-induced changes of molecular indices of dopamine neurotransmission in males are primarily androgen receptor-driven events as estradiol had minimal effect. We conclude that nigrostriatal responsivity to dopamine may be modulated by testosterone acting via androgen receptors to alter gene expression of molecules involved in dopamine signaling during adolescence.Adolescent males have an increased risk of developing schizophrenia, implicating testosterone in the precipitation of dopamine-related psychopathology. Evidence from adult rodent brain indicates that testosterone can modulate nigrostriatal dopamine. However, studies are required to understand the role testosterone plays in maturation of dopamine pathways during adolescence and to elucidate the molecular mechanism(s) by which testosterone exerts its effects. We hypothesized that molecular indices of dopamine neurotransmission [synthesis (tyrosine hydroxylase), breakdown (catechol- Schizophrenia is slightly more common in males than females Dopaminergic transmission involves signaling by five G-protein coupled receptors divided into inhibitory receptors  and excitatory receptors , as well as the regulation of dopamine movement across membranes via dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2). Dopamine is broken down by catechol-O-methyl transferase (COMT) and monoamine oxidase (MAOA and MAOB) enzymes to the main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)  or, following conversion to estradiol by aromatase, to estrogen receptors . Testosterone is also converted to dihydrotestosterone (DHT), a more potent, pure androgen which initiates transcription mainly via ARs Previously, we found that testosterone increased TH in the adolescent substantia nigra In the current work, we investigated which molecular indices of dopamine neurotransmission are modulated by testosterone and whether testosterone actions in the adolescent male rat nigrostriatal pathway are driven primarily by androgenic or estrogenic mechanisms. We achieved this by gonadectomizing 45-day old male rats (adolescence) and replacing endogenous testosterone with either testosterone (T), DHT or 17β-estradiol  until 60 days of age (young adults). Unexpectedly, dopamine was unchanged in the dorsal striatum by gonadectomy or with testosterone replacement, but dopamine turnover was increased by gonadectomy. Gene expression for dopamine transporters (DAT and VMAT2) and protein levels of DAT were increased in the substantia nigra by androgens but not estradiol. DAT protein levels were unchanged in the striatum. We show that, in contrast to our previously reported data in the substantia nigra All animal experiments were approved by the Animal Care and Ethics Committee of the University of New South Wales (Ethics number ACEC10/40) in accordance with the National Health and Medical Research Council of Australia's Code of Practice for the Care and Use of Animals for Experimental Purposes, which also conforms to standard international guidelines. Male Sprague-Dawley rats were used for all experiments . Rats were group housed (3–4/cage) in 12/12 hr light/dark phases with constant humidity and temperature and free access to water and standard rat chow. All surgery was performed under ketamine hydrochloride and xylazine hydrochloride anaesthesia, and all efforts were made to minimize suffering. A total of 78 experimental animals were used.Male rats were gonadectomised at 45 days of age and given continuous replacement testosterone (T), DHT or 17β-estradiol (E) by subdermal silastic implant At 60 days of age, rats were anaesthetized with 60 mg/kg sodium pentobarbital  and decapitated. Brains were removed and tissue blocks containing midbrain and striatum were dissected following a Rat Brain Atlas 3PO4 and 500 µM diethylenetriaminepenta-acetic acid  followed by centrifugation  and supernatant collected and stored (−80°C).Dorsal striatum samples were analysed for dopamine, DOPAC and HVA, using high pressure liquid chromatography with electrochemical detection as previously described High pressure liquid chromatography was performed with a Prominence system  composed of degasser (DGU-20A3), liquid chromatographer (LC-20AD), autosampler (SIL-20A) and communications module (CBM-20A). Samples were injected onto a Gemini C18 column  connected to an electrochemical detector  with an Ag/AgCl reference electrode at a potential of +0.72 V and 40°C. Isocratic mobile phase contained 16% methanol, 84% 0.01 M monobasic sodium phosphate, 0.1 mM ethylenediaminetetraacetic acid, 0.65 mM 1-octane sulfonic acid, 0.5 mM triethylamine at pH 3.4, adjusted with hydrochloric acid (Sigma). Injection volumes were 20–35 µL with a 0.75 mL/min flow rate and a 23.5 min run time.External standards  were run daily  to produce a six-point standard curve for dopamine (0.95–17.07 ng), DOPAC (0.84–15.13 ng) and HVA (1.82–32.79 ng) to quantify samples run on the same day. Peak area analysis was with Class VP7.3 SP1 software (Shimadzu). Aliquots of 20–35 µL of samples, pooled from a subset of thirty-five rats (seven samples from each group) were injected daily and used to normalise between measurements acquired on different days. Dopamine turnover was calculated as DOPAC+HVA/dopamine.Dorsal striatum or substantia nigra tissue blocks were homogenized  and aprotinin 0.015 mM, Sigma) using a handheld electric homogenizer . Protein concentration was determined using the Bradford protein assay (Sigma). An aliquot of each sample was combined and used as a standard and run in duplicate on each gel to allow standardization between blots . Standard curves with between 0.5 and 20 µg substantia nigra or dorsal striatum protein were run and TH and DAT expression was determined to be within a linear range and 3 µg protein/sample was used. SDS-PAGE (10% acrylamide for TH and 8% acrylamide for DAT) was performed and proteins were transferred to nitrocellulose . Primary antibodies were anti-TH , anti-DAT  and anti-β-actin . Secondary antibodies were goat anti-mouse or anti-rabbit horseradish peroxidase conjugated . Immunoreactive bands were detected using the LumiGlo detection kit  on hyperfilm . The immunoblots were scanned and band densities converted to numerical values using ImageJ software . Relative intensities of TH and DAT bands (on separate blots) were normalized to relative intensity of β-actin bands on the same immunoblot.Total RNA was extracted from SN and dorsal striatum samples in 800–1000 µl TRIzol  as recommended by the manufacturer. RNA was quantified using a ND-1000 spectrophotometer  and RNA integrity was assessed with high resolution capillary electrophoresis . Two aliquots of 3 µg RNA from each sample were reverse transcribed with SuperScript III First-Strand Synthesis Supermix and random hexamers, according to the manufacturer's protocol (Life Technologies). The 2 aliquots of cDNA from each sample were pooled and diluted for qPCR. A parallel reaction was performed without reverse transcriptase.Transcripts of interest were measured by TaqMan Gene Expression Assays   on the normalized qPCR, immunoblotting and HPLC data. Immunoblotting data are presented as change of relative intensity compared to the Gdx group ± SEM. Immunoblotting data were normalized to β-actin expression. qPCR data are presented as percent change of mRNA levels relative to the Gdx group ± SEM. Outlier detection of the triplicates obtained from the qPCR raw data excluded measurement errors a priori hypothesis Unless otherwise stated statistical analyses were conducted using SPSS software  and p<0.0001) (p = 0.005) , <0.0001)  and VMAT= 0.005) . Both DAp<0.0001) (p<0.0001) and D2L  p = 0.0001) as well as DRD3 , p = 0.015) mRNA expression in the male rats (p<0.005) and Gdx (p<0.011). E replacement had no effect on any DRD2 mRNA isoform. In contrast, DRD3 mRNA was decreased relative to Gdx by T (p = 0.012) and by DHT replacement (p = 0.004) but was unchanged by E replacement.In the substantia nigra, we also found a significant effect of treatment group on DRD2 pan mRNA , <0.0001) . DRD2 paale rats . DRD2 pap = 0.017 and F = 5.9, df  =  , p = 0.0005, respectively) (p = 0.006). There was only a trend for T to increase DRD1 mRNA expression relative to Gdx (p = 0.064) and E replacement had no effect on DRD1 mRNA. Both T and DHT groups had significantly increased DRD1 mRNA relative to the E group . In terms of DRD5 mRNA, T replacement significantly increased transcript levels relative to Intact (p = 0.003) and Gdx (p<0.001). Both DHT replacement and E replacement significantly increased DRD5 mRNA relative to Gdx .We found a significant effect of treatment group on DRD1 and DRD5 mRNA expression in the substantia nigra , ctively) . DHT repp = 0.311 (p = 0.257 (p = 0.538 (data not shown)].In the striatum, COMT, MAOA and MAOB mRNAs were unchanged by gonadectomy and sex steroid replacement  as well as dopamine transporter  gene expression and DAT protein but decreased DRD3 mRNA. This novel data, combined with our previous findings of increased TH protein and COMT and MOA mRNAs in the substantia nigra Dopamine production is rate limited by TH levels and TH activity. Previous studies in rodent brain have reported both increases and decreases in striatal dopamine in response to sex steroids Gene expression changes in dopaminergic cell bodies can alter protein levels in both presynaptic axon terminals at a distance from the cell bodies and/or locally in somatodendritic fields. Our previously reported increase in TH protein in the region of the dopaminergic cell bodies In support of a local change proximal to dopamine neurons in the substantia nigra, we find that 3 out of 5 dopamine receptor mRNAs are increased in response to adolescent testosterone in the substantia nigra perhaps reflecting changes in available dopamine. We also report increased DRD2 gene expression in response to testosterone in both the region of the dopaminergic cell bodies and in the region of the medium spiny neurons of the dorsal striatum. As such, there are multiple sites at which increases in DRD2 expression could modulate dopamine sensitivity of the nigrostriatal pathway: at the cell bodies and dendrites of dopamine neurons within the midbrain, at