For complex disorders such as alcoholism, identifying the genes linked to these diseases and their specific roles is difficult. Traditional genetic approaches, such as genetic association studies (including genome-wide association studies) and analyses of quantitative trait loci (QTLs) in both humans and laboratory animals already have helped identify some candidate genes. However, because of technical obstacles, such as the small impact of any individual gene, these approaches only have limited effectiveness in identifying specific genes that contribute to complex diseases. The emerging field of systems biology, which allows for analyses of entire gene networks, may help researchers better elucidate the genetic basis of alcoholism, both in humans and in animal models. Such networks can be identified using approaches such as high-throughput molecular profiling  or strategies referred to as genetical genomics, such as the mapping of expression QTLs (eQTLs). Characterization of gene networks can shed light on the biological pathways underlying complex traits and provide the functional context for identifying those genes that contribute to disease development. The multiple genetic, environmental, and behavioral factors that play a role in the development of alcohol use disorders (AUDs) make it difficult to identify individual genes linked to these disorders. Nevertheless, some genetic risk factors  associated with AUDs have been identified within many genes, some of which code for proteins involved in known biological pathways. Despite this progress, it has been exceedingly difficult to determine which genes may be the most relevant to developing therapeutic interventions for alcoholism. The major obstacles in treatment development are that gene–disease associations reveal very little about the underlying biology and that any implicated gene variant explains only a tiny proportion of an individual’s overall risk for an AUD. Recent work focusing on the study of alcohol-related gene networks is helping to shed light on the molecular factors affecting alcoholism and other complex diseases. This article will provide an overview of approaches used to identify or construct gene networks and describe how systems biology approaches are helping to better understand complex traits such as behavioral responses to beverage alcohol  and alcoholism.The predominant experimental strategy used by contemporary geneticists to identify the genetic factors involved in complex traits, such as behavioral responses to alcohol, essentially is an expansion of the gene mapping approach proposed by A similar forward-genetics approach that most often is used for studying animal models of complex traits is called quantitative trait locus (QTL) mapping. A quantitative trait is a phenotype that is determined by several genes, each of which has a variable contribution to the trait. The locations of the involved genes on the chromosomes are referred to as QTLs. QTL mapping studies typically are conducted using inbred strains of mice and their various derivatives. For example, the C57BL/6J (B6) and DBA2/J (D2) inbred mice frequently are used in alcohol research because they clearly differ in various responses to alcohol, including development of functional tolerance , locomotSimilar experiments also can be conducted with special derivatives of inbred strains known as recombinant inbred (RI) mice. These animals are derived by cross-breeding two or more distinct parental strains (which often diverge widely for the trait of interest), followed by inbreeding of the offspring for several generations . Given tThe molecular and genetic resources outlined above have greatly increased the power and resolution of QTL mapping for various behaviors or other traits of interest. Yet despite these advances, the DNA regions identified as QTLs typically still are relatively large and may contain several genes; accordingly, few genes have been validated as contributing to quantitative traits . This difficulty is attributable largely to the lack of sufficient recombination events in existing mouse panels to reduce the size of DNA segments that typically are inherited together  for fine mapping and to the generally small effect size for any single QTG.Because of the technical obstacles impeding their more effective use, both GWASs and QTL mapping studies to date have identified a deluge of disease-associated genetic loci but only few actual causal genes. Moreover, even the most successful studies have failed to place the disease-associated genes in any kind of biological context that would serve to explain the underlying functional biology. Without elucidating the complex interactions of the molecular phenotypes that stand between genetic variation and disease, it will be difficult or impossible to develop new and effective approaches to treating such diseases.The emerging field of systems biology is tackling this immense challenge by studying networks of genes, proteins, metabolites, and other biomarkers that represent models of genuine biological pathways. Studying complex diseases in terms of gene networks rather than individual genes or genomic loci should aid in uncovering disease genes. With this approach, the effects of multiple genes in the network are combined, producing a stronger signal and reducing the number of statistical tests of association that must be performed.These benefits effectively were demonstrated in two recent human association studies that modified the typical GWASs strategy by seeking associations only within groups of functionally related genes, rather than across the entire genome. The first of these studies  discoverNetwork-based approaches to the dissection of complex diseases also can be applied to animal models, yielding experimental results that are more generalizable to humans because the pathways represented by these networks are more evolutionarily conserved than individual genes. This should encourage greater collaboration between researchers studying a common disease in different species. In fact, the biology underlying gene networks is so complex that any hope of deriving novel therapeutics may be entirely contingent on the extent to which scientists with diverse areas of expertise are willing to share and integrate datasets and make the process of interpretation a collaborative one.As the human and mouse genomes were being assembled using the cutting-edge, high-throughput DNA sequencers that made these endeavors possible, new technologies began to emerge that, for the first time, allowed near-comprehensive profiling of other cellular components. The term profiling refers to the measurement of different types of biological molecules, such as DNA to identify polymorphisms, messenger RNA (mRNA) to determine transcript abundance, proteins to identify certain chemical modifications that occur after the initial protein synthesis, and metabolites to evaluate biochemical processes in the cells. Platforms for high-throughput approaches for all these types of molecular profiling have become increasingly commonplace. Concurrently, methods for analyzing data produced by these technologies constantly are evolving, yielding results that are simultaneously more sensitive and more specific. As a result, researchers are better able to appreciate systems-level changes associated with disease.Of these various high-throughput profiling techniques, microarray-based gene expression platforms have featured most prominently in biomedical research to date. Through an unbiased profiling of the transcriptome—that is, a measurement of all mRNA molecules produced within a cell or tissue sample—microarray expression studies allow researchers to identify patterns of gene expression associated with a disease. In some cases, such patterns can better define a complex phenotype by identifying disease subtypes. For example, microarray analysis of breast cancer tumors identified gene expression signatures that predict patient prognosis and therefore help physicians tailor treatment regimens . From a Genomic approaches have been applied to alcoholism directly by studying postmortem human brain tissue isolated from alcoholics and matched control subjects using gene expression microarrays. This has revealed novel information about changes in the brain’s transcriptome that are associated with chronic ethanol consumption. One of the findings was a significant deregulation of genes encoding proteins that synthesize and maintain myelin, the substance that forms a sheath surrounding the long extensions  of nerve cells and that is essential for effective nerve signal transmission . HoweverAnimal models can assist greatly in this analysis by allowing for experiments that are far more detailed and informative but too invasive to ever be performed with humans. Although animal models could never replicate a phenotype as complex as alcoholism, they can mimic certain facets of the trait, which then can be associated with specific expression signatures using gene expression microarrays. For example, a genetic predisposition for alcoholism may entail a stronger-than-average preference for alcoholic