Chloroplasts are semiautonomous organelles which possess their own genome and gene expression system. However, extant chloroplasts contain only limited coding information, and are dependent on a large number of nucleus-encoded proteins. During plant evolution, chloroplasts have lost most of the prokaryotic DNA-binding proteins and transcription regulators that were present in the original endosymbiont. Thus, chloroplasts have a unique hybrid transcription system composed of the remaining prokaryotic components, such as a prokaryotic RNA polymerase as well as nucleus-encoded eukaryotic components. Recent proteomic and transcriptomic analyses have provided insights into chloroplast transcription systems and their evolution. Here, we review chloroplast-specific transcription systems, focusing on the multiple RNA polymerases, eukaryotic transcription regulators in chloroplasts, chloroplast promoters, and the dynamics of chloroplast nucleoids. Chloroplasts are believed to have arisen from an endosymbiotic event between a photosynthetic cyanobacterium and the ancestral eukaryotic cell. Although chloroplasts of modern plants and algae have retained the genome of the symbiont, that genome has markedly shrunk over endosymbiotic evolution. Many chloroplast-encoded genes were lost or transferred to the nucleus soon after endosymbiosis. Thus, chloroplast genomes of extant land plants have only 50 protein-coding genes involved in photosynthesis, gene expression, lipid metabolism and other processes, 30 tRNA genes and full sets of rRNA genes. In spite of their small genomes (0.15 Mbp in land plant chloroplasts versus 3 Mbp in cyanobacteria), chloroplast gene expression is regulated by more complex systems compared to the simple prokaryotic regulatory system. Chloroplast gene expression is mediated by two distinct types of RNA polymerase (RNAP) and is highly dependent on post-transcriptional regulation, such as the processing of polycistronic transcripts, intron splicing and RNA editing. Moreover, recent RNA-seq analyses of chloroplast transcripts identified unexpected diversifications of RNA molecules, such as non-coding and antisense RNAs . HoweverArabidopsis thaliana have suggested that 60% of the chloroplast proteome may have been newly acquired from the nuclear genome of host cells after the endosymbiotic event  and nucleoid proteins at an early stage of their evolution. Genomics and proteomics analyses of chloroplast proteins in ic event . Indeed,Figure 1A) However, all genes for chloroplast sigma factors have been transferred to the nuclear genome, whereas genes for core subunits are typically retained in the chloroplast genome as rpoA, rpoB, rpoC1, and rpoC2.Chloroplast gene expression is largely dependent on prokaryotic machineries derived from the ancestral cyanobacterium. The bacterial multi-subunit RNAP is composed of a core Rpo complex, which has the catalytic enzyme activity, and a sigma factor, which recognizes promoter sequences . ChloropHordeum vulgare), iojap mutants of maize and tobacco mutants with disrupted rpo genes generated by gene targeting using chloroplast transformation . RpoTmp and RpoTp likely represent nuclear encoded RNAP (NEP) enzyme in chloroplasts . Among  merolae , and theydomonas . The HLPvolution . Howeverns of HU . Recentlns of HU . SWIB4 tnucleoid . These fFigure 1B). Human mitochondrial nucleoids have been shown to form layered structures, the central core involved in replication and transcription, and the peripheral region where translation and complex assembly may occur . In fact, land plants have a number of novel host cell-derived transcription regulators and DNA-binding proteins that are involved in the regulation of chloroplast transcription. Thus, it seems likely that chloroplast transcription is mediated by a hybrid system of prokaryotic and eukaryotic origin. Further molecular characterization of pTACs and plastid nucleoid proteins would provide novel insights into the unique plastid gene expression system and as yet known mechanisms of plastid differentiation.Recent proteomic and transcriptomic researches and the development of novel ChIP and imaging technologies have advanced the understanding of the molecular basis of RNAP complexes and nucleoid architecture. In land plants, neither the nuclear nor chloroplast genome encodes prokaryotic transcription factors and nucleoid proteins, whereas chloroplasts retain prokaryotic-type RNAP (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Age-related cognitive impairments often include difficulty retrieving memories, particularly those that rely on executive control. In this paper we discuss the influence of the prefrontal cortex on memory retrieval, and the specific memory processes associated with the prefrontal cortex that decline in late adulthood. We conclude that preretrieval processes associated with preparation to make a memory judgment are impaired, leading to greater reliance on postretrieval processes. This is consistent with the view that impairments in executive control significantly contribute to deficits in controlled retrieval. Finally, we discuss age-related changes in sleep as a potential mechanism that contributes to deficiencies in executive control that are important for efficient retrieval. The sleep literature points to the importance of slow-wave sleep in restoration of prefrontal cortex function. Given that slow-wave sleep significantly declines with age, we hypothesize that age-related changes in slow-wave sleep could mediate age-related decline in executive control, manifesting a robust deficit in controlled memory retrieval processes. Interventions, like physical activity, that improve sleep could be effective methods to enhance controlled memory processes in late life. Age-related cognitive impairments often include decline in executive control critical for strategic controlled memory retrieval –4. VolumGiven the vast amount of information stored in memory, successful goal-directed memory retrieval depends on executive control to select appropriate memories among competing representations , 20, 21.Dissociations in performance on different memory tasks provide evidence that executive control deficits may partly underlie memory deficits in older adults. Older adults show intact recognition memory, a relatively automatic retrieval process, but detailed memory retrieval dependent on the PFC is impaired , 39–43. preretrieval processing. An early selection strategy is often associated with engagement of a retrieval mode  study conducted by Wolk et al.  also fouIt should be noted that this study tested recognition memory, which, as a more automatic memory process, is typically spared in older adults and depends less on PFC function and executive control. Postretrieval processing has also been shown to be engaged in both item and detailed recognition memory tasks , 67, 68.The “late selection” model of aging is consistent with other theories of cognitive decline posited outside the realm of memory retrieval , 70, 71.Though evidence for an age-related decrease in “early selection” has been shown in other areas of cognition , 71, theHaving established that age-related changes in executive control contribute to memory deficits, we can ask which factors result in this specific pattern of impairments. Several factors including sleep have been shown to influence PFC function and executive control in both young and older adults , 79–82. In addition to being more sleep-deprived than younger adults, older adults also show a decline in sleep efficiency, sleep continuity, and slow-wave sleep, , 83–88. In the next sections, we will review studies involving sleep and sleep deprivation and how they relate to deficits in controlled retrieval and executive control processes involved in memory retrieval . We propose that age-related decreases in slow-wave sleep may drive age-related changes in PFC function and in turn controlled retrieval.Sleep deprivation influences performance on a variety of cognitive tasks , 18, 19.Significant decreases in frontal lobe metabolism have been demonstrated in studies of sleep deprivation , 98, 99.It is hypothesized that sleep-deprived young adults may serve as an experimental model for age-related cognitive decline . SimilarCognitive impairments that arise from sleep deprivation are often found for executive control tasks , 106–109Also testing a Go-NoGo paradigm in young adults, Breimhorst et al.  examinedSleep deprivation also appears to influence task-switching processes. Task-switching involves cognitive flexibility and inhibition of irrelevant task-sets and is often considered a model paradigm of executive control , 115. CoHarrison and Horne  revealedAlthough multiple aspects of sleep deprivation, including a lack of all sleep stages, and increased stress and fatigue on the part of the subject, may contribute to these impairments, the lack of slow-wave sleep affecting PFC restoration is a possible mechanism by which these impairments in executive control occur . TogetheThe role of sleep in the offline strengthening of memories through consolidation and integration is a well-established phenomenon in both humans and animals –123, andIn terms of episodic memory, there are very few studies that have investigated how different retrieval strategies and stages are influenced by sleep. Nonetheless, these studies have demonstrated specific impairments in retrieval processes that depend on the PFC . HarrisoSupporting the notion that memory deficits in older adults and sleep-deprived young adults are similar, Nilsson et al.  found siRecognition memory is primarily uninfluenced by sleep deprivation, however similar to frontal lobe patients , 32, 131under-recruitment of PFC. Mograss et al. , 105 invOverall these sleep-deprivation studies of memory retrieval suggest that more controlled retrieval processes are impaired with sleep deprivation compared with more automatic ones and this dissociation may be driven by a breakdown in PFC function.These results point to the possibility that age-related decreases in sleep contribute to executive control deficits. Conversely, because there is more to age-related sleep changes than a mere overall decrease in sleep, the impact of age-related sleep changes on cognition may not be completely comparable to sleep deprivation in young adults. Further, while many older adults exhibit marked impairments in cognitive performance and changes in brain activity, some older adults show little cognitive decline. These individual differences in cognitive decline may be explained, at least partially, by individual differences in sleep. Determining the way in which sleep plays a role in age-related cognitive decline may shed light on why some older adults but not others exhibit impairments.Inadequate sleep is very common among the adult population . AccordiGiven the importance of slow-wave sleep in PFC restoration, decline in slow-wave sleep is a critical age-related sleep change that may contribute to impairments in executive control relevant for memory retrieval. Both human and animal studies have shown a decrease in slow-wave sleep with age , 139–142It should be noted that several reports suggest that older adults are more resilient to sleep deprivation than young adults , 144, suA few studies have suggested that individual differences in slow-wave sleep are related to executive control abilities in older adults. Anderson and Horne  examinedIn an earlier study, Crenshaw and Edinger  investigTo corroborate these findings, a more recent study, Nebes et al.  showed tBased on the studies reviewed above, there is clearly some support for the hypothesis that age-related changes in sleep contribute to decline in PFC and executive control and this may affect controlled memory abilities. Should future research support this model of cognitive aging, treatments aimed at improving slow-wave sleep in healthy older adults could improve executive control, potentially leading to improvements in memory. Sleep disruption and slow-wave sleep in young and older adults appear to have the capacity