the pre-synaptic dopamine terminals in the striatum and at the post-synaptic neurons in the striatum. DRD2 activation at the dopaminergic cell bodies results in the attenuation of dopaminergic neuron excitability via feedback inhibition. DRD2 and DRD3 are considered autoreceptors in dopaminergic neurons where DRD2 at the presynaptic terminal provides inhibitory control over dopamine release and DRD2 and DRD3 control electrical activity of the dopamine neurons at the cell body. The testosterone-induced changes we report in DRD2 gene expression in the nigrostriatal pathway at adolescence are of particular interest as DRD2 receptors are the target of all antipsychotic treatments for schizophrenia, with DRD2 receptors in the dorsal striatum suggested to be the most responsive to changes in tissue dopamine levels Crucial to developing new drug targets for therapy in dopamine-related neural disorders is knowledge of the underlying mechanism(s) driving the changes in dopamine regulating proteins. The majority of the gene expression changes reported here are only induced by DHT and testosterone, and not by estradiol, indicating that in adolescent males androgen receptor, not ERα, activation is critical for these responses. It is less clear whether ERβ is involved or not as DHT, via conversion to 3β-diol has a high affinity for ERβ It is important to note the lack of detectable differences in gene expression between intact and gonadectomised rats. Differences in dopamine pathway-related gene expression between intact and gonadectomised rats in our study may be subtle and difficult to detect due to differences between the natural gradual increase in testosterone over adolescence and the immediate, supraphysiological, steady state levels achieved with implants. Intact animals may require a longer exposure to high testosterone to allow the changes we see in gene expression, that we report in sex steroid replaced animals, to occur. The lack of detectable differences between intact and gonadectomised rats could occur if gonadectomy had occurred after the adolescent increase in testosterone. Although androgen-dependent physiological changes  at adolescence are reported in male Sprague-Dawley rats as early as day postnatal day 38 A limitation of this study is that we do not know exactly how the dopamine receptor mRNAs are translated into protein as there are different synthetic pools of dopamine receptor mRNA  and protein measurements of the receptors in the striatum would reflect a mixture of receptors from different mRNA pools and would not distinguish the source or the cellular location (post-synaptic versus pre-synaptic) of any protein changes in the striatum, making conclusions about protein expression in the dorsal striatum difficult to interpret. However, gene expression data provides valuable information about differential control of these molecules at the mRNA level at different points in the nigrostriatal pathway. In contrast, protein measurements of DAT and TH are more informative as the primary source of these proteins is the dopamine neurons in the nigra, allowing conclusions to be drawn regarding changes in protein expression in the somatodendritic and terminal fields of the nigral neurons. An important follow up to this study will be to determine whether these observed gene expression and protein changes in dopamine-related molecules lead to changes in behavior in response to dopamine-potentiating drugs such as amphetamine.In general, our data support the hypothesis that the ability of the nigrostriatal pathway to respond to dopamine is modulated by circulating testosterone levels during adolescence. Although the current data does not allow us to draw conclusions regarding the functional outcomes of observed changes it is feasible that in individuals with an underlying susceptibility to schizophrenia the pubertal increase in circulating testosterone at adolescence may serve as a trigger for the presentation of dopamine-related psychosis. Studies in humans suggest that increased testosterone increases striatal dopamine. Studies in women using functional magnetic resonance imaging support the hypothesis that exogenous testosterone affects dopaminergic activity We conclude that the testosterone-induced, AR-driven modulation of molecular indices of dopamine responsivity of the nigrostriatal pathway may involve regulation of dopamine feedback inhibition in the somatodendritic field and post-synaptic dopamine action in the terminal field. Testosterone has widespread effects including regulating mRNA and some protein levels of molecules involved in pre-synaptic dopamine synthesis, dopamine reuptake and dopamine packaging, dopamine breakdown and dopamine reception. Although the functional outcome of these molecular changes have not been measured this knowledge provides clues to the coordinated multi-faceted level of sex steroid control over dopamine neurotransmission to target for drug development, whereby dopamine responsivity of the nigrostriatal pathway can be modulated, perhaps by changing androgen and/or estrogen receptor activation.


Successful drug discovery often requires optimization against a set of biological and physical properties. We describe our work on multi-parameter approaches to ligand-based de novo design and studies that demonstrate its ability to successfully generate lead hops or scaffold hops between known classes of ligands for some example receptors. We describe a multi-criteria scoring function incorporating molecular shape similarity, molecular fingerprint similarity, and a number of popular “Lipinski-like” molecular properties.Muse is based on an evolutionary algorithm that operates on an initial population of structures to invent new structures with improved scores. Muse is unique in that it has the ability to work with any user-defined scoring function that provides results in the form of a numerical evaluation of designed structures.Several retrospective studies on various targets demonstrate the ability of the above mentioned approach to generate novel ideas that are not only appealing to design scientists but are also validated by comparison to compounds known to demonstrate activity at the desired biological target. Specific examples where this approach has generated either significant modifications of existing molecular frameworks or structurally new molecular templates relative to design starting points  will be provided.


Improved survival among HIV-infected individuals on antiretroviral therapy (ART) has focused attention on AIDS-related cancers including Kaposi sarcoma (KS). However, the effect of KS on response to ART is not well-described in Southern Africa. We assessed the effect of KS on survival and immunologic and virologic treatment responses at 6- and 12-months after initiation of ART.We analyzed prospectively collected data from a cohort of HIV-infected adults initiating ART in South Africa. Differences in mortality between those with and without KS at ART initiation were estimated with Cox proportional hazard models. Log-binomial models were used to assess differences in CD4 count response and HIV virologic suppression within a year of initiating treatment.3) and proportion on TB treatment (37% vs. 30%). In models adjusted for sex, baseline CD4 count, age, treatment site, tuberculosis and year of ART initiation, KS patients were over three times more likely to have died at any time after ART initiation  than those without KS. The increased risk was highest within the first year on ART  and attenuated thereafter . Those with KS also gained, on average, 29 fewer CD4 cells (95% CI: 7–52cells/mm3) and were less likely to increase their CD4 count by 50 cells from baseline  within the first 6-months of treatment.Between January 2001–January 2008, 13,847 HIV-infected adults initiated ART at the study clinics. Those with KS at ART initiation  were similar to those without KS  with respect to age (35 vs. 35yrs), presenting CD4 count (74 vs. 85cells/mmHIV-infected adults presenting with KS have increased risk of mortality even after initiation of ART with the greatest risk in the first year. Among those who survive the first year on therapy, subjects with KS demonstrated a poorer immunologic response to ART than those without KS. Immunosuppression and co-infections with certain oncogenic viruses appears to increase the risk of some cancers in HIV-infected patients 3 and with skin or lymph node involvement only and no other symptomatic disease The World Health Organization (WHO) estimates that just over 5 million HIV-1-infected people were receiving antiretroviral therapy (ART) in sub-Saharan Africa by the end of 2010, for an estimated coverage of patients eligible for ART of 56% The prognosis of KS is not well defined in resource-limited settings and its influence on response to ART is unclear. In particular, the excess mortality related to KS among those on ART is not well described. We used cohort data from two large urban HIV care and treatment programs in Johannesburg and Cape Town, South Africa, to assess the effect of KS on survival, loss to follow-up and immunologic and virologic responses to ART.This analysis was nested within ongoing cohort studies of routine ART outcomes at the sites in Cape Town and Johannesburg. Use of data from the Themba Lethu and Khayelitsha sites were approved by the Human Research Ethics Committee of the University of the Witwatersrand and the Ethics Committee of the University of Cape Town, respectively. The pooling of data in IeDEA-SA was approved by Ethics Committees at the Universities of Bern and Cape Town. Individual patient consent was not needed, consistent with the South African Medical Research Council’s Guidelines on.Ethics for Medical Research and the Declaration of Helsinki. As this was a retrospective analysis of routine clinical service records, no additional data collection or procedures were undertaken from or on patients, all patient information was entered into the database using coded identification numbers, and no information that could reveal patient identity was available in the analytic datasets.www.iedea-sa.org) Data for this study came from two HIV treatment cohorts: the Themba Lethu Clinic in Johannesburg 3 or a WHO stage 4 illness (such as KS) regardless of CD4 count. We found 13,847 patients were eligible for the current analysis.We included HIV-positive treatment naïve patients ≥18 years of age who initiated ART at a study clinic between 01 January 2001 and 31 December 2007. We limited the analysis to patients starting standard South African public sector first-line ART regimens (stavudine [d4T] or zidovudine [AZT] with lamivudine [3TC] and either efavirenz [EFV] or nevirapine [NVP]) 3 at 6 months and >100 cells/mm3 at 12 months after ART initiation). LTFU was defined as having not attended the clinic in the previous 4 months. Mortality is ascertained through active tracing of patients who do not return to the clinic, and data for those lost was also verified at the end of 2010 with the South African National Vital Registration system for patients in whom a civil identification number was available  all-cause mortality; 2) loss to follow up (LTFU); 3) failure to achieve virologic response at 6- and 12-months on ART ; and 4) failure to achieve immunologic response (CD4 count increase of >50 cells/mm3 and >100 cells/mm3) and VL suppression (<400 vs. ≥400) by 6- and then by 12-months on treatment respectively. All models were adjusted for age, gender, baseline CD4 count (at ART initiation), tuberculosis treatment status, time period (year of ART initiation) and initiating treatment site.Baseline characteristics for each group were stratified by KS status and summarized as proportions or medians with interquartile ranges. Cause-specific Cox proportional hazard models were used to estimate the effect of KS on mortality and loss to follow up on ART at each time period considered. Person-time was calculated from the date of ART initiation to the earliest of: 1) death or loss to follow up; 2) transfer to another facility; or 3) end of study period (31 December 2008). We used mixed linear models with a random intercept and an unstructured correlation matrix to estimate CD4 trajectories over time, accounting for repeated observations on an individual. Time was specified as a quadratic function. Models for those with KS and those without KS were fit separately to allow for different curves by exposure group. The association of KS with change in CD4 count from baseline to 6 and 12 months was estimated using a multivariate linear generalized estimating equation model. Additionally, log-binomial regression was used to estimate the impact of KS status on CD4 response (>50 cells/mm3) but those with KS were about twice as likely to have a CD4 count in the 200–350 cells/mm3 category (12.3% vs. 7.2%). The proportion on TB treatment was also higher among those with KS (37% vs. 30%). As expected, patients with KS were more likely to be male than other patients (49% vs. 36%). Those without KS had received a median of 19.1 months of ART (IQR: 7.8–32.0) compared to 12.3 months (IQR: 2.3–29.8) among those with KS.A total of 13,847 patients met eligibility criteria, including 247 individuals (1.8%) diagnosed with KS at baseline (i.e. 6 months prior to up to 6 months after ART initiation). The prevalence of KS was slightly greater in Khayelitsha than at Themba Lethu (2.2% vs. 1.5%). The patient characteristics at initiation of ART are described in Vital status outcomes were ascertained for 13,065 (94%) of the 13,847 subjects (95% for those with KS and 94% for those without KS). Of these, 10%  died and 14%  were LTFU at some point after ART initiation . Median A greater proportion of individuals with KS died after ART initiation compared to those without KS (27% vs. 10%). The rate of LTFU after ART initiation was 13.0/100 py among those with KS compared to 3.9/100 py among those without KS. Individuals with KS had higher mortality rates at all durations after ART initiation compared to those without KS: 28.3/100 person-years (100 py) vs. 7.4/100 py within the first year and 4.1/100 py vs. 1.8/100 py after the first year.Cumulative incidence curves showed higher incidence of mortality for those with KS after ART initiation with the greatest differences in mortality occurring within the first year on treatment . The risWe also analyzed the effect of time of KS diagnosis in relation to ART initiation on mortality. The mortality rate after ART initiation was greater among those diagnosed with KS before ART initiation than those diagnosed with KS after ART initiation (14.2/100 py vs. 9.8/100 py) though both of these were greater than the proportion who died among those without KS (3.9/100 py). The hazard of death among those diagnosed with KS before ART initiation was higher than the hazard among those diagnosed with KS after ART initiation (HR = 4.14 95% CI 2.97–5.77 vs. HR = 2.61 95% CI 1.47–4.62) comparing both groups to those without KS.A greater proportion of individuals with KS were LTFU after ART initiation compared to those without KS (18% vs. 14%). The rate of LTFU after ART initiation was 8.8/100 py among those with KS compared to 5.5/100 py among those without KS. Among those with KS, the rate of LTFU was greatest in the first year after initiation of ART (13.6/100 py in the first 12 months vs. 7.0/100 py after 12 months). In adjusted proportional hazards models there was little evidence for a difference in the rate of LTFU in those with KS compared to those without, both in first year  and after a year on treatment . In competing risks analyses with death as the competing event, the rate of LTFU was closely similar but attenuated .3 compared to 18.1% (95% CI: 17.5–19.1) of those without KS (3 (IQR 58–164 cells/mm3) among the KS group and 121 cells/mm3 (IQR 66–190 cells/mm3) for those without KS. Patients with KS gained, on average, 29 fewer CD4 cells (95% CI: 7–52 cells/mm3) than those without KS over the same time period. Among the 11,667 patients who survived to a year on treatment, CD4 count values were available for 7,157 (62%) of subjects. 29.9% (95% CI: 21.4–39.6%) of KS patients failed to achieve a 100 cell increase in CD4 count compared to 23.3% (95% CI: 22.3–24.3%) of patients without KS. The median increase in CD4 count by 12 months on ART was 150 cells/mm3 (IQR 90–225 cells/mm3) among the KS group and 175 cells/mm3 (IQR: 105–260cells/mm3) for those without KS.Among the 12,337 subjects alive and in care at 6 months on treatment, CD4 count values were available for 8,676 (70%) of these (63% of those with KS and 70% of those without KS). By 6 months on treatment, nearly a quarter of patients  with KS had failed to achieve a CD4 increase of ≥50 cells/mmthout KS . The med3) than those without KS over the first year of ART. The predicted CD4 trajectories from start of ART suggested some advantage for those without KS  and 100 cells/mm3 increase  in CD4 count at 6- and 12-months on treatment respectively  was an indication for initiation of ART with CD4 count ≥200 cells/mm3 at a time when the ART eligibility criteria were otherwise <200. Second, after initiation of ART, those with KS were less likely to increase their CD4 cell counts by 50 and 100 cells at 6 and 12 months on treatment respectively. The KS group also had a smaller mean increase in CD4 cell count at both time periods than those without KS though the actual difference in CD4 gain was small between the groups. This may be due to differences in disease stage at treatment initiation Our findings need to be considered in light of the study’s limitations. First, the prevalence of KS is likely to be underestimated in these cohorts due to the remote location of oncology services from the HIV outpatient clinics and other reasons explored above. Current linkage projects in collaboration with the National cancer registry are underway which aim to improve ascertainment of cancers among HIV treatment cohorts. Second, high rates of lost to follow up in these cohorts may introduce the possibility of selection bias, though we did not find significant differences in rates of LTFU between the groups. The rate of LTFU may, however, have led to underestimations of the mortality rates due to different mortality rates between patients lost to follow-up with and without KS. As previously noted, anywhere from 20–50% of those lost from HIV care are actually deceased Despite effective ART, clinical KS remains a poor prognostic factor in ART treatment outcomes. High rates of mortality and loss to follow up confirm that KS remains a significant problem in low and middle income countries. Insufficient awareness at primary care level leads to under-diagnosis of KS, especially in its early stages, and result in patients often presenting with advanced disease. Delays in initiation of treatment for AIDS-related malignancies such as KS remain a problem in settings where oncology services and chemotherapy are offered at specialist tertiary centres often remote from primary care services and unable to cope with the high patient burden. Under-diagnosis of disseminated disease and limited chemotherapy options are also likely to be negatively impacting on survival and need to be addressed to improve outcomes for HIV-infected patients presenting for treatment with KS. In particular, chemotherapeutic options, such as paclitaxel, are not readily available in resource-limited settings due to high cost. Future research efforts may focus on investigating alternate chemotherapy drugs, including etoposide


