Copy number variants (CNVs), defined as losses and gains of segments of genomic DNA, are a major source of genomic variation.In this study, we identified over 2,000 human CNVs that overlap with orthologous chimpanzee or orthologous macaque CNVs. Of these, 170 CNVs overlap with both chimpanzee and macaque CNVs, and these were collapsed into 34 hotspot regions of CNV formation. Many of these hotspot regions of CNV formation are functionally relevant, with a bias toward genes involved in immune function, some of which were previously shown to evolve under balancing selection in humans. The genes in these primate CNV formation hotspots have significant differential expression levels between species and show evidence for positive selection, indicating that they have evolved under species-specific, directional selection.These hotspots of primate CNV formation provide a novel perspective on divergence and selective pressures acting on these genomic regions. Copy number variants (CNVs) are gains or losses of genomic material, and are now known to constitute a major source of genetic polymorphism in humans . High rePan troglodytes) [Macaca mulatta)[CNV hotspots are highly plastic genomic regions where mutations leading to copy number differences between individuals occur more frequently than expected ,12. Amonlodytes) ,13 and r mulatta). We haveIn order to identify primate hotspots for CNV formation, we compiled CNV datasets for human, chimpanzee and rhesus macaque. We used two recently published CNV discovery studies in humans ,15 to as2 ratios significantly deviating from 0  to map the rhesus macaque CNVs onto the human genome reference sequence . This observation is consistent with studies on the recurrence of CNV formation among different individuals  Figure . Of the To quantify and understand the evolutionary mechanisms through which the primate CNV hotspots evolved and to delineate their possible functional impact, we collapsed the 170 HCR CNVs into a manually curated, non-redundant list of 34 primate CNV hotspot regions , within a species. Loci with lower κ values are more likely to have evolved under positive selection. Using this measure, we found that approximately 36% of the genes located within HCR CNVs are positively selected, as opposed to only 9% of the genes that overlap with H CNVs .Most HCR CNVs are associated with genes, and primarily with gene families  are indeed riddled with HCR CNVs and are known to evolve under balancing selection [In addition, we found that at the nucleotide level, HCR CNVs are much less conserved than CNVs found only in humans Figure . Given telection ,20, it iHLA, PHDB, and LILR families). As such, the evolution and maintenance of primate CNV hotspots may be a response to diverse environmental pressures acting on the genes residing in these hotspots. The maintained plasticity may then provide the mutational flexibility for these genes to adapt rapidly to changing selective pressures. Therefore, it is not surprising to see that multiple immune system-related genes are variable in copy number across primates, possibly resonating with the 'Red Queen hypothesis': that the constant diversification of the host immune system genes and the parasite defense genes is in response to changes in each other's defenses [In this study, we identified 170 human CNVs located within 34 primate hotspot regions of CNV formation. The structurally plastic hotspots appear to have remained active in the three lineages despite being separated by over 25 million years of evolution. The majority of primate hotspots overlap with functional genomic elements, especially genes related to immunity. A significant portion of these genes that overlap primate hotspots appear to have evolved under positive selection Figure  and somedefenses .For example, we observed a significant enrichment of HCR CNVs in a chromosome 19 region corresponding to the leukocyte receptor cluster (LRC). In humans, this 1 Mb region encompasses several families of immunoglobulin (Ig)-like receptor genes, including gene clusters encoding multiple leukocyte Ig-like receptors (LILRs), leukocyte-associated Ig-like receptors (LAIRs) and killer-cell Ig-like receptors (KIRs). The KIRs have a multifaceted role in two processes, immune defense and reproduction, and interact with cell-surface molecules encoded by the MHC class I locus, another region that displays rapid evolution and copy number variation. These epistatic interactions likely require the co-evolution of MHC and KIR, similar to the co-evolution of parasitic and host defenses described above. Under ever-changing pathogenic pressures, more of this variation could be maintained, especially among primates, which, due to their complex social dynamics, have higher pathogenic transfer rates . TherefoPGA family) are precursors for pepsin (a major digestive enzyme) and may be involved in local environmental adaptation of primates [CHYS1, involved in wound healing, are also noteworthy. More surprising are gene families such as PHDB and CBX, which may be involved in neural function [Other gene ontological categories are represented, albeit less frequently, in the observed primate CNV hotspots. For instance, the pepsinogens , especially when exposed to genetic and environmental variability [In their classic paper, King and Wilson  recogniziability . Hence, PTEN acts as a sponge for microRNAs. As such, the deletion of the pseudogene subsequently increased the number of microRNA molecules, which can, in turn, negatively regulate the expression of the parental gene [In addition, two recent studies demonstrated that copy number variation in one locus affects the expression levels in other loci. One of these studies showed that the expression level of a gene can be changed through alteration of the copy number variation of another gene that shares the same promoter region . The othtal gene .This study provides a critical framework for describing and delineating the functional, biomedical and evolutionary impact of hotspots of CNV formation in primates. Our results underscore the significance of copy number variation as a widespread source of genomic variation among primates, and the implication of natural selection acting on these regions indicates that CNVs have contributed to the evolution of quantitative traits in primates.2 values are provided in Table S2 in Additional file The existing data for rhesus macaque CNVs  is limitet al. [We analyzed the patterns of these primate hotspots with respect to the human reference genome, due to the high level of accurate annotation of the human genome assembly compared to the draft chimpanzee and rhesus macaque reference sequences. We compiled a non-redundant human CNV dataset using data from two recent, high-resolution studies that had an effective resolution of 450 bp ,15. Thiset al. .To compare the locations of macaque and human CNVs, we used the Lift-Over tool developed by the UCSC Genome Bioinformatics Group to convert rhesus CNV loci to human coordinates (hg18). This computational tool utilizes the BLAT algorithm to align orthologous sequences between species . Using tFor statistical calculations and visualization, we used the R statistical package.aCGH: array comparative genomic hybridization; bp: base pair; CNV: copy number variant; HC: human chimpanzee hotspot; HR: human rhesus macaque hotspot; HCR: human chimpanzee macaque hotspot; kb: kilobase; KIR: killer-cell Ig-like receptor; Mb: megabase; MHC: major histocompatibility complex; NEPRC: New England Primate Research Center.OG and PLB carried out the experiments, conducted most of the preliminary analyses and planned the subsequent analyses. OG, PLB, RCI, QZ and XS conducted the population genetics analyses. REM designed the species-specific arrays. II conducted the frequency distribution analyses. EJW conducted initial selection analyses. AGC helped in planning the selection analyses and writing. OG, WEJ and CL conceived and wrote the study. All authors read and approved the final manuscript.Supporting tables. The tables of the CNVs considered in the study and summaries of additional analyses.Click here for fileSupporting figures. Supporting figures for the manuscript.Click here for fileSupporting methods. Additional description of methodologies described in the manuscript.Click here for file