to influence controlled memory retrieval. However, the influence of these factors on controlled retrieval remains unclear. Future research should examine whether pre and postretrieval processing are differentially influenced by sleep deprivation, sleep treatment, or individual differences in slow-wave sleep in recall and recognition memory paradigms. This would answer the question of whether the mechanisms underlying impairments in memory retrieval are similar between older adults and sleep-deprived young adults. Given the “nonselective” frontal recruitment found in both older and sleep-deprived individuals, insufficient inhibitory and filtering processes prior to retrieval is a possible mechanism driving sleep-related impairments in controlled memory retrieval. To the extent that sleep deprivation can be thought of as an experimental model for cognitive aging, we would expect that sleep-deprived subjects would rely more on postretrieval processing, and less on preretrieval processing , 49, 76.Having concluded that age-related changes in sleep may contribute to the pattern of cognitive deficits displayed by older adults, we can next ask whether sleep acts as a mediating factor for other variables that influence cognition. For example, physical activity interventions have been shown to improve executive control in both young and older adults , 152. ThThis paper has focused on the influence of sleep on memory and cognition in healthy aging, but in the interest of informing interventions in healthy older adults, it is worthwhile to acknowledge the efficacy of treating sleep-related disorders and its impact on cognition. Treatment of obstructive sleep apnea syndrome with continuous positive airway pressure has been associated with significant improvements in performance on executive control tasks  associated with changes in sleep, and (b) that sleep deprivation and aging reveal similar patterns of deficient cognition and brain activity, it is conceivable that sleep-deprived young adults may serve as a model for cognitive deficits found in older adults . AlthougThe way in which age-related decreases in slow-wave sleep affect the PFC may shed light on whether similar mechanisms underlie cognitive deficits resulting from aging and sleep deprivation. For instance, age-related changes in slow-wave sleep may have an immediate or gradual cumulative impact on PFC structure and function. It may be that chronic diminished slow-wave sleep over time brings about changes in the PFC seen with advanced age. Alternatively age-related reductions in slow-wave sleep could have an immediate effect, similar to experimentally induced sleep deprivation. Examination of individual differences in slow-wave sleep among healthy older adults may prove effective in revealing the specific processes influenced by age-related changes in sleep and more directly address whether age-related changes in sleep robustly affect cognitive decline. Overall, given that older adults show a wide range of variability in cognitive performance, it is necessary to determine whether there is also a wide range of variability in sleep changes associated with age, and whether the individual differences in cognition and sleep covary. The results from these studies may inform interventions aimed at preventing or reversing cognitive decline. Intervention studies with sleep treatments may address whether age-related changes in sleep directly affect cognition in older adults. Further, exercise intervention studies examining effects on both sleep and cognition may answer questions regarding the mechanisms underlying the relationship between exercise and cognition. These interventions may also serve as a useful technique for improving sleep-related cognitive decline in older adults.  Old/new effects. Differences between brain activity elicited by correctly categorized studied items and brain activity elicited by correctly categorized new items presented at test. Retrieval success. Refers to old/new effects typically found in parietal regions reflecting successful memory recovery. Retrieval attempt. Memory search process prior to the memory decision. Retrieval orientation. The differential processing of retrieval cues based on the sought after information to maximize retrieval success. Slow-wave sleep. Non-REM sleep stages (3 and 4) in which low-frequency delta EEG activity is the highest. Sleep quality. Sleep measures based on time spent lying in bed, including sleep efficiency (proportion of time spent lying down asleep), wake after sleep onset and sleep latency . Sleep efficiency. Proportion of time spent asleep versus time spent lying down. Executive control. Mechanism responsible for goal-oriented processes that involve selection of relevant and inhibition of irrelevant information and actions and the monitoring and updating of information. Preretrieval processing. Processing that takes place in preparation for memory retrieval to filter irrelevant mnemonic information and constrain the memory search space. Postretrieval processing. The monitoring or updating of information following retrieval as relevant for task demands. Episodic memory retrieval. Recovery of memories for personally experienced events usually involving some recollection of details. In an experimental paradigm, studied information must exceed working memory capacity and be cleared from working memory prior to retrieval. Semantic memory retrieval. Recovery of information from knowledge about the world . Obstructive sleep apnea syndrome. Sleep disorder characterized by frequent breathing cessation leading to brief arousals.


Aedes aegypti mosquitoes to decrease dengue virus transmission: population reduction and population replacement. Here we model releases of a strain of Ae. aegypti carrying both a gene causing conditional adult female mortality and a gene blocking virus transmission into a wild population to assess whether such releases could reduce the number of competent vectors. We find this “reduce and replace” strategy can decrease the frequency of competent vectors below 50% two years after releases end. Therefore, this combined approach appears preferable to releasing a strain carrying only a female-killing gene, which is likely to merely result in temporary population suppression. However, the fixation of anti-pathogen genes in the population is unlikely. Genetic drift at small population sizes and the spatially heterogeneous nature of the population recovery after releases end prevent complete replacement of the competent vector population. Furthermore, releasing more individuals can be counter-productive in the face of immigration by wild-type mosquitoes, as greater population reduction amplifies the impact wild-type migrants have on the long-term frequency of the anti-pathogen gene. We expect the results presented here to give pause to expectations for driving an anti-pathogen construct to fixation by relying on releasing individuals carrying this two-gene construct. Nevertheless, in some dengue-endemic environments, a spatially heterogeneous decrease in competent vectors may still facilitate decreasing disease incidence.Two basic strategies have been proposed for using transgenic  Aedes aegypti (Linnaeus), results in approximately 390 million infections each year Ae. aegypti is believed to counteract population suppression efforts , and the model dynamics have been shown to be consistent with the major spatial and temporal patterns of pupal abundance data at the container-level from that location Ae. aegypti population in Iquitos, we believe our main conclusions could apply to other populations in tropical urban environments with limited seasonal variation.Field releases of transgenic mosquitoes, however, occur in spatially heterogeneous environments e.g.,  with theAe. aegypti that incorporates a range of processes, including temperature-dependent survival rates, container-level development and nutrient dynamics, and the impact of water level fluctuations on egg hatching  in all containers are described by the equations in Skeeter Buster is a spatially-explicit, stage-structured model of hatching . Skeeterhttp://www.skeeterbuster.net). For a more thorough description and justification of the features, major assumptions and components of the model, see In Skeeter Buster, mosquitoes emerge from containers as adults and occupy sites (i.e. houses) where the containers are located. The identically sized sites are arranged on a rectangular lattice, and each site's neighbors are assumed to be described by its Moore neighborhood Ae. aegypti population dynamics. Thus, although most parameters in Skeeter Buster are based on laboratory or field studies from a variety of locations ; this, however, did not substantially change the results, so we do not present the outcome of the four-locus cases.As in Transgenes may impose fitness costs due to the expression of transgene sequences or insertional mutagenesis. For simplicity, we only analyze the case where the anti-pathogen gene carries a fitness cost, and do not model such costs associated with the female-specific lethal construct. Fitness costs could, in principle, affect any life stage, but there is no empirical data suggesting a focus on any specific stage. To retain consistency with earlier work Ae. aegypti incapable of transmitting dengue Most transgenic release programs currently emphasize the exclusive release of male mosquitoes e.g.,  to minimTo model the release of transgenic adults, we dynamically add cohorts of mosquitoes homozygous for both the female-specific lethal gene and the anti-pathogen gene at specified dates to specific sites. Because very large releases of mosquitoes carrying the female-specific lethal gene can cause extinctions in closed populations, particularly when population sizes are small Ae. aegypti eggs are desiccation resistant and can remain viable for long periods of time. Consequently, storing and distributing transgenic mosquitoes at the egg stage may be an attractive alternative to the distribution and release of short-lived transgenic adults that would require planning releases to coincide with individuals emerging as adults. Releases of transgenic eggs, following th site along a regular grid, and random point-source releases at 10% of the sites chosen at random.In addition to varying the number, developmental stage, and sex of individuals released, we compare distinct spatial release patterns. Such comparisons can help inform the spatial configuration of releases that actual release programs should strive to reproduce. We model mosquitoes released homogenously into all sites, a regular point-source releases at every 10The comparison between homogenous and point-source releases also allows us to model endpoints along a continuum of mosquito dispersal behavior immediately following releases. The assumption that the dispersal behavior of recently released insects is comparable to the dispersal behavior of resident mosquitoes is standard in spatially-explicit models of transgenic control populations for vector populations e.g., . NeverthIn addition to the comparison between homogenous and point-source releases, we considered two different point-source configurations: a regular release pattern, where the distance between release sites is maximized, and a random release pattern, where release sites are selected at random. For instance, if sites vary in the amount of access individuals implementing the releases can expect to obtain, or if cost considerations require releases to be carried out near distribution centers located haphazardly throughout a neighborhood, the distributions of release sites may appear random with respect to the location of breeding sites. For simplicity, and to highlight the contrast between releases at a limited number of sites and releases at all sites, we assumed that the release sites would remain fixed throughout the duration of releases.The duration of releases was also varied across the different release strategies. As noted in Finally, because strong population reductions can amplify the