Experiments in systems neuroscience can be seen as consisting of three steps: (1) selecting the signals we are interested in, (2) probing the system with carefully chosen stimuli, and (3) getting data out of the brain. Here I discuss how emerging techniques in molecular biology are starting to improve these three steps. To estimate its future impact on experimental neuroscience, I will stress the analogy of ongoing progress with that of microprocessor production techniques. These techniques have allowed computers to simplify countless problems; because they are easier to use than mechanical timers, they are even built into toasters. Molecular biology may advance even faster than computer speeds and has made immense progress in understanding and designing molecules. These advancements may in turn produce impressive improvements to each of the three steps, ultimately shifting the bottleneck from obtaining data to interpreting it. PLoS Computational BiologyThis is an “Editors' Outlook” article for Moore's law has characterized progress in microprocessor techniques see , dashed.Meanwhile, simple computers have gotten cheaper over time. This decrease in costs was so dramatic that many of today's toasters contain microprocessors for time-keeping, switching on or off heat, light feedback of heating state, and the handling of key presses. This makes building toasters simpler and ultimately cheaper. When computers were invented, toasters were certainly not an expected application of computing.Importantly, the speed of computers has increased dramatically faster than the number of neurons that can be simultaneously recorded Molecular biology has seen accelerating progress over the last decades. One readily quantifiable cost in molecular biology is that of sequencing DNA. The development of a host of different methods has allowed the cost of sequencing each base pair to dramatically decrease over time , solid. From a computational perspective, a central objective of neuroscience is to understand how neurons convert their inputs into outputs and collectively produce action based on stimuli and internal processes, such as memory and attention. This leads to what I would call the three central steps of experimental approaches in systems neuroscience. (1) Select the signals that are important for a given neuroscience question. As long as we cannot approach understanding the entire brain at the same time, it is highly useful to select what to stimulate and what to measure. (2) Get stimuli into the brain. To understand what neurons do, inputs need to be defined or known. (3) Get data out of the brain. Only large amounts of data allow meaningful statistical inferences. Virtually all experimental approaches to systems neuroscience can be phrased in these terms.It is interesting to ponder a few well-known examples. In a typical single-cell visual cortex experiment Each of these steps has its own criteria for being maximally useful. For the selection step (1), we would like to select all the relevant signals and nothing else. For the stimulation step (2), we want to be able to get in large amounts of well defined data with high information bandwidth and low noise Each of the currently used approaches has limitations along the three steps. For example, fMRI has a huge number of channels, and can select essentially any brain area. Yet, it seems unlikely that it could be used to record only from certain cell types, such as interneurons Molecular biology is starting to offer powerful solutions to overcome limitations of the three steps (1) The selection step. There are many different levels of selection. We might want to select individuals that have certain diseases for which there are genetic markers. In this case, we can select these individuals through genetic tests (2) The stimulation step. Neurons are traditionally probed with external stimuli , or through electrical or magnetic stimulation methods. Molecular approaches, though relatively new and less frequently employed, allow several clear advantages. First, they can be utilized far more selectively. For example, specific ligands can selectively activate or inactivate certain cellular mechanisms, including those that only exist in certain types of neurons , upper. (3) The “data out” step. Getting data out of neurons is traditionally done using either existing signals (as in intrinsic imaging), secondary signals like blood flow (as in fMRI), dyes , or electrical or magnetic recording . Using molecular techniques, it is possible to have neurons express the dye used to monitor them. Molecular approaches promise some advantages due to the possibility of “clean” selection (see above (1)). For example, it is possible to express a dye only in those neurons of interest , upper. All these developments in molecular biology already make it a major driving force in neuroscience. However, in the same way that exponential growth in computer science has brought us better toasters, I expect that molecular biology will provide refinements of the three steps that are currently hard to imagine. Some past predictions of computer abilities  have been rather unimpressive, whereas others have been rather precise . While I am a computational neuroscientist with limited background in molecular biology, I still want to go out on a limb and make some predictions of developments we may see.The readout of data is currently done using physics, thin wires, and optics, and it may be expected that molecular approaches, aided by the decay in cost of DNA sequencing, may offer new approaches. I can see two major classes of experimental questions. Connectivity: I want to know how neurons and brain areas are wired up To solve the connectivity problem, Tony Zador Lastly, it seems that the step of recording neural activity can also be reduced to DNA sequencing. When a cell divides, it naturally copies its entire DNA using DNA polymerase. The movement of the polymerase along the DNA template could be engineered to be essentially a molecular ticker tape, such that the environment at that point in time is recorded in the DNA sequence , Zurich, Switzerland, where he worked on cat electrophysiology and neural modeling. He received postdoctoral training in London where he worked on motor control and Bayesian statistics. Another postdoc, partially funded by a German Heisenberg stipend, followed at MIT where he worked on cognitive science and natural language processing and deepened his knowledge of Bayesian methods. Since 2006 he has worked for the Rehabilitation Institute of Chicago and Northwestern University, where he received tenure and promotion to associate professor in 2011. His group is broadly interested in uncertainty, using Bayesian approaches to model human behavior and for neural data analysis. Dr. Kording is a Deputy Editor with PLoS Computational Biology.


Kaposi Sarcoma (KS) is a malignancy of endothelial skin cells with multifocal localization on the skin, lymph nodes and visceral organs. Although all clinical variants are associated with HHV-8 infection, specific differences in the clinical onset and in the natural history of AIDS-KS and Classic-KS have been described. The present randomised prospective-observational study aimed to investigate whether the ultrasound pattern and color Doppler flow imaging of vascularisation of skin lesions of patients with Classic KS (CKS) or AIDS-KS could provide useful information to the evaluation of clinical activity of the disease.Cutaneous lesions of 24 patients with histologically confirmed KS were investigated using very high frequency ultrasound probes; 16 patients had CKS and 8 had AIDS-KS. HHV-8 infection was confirmed in all patients by investigating the specific humoral response to viral antigens. Immunological and virological parameters were also assessed to monitor HIV or HHV-8 viral infection. For each patient, a target skin lesion was selected on the basis of size (diameter from 0.4 to 2 cm). Each lesion was analyzed in terms of size, depth and color Doppler pattern.The B-mode ultrasound patterns of skin lesions did not differ when comparing CKS patients to AIDS-KS patients, whereas the color Doppler signal, which is associated with vascular activity, was detected in the KS lesions of 6/8 AIDS-KS patients (75.0%) and in 2/16 CKS ; the latter two patients showed a clinically progressive and extensive disease stage (IV B).Our preliminary results suggest that small cutaneous KS lesions - in both CKS and AIDS-KS patients- display similar B-mode ultrasound patterns . However, the color Doppler signal, which is associated with endothelial activity and angiogenesis, which play a substantial role in KS progression, could constitute a useful tool for evaluating disease activity. Kaposi's Sarcoma (KS) is a tumour affecting mainly the skin, with multifocal expression and possible lymph nodal and visceral involvement . ClassicIn Italy, the most commonly observed clinical variants are CKS, typically found in persons over 60 years of age, and the epidemic form, AIDS-KS, which affects younger persons with HIV infection. In HIV-positive persons, KS constitutes an AIDS-defining condition . AnotherThe clinical onset of KS is characterised by violaceous macules and papules, which over the course of months or years tend to merge into plaques and nodules (in some cases ulcerated), which are associated with a characteristic oedema, particularly evident in the lower limbs. However, definitive diagnosis is based on histopathological evidence of spindle cell and the presence of HHV-8 latency associated nuclear antigen (LANA), in spindle cells and vascular or lymphatic endothelial cells .The clinical progression of CKS is generally slow and not very aggressive, although cases with rapidly growing lesions, with signs of local invasiveness, can be observed, as well as forms that fail to respond to physical or systemic treatment. By contrast, the natural history of AIDS-KS, which can affect mucous membranes, lymph nodes, the gastrointestinal tract, and the lungs, is more aggressive, particularly in untreated HIV-infected individuals .Diverse classification methods have been proposed, based on the clinical aspects and localization of lesions, which can also be assessed by roentgen-ray study, gastroscopy, and total body TC -10. To dOther non-invasive diagnostic techniques, in particular, telethermography and confocal microscopy, could be complementary to traditional staging instruments ,15. ReceOver many years of ultrasound activity, we observed that skin lesions in patients with CKS were structurally more homogeneous and with a lower signal at the color power Doppler, compared to similar lesions in patients with AIDS-KS, which were less homogeneous and showed more intensive signals. Based on these observations, and after having obtained the consensus of the Ethics Committee, we conducted a randomised prospective-observational study, in which we used ultrasound to evaluate the morphology and vascularisation of erythematous-papular-angiomatous