beverages. This particular facet of alcoholism is captured by rodent models that selectively were bred to maximize a penchant for or aversion to ethanol, such as the aptly named high-alcohol preference (HAP) and low-alcohol preference (LAP) mice . In ordeAnother important facet of a genetic predisposition to alcoholism is a comparatively blunted sensitivity to the effects of ethanol. Studies have shown that people who initially are less sensitive to acute ethanol exposure are more likely to have a family history of alcoholism and are at greater risk for developing an AUD , 1994. AIn examining the responses to acute or chronic alcohol exposure in rodent brains, these and numerous other genomic studies have enhanced the understanding of the “ethanol transcriptome” and provided a more comprehensive picture of the genes and molecular pathways that contribute to specific facets of AUDs than what is possible with studies of postmortem human brains . MoreoveThe previous section mentioned several studies that used gene-expression microarrays to define lists of genes responding to ethanol or otherwise relevant to AUDs. Although these studies have provided important biological insights, the question of how such lists can be used to further advance understanding of a complex disease is not easily answered. Network-based approaches can greatly improve the interpretability of differential gene-expression results by providing information about the relationships between genes.Networks are systems of interconnected components. For example, the World Wide Web is a global network of computers sharing documents connected by hyperlinks; road maps are renderings of city networks connected by highways; social networks are groups of people connected through friendships; cellular signaling pathways are groups of proteins connected through molecular interactions; et cetera. Placing such complex systems within a network framework makes it possible to formally analyze the relationships that constitute these systems. Gene networks typically are visualized as mathematical graphs—that is, a collection of vertices and edges, where genes are represented by nodes and the lines connecting the nodes indicate that some relationship exists between the genes.Many published network analyses of gene groups use information about pre-existing biological relationships, which may be derived from sources such as literature co-citation analysis , protein–protein interaction databases, or gene ontology groupings. Some commercial tools are available for such studies, such as Ingenuity Pathway Analysis . However, although these sources provide categories for interpreting the genomic data, they also force such interpretation into the mold of pre-existing information, thereby partially defeating the goal of genomic studies.Genomic data collected with high-throughput molecular profiling presents the opportunity to derive novel gene–gene interactions. The maturity of gene expression microarrays relative to similar technologies designed to measure other molecular phenotypes on a genomic scale has meant that gene networks primarily are rendered as gene coexpression networks. In the context of gene coexpression networks, links between nodes typically indicate that the expression levels for two genes are strongly correlated with one another across whatever conditions an experiment entails . Each link in a gene network essentially represents a testable hypothesis that can be validated through follow-up molecular experiments. Indeed, coexpression networks have been used to identify protein interactions that are novel  and consVarious novel and innovative methods exist for generating gene coexpression networks. Although a comprehensive review of these methods is beyond the scope of this article, a few select methods are described in more detail in the Wolen and colleagues (in press) have attempted to better define the meso-corticolimbic reward pathway’s transcriptional response to acute ethanol exposure by expanding the original B6/D2 study  to incluA variety of calculations can be used to gauge the relative importance of a particular gene to the network as a whole . The simMbp, Mobp, Mal, and Plp1 is of particular interest, because these genes all play a role in the formation and stabilization of myelin. In addition, a parallel analysis was conducted using microarray data only from the prefrontal cortex of the same BXD strains after they had received an injection of 1.8 g/kg ethanol into their abdominal cavity. This analysis revealed that the myelin gene network persisted but underwent minor topographical modifications. Most notably, additional connections were detected between Plp1 and two additional genes called Mog and Lpar1. The absence of Mog in the original network probably was an artifact of the method used to form these networks, and the gene likely should have been included. Plp1 and Lpar1, in contrast, were effectively unrelated at baseline and only showed evidence of coregulation after ethanol treatment, suggesting this is a genuine molecular response to ethanol (see anol see .Lpar1 gene encodes a receptor for lysophosphatidic acid (LPA), a signaling molecule containing phosphate and lipid components . Regulation of Lpar1 is critical for proper nerve-cell formation , including in a brain region called the hippocampus in adults  that occurs after nerve injury  that occurs after nerve injury (Lpar1 is brought into this network by ethanol exposure suggests the intriguing possibility that this gene may play a role in the loss of white matterThe n adults . In addie injury . The fachttp://grappa.eecs.utk.edu/grappa/root). Paracliques can contain members with missing links. Therefore, paracliques provide an attractive compromise by augmenting coexpression with genes whose correlational relationships to a network are strong, but permissibly imperfect with a proportion of the network. This proportion, called the proportional glom factor, is a user-defined parameter.Various methods exist for generating gene networks. As mentioned in the main text, the simplest method for constructing gene coexpression networks involves calculating Pearson correlations for all pair-wise genes and applying a hard threshold to determine which genes should be connected. The robustness of these networks, initially referred to as “relevance networks,” can be assessed through an approach called permutation testing . A more Mog gene from the myelin network described in the main article and depicted in Caenorhabditis elegans . This largely is related to technical constraints, because whole-proteome expression profiling currently cannot be done with the same degree of sensitivity, coverage, and throughput as mRNA profiling.Saccharomyces cerevisiae . These are proteins that bind with regulatory DNA regions that are located in front of a gene. Only when the TF binds to the corresponding DNA sequence can the first step in the process of gene expression—transcription—begin. The interaction between the TF and the DNA involves a certain part of the TF called the TF DNA–binding domain that allows the TF to recognize and bind with specific regulatory DNA sequences. Through this mechanism, certain TFs only may activate the transcription of specific sets of genes. Accordingly, a polymorphism at the DNA-binding domain of a certain TF can affect the TF’s ability to recognize and bind its recognition sites, causing altered expression of all genes regulated by this TF. In other words, the abundance of all transcripts from those genes would co-vary with the TF polymorphism. Such a case might be recognized by a clustering of trans eQTLs at the site of the causal polymorphism, sometimes referred to as a regulatory hotspot. The identification of trans eQTL clusters can be a powerful approach for identifying key regulators underlying a complex trait of interest.A trans eQTL cluster. These findings provide preliminary evidence that acute ethanol–responsive genes comprise a handful of gene coexpression networks in the pre-frontal cortex and that a key regulator of these networks resides on chromosome 7. A more extensive analysis of this type has recently been completed .trans eQTL clusters often have biological functions that have been conserved among species, suggesting that these hotspots may have a biological relevance. Accordingly, the search for trans eQTLs may allow researchers to identify biological functions associated with complex traits through defining quantitative trait gene networks (QTGNs). trans eQTL cluster on distal mouse chromosome 1 and identified a candidate gene (Fmn2) that they propose has a major influence on the expression of linked gene networks. Moreover, a diverse group of phenotypic QTLs seemed to be located in the same region, including several related to ethanol.The genes comprising cis eQTL-linked genes that also were strongly correlated with FPM, the researchers were able to identify two novel obesity candidate genes.The integration of eQTL and classical QTL data enables identification of key markers of disease-causing variants. The effectiveness of this approach was demonstrated by a genetical genomics analysis of liver expression data from a population of mice placed on a high-fat diet . The purcis eQTLs that overlapped previously mapped behavioral QTLs for either alcohol preference . These studies used the BXD panel, so that the genetic regulation of ethanol-induced expression changes and behavioral responses also could be examined. Similar to results shown in