contribution of wild-type migrants to gene frequencies in subsequent generations, we also assessed the effect of immigration by gravid females that lack transgenes into the population. Preliminary examination (not shown) indicated the qualitative effects of immigration to be consistent across different release strategies; thus, we illustrate our results for a release strategy consisting of the release of adult males in 10% of the sites over three years, with each release site regularly spaced apart. We model immigration by dynamically adding gravid wild-type females into randomly selected sites in the urban arena.We found that although releasing transgenic male mosquitoes caused considerable population reduction  of sites spread uniformly apart. We found that only if releases can be maintained for longer periods of time could the frequency of adult females carrying the anti-pathogen gene be raised to comparable long-term levels as when mosquitoes were released everywhere  and evenIncreasing the number of eggs released could correspondingly increase the number of runs where the frequency of the anti-pathogen gene was relatively high. However, we found that increasing the number of released eggs also substantially amplified the variability between runs . This inWe found that immigration by gravid, mature wild-type females could reduce the long-term frequency of females carrying the anti-pathogen gene considerably across a range of release numbers. Increasing both the number and frequency of daily migration events lowered the average frequency of females carrying the anti-pathogen gene across runs. Moreover, the effects of wild-type migrants were most pronounced when higher numbers of transgenic mosquitoes were released per week . This ocAe. aegypti populations by releasing engineered male mosquitoes carrying conditionally lethal elements is unlikely to be a realistic objective for large, heterogeneous urban areas Eliminating entire Our analysis yielded insights that could not have been anticipated without explicitly considering the effects of demographic stochasticity and genetic drift at small population sizes, and how these processes affect the genetic dynamics across space and time during population recovery. Our key finding is that the strong effects of genetic drift during periods of very low population abundance and the local persistence of wild-types strongly constrain the likelihood that such a release program could drive the anti-pathogen gene to fixation e.g., . These eIn contrast to studies with general deterministic models, results from the stochastic, spatially-explicit model used here provide a foundation for quantifying the uncertainty surrounding different release strategies. For instance, As with all modeling approaches, however, we excluded certain considerations in the interest of tractability and space. For instance, depending on the specific anti-pathogen transgene being considered, the fitness cost may operate during different stages of the mosquito life cycle than those considered here. Our model is not meant to describe the outcome of releases involving any specific anti-dengue transgene. If experiments were to show that the fitness costs associated with a promising anti-dengue transgene operate at different stages of the life cycle, then re-evaluating our model conclusions in light of such hypothetical results would be appropriate. If such fitness costs operate primarily at later life stages , then containers consisting of exclusively transgenic individuals might experience higher juvenile density dependence and potentially be less productive.Ae. aegypti populations can exhibit considerable micro-scale heterogeneity  have been released in field settings . Nevertheless, such female releases are not unprecedented. For instance, female mosquitoes infected with the intracellular bacterium gs e.g., . In the Wolbachia infection in all female mosquitoes have undergone field trials . The biology of Ae. aegypti occurs within local sites, represented here with four white rectangles. In our model, eggs (E), larvae (L), pupae (P) develop within individual containers, represented by smaller white triangles, squares and hexagons square, and hexagonal polygons that could potentially describe different container types. Grey circles represent individual adult females, and orange polygons and lines characterize male adult populations and their biology. Solid lines represent development, and dashed lines represent oviposition. Dash-dotted lines represent dispersal between sites, with blue dash-dotted lines representing adult female dispersal and green dash-dotted lines representing the movement of containers between sites. Although adult dispersal and container movement can occur among all sites in the Moore neighborhood, for clarity we only depict dispersal between specific sites in this schematic. Mating occurs among adult males and females in the same site, represented by the red arrows.(TIF)Click here for additional data file.Figure S2The frequency distribution across model runs for adult females carrying an anti-pathogen gene when transgenic adult mosquitoes are released according to a point-source release pattern at regularly spaced sites. Model runs resulting in population extinction are omitted from all panels, and the number at the top of each frequency bar represents the proportion of runs resulting in population extinction. The different panels represent releases of adults for (A) three years with only female adult releases, (B) a single year of only female adult releases, (C) three years of male and female releases, and (D) a single year of male and female releases. The frequency distribution for the female-only and male and female releases are quite similar, suggesting a limited impact of released males when females are released. Lines between the frequency bars represent changes in the average frequency of adult females carrying an anti-pathogen gene across runs that do not result in extinction. As in (EPS)Click here for additional data file.Figure S3Illustrative examples of the genotypic dynamics of adult females through space and time given the same spatial configuration of potential breeding sites across replicate model runs.(EPS)Click here for additional data file.Figure S4The genotypic dynamics through space given the same spatial configuration of potential breeding sites as in  The spatial trajectories of recovery differ considerably between runs, reflecting the influence of demographic stochasticity, spatial heterogeneity, and genetic drift; however, there is less variability between replicates for the overall frequency of the anti-pathogen allele. Nevertheless, even two years after releases end, considerable spatial differences persist between model runs in sites that have a high frequency of carriers of the anti-pathogen allele as a consequence of limited long-range dispersal and the use of non-periodic boundary conditions.(EPS)Click here for additional data file.Table S1A summary of initial conditions for the model.(PDF)Click here for additional data file.


In many neurological disorders strategies for a specific delivery of a biological activity from the periphery to the central nervous system (CNS) remains a considerable challenge for successful therapy. Reporter assays have established that the non-toxic C-fragment of tetanus toxin (TTC), provided either as protein or encoded by non-viral naked DNA plasmid, binds pre-synaptic motor neuron terminals and can facilitate the retrograde axonal transport of desired therapeutic molecules to the CNS. Alleviated symptoms in animal models of neurological diseases upon delivery of therapeutic molecules offer a hopeful prospect for TTC therapy. This review focuses on what has been learned on TTC-mediated neuronal targeting, and discusses the recent discovery that, instead of being merely a carrier molecule, TTC itself may well harbor neuroprotective properties. Neurodegenerative disorders are a group of pathological conditions that typically originate from progressive dysfunction and loss of neurons or synaptic contacts in defined areas of the nervous system. For example, Alzheimer’s, Parkinson’s and Huntington’s diseases affect distinct parts of the central nervous system (CNS), whereas motor neuron diseases such as Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) initiate peripherally by a loss of motor neural connection with the muscle. Although major steps in understanding the pathology and molecular mechanisms underlining these disorders have been taken, effective treatment has only been described in few. Besides the obvious task of finding the appropriate molecules or interventions with beneficial effect, one of the greatest challenges in development of treatments for neurodegenerative conditions relies on the fact that systemic delivery of potential therapeutics, such as growth factors or neurotrophins, may be ineffective due to short half-life and low bioavailability at the target tissue, as well as having potentially hazardous side effects ,2. The ein vitro and in vivo [Non-toxic tetanus toxin C-fragment (TTC) has been exploited as a molecular tool to specifically target motor neurons and has greatly facilitated studies of neuronal processes such as endocytosis, synaptic sorting and retrograde transport  in vivo . Molecul in vivo . This reClostridium tetani, an anaerobic bacterium whose spores are commonly found in soil and animal waste. TeNT is a causative agent of tetanus, a potentially fatal condition that affects the nervous system and is characterized by painful, uncontrolled muscle contractions [N and HC. The HN is thought to facilitate the translocation of the toxic L chain from endosomal compartment to the cytosol or otherwise modulate its trafficking route [C  is required for neuronal cell binding and retrograde transport [Tetanus neurotoxin  is a protein produced by ractions . It is sractions . Althougractions ,8. The Lractions ,10. Protng route . The 50 ransport ,13,14,15Neuronal transport , which nin vivo were limited by development of clinical tetanus and rapid death of the experimental animals [in vivo investigation of TTC internalization by neurons and their retrograde transport from the periphery to the central nervous system. Indeed, TTC has become an important tool to investigate neuronal physiology in a normal context and in conditions where neuronal function is affected by a disorder. Early studies of transsynaptic passage of radioactively labeled TeNT  animals ,30. Sepain vitro and in animal models of neurodegenerative diseases. Several molecules, including reporter genes and potential therapeutic molecules, have been successfully transported into neurons by coupling them with TTC . The usein vivo, the most common being intramuscular injection.Natural capability of TTC to employ neuronal retrograde transport machinery and transcytose to connecting higher order neurons has been exploited to investigate neuroanatomy and to trace physiological and molecular mechanisms of retrograde axonal trafficking. The applied methods include chemical or genetic conjugation of TTC to a histochemically of fluorescently detectable reporter molecules, as well as direct labelling of TTC with fluorochromes. Fluorochrome-labelled or chemically conjugated TTC-tracers may be delivered as protein injection. On the other hand, genetic fusion proteins have been administered either as a recombinant protein or as non-viral “naked DNA” plasmid . In the Xenopus [More than 20 years ago it was shown that intramuscular administration of recombinant TTC conjugated with horse radish peroxidase (HRP-TTC)  could reXenopus  and in mXenopus . It was Xenopus  and thatXenopus . Block oTo facilitate direct microscopic visualization, fusion protein of TTC and green fluorescent protein (TTC-GFP) has been successfully delivered to the neurons by using adenoviral vectors or intracerebral crafting of transfected neural cells , by tranin vivo retrograde transport to the CNS after intramuscular injection, as well as ex vivo histological mapping of the fate