skin lesions in outpatients of the Infective Dermatology Division of the San Gallicano Institute, who we subsequently referred to the Radiology Department.The study population consisted of patients - with final diagnosis of KS - who presented at the San Gallicano Dermatology Institute in Rome- Italy - for the first time in 2010 and who had not been previously diagnosed or undergone to any treatment.A total of 24 patients with a final diagnosis of KS were included in the study, of whom 16 had CKS  and 8 had AIDS-KS . All patients underwent complete clinical staging. For HIV-negative patients, we used the clinical classification criteria of Brambilla  and thatHistological examination of all of the lesions studied by ultrasound was performed on hematoxylin/eosin-stained tissue sections (4 μm) of biopsy samples, fixed in 10% buffered formaline and embedded in paraffin. Sections were also processed for immunohistochemical analysis of the expression of the endothelial associated antigens CD31, CD34 and podoplanin, a transmembrane mucoprotein described in a variety of lymphovascular neoplasms, including KS ,21 , under blind conditions. The images were stored on digital support and then re-evaluated in consensus by both. The ultrasound examination was performed with My-Lab 70 XVG , using a high-frequency linear array probe (18 Mhz); for lesions with a diameter of less than 1 cm, a MyLabOne  was also used, with a linear array probe of 22 Mhz. The settings of the devices were optimized for slow flows and superficial lesions. Written informed consent was obtained from patients. A copy of written consent is available for review by the Editor-in-chief of this journal.A total of 24 lesions (one per patient) were clinically observed and successively evaluated with ultrasound; of these, 16 were CKS, localised on the lower limbs Figure , 4.According to the ultrasound, in 15 of the 16 patients with CKS, the lesions, whether plaque-like or nodular, appeared to be solid and homogeneously hypoechoic, whereas in 3 of the 8 patients with AIDS-KS, the lesions were hypoechoic yet dishomogeneous , there were internal signals , we can conclude that it allows clinically similar pathologies  to be excluded. Moreover, the ultrasound pattern observed in this study differs from that reported in previous studies. Although we evaluated a limited number of patients in a single clinical centre, our results show that small CKS lesions are relatively uniform, superficially, hypo echoic, and with well defined contours; they are usually located between the epidermis and the dermis and lack color power doppler signals in the less aggressive forms, whereas vascularisation is evident in the rapidly evolving forms.In patients with AIDS-KS, the ultrasound pattern in B-mode was similar to that for the other group, although, according to the color power Doppler, the lesions were all hypervascular. This finding is consistent with the presence of marked neoangiogenesis in the HIV-related variants, which is closely related to the activity of the HIV-1 virus on the endothelial cells ,25. HoweThus in our opinion, in patients with CKS, ultrasound evaluation of lesions with the color power Doppler study could be used as a non-invasive diagnostic technique for distinguishing between forms with rapid clinical progression - thus requiring therapy - and less aggressive forms, requiring only follow-up. Although this proposal needs to be evaluated with additional studies, including larger number of patients, given its low cost and non-invasiveness, this technique could be immediately used, at least in experienced centres, and included in the diagnostic-therapeutic course for KS.The authors declare that they have no competing interests.FMS conceived of the study and participated in its design and coordination. AL made the clinical diagnosis and the follow up of patients. FE performed the ultrasound and color Doppler analysis.PCF carried out the immunological and virological determinations. CC performed the histological diagnosis. ADC coordinated the study. All authors read and approved the final manuscript


The electrochemical behavior and the response of the designed materials to the presence of dopamine and ascorbic acid without any protective membranes were studied by cyclic voltammetry and constant-potential amperometry techniques. The catalytic effect towards dopamine detection was proved for the synthetic zeolite-modified graphite-epoxy composite quasi-microelectrode, allowing increasing the sensitivity and selectivity for this analyte detection, besides a possible electrostatic attraction between dopamine cation and the negative surface of the synthetic zeolite and electrostatic repulsion with ascorbic acid anion. Also, the synthetic zeolite-modified graphite-epoxy composite quasi-microelectrode gave the best electroanalytical parameters for dopamine detection using constant-potential amperometry, the most useful technique for practical applications.The present paper aims to miniaturize a graphite-epoxy and synthetic zeolite-modified graphite-epoxy composite macroelectrode as a quasi-microelectrode aiming  The mixture of zeolite and graphite has been proposed in various forms since the 1980s , leadingCombining sensitive materials with smaller geometries of the working electrodes, important sensing devices can be obtained for the monitoring of compounds in the cells of living organisms, of which the simultaneous determination of ascorbic acid and dopamine can serve as a typical example.In this context, Wang and Walcarius  achievedAlthough, miniaturization leads to some important advantages, e.g., decreased ohmic drop of potential , establiin vitro and also, envisaging in vivo simultaneous electrochemical detection of dopamine and ascorbic acid neurotransmitters, or dopamine detection in the presence of ascorbic acid. The electrochemical behavior and the response of the designed materials in direct relation with their size to the presence of ascorbic acid and dopamine without any protective membranes were studied by votammetric and amperometric techniques. To the best of our knowledge, no synthetic zeolite-modified quasi-microsensors suitable for DA detection in the presence of AA have been reported before.The present paper focuses on the miniaturization of a graphite-epoxy and synthetic zeolite-modified graphite-epoxy composite macroelectrode as a quasi-microelectrode aiming at 2.2.1.Synthetic graphite powder used in this study was provided from Sigma-Aldrich , with particle dimension <20 μm. A three-component epoxy resin, epoxy resin , epoxy hardener  and epoxy accelerator  were purchased from Sigma-Aldrich and used as received. Natural zeolite was used as Si source for synthetic zeolite preparation using sodium hydroxide solution and sodium aluminate as aluminium source in accordance with a previously reported method . The surL-Ascorbic acid  solutions were freshly prepared prior to each experiment and dissolved in 0.01 M HCl solution, as well as protected against light exposure during experiments.Phosphate saline buffer solution (PBS) supporting electrolyte was prepared from dihydrogenphospate, sodium chloride and sodium hydroxide of analytical reagent grade (Sigma-Aldrich). Dopamine-hydrochloride (Sigma-Aldrich) solutions were freshly prepared prior to each experiment, dissolved in PBS and protected from light exposure during the experimental procedures. Potassium ferrocyanide and potassium ferricyanide were purchased from Sigma-Aldrich.2.2.Experimental investigations were carried out using an AUTOLAB potentiostat/galvanostat PGSTAT302 , controlled by a PC and the General Purpose Electrochemical System (GPES) software ver. 4.9. All the electrochemical measurements were carried out in a Metrohm three electrode cell equipped with working electrode, a platinum counter electrode and respectively, a Ag/AgCl reference electrode. Before using, working electrode was first cleaned mechanically using fine abrasive paper, 0.3 mm alumina powder , then was washed with distilled water for 5 min.All voltammetric experiments were carried out at room temperature (25 °C) using an eDAQ QuadStat four-channel potentiostat  controlled with an eChem software for cyclic voltammetry in a three-electrode electrochemical cell, carbon composite working electrodes, Ag/AgCl reference electrode and Pt auxiliary electrode. A magnetic stirrer provided the convective transport during the voltammetric experiments.−1 and 50 mV·s−1 for macroelectrodes.For detection experiments, the 50 mM PBS supporting electrolyte at pH 7.4 was placed in the cell, and solutions of ascorbic acid and dopamine were employed by the standard addition method. The scan rate employed for the cyclic voltametric detection experiments for quasi-microelectrodes was 250 mV·s2.3.in vivo applications, this sensor is named a quasi-microsensor [−4 cm2 and the final length of the sensor used for all electrochemical detections was approximately 5 cm.The design of the quasi-microsensor is presented in rosensor . Its pla2.The design of the macrosensor was similar, using a Cu wire as electrical contact inserted in an PVC tube (i.d. = 2.5 cm). The geometrical area of the macrosensor surface with disk geometry was 0.196 cmGraphite-epoxy (μ-GEC) and synthetic zeolite-modified graphite-epoxy composite (μ-SZ-GEC) quasi-microelectrodes were constructed and optimized according to Reference . Graphit3.3.1.3 supporting electrolyte recorded at different scan rates using the Randles-Sevcik equation:Cyclic voltammetry (CV) was used to determine the electroactive areas of all four M-GEC, M-SZ-GEC, μ-GEC, μ-SZ-GEC sensors by a classical potassium ferrocyanide system in 1 M KNO−6 cm2·s−1) for the case of macroelectrodes  and higher for the case of quasi-microelectrodes .As can be seen in 2) and for M-SZ-GEC (0.135 cm2) were lower than the geometrical area of 0.196 cm2, while for the quasi-microsensors the calculated electroactive areas had a higher value than the geometrical one equal with 2.5 × 10−4 cm2. The decreased electroactive surfaces for both macroelectrodes compared to their geometrical areas are dueto the distribution of graphite within the insulating materials. For the case of