Body fat deposition and excess free fatty acid (FFA) metabolism contribute to dyslipidemia and the adverse health consequences of obesity. Individuals with upper body obesity have impaired functioning of adipocytes, the primary fatty acid storage site. Excess visceral fat is strongly associated with impaired suppression of FFA release in response to insulin, as well as with hypertriglyceridemia and low concentrations of high density lipoprotein (HDL) cholesterol. High FFA concentrations can induce insulin resistance in muscle and liver. Furthermore, failure of hyperinsulinemia to normally suppress FFA is associated with impaired carbohydrate oxidation and muscle glucose storage, reduced hepatic insulin clearance and elevated triglycerides. Understanding the impact of body fat distribution on FFA metabolism and dyslipidemia is critical for determining the link between overweight and obesity and cardiovascular disease risk. In the current review, we will explore the relationship between adipose tissue, body fat depots, and FFA metabolism. Independent of total body fat, body fat distribution is an important and established risk factor for cardiovascular disease (CVD). Clinical measures of abdominal obesity, also referred to by some as upper body obesity, such as waist-to-hip ratio and waist circumference, are directly associated with progression of atherosclerosis  and the The majority of adults with abdominal obesity as defined by waist-hip ratio also have disproportionate amounts of omental and mesenteric  fat. Visceral fat is considered by many to behave as an ectopic fat depot, accumulating triglycerides (TG’s) when body fat storage needs exceed the capacity of subcutaneous fat depots to function normally . AbnormaVisceral adiposity is strongly associated with the metabolic abnormalities that increase CVD risk. However, the explanation for this association is incompletely understood. One hypothesis is that visceral fat directly produces substances that cause the metabolic derangements associated with increased CVD risk in obesity ,6,7. AnoOne of the classic characteristics of obesity is dyslipidemia characterized by high levels of TG’s in very-low-density lipoproteins (VLDL) and low levels of high-density lipoprotein (HDL) cholesterol . This tyAdipose tissue plays a dynamic and critical role in fuel metabolism. Adipocytes produce and secrete a broad array of proteins and other molecules such as leptin, adiponectin, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) . Fat stoFat depots can broadly be functionally and anatomically grouped into lower body fat, upper body subcutaneous fat, and visceral/intra-abdominal fat. In normal weight adults, upper body and lower body subcutaneous fat comprise the majority of total body fat . Lower bObesity is often associated with impaired functioning of adipose tissue. If lipolysis per kilogram of fat were the same in obese and non-obese individuals, the increase in plasma FFA concentrations would be much more striking in obesity. Fortunately, there appears to be some down-regulation of lipolytic rates per kilogram of adipose tissue such that in obesity plasma FFA are only 20%–30% greater than normal in the absence of type 2 diabetes ,23,24,25Excess FFA contributes to the adverse health consequences of obesity. It is not as simple as merely greater rates of whole body lipolysis leading to adverse metabolic consequences, however. For example, adipose tissue FFA release is significantly (~40%) greater in women than men relative to energy needs and fat oxidation , yet FFACompared with lean women, women with upper body obesity have lower basal adipose tissue lipolysis per kilogram of fat, but systemic FFA release is greater because of their higher fat mass . FFA relUpper body obesity is associated with increased FFA release in both the postabsorptive and postprandial states, however, we believe the true hallmark of upper body/visceral obesity is the impaired suppression of FFA release in response to insulin  and mealExpanded visceral fat depots and hyperinsulinemia do have adverse effects on the liver. The liver takes up a large proportion of FFA entering the splanchnic bed through the portal vein ,42, withThe dyslipidemia of obesity is characterized by hypertriglyceridemia, an established independent risk factor for CVD . Each 90One function of VLDL is to serve as a means for the liver to export excess energy in the form of triglycerides. VLDL assembly is, therefore, dependent upon the availability of TG . HepaticAs noted above, is appears that omental and mesenteric fat, because of their portal vein venous drainage, play an important role in abnormalities of hepatic lipid metabolism. There is evidence that visceral fat depots are more resistant to insulin than leg fat and upper body subcutaneous fat , and thaDyslipidemia contributes substantially to the CVD risk associated with obesity. In obese adults, adipose depots contain over 50% of total body cholesterol . ChangesLipoprotein metabolism is partially regulated by the triglyceride lipases lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL). High post-heparin LPL activity has been associated with high plasma concentrations of HDL cholesterol ,65,66 anExpanded fat depots may be adversely effecting serum concentrations of HDL cholesterol by influencing HDL metabolism. The overproduction of FFA and VLDL-TG reduces plasma HDL-cholesterol concentrations . ElevateObesity and pathologically increased fat depots adversely affect the functionality and effectiveness of HDL particles. Expanded visceral fat depots, for example, decrease the anti-apoptotic activity of HDL particles .Obesity, and specifically upper body/visceral obesity, is associated with insulin resistance, dyslipidemia and increased cardiovascular risk. Abnormal regulation of subcutaneous adipose tissue lipolysis in those with visceral obesity is likely to play an important role in these abnormalities, although dysregulation of adipokine secretion may also be important. Increased visceral fat, as well as being a good marker for subcutaneous adipose tissue insulin resistance, may also selectively affect hepatic metabolism via greater portal vein FFA delivery. In contrast, insulin resistance in skeletal muscle and abnormal insulin secretion is largely driven by abnormal upper body subcutaneous adipose tissue lipolysis. It will be interesting to determine if treatments that selectively improve the regulation of upper body subcutaneous fat or visceral fat may have differential benefits on lipids.