Attempts to improve cognitive function in patients with brain disorders have become the focus of intensive research efforts. A recent emerging trend is the use of so-called cognitive enhancers by healthy individuals. Here, we consider some of the effects – positive and negative – that current drugs have in neurological conditions and healthy people. We conclude that, to date, experimental and clinical studies have demonstrated relatively modest overall effects, most probably because of substantial variability in response both across and within individuals. We discuss biological factors that might account for such variability and highlight the need to improve testing methods and to extend our understanding of how drugs modulate specific cognitive processes at the systems or network level. N-methyl-D-aspartate (NMDA) receptor antagonist] are now standard treatments In the last decade, pharmacological treatments aimed at improving cognitive function across a range of brain disorders have been explored and have even become established in clinical practice In chronic mental disorders such as schizophrenia, cognitive deficits are a separable feature from positive  and negative  symptoms, with current antipsychotic treatments having little, if any, impact on cognitive impairments. A wide range of compounds is therefore being assessed for cognitive enhancement in this disorder Although most of the reported positive effects of such drugs have been modest in magnitude overall and are highly variable across individuals, they have had an enormous impact, stimulating interest in cognitive enhancement not only for patients with brain disorders, but also for healthy individuals. Compounds such as methylphenidate and modafinil are used by students in pursuit of better grades, military personnel who need to remain awake for long missions, elderly individuals afraid of cognitive decline and even university academics keen to maintain their performance Here we focus on what aspects of cognition are enhanced, the magnitude of these effects and possible mechanisms underlying variations in response across individuals. Our aim is to highlight key common themes across studies of clinical populations and healthy individuals, using examples that highlight these principles. Other recent reviews provide excellent discussions of ethical issues in cognitive enhancement What exactly do cognitive neuromodulators do? It might be tempting to assume a selective one-to-one mapping between a specific neurotransmitter system and a particular cognitive function. For example, dopamine has been strongly linked with working memory (WM) and attention To add to the complexity, neurotransmitters act via a suite of different receptor systems. Thus, dopamine acting at D1 receptors can have very different – even opposing – effects to that of its actions at D2 receptors Finally, there is increasing evidence that several neurotransmitters might have different modes of action when released in a tonic, sustained manner compared to phasic release How do drugs currently used as enhancers produce their beneficial effects? Is it through multiple effects on several different cognitive processes or do they enhance one cognitive mechanism – such as arousal or improved sustained attention – through which they lead to better performance across a battery of tests? For studies in clinical populations, the difficulty is that many standard cognitive test batteries used in clinical trials are very unlikely to be sensitive enough to answer questions on the specificity of cognitive modulation .For example, AChEIs such as rivastigmine and donepezil are now widely used to treat Parkinson's disease dementia (PDD) and the related condition of dementia with Lewy bodies (DLB). Many clinical trials have reported modest global beneficial effects of such drugs on bedside cognitive screening tests Similar considerations as for AChEIs also apply to modafinil, which has become a popular drug for cognitive enhancement in healthy individuals. Although its precise mechanism of action remains to be established, modafinil is used as a wake-promoting agent for the treatment of narcolepsy, a disorder associated with excessive daytime somnolence. Analysis of the effects of modafinil in healthy subjects has revealed improvements in attention, memory and executive function in sleep-deprived individuals It is possible that neuroimging studies might contribute to identification of the mechanisms underpinning improvement on cognitive tests, including arousal. Although early studies assessed changes in brain activity on drug administration A major issue in assessing cognitive enhancement studies is the problem of effect size. First, in studies of healthy subjects, there is no universal, standard battery of tests that has been agreed on, so comparisons across studies are not easy. It is not possible to compare effect sizes for different drugs if the tests used differ in the level of difficulty or method of measurement (e.g. reaction time vs error rate). Overall, however, the effects of cognitive enhancers such as methylphenidate, modafinil and AChEIs in healthy individuals seems to be quite modest according to recent systematic reviews Nevertheless, even for these relatively crude measures, studies in clinical populations have revealed significant effects of long-term drug use that have led to changes in practice. For example, one of the remarkable changes in the management of neurological conditions in the last decade has been the advent of treatment for cognitive deficits in neurodegenerative conditions, initially in Alzheimer's disease with AChEIs Although these trials have now led to widespread clinical use of rivastigmine, it is important to keep the effect size in perspective. In the larger study, rivastigmine produced only a mean 2-point improvement on the ADAS-Cog battery , which hOf course, effect sizes vary across individual patients. Indeed ∼40–80% of PDD or DLB patients might not show a response to treatment on such clinical measures, but other individuals show a very strong improvement By contrast, a first glance might indicate far more substantial effect sizes in treatment trials of ADHD, for which several drugs that target the catecholaminergic system are used in clinics. For example, a recent study using high levels of the α2 noradrenergic agonist guanfacine demonstrated a 12-point mean improvement compared to placebo on a rating scale with a range of 54 points b. HoweveLike all drugs, those used with the aim of enhancing cognition can have side effects via body systems other than the brain. Thus, both AChEIs and methylphenidate frequently cause gastrointestinal upset or nausea, sometimes leading patients to discontinue medication altogether. These effects have the potential to offset any positive effects of the drug on overall performance, and also need to be borne in mind by anyone contemplating use of such drugs for non-medicinal purposes. More important from a cognitive neuroscience perspective is the ability of some drugs to impair certain aspects of cognition while simultaneously enhancing others in the same individual.Thus, rivastigmine in healthy elderly subjects can improve learning on a motor task and making associations between symbols and digits, but can at the same time impair verbal and visual episodic memory Indeed, this conclusion might well be applicable to recent reports that some PD patients on dopaminergic agonists developed impulsive behaviours such as gambling, compulsive shopping and hypersexuality A major theme that has emerged from studies of neurological patient groups is that there is a great variability of response, with many individuals not responding to treatment on (relatively crude) clinical measures, whereas others show a very strong improvement, for example in response to AChEIs The same issue has arisen in investigations in healthy individuals: some subjects respond, whereas others might show little or no benefit. As we discuss below, recent investigations have begun to question whether such differences in outcome might depend on genotype and/or the baseline level of cognitive function. These considerations also raise concerns about what has become the standard method of performing clinical drug trials. Large-scale randomized controlled trials offer protection from false positive findings, but they also have the potential to discard the fact that some subgroups might benefit from a compound, whereas others might not.What might be the cause of such variations in response? Several studies on the effects of dopaminergic drugs on WM in healthy volunteers support the conclusion that those who benefit most are low performers, such as those with low WM capacity or span. Thus, methylphenidate or dopamine receptor agonists such as