quasi-microsensors, the results suggest that the sensors present a large number of conducting microzones and that the majority of their surface is electroactive [versus the quasi-microelectrodes. Also, it must be noticed that the presence of zeolite insulating material decreased further the electroactive surface area.The calculated electroactive areas for M-GEC  and ascorbic acid (AA) in the presence of each other on untreated carbon surfaces is a problem due the the proximity of their oxidation potential values.in vivo real concentration for both targetcomponents [mponents . The resΔE = 0.164 V) was obtained, compared to M-GEC composite (ΔE = 0.147 V). Although the peak separation is less than 0.200 V, the higher value is recommended for this particular situation, and the results are promising taking into account that simultaneous detection of dopamine in the presence of ascorbic acid is very hard to achieve in normal conditions, with simple sensing devices (without improved electroactive surfaces or any protective membranes).The electroanalytical results for both composite materials are summarized in −1·cm−2 for M-GEC and 0.075 nA·μM−1·cm−2 for M-SZ-GEC) while a significant increase of sensitivity was noticed for direct detection of DA at the M-SZ-GEC.Based on these results, it can be observed that very close sensitivity values were obtained for the case of AA detection at both composite macrosensors (0.076 nA·μMTaking into account the results for simultaneous detection (of AA and DA) at macroelectrodes, the individual detection experimental response was investigated at quasi-microelectrodes having the same composition (μ-GEC and μ-SZ-GEC). −1·cm−2 was obtained for the μ-GEC sensor, while a lower sensitivity in terms of possible rejection effect due to the presence of negatively charged synthetic zeolite in the composite matrix was observed for the μ-SZ-GEC sensor (554.11 nA·μM−1·cm−2). A more positive oxidation potential of AA at μ-SZ-GEC sensor  was observed in comparison with the bare graphite-epoxy composite quasi-microsensor   and the ring closure forming leucodopaminechrome (peak 3) were identified. The last one is not good evidenced, avoiding a further leucodopaminechrome oxidation.By adding increasing concentrations of DA, an increase in anodic peak oxidation is observed at the two composite microelectrodes under investigation, as can be seen in −1·cm−2, was also obtained for the μ-SZ-GEC quasi-microelectrode.Under the investigated concentration range between 125 μM and 1.5 mM, the linear dependences between the anodic peak current density and DA concentration were obtained see , responsUnder these experimental conditions, the catalytic effect towards DA oxidation was proved for μ-SZ-GEC quasi-microelectrode, where the use of zeolite modified electrodes allowed increasing the sensitivity and selectivity for this analyte detection, besides a possible electrostatic attraction between DA cation and the negative surface of the synthetic zeolite.To compare the macro and quasi-microelectrode behavior towards DA detection in the presence of AA, It can be noticed that the μ-SZ-GEC quasi-microelectrode allowed reaching much higher sensitivity for both AA and DA without improving peak separation. Nevertheless, by comparison of the results of DA individual and simultaneous detection see  and 4, t3.3.Constant-potential amperometry (CPA) is a very useful technique for practical applications and offers the best temporal resolution among available techniques. Taking into consideration that in the extracellular fluid of the central nervous system, DA concentrations are present at a level from nanomolar to micromolar range (0.01–1 μM) CPA should regarded as the most viable.vs. Ag/AgCl on M-SZ-GEC macroelectrode by continuous adding 10 μM DA (−1·cm−2.10 μM DA  in compa10 μM DA  by contiIn-vitro detection of low dopamine concentrations must be regarded as a starting point for improving the quasi-microsensors for further in vivo experiments.The electroanalytical parameters determined for μ-SZ-GEC quasi-microelectrode using CV and CA are presented in 4.Graphite-epoxy and synthetic zeolite-modified graphite-epoxy composite macroelectrodes and quasi-microelectrodes exhibited different behaviors regarding their electroactive surface areas, which were lower than the geometrical ones for the macroelectrodes. For the composite quasi-microelectrodes, the electroactive areas were higher. The distribution graphite within the insulation epoxy resin and the distances between the conductive islands should be responsible for these behaviours.−1 ·cm−2 was achieved with synthetic zeolite-modified graphite-epoxy composite quasi-microelectrodes versus 0.58 nA ·μM−1·cm−2, which was obtained at the same macroelectrode composition.The quasi-microelectrodes consisting of the same composition as macroelectrodes allowed achieving better sensitivities for DA detection. The sensitivity of 2,044 nA ·μMUnder the above-presented experimental conditions, the catalytic effect towards dopamine detection was proved for the synthetic zeolite-modified graphite-epoxy composite quasi-microelectrode, allowing increased sensitivity and selectivity for detection of this analyte, besides a possible electrostatic attraction between the dopamine cation and the negative surface of the synthetic zeolite and electrostatic repulsion with ascorbic acid anion.The results of dopamine individual and simultaneous detection on synthetic zeolite-modified graphite-epoxy composite quasi-microelectrode showed that the presence of ascorbic acid onlyincreased by 0.8% the voltammetric signal corresponding to dopamine detection, which means that ascorbic acid did not interfere the dopamine detection. Also, synthetic zeolite-modified graphite-epoxy composite quasi-microelectrode gave the best electroanalytical parameters for dopamine detection using constant-potential amperometry, the most useful technique for the practical applications.


We did not expect that research on the molecular mechanism of algal phototaxis or archaeal light-driven ion transport might interest readers of a medical journal when we conceived and performed our experiments a decade ago. On the other hand, it did not escape our attention that channelrhodopsin is helping an ever-increasing number of researchers to address their specific questions. For example, the channelrhodopsin approach is used to study the molecular events during the induction of synaptic plasticity or to map long-range connections from one side of the brain to the other, and to map the spatial location of inputs on the dendritic tree of individual neurons. The current applications have been summarized in a number of recent reviews  and channelrhodopsin-2  is fine-tuned by the distance of the negatively charged counter ion that together form the active site  of red light-absorbing rhodopsins even when synthetic retinal analogues are used as chromophores.Our expectations for future applications of ChR are high. However, ChRs show clear limitations, such as the small conductance. We may be able to widen the pore by molecular engineering, but presumably at the cost of destabilization and thermal activation in darkness. Selectivity can be changed towards higher or exclusive HDespite these limitations, engineering of ChR and other microbial rhodopsins will progress and, moreover, countless ChR variants will be discovered from the hundreds of new algal genomes sequenced. Better solutions for targeting ChRs into membrane subareas will be found, directing them into organelles, making them bimodal switchable, controlling expression more accurately, and guaranteeing better turnover and photostability for retinal prosthesis and vision in bright light. ChRs will be further optimized for two-photon microscopy and many novel unprecedented variants will be identified. Moreover, ChRs may become commonly used analytical tools or even therapeutics for treating specific diseases.


Haemophilic arthropathy (HA), which shares some clinical and biological injury characteristics with rheumatoid arthritis (RA), is characterized by chronic proliferative synovitis and cartilage destruction. Anti-Fas mAb specifically targets the Fas molecule, which is expressed and activated on the cell surface of inflammatory synovial cells and plays a key role for induction of apoptosis. Caspases are the final executioners of apoptosis and their activation requires proteolytic processing of inactive zymogen into activated fragments.To evaluate the effects of anti-Fas mAb on HA synoviocytes and its capacity of inducing apoptosis analysing caspase 3 activity.HA synoviocytes were incubated with IgM 1000 ng/ml (control), TNFalpha 10 ng/ml, FGF 10 ng/ml, CH11 100 ng/ml (positive control of apoptosis) with or without anti-Fas mAb at different concentrations  for 24 h. RA and healthy synoviocytes were used as controls. To measure cell proliferation/citotoxicity, the WST-1 assay has been performed. Caspase 3 activity has been evaluated with ELISA kit and western blot.Anti-Fas mAb induced a citotoxic effect in HA , healthy  and RA synoviocytes  reaching a maximum effect at 1000 ng/ml. After stimulation with anti-Fas mAb combined with TNFalpha, there was a citotoxic effect on healthy , RA  and HA synoviocytes . After stimulation with anti-Fas mAb combined with FGF, there was a citotoxic effect on healthy, RA and HA synoviocytes . Caspase 3 levels were increased in HA synoviocytes after anti-Fas mAb treatment in a dose-dependent manner, even after co-stimulation with TNFalpha . CH11 induced an increase of caspase 3 levels in HA synoviocytes more than RA synoviocytes. Western blot showed that HA synoviocytes had higher levels of activated caspase 3 compared to RA synoviocytes after stimulation with Anti-Fas mAb, CH11 and co-stimulation with TNFalpha.Anti-Fas mAb has a dose-dependent citotoxic effect on HA synoviocytes, even when associated with TNFalpha and FGF. Anti-Fas mAb is effective in increasing caspase 3 levels in HA synoviocytes in a dose-dependent manner. HA synoviocytes show higher levels of activated caspase 3 compared to RA synoviocytes. Our results suggest that anti-Fas IgM mAb may favour the induction of apoptosis in HA synoviocytes.