Blood coagulation is a complex biological mechanism aimed to avoid bleeding in which a highly regulated and coordinated interplay of specific proteins and cellular components respond quickly to a vascular injury. However, when this mechanisms occurs in the coronary circulation, it has not a “protective” effect, but rather, it plays a pivotal role in determining acute coronary syndromes. Coagulation recognizes Tissue Factor (TF), the main physiological initiator of the extrinsic coagulation pathway, as its starter.Since TF:VIIa complex is the critical point of the blood coagulation cascade, it is a pharmacological attractive issue for the development of agents with anti thrombotic properties that can exert their activity by inhibiting complex formation and/or its catalytic activity. In fact, it is intuitive that an antithrombotic agent able to inhibit this initial step of the coagulation pathway has several theoretical, extremely important, advantages if compared with drugs active downstream the coagulation pathway, such as FXa or thrombin. The present report gives a brief overview of TF pathophysiology, highlighting the most recent advances in the field of inhibitors of the complex TF/VIIa potentially useful in cardiovascular disease. Blood coagulation is a complex biological mechanism aimed to avoid bleeding in which a highly regulated and coordinated interplay of specific proteins and cellular components respond quickly to a vascular injury. However, when this mechanism occurs in the coronary circulation, it has not a “protective” effect, but rather, it plays a pivotal role in determining acute coronary syndromes .Coagulation recognizes Tissue Factor (TF), the main physiological initiator of the extrinsic coagulation pathway, as its starter. Indeed, several experimental and clinical studies indicate that TF plays a pivotal role in the pathophysiology of acute coronary syndromes: it triggers the formation of intracoronary thrombi following endothelial injury -5. TF isSince TF/VIIa complex formation represents the critical point of the blood coagulation cascade, it is an attractive tool for the development of agents with anti thrombotic properties, which could inhibit complex formation and/or its catalytic activity. Indeed, it is intuitive that an antithrombotic agent able to inhibit this initial phase of the coagulation pathway has several theoretical, extremely important, advantages if compared with drugs active downstream the coagulation pathway, such as FXa or thrombin, since this pathway is specifically blocked right from the beginning.The present report gives a brief overview of TF pathophysiology, highlighting the recent advances in the field of inhibitors of the TF/VIIa complex useful in cardiovascular disease treatment.1). It is intuitive that TF is normally not exposed to circulating blood to avoid its improper interaction with other circulating coagulation factors. Vice versa, vascular injury, through physical damage of the endothelial layer of the blood vessel, causes the exposure of TF to circulating blood, making it accessible to circulating factor VII (FVII) [via limited proteolysis [2). Activated FVII binds Factor X (FX), that, in turn, is converted in its activated form (FXa). Then, the extrinsic pathway continues, leading to thrombin activation and clot formation [via an auto activation reaction [Tissue Factor (TF), also known as thromboplastin or CD142, is a glycosylated transmembrane protein consisting of a single polypeptide chain  . This glI (FVII) -16. ThisI (FVII) . Once boteolysis -19 . TFPI is composed of three Kunitz-type protease inhibitor domains: the first Kunitz domain reacts with the active site of FVIIa in the TF:VIIa complex [in vivo infusion of heparin increases the circulating levels of TFPI in plasma 2- to 4-fold [in vivo represents the carrier-free molecule, which might be biologically most active [Pathophysiology of coagulation is tightly regulated by another important protein known as Tissue Factor Pathway Inhibitor (TFPI), that is the endogenous inhibitor of the extrinsic coagulation pathway; specifically, TFPI is a potent inhibitor of the TF/FVIIa complex, and its action is related to the presence of FXa   complex , while t complex , 38. Muc complex -40; this complex . The in o 4-fold , 43. Thet active , thus suSeveral experimental and clinical studies have clearly demonstrated that the complex TF/FVIIa is the key initiator of the coagulation cascade in cardiovascular disease .TF expression represents the link explaining the relationship existing among several chemical mediators and the pathophysiology of coronary artery disease as well as of its main complication represented by acute coronary syndromes. In fact, it has been demonstrated that these mediators might exert their effects by inducing TF expression. Specifically, inflammatory markers such as C-Reactive Protein or neopterin as well as smoke-derivative substances might play an important role in acute coronary syndromes since it has been demonstrated that they are active partaker in triggering coronary TF-mediated coagulation , 45, 46.in vivo hearts subjected to ischemia and reperfusion, a condition associated with a production of oxygen free radicals in large amounts, a marked increase in TF activity occurred. This increase was accompanied by a significant impairment of coronary flow during reperfusion and possibly contribute to the occurrence of reperfusion injury [Moreover, oxygen free radicals, endogenously generated upon post-ischemic reperfusion, induce TF-mRNA transcription and expression of TF procoagulant activity. As seen in in ex vivo and n injury .et al, analyzing gene expression in coronary plaques from patients with stable or unstable angina using gene arrays, demonstrated higher TF expression in unstable angina samples [et al demonstrated that systemic TF activity in acute myocardial infarction has an unfavorable prognostic value and, as a marker for altered coagulation, it might predict the atherothrombotic risk [Immunohistochemistry studies have demonstrated that TF can be detected in several cell types, such as monocytes, foam cells, and fibroblasts isolated from human atherosclerotic coronary and carotid plaques , 48. Int samples . Furthertic risk .et al. showed that polymorphisms in the factor VII gene promoter on activated factor VII levels may modulate the risk of myocardial infarction in males with advanced coronary artery disease [et al. have recently demonstrated that raised FVII and leptin levels in coronary artery disease (CHD) patients were independently associated with insulin resistance; this was not observed among the non-CHD subjects [Plasma levels of FVIIa seem to be an another key point of the coagulation cascade. Bozzini  disease . In addi disease . Moreovesubjects .Taken together, these data clearly underline the importance of TF:FVIIa as one of the main determinant of human atherosclerotic plaque thrombogenicity.2), recombinant human TFPI might be useful in patients with acute coronary syndromes. Thus, this protein has been successfully expressed in a variety of hosts, including bacteria, and has been shown to be effective in preventing thrombus formation in a variety of experimental models. Haskel et al. [et al. [et al. [in vivo gene transfer technologies has created powerful new tools to transfer foreign genes into the cells of a variety of organs, including the vascular wall. Different vectors have been developed to efficiently transfect target cells, including retroviral, adenoviral, and direct DNA transfer. Therefore, giving