bromocriptine improve WM updating or retrieval in people who were low performers on study entry, but can actually impair performance in participants with high baseline WM spans One possible explanation for such contradictory effects might reside in the classic inverted U-shaped relationship between cognitive performance and dopamine receptor (particularly D1 receptor) stimulation . Such efHowever, until recently, direct evidence in favour of this concept has been lacking in humans. New findings reveal that dopamine synthesis capacity in the caudate nucleus of the basal ganglia is lower in individuals with low WM spans compared to those with high spans More recently, it was demonstrated using radioligand positron emission tomography (PET) imaging that individuals with small levels of dopamine release induced by methylphenidate improved on a reversal learning task The effects of methyphenidate on spatial WM in healthy subjects are also most prominent in individuals with the lowest performance COMT gene seem to be associated with variability in human WM performance and associated brain activity, presumed to be via its putative influence on cortical dopamine levels Genetic predictors of individual variability in response to treatments aimed at improving cognitive function would clearly be beneficial in effective targeting of therapeutic strategies. These effects might result directly from variations in efficiency of drug targets or indirectly via metabolic pathways or other risk genes. Several studies have suggested a role for polymorphisms in the catechol-O-methyltransferase (COMT) enzyme-coding region on chromosone 22 in WM Amphetamine responses might interact with COMT activity. When performing a test of cognitive flexibility – the Wisconsin Card Sorting Test – those with the higher-activity COMT Val-Val genotype improved, whereas those with the lower-activity Met-Met genotype deteriorated after a single dose of amphetamine. An inverted-U relationship between predicted cortical dopamine levels and performance is consistent with these findings .Variations in COMT and the dopamine transporter gene (DAT) are both obvious candidates for modulation of response to psychomotor stimulant treatment in a condition such as ADHD. DAT is a major target of methylphenidate and amphetamine, and many treatments for ADHD, including the noradrenaline transporter inhibitor atomoxetine, are thought to increase cortical dopamine levels apoE4), an allele of apolipoprotein E, which is involved in lipoprotein processing in cells, increases the risk of developing dementia later in life. Perhaps paradoxically, young healthy carriers of this genotype, who have a higher risk of cognitive decline later in life, actually show better performance on decision-making and prospective memory tasks compared to their apoE3 counterparts apoE4 carriers, producing greater cognitive benefits in these individuals than in apoE3 carriers on these tasks Apoliprotein E4  might show greater response to donepezil and rivastigmine One area that is likely to develop in cognitive enhancement research is investigation of the interaction between drugs and behavioural approaches to improve cognition. There has been a great deal of recent interest in the potential for cognitive training, for example on WM tasks, to improve performance not only on these paradigms but also to generalize to other tasks in healthy people, as well as those with brain conditions such as ADHD An important question for future studies will be whether there can be synergistic effects of behavioural training and cognitive-enhancing drugs. Such synergism has been demonstrated for learning of new material and levodopa in healthy subjects It would probably be fair to say that we are still in the first generation of studies to examine the potential for cognitive enhancement in humans. In both healthy individuals and many patient groups, the overall effects of drugs generally seem to be modest. However, there is evidence that there might be more significant effects in subgroups, such as those whose baseline performance is poorest or individuals with a particular genotype. Moreover, new drugs aimed at enhancing the phasic response of neurotransmitter systems, such as direct nicotinic agonists for the cholinergic system


The stubbornly high incidence of new HIV infections belies the overwhelming evidence showing that sustained highly active antiretroviral therapy (HAART) has the power to dramatically reduce the spread of HIV infection and forever change the face of this devastating epidemic. One of the main contributors to this public health paradox is the ongoing HIV epidemic among substance users who contribute significantly to HIV infection rates through injection drug use and high-risk sexual behaviours. Current evidence clearly shows that, in order to fill this gap, we need to integrate substance abuse treatment with HIV treatment programmes and provide substance abusers with universal access to HIV treatment through a focussed effort to seek, test, treat, and retain hard-to-reach high risk individuals. These aims will require structural changes in the health care system to overcome many of the obstacles that have inhibited the merging of substance abuse treatment with HIV programmes for far too long. As this special issue demonstrates, there is now ample evidence that sustained highly active antiretroviral therapy (HAART) leads to significant reductions in the spread of Human Immunodeficiency Virus (HIV) infection. Vigorous deployment of HAART has practically eliminated vertical HIV transmission in the developed world and even curtailed horizontal transmission among serodiscordant heterosexual couples. And expansion of HAART coverage, particularly among high risk populations, further reduces HIV transmission .However, the incidence of new infections - estimated at about 55,400 per year in the past decade - has failed to reflect HAART’s clinical efficacy and promising outlook . The maiAddiction research has uncovered critical information about the impact of substance abuse behaviours on the risk of HIV transmission by shedding light on the acute and long-term deleterious effects of drugs of abuse on cognitive control, particularly as they interfere with normal frontocortical activity. It has been known for over two decades that the rewarding effects of drugs abused by humans are associated with increases in dopamine (DA) in the nucleus accumbens (NAc), which is a central hub in the brain’s reward network . Not surKnowledge of the connections between substance abuse and HIV transmission, whether through direct (contaminated needles) or indirect (increased impulsivity leading to high risk behaviours) means should be a powerful incentive for incorporating HIV prevention and treatment in substance abusing populations . UnfortuThe gap in the treatment of substance use disorders also cripples our efforts to combat the HIV epidemic by shrinking the reservoirs that persist among high-risk populations. An example is our failure to fully harness the unique therapeutic opportunities that exist among incarcerated individuals, more than half of whom have a history of substance use . Indeed,The global gap is similarly disconcerting: in the five low-income and middle-income countries that contain nearly half of all IDUs infected with HIV, IDU access to ORT ranges from 0% (Russia) to 4% (China) . This laSubstance abusers, and IDUs in particular, are presently much less likely to receive HAART, despite their higher rates of HIV infection . This gavis á vis the HIV epidemic should reflect the solid new understanding that a) injecting and non-injecting drug abusers can successfully undergo HIV treatment; b) many substance abusers adhere to antiretroviral therapy as well as people who do not inject or take drugs; and c) injecting drug users who undergo substance abuse treatment are more likely to obtain and stay in treatment for their HIV infection. The combined evidence makes a compelling case for integrating substance abuse treatment with HIV treatment programmes and for providing substance abusers with universal access to HIV treatment. At the moment, the most effective strategy for advancing this agenda appears to be a focussed “seek, test, treat, and retain” effort to aggressively seek out high-risk, hard-to-reach substance abusers, offer them HIV testing and access to HIV treatment when medically appropriate, and provide the necessary support to help them stay in treatment – both for HIV as well as substance abuse [In short, our efforts to improve public health outcomes ce abuse . Achievi