Expression of WRAP53 protein has oncogenic properties and it is up regulated in several types of tumors.We examined expression of WRAP53 protein in rectal cancers and analyzed its relationship to the response to preoperative radiotherapy and patient survival. The WRAP53 protein was examined by immunohistochemistry in normal mucosa, primary tumors and lymph node metastases from 143 rectal cancer patients participated in a Swedish clinical trial of preoperative radiotherapy.Frequency of WRAP53 protein expression was increased in primary rectal cancer compared to the normal mucosa (p < 0.05). In non-radiotherapy group positive WRAP53 in primary tumors  or metastases , was associated with poor prognosis independently of stages and differentiations. In radiotherapy group, positive WRAP53 in the metastasis correlated with better survival (p = 0.04). An interaction analysis showed that the correlations of WRAP53 with the prognostic significance with and without radiotherapy in the metastasis differed (p = 0.01). In the radiotherapy group, expression of WRAP53 in metastases gave a better outcome , and an interaction analysis showed significance between the two groups (p = 0.01).WRAP53 may be a new biomarker used to predict prognosis and to select suitable patients for preoperative radiotherapy. Colorectal cancer is one of the most common types of cancer worldwide , and 50%WRAP53 gene (for WD40-encoding RNA antisense to p53) encodes a regulatory RNA essential for p53 function upon DNA damage [WRAP53 also encodes a protein for maintenance of nuclear organelles called Cajal bodies [WRAP53 knockdown triggers apoptosis of cancer cells [WRAP53  have been associated with an increased risk for developing breast [A damage . WRAP53 l bodies -10. WRAPer cells . Common g breast  and ovarg breast . HoweverIn this study, we examined WRAP53 protein in biopsies, and surgical specimens from distant normal mucosa, adjacent normal mucosa, primary tumor and lymph node metastasis from the patients participated in a Swedish rectal cancer clinical trial of preoperative radiotherapy  .The sample profile of the rectal cancer patients showed in Figure The data for terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay  and survTissue arrays sections (5-μm) were deparaffinized, rehydrated and cooked  for 5 minutes, incubated in peroxides block  for 5 minutes and in protein block (Dako) for 10 minutes, the sections were incubated with rabbit α-WRAP53-C1 (WRAP53-483) polyclonal antibody  at 1:500Expression of WRAP53 protein was subjectively examined by two researchers (Zhang H and Wang DW). The investigators scored the sections in a blinded fashion without knowledge of clinicopathological data. The expression was scored as negative (<5%), weak, moderate and strong, and in statistical analyses, we divided cases with the staining into a negative (negative + weak staining) and a positive group (moderate + strong staining) due to the similarity of clinicopathological features in the cases with negative and weak staining versus the cases with moderate and strong staining.The differences in the frequency of WRAP53 expression and its association with clinicopathological/biological factors were analyzed by McNemar’s or Chi Square. The relationship between the WRAP53 and survival were analyzed by Cox’s proportional hazard model. The patient survivals were further analyzed by multivariate and interaction methods.Immunohistochemistry was carried out in primary rectal cancers (n = 143), along with available distant (n = 118) and adjacent (n = 81) normal mucosa as well as metastasis in the lymph node (n = 49) to examine the expression and localization of the WRAP53 protein. Figure In whole group of the patients, the WRAP53 expression was significantly increased from the distant (p < 0.0001) or adjacent mucosa (p < 0.0001) to the primary tumor Figure A, B, D. WRAP53 expression was further analyzed in patients who did not receive radiotherapy or the patients received radiotherapy. In the non-radiotherapy group, WRAP53 expression was significantly increased from the distant (p = 0.03) or adjacent mucosa (p = 0.03) to the primary tumor Figure E. The WRWe observed an increased number of WRAP53 positive cases in the non-radiotherapy group  although univariate analysis did not show a significant relationship . In the WRAP53 positive group, radiotherapy did not play a prognostic role in primary tumors . In the radiotherapy group, among 5 patients with non-distant recurrence, 100% showed WRAP53 positive expression, while in 16 patients with distant recurrence, only 56% had positive WRAP53 (p = 0.07).Based on the above results, especially the relationship of WRAP53 with radiotherapy and survival, we asked whether WRAP53 might function through an apoptotic pathway. We first analyzed whether WRAP53 was related to apoptosis and the survivin protein in the primary tumor samples (we had no available data of apoptosis and survivin expression from the lymph node metastasis).In the radiotherapy group, the WRAP53 expression was positively related to apoptosis (p = 0.04) and negatively correlated to survivin (p = 0.002), namely, the tumors showing positive WRAP53 after radiotherapy, had a higher frequency of apoptosis and low frequency of surviving (data not shown). There was no such evidence in the non-radiotherapy (p > 0.05).To our knowledge, this is the first report concerning WRAP53 expression and its association with prognosis in rectal cancer. We found that WRAP53 was increased from normal mucosa to primary rectal cancers. Considering previous reports on oncogenic properties of WRAP53 overexpression of WRAP53 contributes to malignant transformation , the enhThe patient material investigated here is from a Swedish clinical trial of preoperative radiotherapy in rectal cancer patients . TherefoThe next question is whether the apoptosis pathway is involved in the association of WRAP53 with radiotherapy. Indeed, in rectal cancers with radiotherapy, there was a relationship of positive WRAP53 with increased apoptosis and decreased survivin. Moreover, WRAP53 positive colon cancer cells underwent spontaneous apoptosis upon reduction of WRAP53 expression. WRPA53 knockdown has been reported to result in a significant decrease in p53 mRNA and suppression of p53 induction upon DNA damage . NotablyWRAP53 protein may be a potential “oncoprotein” in rectal cancer development, and involved in induction of apoptosis in response to radiotherapy. We propose WRAP53 as biomarker for selecting suitable patients for preoperative radiotherapy.The authors declare that they have no competing interests.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/294/prepub


L-dopa is the most effective, currently available treatment for Parkinson's disease (PD), but it leads to the development of involuntary movements known as L-dopa-induced dyskinesia (LID) in the majority of patients after long-term use. Both gene and cell therapy approaches are the subject of multiple ongoing studies as potential ways of relieving symptoms of PD without the complication of dyskinesia. However, the spectre of dyskinesia in the absence of L-dopa, the so-called “off-phase” or graft-induced dyskinesia (GID), remains a major obstacle particularly in the further development of cell therapy in PD, but it is also a concern for proponents of gene therapy approaches. LID results from nonphysiological dopamine release, supersensitivity of dopamine receptors, and consequent abnormal signalling through mechanisms of synaptic plasticity. Restoration of physiological circuitry within the basal ganglia loops is ultimately the aim of all cell and gene therapy approaches but each using distinctive strategies and accompanied by risks of exacerbation of LID or development of “off-phase”/GID. In this paper we discuss the details of what is understood regarding the development of dyskinesias with relevance to cell and gene therapy and potential strategies to minimize their occurrence. L-dopa is the most effective treatment for Parkinson's disease currently available and for many patients can provide effective relief of symptoms for many years after diagnosis. In most patients, L-dopa treatment leads to a “honeymoon” period during which the motor symptoms are well controlled. However, after 5 years of treatment, approximately 40% of patients will develop fluctuations in symptom control in response to the drug, as well as involuntary movements known as “L-dopa-induced dyskinesias” (LID) . These cTo improve upon the limitations of currently available therapies, studies are being performed to assess the role of either gene therapy or cell therapy to provide PD symptom relief without the complication of dyskinesia. Cell therapy trials have been seriously hindered by reports of dyskinesia occurring in the absence of L-dopa—the so-called “off-phase” or graft-induced dyskinesia (GID) , 6. TherRisk factors for the development of LID include younger age , 8, doseThis model, is however, inconsistent with several clinical and experimental findings. During LID in the nonhuman primate model of PD, there is decreased rather than increased metabolic activity in the ventral anterior (VA) and ventrolateral (VL) thalamic nuclei, regions of the thalamus that receive input from the GPi . FurtherThe pathophysiological changes that underlie the development of LID must therefore be far more complex. Recordings taken from PD patients undergoing DBS surgery have revealed that during periods of LID there is an increase in 4–10 Hz activity in the contralateral STN, suggesting that there is an abnormal pattern of oscillatory activity throughout the basal ganglia . The cau∼60% of neurons, are lost. Until this point, administration of L-dopa does not alter the concentration of striatal dopamine, but beyond the 60% deficit, the concentration of dopamine in the striatum increases 3-fold after L-dopa administration [The surviving dopaminergic neurons in the progressively denervated striatum, sprout branches that successfully compensate for the neurodegenerative process until stration . While Lstration –23. Thesstration .The role of serotonergic neurons in the development of LID has been the subject of particular study. In rats with 6-hydroxy dopamine lesions, approximately 80% of the peak dopamine (DA) efflux measured in the striatum following the administration of L-dopa originates from serotonergic neurons –28.This Under conditions of chronic denervation, dopamine receptors develop supersensitivity, involving an increased expression of receptors on the postsynaptic membrane of medium spiny neurons , 31. D1 Physiological dopaminergic input from nigrostriatal neurons onto the striatal medium spiny neurons plays an important role in the potentiation and depotentiation of synapses of the corticostriatal pathway. Repetitive stimulation can cause either a long-lasting increase in synaptic strength known as long-term potentiation (LTP), or an enduring decrease known as long-term depression (LTD), a phenomenon known as synaptic plasticity , 40. It Disruption of normal synaptic plasticity is strongly linked to the appearance of dyskinesia –45. It iL-dopa can restore normal synaptic plasticity among individuals free of dyskinesia, but not when dyskinesias