the feasibility of transfecting the arterial wall with foreign genes, it is of no surprise that TFPI has been the focus of several studies in this field. The main theoretical advantage of increasing local TFPI concentrations by gene transfer to the arterial wall is that therapeutic TFPI levels can be achieved only where they are needed, i.e., at the damaged arterial site where TF is exposed, without concomitant, potentially dangerous systemic effects. Thus, starting from these considerations, several studies have clearly demonstrated the antithrombotic efficacy of arterial TFPI gene transfer in different models of intravascular thrombosis [Since TFPI has an important role in modulating the activity of the TF:FVIIa complex .Recently, human recombinant FVIIa in which the active site of FVIIa is blocked with a covalent inhibitor, such as chloromethylketone (known as rFVIIai), has been produced and successfully used to block TF:VIIa procoagulant activity. rFVIIai retains its TF binding capacity but is enzymatically inactive. This molecule exerts its antithrombotic effect by competing with native factor VIIa (FVIIa) for TF binding. Since it has a significantly higher affinity to TF than native FVIIa, it avoids that coagulation cascade could proceed downstream -64  . On the in vitro and in vivo at very low concentrations [3). In particular, administration of AP-1 to rabbits with recurrent thrombosis of the carotid artery was associated with a complete inhibition of thrombosis without a concomitant prolongation in systemic hemostatic parameters or an alteration in platelet aggregation [Another potential option to interfere with TF/FVIIa complex is represented by antibodies directed against TF: binding of these antibodies prevents FVII interaction with its natural ligand. One of the first antibody able to interfere with TF/ FVIIa complex was AP-1, a monoclonal antibody raised against rabbit TF. AP-1 has been proven to block TF-procoa-gulant activity trations  regation . The samregation .Recently, it has been developed a chimeric mousehuman monoclonal antibody directed against TF and known as ALT836. This antibody binds to TF at its FX binding site. In patients with stable coronary artery disease enrolled in the PROXIMATE-TIMI 27 trial, this antibody had an interesting dose-dependent anticoagulant effect without any significant side effect such as bleeding .Another potent TF:VIIa complex inhibitor is known as XK1. It is a chimeric protein which consists of the light chain of FXa linked to the first Kunitz domain of TFPI . Other hin vivo after parenteral administration, but research of an orally bio-available drug still remains an undiscovered field. Comforting results observed in preclinical models and clinical trials [As reported above, TF/FVIIa inhibitors have been successfully tested l trials , 82 in wMoreover, since TF/FVIIa inhibitors might be useful also in preventing other TF-mediated phenomena, such as inflammation and cell proliferation, these molecules should be tailored to exert their effects only where they are needed, without affecting the physiological haemostasis.In interventional cardiology area, particularly attractive could be the engineering of a balloon coated with an inhibitor of TF/FVIIa, such as rFVIIai able to permit local drug release at site of injury/thrombosis, during percutaneous transluminal coronary angioplasty (PTCA). Alternatively, a TFPI-coated balloon might be used in the same clinical setting, in order to increase local TFPI concentrations and obtain a local antithrombotic effect. Finally, a fascinating hypothesis might be that of a balloon coated with genes codifying for TFPI, to directly obtain gene transfer to the arterial wall, in order to facilitate the stabilization of active atherosclerotic plaque before stent deployment.TF:VIIa complex represents the “critical point” of the extrinsic pathway of blood coagulation. Thus, it is intuitive why it has become an attractive tool for the development of newer antithrombotic agents able to prevent complex formation or to inhibit its catalytic activity. This kind of antithrombotic therapy has several theoretical advantages if compared with other interventions directed against other “downstream” components of the coagulation cascade, such as heparin and its derivatives or direct antithrombin agents.Indeed, although potent synthetic inhibitors of TF/FVIIa had been discovered and tested in animal models, any of these have advanced into clinical trials. The systemic effects observed and, specifically, the marked elongation of bleeding time observed in experimental studies have highlighted that safety, effective dose and route of administration are the main issues to resolve. On the other side, while the local delivery of genes or drugs during interventional procedures seems very intriguing, the feasibility of this approach needs to be demonstrated.However, in spite of these considerations, TF:FVIIa complex still remains a challenging and important target to study and develop future generations of antithrombotic agents.


Platelets contribute to vascular homeostasis and angiogenesis through the release of multiple growth factors, cytokines and nucleotides, such as ATP and ADP. Recent reports have demonstrated a marked growth-promoting effect of total platelet extracts and selected platelet growth factors on therapeutic angiogenesis. However, since endogenous adenine nucleotides are rapidly degraded during the platelet isolation and storage, we examined whether supplementing a platelet-derived extract with exogenous adenine nucleotides would augment their pro-angiogenic effects.vasa vasorum endothelial cells (VVEC) were used to examine the effects of dialyzed platelet-derived soluble extracts and extracellular adenine nucleotides on proliferation, migration and tube formation. In addition, an in vivo Matrigel plug assay was used to examine the effects of platelet extracts and adenine nucleotides on neovascularization of plugs subcutaneously placed in 50 ICR mice. The number of vascular structures in Matrigel plugs were evaluated by histological and statistical methods.Pulmonary artery Platelet extracts (6.4-64 μg/ml) significantly induced DNA synthesis and at a concentration of 64 μg/ml had a biphasic effect on VVEC proliferation (an increase at 48 hrs followed by a decrease at 60 hrs). Stimulation of VVEC with platelet extracts also significantly  increased cell migration and tube formation on Matrigel. Stimulation of VVEC with extracellular ATP (100 μM) dramatically (up to ten-fold) increased migration and tube formation on Matrigel; however, no significant effects on cell proliferation were observed. We also found that ATP moderately diminished platelet extract-induced VVEC proliferation (48 hrs) and migration, but potentiated tube formation. Neither ATP, or a mixture of non-hydrolyzable nucleotides  induced vascularization of Matrigel plugs subcutaneously injected in mice, however, the combination of these nucleotides with platelet extracts dramatically increased the number of functional capillaries in the Matrigel plugs.Data from this study suggest that platelet-derived growth factors and extracellular nucleotides represent important regulatory signals for angiogenesis. Supplementation of platelet extracts with exogenous adenine nucleotides may reveal new possibilities for therapeutic angiogenesis and tissue regeneration approaches. Angiogenesis is a fundamentally important process occurring under both physiological and pathological conditions, including embryonic development, tumor