Tendon healing is fraught with complications such as reruptures and adhesion formation due to the formation of scar tissue at the injury site as opposed to the regeneration of native tissue. Stem cells are an attractive option in developing cell-based therapies to improve tendon healing. However, several questions remain to be answered before stem cells can be used clinically. Specifically, the type of stem cell, the amount of cells, and the proper combination of growth factors or mechanical stimuli to induce differentiation all remain to be seen. This paper outlines the current literature on the use of stem cells for tendon augmentation. Tendons function to transmit force from muscle to bone, with the ultimate effect of actuating motion. Tendon tissue has been designed to endure large tensile loads. When tendons are injured, normal tendon histology is not restored and, therefore, neither is function. Tendons heal with an intervening layer of scar tissue. This scar tissue has material properties that are inferior to native tendon. This makes surgical repairs of torn tendons prone to failure. It also makes them susceptible to adhesion formation due to excess fibrous tissue formation. Therapies that can augment regeneration of normal tendon and limit the amount of scar tissue that is formed in response to injury may improve clinical outcomes.Stem cells have great promise in enhancing the biologic healing process since they provide a self-renewing population of pluripotent cells. However, several questions still remain before stem cells can be used clinically for augmenting tendon healing. Specifically, the type of stem cell, the amount of cells, the combination of growth factors and mechanical stimuli, and the ideal delivery vehicle all still need to be determined. The purpose of this paper is to outline the current research on developing a clinical stem cell therapy for the augmentation of tendon healing.Stem cells are defined as a population of cells that can self-renew through symmetrical mitotic division, form daughter cell lines, and generate a broad range of tissue lineages through terminal differentiation , 2. StemMSCs  are defined by their ability to self-renew and their multipotentiality . MSCs ar2 (PGE2), BMP-2, BMP-12 and -13 TGF-β3, and platelet-rich plasma releasate are proposed to be important mediators for promoting stem cell differentiation into tendon tissue as opposed to adipocyte and osteocyte formation [Bi et al. identified a tendon progenitor stem cell (TPSC) population in both mice and humans . A greatormation , 18. Tenormation . IncreasMSCs have been isolated from a number of different tissue sources. The most common sources of MSCs for musculoskeletal applications are bone marrow and adipose tissue due to their accessibility and the ability to obtain large numbers of viable cells . Bone-maAs MSCs possess the potential to differentiate into tenocytes, MSC application to torn tendons is proposed to recapitulate tendon development signals and improve tendon healing capabilities. MSC-mediated tendon regeneration has been studied in numerous animal tendon models.The addition of MSCs to semitendinosus tendons in bone tunnels during anterior cruciate ligament (ACL) reconstructions promoted improved healing with higher failure loads and stiffness after 8 weeks . HealingAwad et al. demonstrated that bone-marrow-derived stem cells delivered to a patellar tendon wound site resulted in improved mechanical and some histological properties . The addAnother study used a rabbit Achilles model with a 1 cm tendon defect that was augmented with MSCs suspended in collagen gel that had been contracted onto pretensioned sutures . MSC treA number of animal studies suggest that simply delivering stem cells alone not appear to improve tendon healing. Gulotta et al. showed that the application of bone-marrow-derived MSCs alone was insufficient to improve rotator cuff healing in a rat model . The effA few studies have tried to address the effect of different stem cell densities. MSCs were seeded onto collagen matrices and contracted onto sutures at different cells densities of 1, 4, and 8 million cells/mL prior to implantation into rabbit patellar tendon defects . The repSome studies have tried to address whether stem cells mediate a superior effect on tendon healing compared to alternative types of differentiated cells. Hankemeier et al. used a patellar tendon defect model to compare the effects of stem cells and human fibroblasts that were both delivered using a fibrin matrix . Stem ceWhile most animal studies have shown encouraging results with the simple application of untreated stem cells, a growing consensus exists that optimal therapy will include treatment of these cells with growth factors. Many studies have demonstrated that mesenchymal stem cells differentiate in response to bone morphogenetic proteins, transforming growth factors and fibroblast growth factor .Gulotta et al. have studied the effect of various growth or differentiation factors and stem cells on tendon healing in a rat model of rotator cuff repair –37. TheyIn another study, investigating the role of bone morphogenetic proteins, Rui et al. modeled calcifying tendinopathy with an in vitro model that applied uniaxial cyclic loading to tendon-derived stem cells grown on plates coated with collagen type I . IncreasWhile growth factor therapy in addition to MSC seems promising, the question remains as to what factor yields the best results. The various possible combinations of factors are daunting, which has led researchers to pursue platelet rich plasma (PRP) as a way of delivering multiple factors in a single therapeutic agent. PRP has been shown to influence the behavior of stem cells. Using tendon stem cells derived from rabbit patellar tendons, Zhang and Wang demonstrated that increased PRP releasate caused tendon stem cells to become larger and elongated . PRP relIn addition to growth factors, altering the mechanical environment is another way to induce stem cell differentiation into tenocytes. The mechanical loading of stem cells appears to guide differentiation. Sharma and Snedeker combined mechanical and molecular cues by culturing bone marrow stromal stem cells on a hydrogel matrix with a gradient of mechanical compliance as well as gradients of the ligands fibronectin and collagen type I . These cAs work progresses on the development of biomimetic scaffolds for tendon tissue, the mechanical loading of tissue has also been demonstrated to influence stem cell differentiation . Zhang aWhile mechanical signals have been shown to affect mesenchymal stem cells fate, the characteristics of an effective loading regimen for tendon-to-bone healing have yet to be fully determined. Sen et al., using an in vitro model in which mesenchymal stem cells underwent two twenty-minute episodes of low intensity vibration or high magnitude strain, demonstrated that adipogenesis was suppressed and osteogenesis was amplified when there was at least a one-hour refractory period between bouts . The gron = 213) or intervertebral discs (n = 13) [A number of clinical applications for augmenting tissue healing with stem cells in humans have been reported. MSCs have been used for human craniofacial tissue regeneration with varying reports of success . Orthopa(n = 13) . PatientDespite the promising MSC-mediated effects on tendon healing noted in a number of animal studies, there are no clinical studies examining the efficacy and safety of stem cells on human tendon repair. However, other cellular therapies have been shown to clinically improve tendon healing. Clarke at al. applied skin-derived tenocyte-like cells to 33 patients with patellar tendinopathy and compared it to 27 patients who were treated with plasma . The groA number of issues regarding the clinical application of stem cells still need to be addressed. The method of stem cell aspiration may affect the viability and function of the cells. Stem cell therapies typically require in vitro culturing and expansion of cells to obtain sufficient numbers of viable cells prior to reimplantation . This raThe source of stem cells may help to determine stem cell effectiveness. Locally derived stem cells may most likely to be effective in promoting tendon regeneration, particularly in view of the importance of tendon “niches” in tenocyte differentiation humans . ProgeniEffective translation of promising stem-cell-mediated tendon healing in animals has not yet been proven in the clinical setting. A number of concerns exist about MSC use within humans, such as tumor-like growth of MSCs and modulation of the immune system. Regardless of the source of MSCs, there is a risk of differentiation into undesirable lineages which could result in ectopic tissue formation and calcium deposition. Systemic injections of allogeneic MSCs have been shown to disseminate to a number of organs in baboons . Some ofStem cells are an attractive option for the augmentation of tendon repairs. Embryonic stem cells appear to offer the best differentiation potential, but their use is controversial, and no studies have evaluated their clinical usefulness. Several studies have evaluated the use of MSCs with some success, but profound results have been lacking when these cells are applied untreated. Molecular cues, such as the addition of BMP's, MT1-MMP, and scleraxis, and mechanical cues such as changes in strain and nanotopography, have shown promising results in their ability to drive MSCs into tenocytes differentiation. Utilizing this knowledge to develop a clinically useful therapy has yet to be accomplished, but our understanding of stem cell biology continues to expand with the hope of one day finding one.