and transport rate of the tracer. Because of the non-toxicity and non-invasive administration, tracers of this class may become important agents for neurography in humans. For example, they may ease neuronal detection in complicated surgical procedures, aid visualization of neuronal circuits after injury, or function as a marker to evaluate therapeutic effects of neuroprotective of neurotrophic treatments.As for neuroanatomy, TTC has served as an important tool to investigate mechanistic aspects of neuronal traffic. Using paramagnetic iron beads coupled with TTC, it was shown  that smai.e., in a depolarization- and calcium-dependent manner [Less is known about the mechanism of how TTC and its fusion variants are transported from the motor neuron dendrites to the more central interconnected axons. The existing data suggests that the internalization is mechanistically different in the presynaptic terminals of the NMJs and in those of centrally located axonal projections, such as inhibitory interneurons . For exat manner ; whereast manner  and is nt manner . The quet manner ,23,25,26t manner . HoweverLost or altered metabolic enzyme activity underlies many neurodegenerative and neuromuscular diseases, including classical MNDs, some forms of peripheral neuropathy and neuronal lysosome storage diseases. When a single gene product is affected negatively by the condition, enzyme replacement therapy may offer a promising approach to compensate insufficient biological activity.in vitro [Tay Sachs disease (TSD) is an autosomal recessive human GM2 gangliosidosis characterized by lipid accumulation in the lysosomes of the nervous tissue and progressive deterioration of the brain and the spinal cord. TSD is caused by inactivating mutations in the gene for lysosomal GM2 ganglioside-degrading enzyme beta-hexosaminidase (HEXA) and represents an attractive target for enzyme replacement therapy. Human placental HEXA conjugated with TTC  was found to be efficiently internalized by rat brain cells in vitro . Furtherin vitro .In vitro, SOD1-TTC molecules are ineffective in protection of cells from starvation-induced oxidative stress, possibly due to their location in the vesicles [in vivo effects have not yet been reported. Because SOD1 mutants associated with ALS are not hypomorphic but instead gain-of-function, wild-type SOD1 expression in ALS may not be beneficial at least in familial ALS [Cu/Zn superoxide dismutase (SOD1) is a cytosolic enzyme that converts superoxide radicals to molecular oxygen and hydrogen peroxide and thus protects cells from oxidative stress. SOD1 is mutated in 20% of the familial ALS patients  and incrvesicles . Althouglial ALS ,55. Howelial ALS ,57 and, in vitro, it is not released from the endosomal containment [Although TeNT entry to the CNS is thought to be primarily mediated by binding to the motor neuron terminals at the NMJ, all neurons seem to be capable of binding both TeNT and TTC. Recombinant SOD1-TTC fusion protein used for a previous study  was alsotainment .Spinal muscular atrophy (SMA), the most common genetic cause of infant mortality , is a reNeuronal survival is dependent on constant exposure to neurotrophins which also mediate differentiation, growth, and apoptosis of neurons by binding to two types of cell surface receptors, the Trk tyrosine kinases and the p75NTR ,64. As min vitro and in vivo. CT1 improves motor function, increases survival and protects NMJs in progressive motor neuropathy (pmn) mice, after adenoviral or naked DNA mediated delivery [in vitro, whereas the glia, hepatocytes or cardiomyocytes did not bind or uptake the recombinant protein. CT1-TTC promoted motor neuron survival in a dose-dependent manner in vitro and induced typical CT1 responses, including IL-6 secretion and transcriptional activation of its known target genes. This implies that TTC does not interfere with the endogenous function of the cytokine and that CT1-TTC could serve as a candidate for in vivo studies. TTC-conjugation may be particularly relevant to CT-1 since, unlike some other neurotrophic factors, it is not transported to neuronal dendrites and may be subjected to rapid lysosomal degradation [Cardiotrophin-1 (CT1) is a cytokine of IL6 family that can increase motor neuron survival delivery ,66. Addidelivery . Howeverdelivery . Geneticradation , both ofin vitro [in vivo are still pending.Apoptosis plays a key role in death of neurons in neurodegenerative diseases . Findingin vitro . ImportaSeveral neurotrophic factors and growth factors have been suggested for the treatment of motor neuron diseases. In ALS patients, however, the repeated subcutaneous injection of these factors as recombinant proteins may be complicated by their systemic toxicity due to their tropism to a variety of tissues and a poor bioavailability at the motor neuron terminals ,74,75. Tin vitro and in vivo neuronal retrograde transport [Insulin-like growth factor 1 (IGF-1) promotes growth of both skeletal muscles and motor neurons, and muscle-specific expression of IGF-1 can provide trophic support to the motor neurons. IGF-1 has been shown to decrease disease pathology and progression in animal models of ALS  and spinransport . Furtherransport ,80. Howeransport . This isransport , suggestGlial cell-derived neurotrophic factor (GDNF) potently promotes the survival, proliferation and differentiation of various types of neurons, including motor neurons ,85,86,87Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of growth factors related to the canonical nerve growth factor, NGF. Genetic fusion of BDNF and TTC (BDNF-TTC) can induce neuronal survival Akt kinase pathway in mouse cortical culture neurons and inhibit apoptosis in mouse neuroblastoma cells . AlthougSignaling through phosphatidylinositol 3-kinase (PI-3K), followed by PI-3K-mediated activation of serine/threonine kinase Akt is the key regulator of neuronal survival ,100,101.Binding of TTC to rat brain synaptosome activates Trk-dependent signaling, which mimics the action of their natural neurotrophic ligands ,103. TTCin vitro studies. Parkinson’s disease is caused by degeneration of nigrostriatal dopaminergic neurons which results in dopamine deficiency and clinical features involving slowness of movements, tremor and rigidity. Parkinsonism can be induced in experimental animals by 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), whose metabolite MPP+ specifically destroys dopaminergic neurons. In cerebellar granule neurons in vitro, TTC protects against MPP+ by enhanced protection against apoptosis [in vivo by MPP+, striatal administration of TTC before MPP+ infusion resulted in significant amelioration of motor deficits and restored the dopamine levels to those of controls [The evidence pointing towards antiapoptotic properties of TTC is not only limited to poptosis . In a racontrols . These rper se had no effect on motor response, it suppressed characteristic hyperlocomotion in the post-ischemic animal [Neuronal death after ischemia, a condition that temporarily deprives the brain of oxygen and glucose, has been shown to rely on similar mechanisms observed in ALS patients including glutamate mediated excitotoxicity and oxidative stress. Similarly, cardiac surgery may lead to severe oxidative stress due to the formation of oxidation products generated during ischemia and reperfusion . In a moc animal . TherefoThe evidence above infers that TTC-based methods may serve as promising alternatives to viral gene delivery for neuronal gene therapy. However, before these therapies are applicable to humans, problems related to efficient delivery methods and potential immunogenicity of TTC need to be addressed. Additionally, there is a demand for optimization of the non-viral naked DNA method to increase expression levels which, at the moment, may not be sufficient for administration to large muscles . Finally, in future TTC-mediated neuronal gene therapy experiments, it is crucial to include careful controls to investigate the potential TTC-specific or synergistic effects with the passenger molecules.in vitro internalization and transport kinetics as well as activity of the passenger molecule before in vivo animal studies.Although clear benefits of TTC conjugation for efficient and specific neuronal targeting have been demonstrated, it is becoming clear that, the optimal organization of the fusion modules needs to be determined for each molecule. Genetic fusion of trophic factors or enzymes to the TTC may alter their conformation thus undermining the natural properties, for example, by blocking their active site by steric hindrance. Alternatively, the efficiency of the TTC to promote retrograde transport could be affected by the passenger molecule resulting in insufficient internalization or altered trafficking. For example, with different domain organizations of SOD1-TTC, the activity of the enzyme was affected but the internalization and transport in the neurons was not . In contper se is maintained. SOD1 is normally cytosolic, and its vesicular location (as is the case with SOD1-TTC) may prevent its efficient function in free radical defence [The question of how much the passenger protein may influence the intracellular location remains, because the internalized TTC is largely endosomal. TTC-containing retrograde organelles display a neutral pH  which pr defence . The sit defence ; whereas defence  may indi defence ,109. Thein vitro and in vivo, when delivered as protein or naked-DNA using various administration routes. However, membrane binding and internalization of TeNT holotoxin is more efficient than that of TTC alone [et al., 2009 – Abstract 38 at Lysosomal Disease Network World Symposium 2010) provided that these re-designed TTC-molecules retain their neurotropic and retrograde transport properties. TTC is truly neurotropic TC alone . Design TC alone . Most inTC alone . Rapid nTC alone . AdditioTC alone  (see alsAlthough rapid axonal transfer of TTC to the CNS, especially in the large-diameter fibers, has been demonstrated ,116, forin vitro activation of signaling pathways, as well as route and dose of administration in vivo, which may differ between laboratories. In future studies, it is advisable to include TTC itself as a control to preclude its effect from those of putative therapeutic molecules.Because TTC is able to stimulate signaling pathways that increase neuronal survival, the question remains of how much the reported effects in general can be ascribed to the passenger molecules and how much to the TTC-carrier itself. As proposed by Mendieta and coworkers , the antper se contribute to the potential therapeutic properties of TTC? Defects in retrograde transport may characterize many neurodegenerative diseases [Another, perhaps related question is: Does the stimulation of retrograde transport diseases ,117,118 diseases ,119. Thediseases  may faciin vitro [in vitro, where they can elicit the expression of plasmid-encoded gene [in vivo, these vectors have a substantial potential to serve as retrograde transport-exploiting drug/gene delivery vehicles that may reduce the potential hazards associated with systemic drug delivery.Finally, TTC may serve to target non-proteinous molecules, such as DNA or drugs, specifically to neurons. TTC-conjugated nanoparticles made from biodegradable polymers can selectively target neuroblastoma cells as opposed to liver or endothelial cells in vitro . Similarded gene . If showTTC offers a promising tool for specific targeting of neurons. It can be used either as a neuroanatomical and functional tracer, or administered directly by protein injection or by non-viral naked-DNA methods, to carry therapeutic biological activity into the CNS. Growing evidence suggests that TTC may itself harbor neuroprotective properties, which opens attractive avenues for future research on its potential use to alleviate neurological disorders. Future modifications of TTC molecule, as well as development of TTC-based therapeutic interventions, are likely to further enhance its efficacy and applicability as a molecular neurotrophic courier.