are already developed . It has Graft-induced dyskinesia (GID) was first brought to widespread attention following the publication of the randomised sham-surgery-controlled trials of fetal dopaminergic cell transplantation , 6. GIDsPatients receiving open label transplants that predated the Freed and Olanow trials had also experienced GID –55 but tInitial hypotheses were that GID occurred due to excessive dopamine release by the grafts. This has not, however, been supported by functional imaging of the patients in the Olanow trial or the Hagell patient series –57. DyskIt was further proposed that islands of excessive dopaminergic activity might relate to GID . Indeed In the same clinical studies, the severity of GID was not found to correlate with the severity of pretransplant L-dopa-induced dyskinesias (LID). However, a negative correlation was seen between the severity of postoperative “off-phase” GID and preoperative putaminal 18F-Dopa uptake , 57. ThiIn the Freed trial, cells were stored for up to 4 weeks before transplantation, In the Hagell series, the appearance of GID was reported in patients with grafts stored for 1–8 days. However, any hypothetical relationship between tissue storage and GID hypothesis was not supported by the Olanow trial, in which no grafts were stored for >48 hours.In the Olanow trial, the initial significant improvement in the grafted patients compared with sham-operated cases  was lost11-DASB PET). Importantly this excessive serotonergic innervation was seen restricted to the areas of their grafts. There is mounting evidence that serotonergic neurons contaminating the original graft release dopamine in an uncontrolled manner and then lack the ability to reuptake DA and buffer extracellular DA levels leading to GID [It has now been shown that in 2 patients experiencing GID, there was excessive serotonergic innervation in their brains following transplant, (252–285% higher than comparable advanced PD patients). This was measured using functional imaging with ((11)C)-3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)  benzonitrile positron emission tomography  is a critical factor in LID development after graft , 68. It he graft .The appearance of LID after graft may occur via (i) dysregulated DA release from serotonergic neurons themselves, (ii) lack of reuptake of released DA due to insufficient DA neurons, (iii) inhibition of the dopamine transporter (DAT) on DA neurons by 5HT release thus preventing DA reuptake by DA neurons, and (iv) abnormal dopamine receptor supersensitivity  as evidenced by increases in apomorphine-induced rotations , 68.Despite these consistent findings of LID appearance following serotonergic contamination of cell grafts, “off-phase” dyskinesias (GID) do not appear to occur in animal models of PD undergoing cell grafts with the exception of very mild abnormal movements occurring in 2 studies , 71. ThiGene therapy represents an exciting new prospect for the treatment of patients with PD. This technology exploits the properties of viral vectors to invade host cells and incorporate DNA into the host genome. Appropriate modification of viral vectors allows control over which genes are incorporated and within PD research the main focus has been predominantly on genes encoding growth factors or enzymes involved in dopamine synthesis , 75. GenThere are 4 gene therapy programs that have already reached the stage of clinical trial evaluation.AAV2-Neurturin, an analogue of glial-cell-derived neurotrophic factor (GDNF), has been developed in an attempt to provide trophic support to neurons/glia and thus manipulate the progression of PD. Both (GDNF) and Neurturin enhance dopaminergic neuron survival and nigrostriatal function in animal models of PD. Both factors provide protection from 6-OHDA-induced degeneration in rats  and provGAD) into the neurons of the STN offers an alternative therapeutic strategy. Inspired by the effectiveness of STN DBS, the hypothesis emerged that expression of GAD (the rate-limiting enzyme for GABA production) would inhibit overactivity in the STN and would improve off-medication UPDRS motor score. This strategy has been effective in the rat model of PD [Gene therapy consisting of insertion of the glutamic acid decarboxylase gene , the enzyme that converts L-dopa into dopamine, aims to improve the conversion of exogenously administered L-dopa. Since this therapy remains dependent on exogenous administration of L-dopa, “off-phase” dyskinesia is unlikely to occur. However, striatal transfection with AAV-hAADC has been shown to increase LID in primate parkinsonian models if delivered in a nonhomogeneous way , reminisThe ProSavin approach incorporates all 3 enzymes required for dopamine biosynthesis (tyrosine hydroxylase (TH), AADC, and GTP cyclohydrolase L1(GCH1)), with the aim of transfection of nondopaminergic cells so that they may produce and release dopamine endogenously. Again the theoretical concern is that transfected neurons may synthesize dopamine but may not be able to store and release this dopamine in a physiological manner, and an increase in dyskinesia may follow. The pilot phases of this program are using a dose escalation strategy, with careful evaluation of efficacy and dyskinesia severity at each stage, before proceeding with dose increases in subsequent patient cohorts. So far, there have been no concerns regarding “off-phase” dyskinesias. Further addition of the vesicular monoamine transporter 2 (VMAT2) gene to allow dopamine transport by transfected cells has not shown any advantage over the ProSavin approach in laboratory experiments .An additional gene therapy program that has not yet reached clinical trial stage aims to deliver continuous dopamine therapy using AAV-TH-GCH1. By omitting the AADC enzyme, transfected cells would be able to synthesize L-dopa but rely on endogenous AADC activity to produce dopamine. This reduces the risk of uncontrolled dopamine production that cannot be stored or transported physiologically and aims to deliver “continuous dopaminergic stimulation (CDS),” to mimic the advantages observed using other methods of CDS . In rodeAll of the current gene therapy approaches, if successful, could permit a reduction of L-dopa dose and, therefore, achieve greater control of LID. No major concerns regarding “off-phase” dyskinesias have been reported among patients exposed to PD gene therapy. However, dose escalation to try and achieve greater efficacy from these approaches will necessitate continued vigilance with regard to dyskinesia emergence.Our knowledge regarding the underlying causes of LID is growing, and it is clear that the pathophysiological processes are complex, depending on the number of intact dopaminergic terminals, the chronicity, and pattern of administration of L-dopa replacement as well as the possibility of genetic variability in pathways controlling receptor supersensitivity/internalization and synaptic plasticity. There is converging evidence to implicate nondopaminergic neurons, in particular serotonergic neurons releasing dopamine in a nonphysiological manner, as a major contributory factor in LID and also GID.This knowledge is vital in trying to minimize the likelihood of “off-phase” GID developing in PD patients participating in future trials of fetal cell therapy. Careful attention must be paid to selecting the optimal patients phenotype with respect to the severity of their PD and by implication the number of surviving dopaminergic terminals at the time of transplantation. Patients with advanced dopaminergic degeneration will be at greater risk of developing dyskinesia from grafts that contain an excessive number of serotonergic neurons. Patients with less advanced dopaminergic cells loss should be more tolerant of a greater number of serotonin contaminating cells. The relative extent of dopamine/serotonin striatal innervations can be revealed using preoperative functional imaging, to quantify the extent and severity of both dopaminergic and serotonergic innervations in the striatum.The perioperative and intraoperative details are also extremely important. It is possible to manipulate the number of contaminating serotonergic neurons within grafts, through optimisation of the dissection margins in the ventral mesencephalon of the fetal tissue, during graft harvesting. Furthermore, clinical and animal data both suggest that surgical targeting should avoid the ventral putamen. While the type and duration of immunosuppressive regimes may or may not have major relevance for GID development, it is likely that overall cell survival is improved by maintaining immunosuppression for longer than the 6 months adopted in previous cell therapy trials.The use of serotonin (5HT-1A) agonists as antidyskinetic agents is not new. Buspirone, a (5-hydroxytryptamine (5HT 1A) receptor agonist, has previously showed beneficial effects lessening the severity of LID , by dampα2Adrenergic agonists modulate the activity of the direct striatopallidal pathway and have been shown to reduce LID in PD patients but their use in PD patients has been limited by development of side effects [The beneficial effects of Amantadine on LID through its action on NMDA receptors have prompted further study of agents acting on the corticostriatal glutamatergic input evaluating the effects of metabotropic glutamate receptor (mGLUR) antagonists and Adenosine A2A receptor antagonists. AFQ056 is a potent, selective mGLUR5 antagonist that shows antidyskinetic effects in a rodent PD model and has been shown to have significant antidyskinetic effects in 2 small blinded trials . Istrade effects , 93. Giv effects , 37, a f effects ; howeverThe current and future gene therapy and cell therapy programs represent a great source of optimism for patients with PD. However, PD is a heterogeneous disease and only a subset of patients are likely to benefit—perhaps the subgroup of patients with young onset PD that remain with a predominantly motor deficit for many years . In thesDyskinesia development has multiple determinants, and therefore multiple potential opportunities exist to intervene and prevent their occurrence. Some of these processes may be relevant solely as a secondary consequence of nonphysiological dopamine release (presynaptic component) from serotonergic or other neurons, or as a downstream effect of chronic dopamine receptor supersensitivity (postsynaptic component). Consequent downstream changes in synaptic plasticity may account for abnormal oscillatory firing patterns, throughout the basal ganglia circuitry. L-dopa itself  has been identified as having a role in the restoration of normal synaptic plasticity, as evidenced from recordings of patients undergoing high-frequency stimulation of the substantia nigra pars reticulata, in both on- and off-medication states , 97. WheIn parallel with approaches to relieve LID and GID, our understanding of the importance of the ratio of serotonin and dopaminergic neurons allows optimisation of future interventions and accompanying trial design. Our greater understanding of the causes of dyskinesia, either L-dopa related or graft and gene therapy induced, represents a considerable step towards ensuring the success of future gene and cell therapy programs.