Most pathological processes involve complex molecular pathways that can only be modified or blocked by a combination of medications. Combination therapy has become a common practice in medicine. In ophthalmology, this approach has been used effectively to treat bacterial, fungal, proliferative/neoplastic, and inflammatory eye diseases and vascular proliferation. Combination therapy also encompasses the synergistic effect of electromagnetic radiation and medications. However, combination therapy can augment inherent complications of individual interventions, therefore vigilance is required. Complications of combination therapy include potential incompatibility among compounds and tissue toxicity. Understanding these effects will assist the ophthalmologist in his decision to maximize the benefits of combination therapy while avoiding an unfavorable outcome. Combining means and therapeutics to address various pathologic processes is now a familiar topic across many, if not all, medical specialties. There is compelling clinical, technological and commercial justification for combining therapies into fixed and/or unfixed drug combinations or combining a device and a drug to achieve better outcomes. Usually, fixed drug combinations are formulated to address patient compliance and convenience or manufacturing costs. Other fixed therapies encompass the combination and engineering of drug and device into one unit as a means of drug delivery or combining a drug and a physical source of energy  to achieve a localized therapeutic effect. Examples are cardiovascular drug eluting stentsThere are also convincing medical and biological reasons to combine two or more drugs. Different pathologies develop and progress through multiple molecular pathways and therefore, different mechanisms of action and opportunities exist to interfere and partially block such pathways. Capitalizing on such opportunities must be done with extreme care, since combining chemical entities and formulations can be toxic to specific cells, and sometimes to whole organs. Although such combinations may result in additive or synergistic therapeutic actions, they may also cause unfavorable outcomes; therefore stringent pharmacovigilance at all times is warranted.Awareness of the potential hazards of combination products has prompted many regulatory bodies such as the US Food and Drug Administration (FDA), European Medicines Agency (EMA), and the Japan Health Agency to mandate thorough screening of potential benefits and adverse effects. These measures have resulted in new guidelines and policies for fixed therapeutic products.Discussing the broad ramifications of all combination products would go beyond the scope of this paper. Therefore, we focus on combination drugs in ophthalmology and in particular, on intravitreal drug combinations.The eye is a unique structure, in which different tissues  interact and fuse in a compact space with well-defined boundaries. The eye is susceptible to pathologies that can affect individual tissues. Pathological processes may also affect more than one tissue at a time and therefore, multiple drugs could be used to address each layer and tissue accordingly. Our current knowledge of many ophthalmic diseases is still incomplete regarding the exact causative molecular pathways. Current clinical practice categorizes a collection of disorders under single disease entities, e.g., glaucoma, dry eye, age-related macular degeneration. However, in the abovementioned pathologies, varying stages and severities of disease manifestations are the result of a collection of molecular pathways brought about by multi-factorial conditions. As our basic knowledge of these conditions grows, so do our therapeutic strategies to address them. Precise mapping of different molecular pathways allows us to devise approaches for each independent pathway. It is no surprise that no single drug can address all pathways. Therefore, combination approaches have become inevitable, and understanding the chemical and physical interactions of the components remains of paramount importance. Disregarding such considerations may result in irreversible consequences.Combination of drug and device within ophthalmology dates back to the 1970s when the first drug and device combination was approved in the USA. Ocusert  was a pilocarpine-containing, polymer-membrane unit that provided a depot in the conjunctival cul-de-sac to address glaucoma.7Peyman and colleaguesThere are other drug and device combinations in late-stage clinical development. Examples include contact lenses with drug coatings as drug delivery means for seasonal allergies and other anterior segment pathologies13in vitro or in vivo, they may interact. Some drugs change the way others are transported or absorbed in the tissue. Such interactions may occur during chemical and physical intraocular mixing or at the time of mixture of injections.The combination of multiple chemical agents for ocular disorders provides an effective strategy to address clinical needs in acute and chronic conditions. When two or more different drugs are mixed Drug interactions may increase or decrease drug effects. Competitive effects occur during combination of drugs which can be positive or negative, depending on whether the drugs have synergistic or antagonistic effects. Drug displacement interactions are a possibility; this can occur at binding or receptor sites. Alternatively, the combination of drugs can produce a new effect that neither drug produces on its own. The effect of combining drugs within an organ such as the eye can be considered localized. However, one needs to be alert towards the possible additional interactions with systemically dosed agents and thus, adverse interactions elsewhere in the body.Ophthalmology is a specialty that is quite familiar with combinations of different drugs and therapies. As an example, endophthalmitis is a well known (and dreaded) condition that is currently addressed with unfixed combination therapy; at least two different classes of antibiotics are co-administered to provide coverage for both gram-negative and gram-positive bacteria.Next, we will briefly discuss possible physical-chemical interactions between various drugs and comment on their possible applications and/or outcomes for intravitreal combination therapies.It remains almost impossible to predict drug interactions in different locations of an organ or the whole body, as transport and elimination routes and dynamics can vary greatly depending on ongoing physiological and pathological processes. However, there are a few exceptions. One of them is the vitreous body, where locally administered drugs can achieve high concentrations, at least for a certain period of time. It should be noted that the duration of this effect depends on the drug’s half-life, molecular weight, molecular size, electric charge, elimination route, etc.Peyman and colleagues Specific physiochemical reactions for a drug or combination of drugs must be considered when one administers medications intravitreally. Another major consideration is related to the biological effect that a drug or the combination of multiple drugs can induce in ocular tissues. Such a biological effect may be the result of tissue reactions to each individual drug or the products of their combination.in vitro or within the organ. Such adverse effects may manifest as incompatibility, which is described as preventable or reversible precipitation or insolubility and recognizable as crystals, turbidity, or haziness. Incompatibility can be easily caused by acid-base reactions. However, other scenarios are also possible, such as non-dissociated salts of organic ions, salting out, salts of inorganic divalent ions, desolvation of non-ionized organic drugs, and organic ion-inorganic ion salts.17The physiochemical adverse reactions of a single, “approved” drug are usually mapped out and generally deemed safe to apply due to extensive preclinical and clinical testing that is undertaken prior to approval. Nevertheless, vigilance is still required when a single drug is being used “off-label” for a different indication. The challenge becomes even greater when two or more drugs are combined and their combination may cause new physiochemical reactions within the mixture Another possible physiochemical reaction due to combinations of drugs stems from instability, which can result from hydrolysis and oxidation. The combination of chemical entities can cause degradation products and such products are often unstable chemicals that may be less active or inactive therapeutically, but more importantly, they can become toxic products which are not always visually discernable.As mentioned, resultant physiochemical drug interactions can become toxic and therefore, when contemplating new medications for intraocular administration, care should be given to such adverse side effects. Different cell layers of the retina, the endothelium of the cornea, and the crystalline lens are among the ocular tissues that have been reported to be subject to toxicity as a result of drug degradation or complexation products. One example of such an adverse effect was reported during cataract surgery while mixing chondroitin sulfate with hyaluronate sodium .21The neutrality of pH for medical formulations while desirable, is a hard target to achieve for many compounds. Many formulations become unstable and suffer from lack of  solubility if pushed toward a fixed pH. Ciprofloxacin is a fair example that can be stable and soluble at a pH around 4.5, but would start precipitating if pH is altered to 7.0.Episodes of white precipitation on the corneal surface were reported among more than 600 patients with bacterial keratitis, treated with topical ciprofloxacin 0.3%.23All of the above-mentioned examples were related to clinically visible precipitation, however, microprecipitation and/or incompatibility may also occur. A combination of vancomycin and ceftazidime has been routinely injected intravitreally for suspected bacterial endophthalmitis. There are claims of precipitation when these two antibiotics are physically mixed. Also, some investigators have reported pH-dependent precipitation of vancomycin regardless of the presence of ceftazidime in the medium.24+ in the body is cellular metabolism as a result of oxidation of glucose and fatty acids. These hydrogen atoms are the most reactive cations in the body and they become crucial when one considers the functional groups of proteins, which