In contrast to most other cancer therapies, early research with oncolytic viruses (OV) tended to use direct or intratumoural routes of administration, initially motivated by concerns that the major limitation of intravenous delivery would be immune attack against the viruses, whether by complement, cytokine or most critically neutralising antibodies. Even in the absence of preformed circulating neutralising antibodies, antiviral immunity develops rapidly over the course of multiple viral administrations . PreclinGiven these perceived limitations, many have chosen direct administration to maximise intratumoural delivery: H101 (an oncolytic adenovirus with selectivity against p53-defective tumour cells) is approved in China and is given intratumourally for head and neck cancers. Several viruses have been administered into the tumour bed perioperatively for glioma. Measles has been administered intraperitoneally in ovarian cancer. One of the field-leading products is T-Vec (aka OncoVex), an engineered herpes virus. Its recent acquisition by Amgen was a fillip for those in the OV field, and was based on promising phase II data in melanoma where the virus was injected intratumourally into all accessible lesions ; a phaseWe previously recovered replication-competent reovirus from tumour following intravenous infusion in three patients with head and neck cancer,  a findinThese vital observations that two different viruses, administered intravenously, reach tumour tissues strongly support the further development of OV for systemic use in metastatic disease. However, neither of these studies completely addressed the issue of how a systemically administered virus might successfully run the gauntlet of anti-viral immunity.Reovirus is an endemic human pathogen and, therefore, neutralising anti-reovirus antibodies (NARA) are extremely prevalent in the population. In contrast vaccinia is not present in the environment and routine vaccination against smallpox ended in North America in the mid-1970s. In fact only one of the subjects treated with high-dose JX-594, in whom virus was detectable in the tumour, had pre-existing neutralising anti-vaccinia antibodies. In addition it is highly likely that successful virotherapy will require several doses of OV, which will induce high titres of neutralising antibodies in most patients.Against this background, we have recently confirmed successful intravenous delivery of reovirus in a population of pre-immune cancer patients . In a phex vivo-cultured carrier cells is an area that we and others have explored preclinically [in vivo following intravenous administration of neat virus. Further work to characterise and enhance the mechanism of viral carriage is underway, and will be critical to optimising systemic virotherapy.Importantly, all patients in the study had detectable neutralising antibodies before treatment and NARA titres peaked in all patients by the time of surgery. Accordingly, although reovirus was detected by PCR in the plasma of patients shortly after infusion, we were unable to recover replicating virus from plasma. This finding is compatible with NARA functionally neutralising the virus in the circulation. However we recovered replication-competent virus from the peripheral blood mononuclear cell, granulocyte and platelet fractions of the patients’ blood. These data indicate that virus hitchhikes on these circulating blood cells, circumventing the anti-viral immune response to access tumour. Hitchhiking virus onto inically ,8, but hIt is an exciting time to be involved in OV research. Preclinical developments continue to inform the clinical approach and provide novel targeted or armed agents for therapeutic testing . Clinica


Stimulant dependence is associated with neuropsychological impairments. Here, we summarize and integrate the existing neuroimaging literature on the neural substrates of neuropsychological (dys)function in stimulant dependence, including cocaine, (meth-)amphetamine, ecstasy and nicotine dependence, and excessive caffeine use, comparing stimulant abusers (SAs) to nondrug using healthy controls (HCs). Despite some inconsistencies, most studies indicated altered brain activation in prefrontal cortex (PFC) and insula in response to reward and punishment, and higher limbic and anterior cingulate cortex (ACC)/PFC activation during craving and attentional bias paradigms in SAs compared with HCs. Impulsivity in SAs was associated with lower ACC and presupplementary motor area activity compared with HCs, and related to both ventral  and dorsal  systems. Decision making in SAs was associated with low dorsolateral PFC activity and high orbitofrontal activity. Finally, executive function in SAs was associated with lower activation in frontotemporal regions and higher activation in premotor cortex compared with HCs. It is concluded that the lower activations compared with HCs are likely to reflect the neural substrate of impaired neurocognitive functions, whereas higher activations in SAs compared with HCs are likely to reflect compensatory cognitive control mechanisms to keep behavioral task performance to a similar level as in HCs. However, before final conclusions can be drawn, additional research is needed using neuroimaging in SAs and HCs using larger and more homogeneous samples as well as more comparable task paradigms, study designs, and statistical analyses. Substance abuse is characterized by recurring compulsive urges to use drugs, despite long-term negative consequences, which may include a wide range of psychological, social, and medical complications. Moreover, even after treatment and regardless of motivation to quit, relapse is common. In 2008 alone, over 700,000 people in Europe and over 3.5 million people in the United States were seeking treatment for problematic drug use  model of Habitual Behavioral Model emphasizes the importance of a switch from goal-directed behavior to habitual behavior during the development of drug dependence. Habitual behavior would be less sensitive to outcome values and would lead to loss of voluntary control and the development of compulsive behavior, such as compulsive drug use. The switch to habitual behavior would represent a progression from prefrontal cortical to striatal control and a switch from ventral to more dorsal striatal regions  and drut theory ). HoweveWhile early hypotheses were stated from a behaviorist and psychological point of view , and – to a lesser extent – nicotine and caffeine are associated with more severe neuropsychological impairments than alcohol, cannabis, or even opioid dependence , and positron emission tomography (PET). Functional MRI (fMRI) uses blood oxygenation level dependent (BOLD) contrast to visualize differences in regional brain activity, a technique with much higher temporal and higher spatial resolution than PET. Before the introduction of fMRI, [15O] PET was widely used to perform activation studies due to the relatively short half-life of 15O (122 s), permitting repeated task versus baseline scans during a single session. In contrast, the 18F-tracer fluorodeoxyglucose has a much longer half-life (about 110 min) and is therefore primarily used for resting-state studies. The latter were omitted from this review, as were single photon emission computerized tomography (SPECT) studies. Electroencephalography (EEG) studies were also excluded, because of inherent poor spatial resolution. Finally, diffusion tensor imaging provides a visualization of white matter tracts by measuring voxel-based diffusion coefficients of water in brain tissue and fDTI is based on the idea that changes in white matter axonal volume may accompany brain activation patterns , did not have a matched control group (n = 16), re-used an external (nonmatched) control group from a previous study (n = 2), did not match for alcohol and/or cannabis use (n = 7), or included other imaging techniques (n = 9).Searches yielded 107 articles, of which only 40 used functional neuroimaging. Another 27 potentially relevant articles were found through cross-referencing. Of these 67 articles, 37 were excluded because they included only polydrug users (15O] PET. The most frequently studied substance was cocaine (n = 17), followed by nicotine (n = 5), (meth-)amphetamine (n = 4), and ecstasy (n = 4). No studies were found in subjects with excessive use of caffeine compared with low or no caffeine consumers. For several details concerning the reviewed studies , four tables  feedback (reward) after a correct response or negative (monetary) feedback (punishment) following an incorrect response. Tasks that measure reward and punishment sensitivity include the stimulus-response learning task, and the probabilistic reversal learning task (PRLT), and a variety of gambling tasks which focus on processes like risk taking strategies regarding wins and losses, or on learning reward and punishment contingencies. The PRLT is a task in which the individual is required to adapt his or her response to changing contingencies (shifts) to win the largest amount of money. Tasks may feature several reward contingencies, representing high and low reward options or measure response differences during reward and punishment processing.On the PRLT, cocaine abusers made fewer response shifts to changes in reward contingency than HCs, indicating high response perseveration in cocaine abusers  and models with an important role for neurobiological changes in the PFC leading to persistent drug use ., which might be responsible for different findings when using these different paradigms.Attentional bias, craving, and relapse are presumably the most characteristic features of drug dependence. Drug abusers tend to direct their attention unconsciously to stimuli previously associated with drug use. Attentional bias may be due to enhanced sensitivity to drug-related rewards and constitutes a risk for the development of  cue-reactivity, which in turn may elicit craving, that is, a subjective feeling of intense need for the drug, which may ultimately lead to relapse  that occur in the presence of drug-related stimuli, and can ultimately lead to relapse. Cue-reactivity is generally investigated using a cue-exposure or cue-reactivity task. Unlike other neurocognitive tasks, cue-reactivity paradigms employed during functional imaging only require the participant to watch drug-related pictures or videos (without any cognitive effort), although some cue-reactivity tasks include easy binary tasks to control for attention differences, in which baseline trials are usually incorporated requiring similar motor responses.To date, there are no neuroimaging studies on attentional bias in SAs, and therefore, the studies in this paragraph are restricted to those on cue-reactivity.15O] PET. It should be noted that PET has lower spatial resolution than MRI, even when ROI are delineated on co-registered anatomical MRI scans, as in this study. Therefore, rCBF of the nucleus accumbens (NAcc) could not be assessed. A methodological problem was the small HC group  reward pathway), as well as in regions part of the visuospatial attention