progression, and tissue regeneration. Regulation of angiogenesis requires a balance between multiple pro- and anti-angiogenic factors and involves interaction of the vascular wall and circulating blood cells -3. Platein vitro Matrigel tube formation and migration assay  thymidine  for 24 hrs. Incorporated radioactivity was measured using a β-counter as previously described  thymidine incorporation and cell proliferation assays. Data in Figure The effects of platelet-derived growth factors on VVEC mitogenesis were examined using [Cell proliferation assay showed a gradual increase in cell numbers in response to stimulation with 6.4, 16 and 64 μg/ml of platelet extracts for 48 hrs Figure . The stiAnalysis of VVEC migration in the Boyden chamber assay demonstrated that platelet extracts at the concentration of 32 μg/ml increased the number of migrated cells. Noteworthy, clusters of migrated cells were formed on the underside of the transwell membrane , however no distinguishable network formation was observed . Incubation of VVEC in the presence of platelet extracts (16 μg/ml) for 10 hrs resulted in visible cell rearrangements into tube-like networks  evaluated as an increased total length of satisfactory-developed tubes and a decreased length of poorly-developed tubes . The number of tubes per plug was lower than in the control group .Furthermore, the effects of adenine nucleotides on Matrigel plugs vascularization were evaluated. Data in Figure 5p Figure . Notablyin vitro, it was expected that a similar effect would be observed in plug vascularization in vivo. Data in Figure panel e show that subcutaneous injection of Matrigel supplemented with mixtures of non-hydrolyzable nucleotides, ATPγS, ADPβ, MeSATP, and MeSADP  and platelet extracts (58 μg/ml) resulted in the enhanced formation of tubular structures and functional capillaries in Matrigel plugs. In addition, in some plugs, blood infiltrates can be observed, suggesting that some neovessels/immature blood conduits, prone to rupture, might have formed there . The tube numbers in nucleotide -containing plugs were almost the same for each nucleotide concentration, and these numbers were lower than those in the plugs supplemented with platelet extracts only .Since ATP potentiated the effect of platelet extract on VVEC tube formation y Figure . Data iny Figure . Histoloin vitro and in vivo studies suggest that platelet extract and adenine nucleotides promote distinct but likely complementary signaling events, leading to functional neovessel formation.Altogether, the results from our in vitro and in vivo. Our data demonstrated that platelet extracts and extracellular adenine nucleotides exerted similar, but not identical effects on endothelial proliferation, migration and tube formation. The main conclusion from this study is that extracellular adenine nucleotides modulate angiogenic effects of platelet extracts in vitro and, importantly, lead to a greater amount of functional capillary formation in Matrigel plugs in vivo.Development of improved strategies for therapeutic angiogenesis remains an imperative task for treatment of a variety of pathological conditions. Of all the circulating blood cells, platelets are the most abundant sources of endogenous angiogenic regulators. In this study we challenged the hypothesis that combination of platelet-derived factors with extracellular nucleotides, also known as regulators of the vascular growth, would have even more potent angiogenic effects As mentioned above, platelets contain multiple constituents including angiogenic mediators, extracellular matrix components, nucleotides, sugars, growth factors, antimicrobial peptides, and cytokines that can support wound healing and angiogenesis ,14,30,33However, despite a strong evidence of angiogenic effects of platelets, there is also some degree of skepticism about the utility of platelet-derived components for a modulation of angiogenesis due to a high degree of variability in clinical outcomes, which are probably associated with: (i) lack of sufficient release of key growth factors following an incomplete platelet "activation" with a single factor, such as thrombin, during PRP preparation; (ii) failure to separate the platelet-derived growth factors from the bulk of the highly concentrated serum components, which may limit bioavailability; (iii) failure to determine at least the protein concentration of the active platelet extract (separated from the plasma) and using this to establish a proper dosing; and iv) variability in the composition, and therefore activity, of platelet extracts from different individuals that may need to be accounted for in the dosing. Reducing variability in the clinical response to PRP remain as important goal in bringing platelet extract preparation methods to full utility.In the present study we demonstrated that total cell extracts isolated from preserved platelets have an angiogenic potential. Our approach of using total platelet extracts assumed that a crude extract isolated by a lysis of platelet pellet, even though lacking the ability of intact platelets to sense and respond to signals in a tissue environment, can still be used to produce a balanced regenerative response in a tissue. According to our established procedure, soluble platelet extracts were prepared by platelet sonication and subsequent stabilization of soluble components through the dialysis of cellular extract against 10 mM HCl in a 3.5 KDa cut-off membrane. Based on some reports, mildly acidic environment  may possibly optimize the stability and activity of platelet-derived factors such as PDGF and TGFβ ,40. Howein vivo models of angiogenesis, as well as combined effects of extracellular nucleotides and platelet-derived growth factors on angiogenesis have not yet been investigated. Our present study provides new evidence that platelet-derived extracts and adenine nucleotides exerted diverse, but complementary angiogenic effects in isolated VVEC. Platelet extract induced DNA synthesis and affected proliferative responses in VVEC. The proliferative response was at its highest at 48 hrs, followed by a decrease at 60 hrs. This biphasic effect may suggest a possibility that the initial increase in VVEC proliferation may be followed by more long-term regulatory signals leading to endothelial differentiation, stabilization and/or morphological changes. Strikingly, extracellular ATP, despite strong effects on DNA synthesis, did not induce VVEC proliferation and to some extent diminished proliferative responses induced by platelet extracts measured at 48 hrs, again, suggesting prolonged and more complex integrative signaling events underlying the action of extracellular nucleotides and platelet-derived angiogenic factors.A variety of pathological conditions in the vasculature is accompanied with elevated levels of extracellular ATP and ADP, platelet immobilization, activation, and their adhesion to endothelial cells, with ultimate endothelial cell activation. These events may possibly lead to angiogenesis and initiation of vascular regeneration processes ,41-44. Ain vitro studies demonstrated that both extracellular ATP and platelet extracts exert angiogenic effects in VVEC, but only morphogenetic effect was potentiated in response to the combined action of extracellular ATP and platelet extracts. Further investigation is necessary to define molecular mechanisms contributing to intracellular signaling cross-talk initiated by purinergic receptors and receptor tyrosine kinases in VVEC.Our study also revealed differences in