are negatively charged and can react with them. Such reactions may cause alterations in the structural conformation of the protein and consequently its behavior. Many new drugs in development are protein-based and therefore such possible reactions with other drugs become noteworthy. Less is known about the buffering capacity of the vitreous in general, until recently.As the number of intraocular injectable candidates increases, more investigations are necessary to study such physiochemical interactions. However, designing such studies under conditions that would mimic the physiological environment of ocular tissues remains challenging. A cornerstone of maintaining such an environment is preservation of a constant internal environment in terms of temperature, fluid volume, and fluid consistency, collectively called homeostasis. One important factor in homeostasis is the maintenance of a relatively constant pH. Many buffering systems are employed in the body to support pH levels. Examples are bicarbonate in the interstitial fluid, phosphates, and proteins, which are also present intracellularly. An important source of Hin vivo using electroretinography. Toxicity in the anterior segment such as lens opacification can easily be revealed with slit-lamp biomicroscopy. Corneal toxicity usually manifests as endothelial cell dysfunction which can be shown using a corneal stress test.26It has been demonstrated that intraocular injections of drugs can cause toxicity to different tissues. Such toxicity is usually shown by means of postmortem histologic studies in animal models. Toxicity to retinal cell layers can be shown Less risky methods for testing the biological effects of combination (and single) drugs in preclinical settings include bacteria, specialized cell cultures, ocular inflammatory receptors, viruses, and proliferative tissues.Currently, therapy for bacterial endophthalmitis consists of combination therapy, a mixture of antibiotics aimed at addressing both gram-negative and gram-positive bacteria. This is the standard of care since such a sight-threatening condition allows no time to explore the nature of the causative organism. Antibiotics usually interfere with bacterial cell wall or ribosomal protein synthesis. Gram-positive bacteria have different cell walls from gram-negative microorganisms and therefore a single antibiotic may be ineffective on the wrong target. The current antibiotic treatment for acute-onset bacterial endophthalmitis includes a mixture of vancomycin for gram-positive coverage and either ceftazidime or an aminoglycoside for gram-negative coverage. Aminoglycosides do not interact with the cell wall but inhibit protein synthesis by binding to the 30S bacterial ribosomal subunit, resulting in misreading of the messenger RNA. Toxicity levels for each of these antibiotics have been determined and they are generally considered to be safe for administration through the pars plana.37When Peyman and colleagues first introduced combination therapy for posterior segment disordersCombinations of steroids and amphotericin B have been investigated for fungal infections which are also typically accompanied by marked inflammation.41in vivo.49In current practice, corticosteroids are important candidates for intravitreal applications. Non-toxic doses of dexamethasone for ocular tissues are close to 400 μgA triple combination of clindamycin, aminoglycosides, and dexamethasone in the infusion fluid has been investigated by our group since 1990 for prophylaxis of bacterial infection in vitrectomy procedures.50A combination therapy, per regulatory guidelines, can include a device and a drug. Steroids have been successfully formulated and incorporated into implantable devices. Retisert is an example of a drug and device combination, engineered as a non-biodegradable intravitreal implant for slow but steady release of fluocinolone acentonide for up to 3 years. The device is sutured into the vitreous cavity, away from the optical axis and acts as a steady drug delivery system.Another interesting vehicle for delivery of steroids is iontophoresis technology. Iontophoresis utilizes an electric current to drive charged ions (drugs) across a barrier such as the sclera or the cornea. Eyegate Pharmaceuticals has recently completed a phase II study on management of anterior uveitis using an iontophoretic drug delivery system for ocular application.Other delivery devices are in development to address on-target transport challenges that new drugs and therapies face to reach the posterior segment. A novel injection device for intravitreal and subconjunctival drug delivery has been developed by Peyman and co-workersViral infections of the posterior segment have also been targeted with combination therapies. Viral infections develop in different stages and most antiviral drugs interfere with the replication stage. They do so by integrating defective nucleoside analogues into the viral genome and thus, targeting specific viral enzymes or proteins. Fortunately the incidence of cytomegalovirus retinopathy is decreasing thanks in part to the employment of advanced therapies for HIV infected individuals. Nevertheless, antiviral therapies for this condition are still being used, and a drug and device combination (Vitrasert) for intravitreal implantation is available for slow release of ganciclovir up to 8 months. This implant was developed before Retisert and its implantation procedure resembles that of Retisert. Antiviral drugs were injected intravitreally before the introduction of implants. Different doses were used to administer each compound, for example, ganciclovir at 2000 μg, foscarnet at 1000 to 2000 μg, and acyclovir at 240 μg. Other antiviral drugs such as trifluorothymidine and vidarabine have also been injected intravitreally. A combination of ganciclovir and foscarnet  can also be used for mixed therapy.Several ocular pathologies could benefit from combination therapies. Diseases such as proliferative vitreoretinopathy (PVR), vascular proliferation in age-related macular degeneration (AMD), diabetic retinopathy, and malignant tumors, exhibit distinctly different stages which can be potential targets for different anti-proliferative, anti-inflammatory, and matrix metalloproteinase inhibiting agents. Current management of vascular proliferation has been dominated by blocking the activity of existing vascular endothelial growth factor (VEGF), while other small and large molecular candidates are being investigated to block the production of VEGF. Targeting VEGF in AMD has probably provided the biggest advance for this ever-growing condition. Currently, the two prominent treatment options for VEGF blockage include intravitreal administration of ranibizumab (Lucentis) or the off-label use of bevacizumab (Avastin). However, other VEGF blocking treatments are in development and probably the closest compound to approval is aflibercept (VEGF Trap), developed by Regeneron Pharmaceuticals. Aflibercept is a fusion protein specifically designed to bind all forms of vascular endothelial growth factor-A .A number of candidates for co-administration with VEGF blocking agents are being investigated clinically and results thus far are promising. These include not only intravitreal combinations of drug candidates but also intravitreal and topical combinations aimed at increasing efficacy and/or decreasing injection frequency and cost, thus ultimately improving compliance. In one clinical study, the combination of topical bromfenac 0.09% , which is an NSAID, and the intravitreal injection of ranibizumab resulted in a threefold decrease in the frequency of injections.56Other therapies that can be combined with anti-VEGF therapy include photodynamic therapy (PDT), transpupillary thermotherapy (TTT), and intravitreal steroid injections. There have been a number of uncontrolled clinical studies where combinations of VEGF therapy with steroids and PDT (as a combo or triple treatment) were investigated, with favorable results. Triple therapy for posterior segment diseases such as wet type AMD has been investigated by a number of clinicians.in vitro during mixing and in vivo upon delivery. Although research on adverse effects of drug/drug and drug/device interactions is regulated for commercial combinations, there is little control over off-label and unfixed combinations devised and utilized by clinicians. More research is warranted on new combination therapies to increase efficacy and safety.Combination therapies are increasingly expanding due to the growth of knowledge about the core pathologic mechanisms of diseases. Ophthalmology has been an area of successful implementation of a number of fixed and unfixed drug combinations, along with drug/device combinations. Investigators have combined different drugs with devices to address challenging needs of chronic conditions and drug delivery problems. Such combination approaches should ideally achieve clear advantages in terms of therapeutic efficacy, lowered toxicity, and increased compliance. There are also risks that need to be mitigated, if possible. Combinations of pharmaceutical compounds may precipitate adverse physical and chemical interactions, both


Hypokinetic symptoms of Parkinson’s disease have been related to the excessive beta-band oscillations and synchrony in basal ganglia and other structures; suppression of these synchronized oscillations is believed to suppress the motor symptoms of Parkinson’s disease . This leHowever, the dynamics of the neural activity in Parkinson’s disease exhibits complex intermittent synchronous patterns , far froWe study the effect of delayed feedback in numerical experiment. When the parameters of our model are such that the dynamics is strongly synchronous, the feedback, in line with earlier studies, exerts desynchronizing action. However, this strongly synchronous dynamics is not physiologically realistic. When the model is tuned to reproduce highly variable temporal patterns observed experimentally, the same delayed feedback tends to increase the synchrony in the system. “Desynchronizing” delayed feedback acting on realistic partially synchronous dynamics boosts rather than suppresses synchronization. The study also indicates that in general, delayed feedback stimulation may not necessarily exhibit desynchronization effect, when acting on a realistic partially synchronous dynamics (the kind of dynamics expected in neuroengineering applications), and sets an example of how to estimate the stimulation effect.