circuitry, compared to HCs  measure cognitive impulsivity by determining the individual's preference for an immediate small (monetary or drug) reward over a larger reward in the future. Using DDTs, some studies have shown that ecstasy use correlates with increased cognitive impulsivity  rather than a specific motor inhibition task showed clear differences between psychostimulant abusers and HCs regarding motor performance, suggesting an association with increased motor impulsivity. While one study showed a significant association between motor performance deficits in chronic crack cocaine abusers and decreased activity in the dorsal striatum  ACC, lateral PFC, and pre-SMA. These findings are corroborated by a volumetric study showing decreased gray matter volume of the ACC in addition to superior temporal regions, and insula in cocaine users amphetamine, and caffeine use. A general methodological issue is that most studies published to date do not sufficiently control for the duration of abstinence (or time since last use). In addition, conflicting findings have been reported regarding rostral ACC, which was found to be less active in one study .Methamphetamine-dependent users displayed higher delay discounting with difficult choices  versus easy choices, resulting in lower activations of the left DLPFC and intraparietal sulcus (IPS) compared with HCs  and the dorsal (control) circuit. Whether similar changes can also be found in other stimulant abuse populations, such as cocaine, MDMA, nicotine, or caffeine abusers is still unknown.Decision making, memory, working memory, attention, cognitive flexibility, conflict monitoring, and planning are often conceptualized as separate elements of executive functioning, generally linked to intact  PFC function (Decision making can be assessed using the Iowa Gambling task (IGT)  steps earlier (Immediate memory (and working memory: WM), is often assessed with the Wechsler Adult Intelligence Scale (WAIS) Digit Span or Memory Span task, requiring the person to remember a string of digits, letters or words. The N-back task is a continuous WM task which requires subjects to indicate whether the current letter matches the one from Attention is a complex process that can be divided in different aspects. For instance, sustained attention is the ability to maintain attention for a longer period on a certain task which can be measured using a sustained attention task, whereas divided attention is the ability to shift attention between different task demands. Cognitive flexibility or “set-shifting” is the ability to shift cognitive set depending on task demands  and is often assessed using the WCST (Planning ability is often measured using the Tower of London (ToL)  could not be assessed in this study, which awaits replication using an fMRI event-related design. In a [15O] PET study by 15O] PET, this latter study is particularly interesting because their decision-making task was specifically designed to exclude the possible confounding effects of differences in working memory load and visuomotor demands and because the task excluded the learning component  during the 2-back condition , and right precuneus compared with HCs amphetamine users  would reflect increased valuation of drug stimuli. Together, impaired prefrontal activity and overactivation of limbic structures would thus result in maladaptive (impulsive and compulsive) behaviors and risky sensations (craving) leading to persistent drug use and relapse into recurrent episodes of maladaptive drug use with long-term negative consequences. Aspects of the Incentive-Sensitization theory, with its emphasis on neurobiological changes paving the way to more persistent drug use, can only be tested in longitudinal/prospective studies of drug users and HCs, that are currently not available, but it stresses the important role of neurobiological changes in areas such as the dorsal PFC, dorsal ACC, and various limbic structures, that is, increased or decreased responsiveness of brain circuits, related to the repeatedly observed changes in the function of these brain areas (regardless of cause or effect). Finally, the Habitual Behavioral Model stresses impulsivity and decision related changes that were observed in the dorsal system  and the more ventral regions . However, with the exception of a single study , 2010. SAs was discussed previously, inconsistencies in findings may at least partly be explained by methodological heterogeneity, stressing the need for similar neurocognitive tasks and experimental procedures. Neurocognitive tasks are continuously altered and improved to meet new research questions. Such continuous modifications, while helpful for further research, also limit comparability across studies, which is problematic when only a limited number of studies in SAs with HCs are available. Note also that test–retest reliability for fMRI designs such as reward paradigms, while acceptable at a group level, are moderate at best for single subjects . More generally, the BOLD fMRI technique has several limitations, such as susceptibility to signal distortion and dropout in the vicinity of bone-air transitions, such as the nasal sinuses, resulting in poor sensitivity to detect activity in, for example, medial OFC. Also, while BOLD fMRI is predicated on the assumption of increased regional perfusion being associated with greater neural activity, this neurovascular coupling may be compromised in elderly people but also following drug intake  data. In studies in which similar performance on neurocognitive tasks was observed between HCs and SAs, or task specifics were manipulated to obtain similar performances, differences in regional activations are usually explained by some kind of compensation hypothesis, stating that higher activations coupled with similar performance may result from decreased neural efficiency coupled with compensatory mechanisms, so-called “compensatory scaffolding”  and possibly also for other psychotropic drugs  and for behavioral addictions (e.g., de Ruiter et al. This review has both strengths and limitations. The main strengths include the careful selection of studies including only papers with SAs and HCs, the thematic ordering of the studies using integrated addiction models as the organizing principle, and the detailed description of the study populations and the tasks that were used in the selected studies. The review also has limitations. First, although we aimed to exclude studies in polysubstance users, most SAs were also smokers so that effects of nicotine could not be excluded. Second, many studies failed to adequately report the duration of substance use, so that correlations between abuse duration and morphological and functional brain abnormalities could not be assessed. Third, gender distribution was often unequal in the study groups, which is likely to be relevant because significant sex differences have been found in brain responses in HCs as well as in patients with stimulant dependence , 2010, tThe findings in this review are potentially important in the development of new interventions for the treatment of patients with a stimulant use disorder as both existing and novel neuromodulation techniques are currently implemented and tested in addiction treatment settings. Existing techniques include EEG neurofeedback (e.g.,


The common preference of cancers for lactic acid-generating metabolic energy pathways has led to proposals that their reprogrammed metabolism confers growth advantages such as decreased susceptibility to hypoxic stress. Recent observations, however, suggest that it generates a novel way for cancer survival. There is increasing evidence that cancers can escape immune destruction by suppressing the anti-cancer immune response through maintaining a relatively low pH in their micro-environment. Tumours achieve this by regulating lactic acid secretion via modification of glucose/glutamine metabolisms. We propose that the maintenance by cancers of a relatively low pH in their micro-environment, via regulation of their lactic acid secretion through selective modification of their energy metabolism, is another major mechanism by which cancers can suppress the anti-cancer immune response. Cancer-generated lactic acid could thus be viewed as a critical, immunosuppressive metabolite in the tumour micro-environment rather than a ‘waste product’. This paradigm shift can have major impact on therapeutic strategy development. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Deregulated proliferation of cancer cells is generally associated with altered energy metabolism. Glucose is a primary source of energy. Under aerobic conditions, normal cells metabolize glucose to pyruvate via glycolysis in the cytosol, and subsequently convert pyruvate to carbon dioxide in the mitochondria for oxidative phosphorylation; under anaerobic conditions, conversion of pyruvate to lactic acid is favoured with relatively low amounts of pyruvate being diverted to the mitochondria. In contrast, cancer cells primarily derive energy from glucose via glycolysis to lactic acid, even under highly aerobic conditions, a property first observed by Otto Warburg The preference of cancers for aerobic glycolysis over the more energy-efficient oxidative phosphorylation pathway has been a subject of major interest since it was first observed in the 1920s Tumours commonly encounter fluctuating oxygen levels, periodically alternating between normoxic and hypoxic conditions Ras, Akt, and MycComponents of glucose uptake and glycolytic pathways can be up-regulated by oncogenes such as Increased glutaminolysis would also have several advantages for cancers: glutamine is the most abundant amino acid in plasma and forms an important additional energy source in tumour cells, especially when glycolytic energy production is low; increased availability of the degradation products of glutamine, ie glutamate and aspartate, as precursors for nucleic acid and serine synthesis; and the insensitivity of glutaminolysis to high concentrations of reactive oxygen species Of major interest is the finding that the reprogrammed energy metabolism plays an important role in cancer growth-promoting angiogenesis. Angiogenic endothelial cells, like tumour cells, are largely dependent on aerobic glycolysis and increased glutaminolysis for energy. The preference for these pathways allows the development of neovasculature from endothelial, tumour-blood-vessel-lining cells under hypoxic conditions. In addition, lactic acid generated by the pathways has been found to markedly promote angiogenesis by increasing the production of interleukin-8/CXCL8, driving the autocrine stimulation of endothelial cell proliferation and maturation of new blood vessels Recent studies indicate that altered energy metabolism can also enhance the growth of cancers by promoting tumour evasion of immune destruction. It is now commonly accepted that a transformed, immunogenic cell can only develop into a tumour if it has the ability to evade the cytotoxic immune response that it evokes The primary cause responsible for the acidic pH and pH-dependent T-cell