extracellular ATP- and platelet extract-induced migratory responses. Extracellular ATP had a more potent effect (~75- fold) on VVEC migration in contrast to the effect induced by platelet extracts (~7-20-fold). However, more dramatic effects (up to 65-fold) can be observed in response to platelet extracts derived some units of preserved human platelets  speeded up the healing process of incision wounds placed on the backs of mice (unpublished observation). However, some variations in minimal effective concentration of platelet extracts were observed among human plasma sources. For instance, platelet extracts used in this study exhibit the highest activity at concentrations between 16 and 64 μg/ml. The functionality of capillaries in Matrigel plugs was evaluated based on microscopic detection of erythrocytes within the capillary lumen, suggesting a link to the systemic circulation. In agreement with the in vitro data, we observed increased formation of tube-like structures in Matrigel plugs supplemented with platelet extract. Despite a number of studies showing angiogenic effects of extracellular nucleotides in various cell systems, the angiogenic effects of extracellular nucleotides in the in vivo Matrigel plug model have not been previously investigated. To our surprise, tube formation in the Matrigel plugs supplemented with extracellular ATP was even lower than the tube formation in the control plugs.Consistent with the results from the It would have been expected that extracellular ATP might be hydrolyzed to adenosine by endothelial and blood cell ecto-nucleotidases, resulting in decreased plug vascularization. Therefore, we evaluated the effect non-hydrolyzable nucleotide analogs, ATPγS, ADPβS, MeSATP, MeSADP. No additional tube formation was observed in response to these nucleotides, suggesting that decreased plug vascularization in response to ATP could not be explained by its hydrolysis to adenosine. Remarkably, we found more cellularity in some Matrigel plugs, containing mixtures of high concentrations of extracellular nucleotides (1 mM). Considering that monocytes/macrophages and endothelial progenitor cells may contribute to neovascularization, and can be attracted by platelet-derived microparticle components ,46-49, wIn conclusion, our study provides new evidence for the angiogenic effects of platelet-derived extracts in combination with extracellular adenine nucleotides. These results shed new light on the role of platelets and extracellular nucleotides in the regulation of vascular hemostasis and angiogenesis. Our findings could be clinically applicable for the optimization of wound healing, tissue engineering or vessel stabilization, important in many pathological conditions, such as those observed in tumor and diabetes complications.The authors declare that they have no competing interests.MR performed Matrigel plug experiments in mice and analyzed Matrigel plug vascularization. HN performed angiogenic assays and analyzed the data. NB isolated VVEC and performed immunohistochemistry. AAS performed migration data analysis. KRS provided lung tissue for VVEC isolation and critical comments for the manuscript. EVG designed the study, performed in vitro experiments and analyzed the data. The manuscript was written by EVG and MR. All authors read and approved the final manuscript.


Short-term plasticity (STP) denotes changes in synaptic strength that last up to tens of seconds. It is generally thought that STP impacts information transfer across synaptic connections and may thereby provide neurons with, for example, the ability to detect input coherence, to maintain stability and to promote synchronization. STP is due to a combination of mechanisms, including vesicle depletion and calcium accumulation in synaptic terminals. Different forms of STP exist, depending on many factors, including synapse type. Recent evidence shows that synapse dependence holds true even for connections that originate from a single presynaptic cell, which implies that postsynaptic target cell type can determine synaptic short-term dynamics. This arrangement is surprising, since STP itself is chiefly due to presynaptic mechanisms. Target-specific synaptic dynamics in addition imply that STP is not a bug resulting from synapses fatiguing when driven too hard, but rather a feature that is selectively implemented in the brain for specific functional purposes. As an example, target-specific STP results in sequential somatic and dendritic inhibition in neocortical and hippocampal excitatory cells during high-frequency firing. Recent studies also show that the Elfn1 gene specifically controls STP at some synapse types. In addition, presynaptic NMDA receptors have been implicated in synapse-specific control of synaptic dynamics during high-frequency activity. We argue that synapse-specific STP deserves considerable further study, both experimentally and theoretically, since its function is not well known. We propose that synapse-specific STP has to be understood in the context of the local circuit, which requires combining different scientific disciplines ranging from molecular biology through electrophysiology to computer modeling. The functioning of the brain is governed by its neuronal connectivity and by the synaptic dynamics of these connections. Learning and information storage in the brain, for example, are widely thought to be due to long-term changes in connective strength, as postulated by Donald Hebb  and otheSynaptic short-term plasticity (STP) is thought to result from a combination of mechanisms, chiefly presynaptic ones, including vesicle depletion and accumulation of calcium in the presynaptic terminal during prolonged high-frequency activity, but desensitization of postsynaptic neurotransmitter receptors also matter  connections from granule cells to Purkinje cells and basket or stellate INs, with similar functional consequences. Whilst PF synapses onto Purkinje or stellate cells exhibit persistent facilitation, PF inputs to BCs display short-term depression following initial paired-pulse facilitation  INs and that may signal postsynaptic identity to presynaptic terminals. Knockdown of Elfn1 in OLM INs led to a marked reduction in facilitation and in an increase of prelease at connections from CA1 PCs, an effect not seen in uninfected neighbor cells  INs exhibit greater facilitation than those to CA1 PCs  INs had less facilitation or even depression. Presynaptic terminal properties can thus differ widely depending on the target cell, even across IN types. Properly determining IN class is therefore vital when investigating target-specific STP (Ascoli et al., Other hippocampal connections show more varied target-specific STP. For example, synapses from CA3 PCs to CA1 prelease, resulting in facilitation. Synapses onto CA3 INs, however, are less facilitating, or can be depressing (Salin et al., In CA3, large boutons of mossy fibers originating from granule cells in the dentate gyrus contact PCs, whilst smaller synapses impinge on INs (Acsady et al., Whilst the studies above often describe STP between excitatory cells, target-specificity has usually been established only by comparison to connections onto inhibitory interneurons. Target-specific STP at different excitatory-excitatory connections is therefore an interesting possibility that has been investigated in less detail, perhaps as the recordings required for direct comparison are more difficult to achieve experimentally. This said, some studies have touched on this topic. In neocortex, stellate cells in layer 