Obstructive sleep apnea is a common disorder acting as a risk factor for the development and progression of cardiometabolic derangements including non-alcoholic fatty liver disease. Recent research data suggest that non-alcoholic fatty pancreatic disease may be a more sensitive marker than non-alcoholic fatty liver disease for early subclinical metabolic risk and may contribute to the progression of subclinical disease to overt type 2 diabetes mellitus.We postulate that obstructive sleep apnea may be a risk factor for non-alcoholic fatty pancreatic disease. It is well known that intermittent hypoxia related to obstructive sleep apnea leads to hormonal derangements. Excessive lipolysis, enhanced lipid synthesis and systemic and local inflammation may favor ectopic fat deposition similarly to non-alcoholic fatty liver disease. Furthermore, it is possible that obstructive sleep apnea can lead to pancreatic beta cell damage via intermittent hypoxia.Future research should focus on the following: first, whether non-alcoholic fatty pancreatic disease is an independent risk factor for the development of metabolic disease including diabetes mellitus or is a simple consequence of obesity; second, the prevalence of non-alcoholic fatty pancreatic disease among people with obstructive sleep apnea and vice versa, which should be compared to the prevalence of these diseases in general population; third, whether coexistence of these conditions is related to greater cardiometabolic risk than either disease alone; and fourth, whether the treatment of obstructive sleep apnea will translate into the resolution of non-alcoholic fatty pancreatic disease.If proven, this hypothesis will provide new knowledge on the complex interplay between various metabolic insults. Second, screening for NAFPD may identify individuals at risk for developing type 2 diabetes mellitus for targeted prevention. Third, screening for the presence of non-alcoholic fatty pancreatic disease in patients with obstructive sleep apnea may help to decrease the incidence of diabetes mellitus through a targeted prevention. Obstructive sleep apnea (OSA) is a disorder that is characterized by complete or partial breathing disturbances during sleep with a minimum prerequisite frequency of five events per hour, each of which should last for at least 10 seconds . Its preRecent data suggests that OSA is strongly associated with cardiovascular, metabolic and even malignant disease and may be an independent risk factor for their occurrence -11. NonaMore recently, however, the fatty infiltration of pancreas or nonalcoholic fatty pancreatic disease (NAFPD) was shown to correlate with metabolic risk . Indeed,From a theoretical standpoint, the presence of NAFPD may explain why obese patients have worse outcomes than their lean counterparts in acute pancreatitis . It is pWe hypothesize that OSA is an extra hit for the development of NAFPD and subsequent metabolic risk. It is interesting to note that OSA may mediate hypoxic damage to pancreatic beta-cells and, thus, lead to type 2 DM . Below wWe hypothesize that OSA may mediate its detrimental cardiovascular and metabolic effects through the development of ectopic fat deposition in pancreatic tissue.Recent data strongly suggest that OSA is an important contributing factor for the occurrence of dyslipidemia ,30. The More recently a study by Minville et al. showed that OSA and more specifically, nocturnal hypoxia was demonstrated to be independently associated with liver steatosis in obese individiuals further supporting the independent interrelationship between OSA and metabolic diseases . FurtherAn important question is whether OSA is independently (adjusted for confounders such as body mass index (BMI), smoking, age, gender, alcohol intake, family history etc.) associated with organ-specific cancers . Of partWe hypothesize that OSA may contribute to the occurrence of NAFPD in a similar fashion as it does to NAFLD. Future studies should assess whether OSA is a true independent risk factor for the occurrence of NAFPD, given that NAFPD may precede NAFLD  and thusThe relationship between metabolic health and fatty infiltration of the pancreas is a well-known phenomenon, which was first described in 1926 by Schaefer, who showed a correlation between the weight of the human body and that of the pancreas . In 1933Several animal studies have investigated the effects of fat deposition in pancreas. Shimabukuro et al. demonstrated that FFA induced apoptosis of pancreatic beta- cells (endocrine cells producing insulin) in Zucker diabetic fatty rats . Mathur Tushuizen et al. studied 12 males with type 2 DM and 24 age- and BMI-matched non-diabetic men . These rSubsequently, scientific studies showed that increased age, obesity, visceral steatosis, hypertension and type 2 DM were associated with the presence of NAFPD -45. NeveMost studies to date have investigated patients at risk for type 2 DM and, thus, the prevalence may be overestimated. Several imaging modalities can be of use in quantifying pancreatic fat such as trans abdominal and trans esophageal ultrasound, computed tomography and magnetic resonance tomography -46,49-51In a pig model Hanukkainen et al. demonstrated that pancreatic fat is associated with insulin resistance and liver fat content . HoweverMore recently two studies from Taiwan were published on the association between NAFPD and metabolic risk. In a first study, Wu and Wang confirmed an intricate association between NAFPD and metabolic risk factors . They reFuture research is needed to clarify whether NAFPD is a simple consequence of obesity or indeed, an independent player in the development of metabolic complications such as type 2 DM. It will also be interesting to investigate whether the presence of NAFPD is related to the severity of clinical pancreatitis. Of greater importance is whether NAFPD represents a risk factor for the occurrence of exocrine pancreatic cancer.We postulate that NAFPD is an early marker of increased cardiac and metabolic risk. NAFPD may precede clinically overt metabolic disease  and, thus, would be a useful marker of cardiometabolic risk in any individual. Therefore, identifying such individuals is of paramount importance since early intervention may reduce the burden of type 2 DM and cardiovascular disease. On the other hand, OSA was shown to be a risk factor for a myriad of cardiac and metabolic complications such as cardiovascular disease, hyperlipidemia, type 2 DM and NAFLD. Of particular note, NAFLD is a spectrum of metabolic syndrome and is related to a greater risk of vascular disorders. It was reported that OSA is a contributing factor for the development of NAFLD, and through this mechanism may lead to a greater risk of type 2 DM. Furthermore, OSA may exert hypoxic damage to pancreatic β-cells and lead to the occurrence of type 2 DM in susceptible individuals.Further studies are needed to clarify whether OSA is related to NAFPD. Studies assessing the prevalence of NAFPD in patients with OSA (and vice versa) may be needed initially. The results of such studies should be adjusted for common confounders such as age, gender, BMI and obesity, metabolic syndrome, type 2 DM, hyperlipidemia and alcohol intake. Subsequently, longitudinal studies of OSA patients will be needed to assess the impact of OSA on pancreatic fatty infiltration and the natural history of NAFPD. Studying the natural history of NAFPD (with and without concomitant OSA) is of paramount importance and such research should give answers to the following question: is NAFPD truly a benign disease or is it associated with subclinical pancreatic inflammation and a resultant increase in the severity of clinical cases of acute pancreatitis, pancreatic insufficiency (both exocrine and endocrine) and pancreatic cancer. In turn, it should be determined whether OSA is a key player in the development of the above complications.These research questions somewhat parallel those which have been asked about NAFLD, a sister disorder of NAFPD. NAFLD was found to be a non-benign disease, with some patients progressing to liver cirrhosis and hepatocellular cancer. Whether the same is true for NAFPD is a matter for future research.OSA is a common disorder acting as a risk factor for the development and progression of various cardiometabolic derangements. Of note, OSA seems to be an independent risk factor for the development of NAFLD. Recent research data suggest that NAFPD may be a more sensitive marker for early subclinical metabolic risk than NAFLD and may contribute to the progression of subclinical disease to overt type 2 DM. We postulate that OSA may be a risk factor for NAFPD as well. OSA may mediate NAFPD via tissue hypoxia, lipolysis and fat deposition in pancreatic tissue with resultant derangements in pancreatic function. Future studies should investigate the association between OSA and NAFPD as well as give us prospective data on the natural history of NAFPD.BMI: Boby mass index; DM: Diabetes mellitus; FFA: Free fatty acids; HIF: Hypoxia inducible factor; IH: Intermittent hypoxia; NAFLD: Non-alcoholic fatty liver disease; NAFPD: Non- alcoholic fatty pancreatic disease; OSA: Obstructive sleep apnea.The authors declare that they have no competing interests.