function-suppressive effect in a tumour micro-environment has been identified as lactic acid In view of the above, researchers are starting to attribute a critical, regulatory role to lactic acid rather than regarding it as a mere metabolic waste product As previously reviewed, there apparently are various ways by which epithelial cancers can suppress an anti-cancer immune response Aerobic glycolysis and increased glutaminolysis are preferred metabolic energy-generating pathways of cancer The restoration of the anti-cancer immune response may be accomplished not only through targeting of the two pathways leading to inhibition of lactic acid production Altered glucose/glutamine metabolism is only one aspect of tumour metabolism. In addition to lactic acid, other metabolites that suppress T-cell function can be generated by tumours via alterations of lipid and adenosine metabolisms, eg indolamine-2,3-dioxygenase, arginase, inducible nitric oxide synthetase, and lactate dehydrogenase, important players in immune escape mechanisms. Pharmacological blockage of such metabolites could lead to improved cancer therapy by rescuing the endogenous immune response against tumour cells Lactic acid, commonly generated by cancers via reprogrammed energy metabolism , has a critical role in their growth as an immunosuppressive metabolite as well as a promoter of angiogenesis. Targeting altered, lactic acid-producing metabolisms of cancers may therefore lead to inhibition of their growth not only via energy/nutrition deprivation, but also through reduction of their immunosuppressive activity in the tumour micro-environment. This strategy, which simultaneously targets two fundamental cancer characteristics , could be effective for the therapy of a broad range of cancers and could be particularly useful in combination with cancer immunotherapy.


Views on the evolution of immunity have been redefined as studies in protostomes, invertebrate deuterostomes, and various vertebrates have elucidated molecular details of host defense  Brent, . These eCiona intestinalis. Only two TLRs can be detected in this species, with presumed hybrid functionality in vitro  is involved.Extensive variation in immune genes traditionally has been equated with the immunoglobulin and T cell receptor gene loci in B and T lymphocytes, respectively, as well as in some MHC loci Hughes, . Recent Drosophila melanogaster (fruit fly) uses complex alternative RNA splicing to diversify the Down's syndrome cell adhesion molecule (DSCAM), a multiexonic receptor implicated in neuronal patterning , a member of the alkaline phosphatase (Alp) family. One possible role for these molecules is the detoxification of LPS, which in turn minimizes innate responses to commensal or beneficial microbial communities (Beumer et al., Phylogenetic considerations, including the use of non-traditional models, have been instrumental in forging new thinking among immunologists (Loker et al.,


Objective. We describe the study design and evaluate the implementation, feasibility, and acceptability of an ecological momentary assessment (EMA) study of illicit drug users. Design. Four sequential field trials targeting observation of 30 individuals followed for a four week period. Participants. Participants were recruited from an ongoing community-cohort of current or former injection drug users. Of 113 individuals enrolled, 109 completed study procedures during four trials conducted from November 2008 to May 2013. Methods. Hand-held electronic diaries used in the initial trials were transitioned to a smartphone platform for the final trial with identical data collection. Random-prompts delivered five times daily assessed participant location, activity, mood, and social context. Event-contingent data collection involved participant self-reports of illicit drug use and craving. Main Outcome Measures. Feasibility measures included participant retention, days of followup, random-prompt response rates, and device loss rate. Acceptability was evaluated from an end-of-trial questionnaire. Sociodemographic, behavioral, clinical, and trial characteristics were evaluated as correlates of weekly random-prompt response rates ≥80% using logistic regression with generalized estimating equations. Results. Study participants were a median of 48.5 years old, 90% African American, 52% male, and 59% HIV-infected with limited income and educational attainment. During a median followup of 28 days, 78% of 11,181 random-prompts delivered were answered (mean of 2.8 responses daily), while 2,798 participant-initiated events were reported . Self-reported acceptability to study procedures was uniformly favorable. Device loss was rare (only 1 lost device every 190 person-days of observation). Higher educational attainment was consistently associated with a higher response rate to random-prompts, while an association of HIV infection with lower response rates was not observed after accounting for differences in trial recruitment procedures. Conclusion. Near real-time EMA data collection in the field is feasible and acceptable among community-dwelling illicit drug users. These data provide the basis for future studies of EMA-informed interventions to prevent drug relapse and improve HIV treatment outcomes in this population. Optimal HIV care requires prompt identification of HIV infection, linkage to HIV care, prolonged engagement in HIV care with regular attendance at appointments, and high levels of adherence to antiretroviral regimens in order to achieve viral suppression . IllicitThe promise of mobile health  technologies for strengthening HIV care delivery has been acknowledged by the United Nations Joint Programme on HIV/AIDS (UNAIDS) strategic plan . Text meIn response to the limited application of EMA approaches to out-of-treatment, high-risk populations, we sought to develop EMA methods for near real-time characterization of illicit drug use occurring in users' natural environments. In this paper, we describe the study design and participants of the EXposure Assessment in Current Time (EXACT) study. Further, we characterize implementation barriers and examine the feasibility and acceptability of using intensive EMA data collection methods among community-dwelling illicit drug users with or at-risk for HIV infection.EXposure Assessment in Current Time (EXACT) study participants were recruited from the AIDS Linked to the IntraVenous Experience (ALIVE) study, an ongoing, community-recruited, observational cohort of persons with a history of injecting drugs in Baltimore, MD . The ALIThe Johns Hopkins School of Public Health approved the study protocol. An existing Certificate of Confidentiality through the National Institute on Drug Abuse was amended to include EXACT study procedures. All participants provided written informed consent. Participants in the EXACT study were informed that involvement (or noninvolvement) in EXACT would in no way affect their participation in ALIVE.Funding for the study was provided through the National Institute on Drug Abuse as part of the Network on Exposure to Psychosocial Stress and Addictive Substances, a component of the NIH's Genes, Environment and Health Initiative .Current EMA methodology generally employs electronic diaries or mobile devices to collect data on both a random and an event-contingent schedule. For random-prompt data collection, EXACT participants responded to an alarm that sounded at a set number of randomly spaced times throughout the day by answering a questionnaire on the device. For event-contingent data collection, participants self-initiated a questionnaire in close temporal proximity to an event of interest ; participants were required to confirm that the event occurred within the last 30 minutes. http://www.satelliteforms.net/). To reduce the street value of handheld devices, all PDA programs were disabled except for study-required applications. In Trial 4, participants were provided an Android smartphone (Motorola Droid X2), running the electronic Mobile Open-source Comprehensive Health Application (eMOCHA), developed at Johns Hopkins School of Medicine [EXACT participants were provided a hand-held electronic device to carry for four weeks for real-time data collection. For Trials 1–3, participants were provided PDAs  running applications developed using Satellite Forms software -based version of the end-of-day questionnaire modified to reflect activities, behavior and events during the prior week. Similarly, at the conclusion of each trial, participants completed an ACASI-based questionnaire designed to reflect the entire four week study period. Hair and/or sweat patch samples were collected weekly for measurement of illicit substances, and blood samples were collected at the end of the trial period to be tested for potential biomarkers of psychosocial stress. Participants received remuneration for attendance at study visits, for providing adequate responses to weekly random-prompts, and for returning devices upon study completion. Participants were informed at entry that loss of two study devices would result in dismissal from the study.Baseline sociodemographic , behavioral , and clinical  characteristics were obtained from the existing ALIVE database. Depressive symptoms were assessed using the Center for Epidemiologic Studies Depression Scale (CES-D) for the 6 months prior to EXACT study entry.Characteristics of participants, days of followup, random-prompt response rates , and device loss rate were examined by trial number. Using a response rate of ≥80% to weekly random-prompts as a dichotomous outcome variable, logistic regression models with generalized estimating equations (GEE) were evaluated to identify the sociodemographic, behavioral, clinical, and trial-related correlates of higher response rates. Analyses were performed using Stata statistical software (version 11). We analyzed data from 109 participants who had at least one week of follow-up. Nine participants were followed less than the full four weeks possible . For 109Overall, the 109 participants provided 3,047 days of observation  with little variability between trials . A totalThe overall proportion of random-prompts responded to was 78%, which translated to an average of 2.84 random-prompt responses per day. The response rate was relatively consistent in Trials 1 and 2 but was lower in trials which targeted more intense, recent drug users . Despite the lower response proportion in Trial 4, the more efficient delivery of prompts by smartphone translated into a greater average number of prompts answered daily . Problems in the PDA studies including technical problems, battery outage, and device loss or damage resulted in the loss of several days' data collection. Further, the data collection software required that the data be manually uploaded using a USB cable from the PDA to the computer where the database was located. The eMOCHA software eliminated this requirement by transmitting the data wirelessly and securely to a server approximately every 15 minutes. Recognizing concerns that exhaustion with responding to device prompts may occur, we examined