4 connect with depressing synapses (Egger et al., per se remains to be shown, they constitute appealing candidates.Target-cell-specific STP predicts that neighboring presynaptic compartments may possess dissimilar release properties, as governed by their postsynaptic partners (see Figure Cadherins are a group of synaptic adhesion molecules widely believed to be involved in e.g., synapse formation (Takeichi and Abe, prelease. In a study by Bozdagi et al. (prelease. Another study, in this case using presynaptic wild-type neocortical neurons paired with embryonic stem cells lacking N-cadherin, showed enhanced short-term depression in response to 50-Hz stimulation (Jungling et al., release (Vitureira et al., i et al. , postsynNeuroligins, another group of postsynaptic cell adhesion proteins with a role in synapse maturation, has also been implicated in transsynaptic regulation of neurotransmitter release (Dean and Dresbach, prelease (Varoqueaux et al., PSD95 postsynaptically, which has previously been shown to accelerate synaptic maturation (El-Husseini et al., prelease. Overexpression of PSD95 or neuroligins reduced paired-pulse ratio, while deletion of the same proteins increased paired-pulse ratio, indicating that either PSD95 or neuroligins can increase prelease. The effect of up-regulating one of the proteins was occluded by down-regulating the other. Overexpression resulted in increased sensitivity to extracellular calcium concentration and in higher glutamate concentration in the synaptic cleft, indicating that more vesicles were released. The effect on release was mediated by presynaptic β-neurexins (Futai et al., prelease, possibly by controlling both the vesicle pool size and the calcium sensitivity. In addition to their individual effects on transmitter release, the cadherin and neuroligin systems can cooperate in regulating release from presynaptic terminals. Stan et al. (prelease.n et al.  found thSynCAM, an immunoglobulin domain–containing homophilic synaptic cell adhesion molecule, that when overexpressed in hippocampal neurons brings about an increase in excitatory spontaneous release (Biederer et al., SAP97, a postsynaptic scaffolding protein, increased vesicle release probability, presynaptic protein content, and the size of the active zone (Regalado et al., Shank1, a synaptic scaffolding protein implicated in autism (Jiang and Ehlers, There are several other molecules that have been suggested to play a part in transsynaptic regulation of presynaptic transmitter release via activation of either the neuroligin or the cadherin system. One example is The above synaptic adhesion molecules may enable cells to signal specifically to their own presynaptic boutons. The regulation of neurotransmitter release could subsequently be achieved via many different presynaptic mechanisms (reviewed in Zucker and Regehr, In the past decade, attention has turned to presynaptic ionotropic receptors in synapse-specific control of transmitter release (Engelman and MacDermott, NMDA receptors are tetrameric ionotropic glutamate receptors that have been implicated in memory formation and in disease states such as pain, neurodegeneration and schizophrenia (Paoletti et al., prelease during high-frequency firing, such as the layer-5 PC complex spike (Larkum et al., per se is unknown.Using NMDA uncaging onto PC axons in combination with paired recordings and pharmacology, we found that in layer 5 of developing visual cortex, preNMDARs are present at connections from PCs to other PCs as well as to MCs, but not to BCs Figure  Buchana. These pOver development, STP switches in several brain regions from high to low probability of release, i.e., from short-term depression to relatively more facilitating short-term dynamics (Bolshakov and Siegelbaum, What is the function of this developmental STP switch? Cheetham and Fox  proposedOne interesting possibility is that the dynamics of different synapse types may mature in a differential manner. In other words, synapse types with similar STP may become dissimilar with age, while those with different synaptic dynamics in the juvenile state may be indistinguishable as the brain reaches maturity. To our knowledge, this possibility has not been extensively explored. One study by Takesian et al. , howevera priori theoretical reason to believe that STP ought to be specific to synapse type at all—STP could for example be heterogeneously determined by active learning rules that control synaptic dynamics (Markram and Tsodyks, In this review, we have discussed the functions and mechanisms of STP, with a specific focus on synapse-specific forms of synaptic dynamics. We should point out that, from a naïve and unbiased point of view, there is no An emerging principle is thus that STP must be analyzed and understood in the context of the local circuit. This requires combining different scientific disciplines ranging from molecular biology through electrophysiology to computer modeling. This also critically requires that cell types be properly identified, which itself can be a major challenge (Markram et al., Still, it is important to recall that STP can also be directly postsynaptically determined, for example due to desensitization of calcium-permeable AMPA receptors (Rozov and Burnashev, But we should re-iterate that the postsynaptic neuron does not solely determine synapse type; target-cell-specific STP is merely the specific focus of this particular review article. Indeed, Gupta et al.  summarizFurthermore, since most synapses seem to undergo developmental switches in STP, as discussed above, this potentially alters the picture of target-specific STP. For example, connections between PCs in neocortex have been observed to change from depressing to facilitating during development (Reyes and Sakmann, The inhibitory circuits activated by target-specific STP are also subject to modulatory and disinhibitory control. For example, monocular deprivation during a critical period leads to a transient reduction in the activity of PV-specific inhibitory circuits in binocular visual cortex, which is permissive for competitive plasticity and ocular dominance shifts (Kuhlman et al., Forms of plasticity other than STP may also be synapse specific and may thus depend on the postsynaptic cell type. For example, long-term plasticity depends on the target inhibitory cell type in neocortex (Lu et al., Another emerging principle is that late-onset INs target the dendrite of local principal neurons, whereas early-onset INs are perisomatically innervating (see Figure Based on a normative theoretical approach, Pfister et al.  found thFmr1 gene knockout results in pathological STP (Deng et al., An involvement of target-cell specific STP in disability and disease has also emerged recently. For example, the Fragile X Mental Retardation Protein FMRP regulates neurotransmitter release presynaptically (Deng et al., In this paper, we have overviewed the possible functions and mechanisms of target-specific STP. We have listed several examples of STP specific to the target cell, and we have found a few common principles, but it is clear that not nearly enough is known about the why and the how, which means the specificity of STP should be investigated considerably more, both experimentally and theoretically. We argue that to understand the function of target-specific STP, researchers have to examine the results in the context of the local circuit, with its many different cell and synapse types (e.g., Pouille and Scanziani, 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.