the response rates and average daily number of prompts answered by week of trial . OverallRegarding event-contingent data, there were 2,798 participant-initiated events reported, representing 70% drug craving and 30% drug use events . Drug crOf 201 devices issued to participants, 15 were lost or damaged which translates to the loss of a device once every 190 days of observation. Only two participants (1.8%) were excluded from continuing in the study because they lost two devices. Monetary incentives were provided for active participation, which averaged about $15 per day of observation. In assessment of participant acceptability , favorabTo understand what factors may contribute to higher levels of active participation in EMA studies, we examined correlates of responding to ≥80% of weekly random-prompts . Of 405 Trials 3 and 4 were associated with lower responses , Model BIllicit drug users are considered a challenging population to identify, recruit, and retain in epidemiological, behavioral, or clinical research. Consequently, many issues of primary concern to this population remain poorly understood, such as why some active drug users are able to maintain cessation and do well on HIV treatment while others have difficulty with drug relapse, ART nonadherence, and virological failure. By employing multiple methods to maximize retention, including strong rapport among clinic staff and participants, mailed appointment reminders, phone calls, tracing through contacts, street tracing, transportation vouchers, and minimal financial remuneration for participation, the ALIVE study has demonstrated the ability to successfully follow an IDU population over >25 years in a design incorporating study visits conducted every six months. Further, ALIVE represents a community-recruited cohort following IDUs outside of the provision of clinical care in research clinics situated in residential and commercial sections of East Baltimore rather than physically colocated in buildings of the Johns Hopkins Medical Institutions. Despite these successes, there clearly is potential for extremely valuable information on the proximate influences of drug users' behavior to be gained by refining the data collection window to minutes instead of months and of locating data collection to occur wherever persons may be, rather than relying on retrospective reporting in a formal clinic setting. The primary objective of the EXACT study was to develop EMA methods for real-time characterization of illicit drug use occurring in users' natural environments.In this paper, we provide strong data supporting the feasibility and acceptability of using EMA methods for data collection among illicit drug using populations. Our recruitment and referral process from ALIVE was highly efficient. With inclusion of a dedicated screening visit, which served to ensure attendance at an additional study visit prior to enrollment and to gauge potential participants comfort level with the technology, we had a 93% rate of study completion. During the process of implementing the study, we were frequently met with healthy skepticism from other researchers regarding the likelihood of success in meeting our objectives. Our study protocol included 5 daily random-prompts, which represents a higher participant burden than many EMA studies. A recurring question from colleagues and IRB reviewers was whether illicit drug users would really answer all those questions. Overall, EXACT participants answered 78% of random-prompts, a response rate comparable to EMA studies performed using similar technologies in varied settings. Prior EMA studies in diverse populations including illicit drug users, chronic pain patients, and smoking cessation or obesity intervention participants achieved response rates to random-prompt surveys ranging from 70–80% –26. In qWe examined correlates of higher response rates and found that higher educational level was a strong and consistent predictor, while age, race, gender, and income level were not associated. Our study population had low overall educational attainment with only 41% completing high school or equivalency. It is unclear whether this association represents more ease or familiarity with the technology among more educated persons or whether education status is a surrogate marker for other factors which may facilitate high responses. In a recent survey conducted in ALIVE, we found that participants with greater education reported increased ownership of smartphones and an increased willingness to receive health-related communications through various technologies. Educational attainment is strongly associated with health literacy, defined as the ability to obtain, process, and understand the basic health information and services needed to make appropriate health decisions , 28. TheWe provided modest weekly incentives to participants for providing more complete responses to EMA questioning. Our lack of association of response rates with income and the finding that higher educational attainment (which would be expected to be associated with higher income) were associated with higher responses provides indirect evidence suggesting that the level of incentives was not coercive to vulnerable participants with limited income. Future studies will need to examine further the efficacy and ethics of providing incentives for improving EMA responses and to establish what type and level of incentives may be most appropriate. One EMA study has supplemented response-weighted incentives with weekly sessions between participants and research staff to review their individual response rates and emphasize compliance to obtain even higher levels of response . In addition to higher educational attainment, our analysis of response rates to the random-prompts indicated that the later trials that selectively recruited HIV-infected participants with more intense recent substance use had lower response rates. This raises concern that the targeted population of out-of-care HIV-infected persons with detectable viremia and active illicit drug use may be less likely to actively participate in similar studies or interventions. However, it should be noted that after accounting for the differential recruitment criteria between trials, the association with HIV infection was no longer observed. Further, the mean number of daily responses was actually higher among HIV-infected participants. Ultimately, larger and longer trials will likely be needed to refine how those populations at greatest risk will engage and respond to EMA studies. Another challenge to using technological devices among drug user populations is concerned that participants might simply sell the devices. To mitigate this potential problem, we clearly delineated to participants during screening and enrollment procedures that the loss of two devices  would result in their exclusion from the study. Further, we provided a small incentive for return of the devices upon completion of the study. Remarkably, only two persons  were excluded for lost devices. Overall, we averaged only one lost device per every 190 days of observation, which we feel represents an effective utilization of these technologies. We acknowledge that the street value for the PDAs is less than for the smartphones; however, in our limited data, there was not a substantial difference in the device loss rate between trials using the different devices. While the PDAs had nonstudy features locked out, we did not implement similar restrictions with the smartphones. Current technologies allow the remote inactivation of smartphones if they are stolen or misplaced which could represent an important deterrent, although this capability was not disclosed to participants nor employed in our study. Contemporary mHealth technologies are evolving at a rapid pace. In our study, the PDA model we used at study initiation was no longer being manufactured after 10 months of study recruitment and was largely obsolete by the date of study completion. Rapid transitions in technology, therefore, represent a significant barrier to successful implementation of mHealth programs, often limiting the interpretability and applicability of data from these studies. Our smartphone platform resulted in more efficient delivery, a higher average number of daily responses, and provided automatic data transfer which both enhanced security and minimized data loss. In moving forward with advancing technology, alternative approaches to trial design must be considered, including designs fitted to the stage of technology or intervention development and adaptive trials that allow for evolving technologies or intervention optimization . In addition to the challenges described above, our study had other limitations. In these field trials designed to provide information on feasibility and acceptability, we followed a limited number of persons for approximately four weeks. It has been well recognized that many intensive data collection interventions may plateau or reach exhaustion within weeks or months, diminishing the level of participation and success of these strategies. Importantly, in our examination of weekly response rates, we did not find evidence for exhaustion. In the future, the broader goal of EMA studies would be to provide longer-term monitoring or intervention through trials that extend beyond our limited time frame. Despite our study being nested within an IDU cohort, many participants were not actively using drugs. In these early studies, we selected for a range of drug use intensity and the resulting cohort overall would not be considered heavy users. Therefore, our findings may not extend to drug users with more intense drug use patterns.Finally, EXACT participants were largely African American from a single urban center, raising concerns regarding the generalizability of our findings. However, our IDU population represents among the most disadvantaged and vulnerable drug using populations, and our demonstration of successful implementation of EMA methods in this population bodes well for efforts to apply these methods to less challenging populations. Further, the aging, African American IDU epidemic in Baltimore is similar to many other urban IDU epidemics in the USA, from Newark to St Louis to Detroit. It is these populations that are at greatest risk for limited access to appropriate drug treatment or HIV care that are most in need of innovative and tailored interventions to improve outcomes. In conclusion, findings from the EXACT study demonstrate that EMA methods are feasible and acceptable approaches for data collection among illicit drug users. As the next important step, interventions that leverage the predictive value of intensive EMA data collection to allow real-time tailored interventions in drug users' natural environments  will need to be developed and rigorously evaluated. The translation of intensive EMA data into tailored EMI holds promise for improving our understanding and our ability to reduce relapse, improve engagement in care, enhance ART adherence, and to intervene on other difficult issues surrounding drug use and HIV care.