Despite recent advances, the treatment of malignant melanoma still results in the relapse of the disease, and second line treatment mostly fails due to the occurrence of resistance. A wide range of mutations are known to prevent effective treatment with chemotherapeutic drugs. Hence, approaches with biopharmaceuticals including proteins, like antibodies or cytokines, are applied. As an alternative, regimens with therapeutically active nucleic acids offer the possibility for highly selective cancer treatment whilst avoiding unwanted and toxic side effects. This paper gives a brief introduction into the mechanism of this devastating disease, discusses the shortcoming of current therapy approaches, and pinpoints anchor points which could be harnessed for therapeutic intervention with nucleic acids. We bring the delivery of nucleic acid nanopharmaceutics into perspective as a novel antimelanoma therapeutic approach and discuss the possibilities for melanoma specific targeting. The latest reports on preclinical and already clinical application of nucleic acids in melanoma are discussed. Melanoma derivates from melanocytes—pigment cells of the skin. Melanoma most commonly arises from epidermal skin melanocytes (cutaneous melanoma), but primary tumors can also be found lining the choroidal layer of the eye  or the mucosal surfaces of the respiratory, genitourinary, and gastrointestinal surfaces. Similar to other tumors, the progression stage of melanoma is predictive for therapeutic success. Early stage melanomas (thin tumors) result in a 97% 5-year survival rate of the patients, after surgical removal . ConversBased on clinical and histopathological features melanoma cancer cells undergo four sequential phases before reaching metastasis . These pPresently, it is generally believed that melanomagenesis instigates from alterations in multiple molecules or pathways rather than a single high-risk melanoma loci. Moreover, melanoma progression is a dynamic process involving several steps, each requiring the activation of different genes. First, normal melanocytes undergo genetic alterations that lead to their transformation into benign nevi. Benign nevi differ from normal melanocytes in that they have initially proliferated in the basal layer of the epidermis; however, they entered a long-term dormant status due to the lack of additional oncogenic alterations. For example, the most frequent activating mutation in the BRAF gene occurs in the same frequency in nevi, where it causes a dormant status called oncogene-induced senescence . AdditioThe most important risk factor for melanoma is UV irradiation upon sun exposure. Whole genome sequencing revealed that melanoma is the tumor type with the most DNA mutations—many being typical for UV-induced mutations . DespiteV600EBRAF leads to an alternative protein structure and to a constitutive active protein. 50%–60% of melanomas contain an activating mutation in BRAF -α-MSH or NDP-α-MSH , through a PEG linker. And importantly, in vitro cell-uptake studies showed the ability of conjugated micelles to selectively bind to MC1-R receptor, and, whether due to multivalent interactions or other factors, the micelles had higher avidity for the receptor than the ligand alone. Nevertheless, further studies  to quantify the uptake of these conjugated micelles are needed to better evaluate the delivery efficiency of this platform. More recently, α-MSH peptide has been conjugated to a nanoplatform based on the heavy chain of the human protein ferritin (HFt) fluoro-2-deoxy-d-glucose (18F-FDG) is the only radioactive probe used in the clinic to detect melanoma. Be that as it may, 18F-FDG is an unspecific positron emission tomography (PET) imaging agent with poor sensitivity towards micrometastatic sites  developed invasive melanomas in the skin following neonatal exposure to carcinogenics.Of special interests are combinatorial strategies involving siRNA delivery as these, similar to other combinatorial therapies, cause the most significant outcomes. Particularly, Poeck and coauthors have used a simple and elegant siRNA design . The autimmunity . In turnimmunity . Interesimmunity . Not onlin vitro studies have reported on the miRNA potential for antimelanoma therapy [While a number of microRNA has been described to play relevant roles in melanoma progression , only fe therapy , 120. Ho therapy , 143 forAs an overview of this section, in vivo distribution needs to be assessed, preferably by several approaches , and magnetic resonance imaging (MRI)). To this end, immunohistochemistry studies may be suitable and very convenient to corroborate and support data collected by different means, but also microscopy  has had its traps [It is of general consensus that the last decade of cancer research significantly expanded our knowledge in tumor development and progression. Unfortunately—similar to the tumor escape shaped by the immune surveillance in an early growth phase—as new therapeutic strategies are applied, tumor cells undergo another round of selection, giving rise to therapy-resistant cells. It is therefore necessary to combine several approaches to attack different paths of tumor escape—a fact that is confirmed by the most significant results reported in studies where such strategies have been used. On this note, nucleic acids deliveries are truly advantageous tools as they allow the systemic delivery of potentially toxic molecules that can be combined with chemotherapy aiming at terminating possible resistant-tumor cells. As an example, recently, Su and collaborators have reported on an antitumor strategy combining TNF-encoding pDNA and chemotherapy . While sts traps .In summary, already a number of promising nucleic acid strategies exist, and these certainly present less hurdles for delivery than their protein counterpart, as they are smaller, less antigenic, and can bypass certain resistance mechanisms. Nevertheless, further improvements in nonviral targeted delivery appear required to increase the efficacy of such therapies. A small final note regarding the potential of miRNA approaches: microRNA therapies can aim at (1) miRNA upregulation, when the target nucleic acid is enrolled in cell homeostasis and is found silenced in tumor cells; (2) miRNA downregulation by antimiRs, when it is upregulated in tumor cells due to its play in cell proliferation; (3) alternatively, miRNA can also have a role in cell-specific transcription in pDNA vectors containing miRNA binding-sites, allowing the expression of the gene of interest in cells, where the miRNA is silenced. All these assets make miRNA undoubtedly a very elegant and flexible tool.


One common practice in drug discovery is to optimize known or suspected ligands in order to improve binding affinity. In performing these optimizations, it is useful to look at as many known inhibitors as possible for guidance. Medicinal chemists often seek to improve potency by altering certain chemical moieties of known/endogenous ligands while retaining those critical for binding. To our knowledge, no automated, ligand-based algorithm exists for systematically ‘swapping’ the chemical moieties of known ligands to generate novel ligands with potentially improved potency. To address this need, we have created a novel algorithm called ‘LigMerge’. LigMerge identifies the maximum (largest) common substructure of two three-dimensional ligand models, superimposes these two substructures, and then systematically mixes and matches the distinct fragments attached to the common substructure at each common atom, thereby generating multiple compound models related to the known inhibitors that can be evaluated using computer docking prior to synthesis and experimental testing. To demonstrate the utility of LigMerge, we identify compounds predicted to inhibit peroxisome proliferator–activated receptor gamma, HIV reverse transcriptase, and dihydrofolate reductase with affinities higher than those of known ligands. We hope that LigMerge will be a helpful tool for the drug design community. Virtual screening approaches enrich a pool of candidate ligands for true binders; only a limited number of the best-scoring compounds are then tested experimentally, leading to greater hit rates and decreased cost , compounds derived from common reactions , compounds with known pharmacological properties , or analogs of known ligands.In part because of the advent of high-throughput screening, many protein receptors are associated with a plethora of experimentally validated ligands . In desiBREED , an algovia virtual screening. LigMerge is implemented in Python and so is easily editable, customizable, and platform independent. A copy can be downloaded free of charge from http://www.nbcr.net/ligmerge/.Here, we present a program called LigMerge that provides a fast and easy way to generate molecular models derived from known inhibitors without the need for information about the receptor. We expect the program will be useful for those designing custom virtual screening, small-molecule databases when many ligands, potent or otherwise, have been identified experimentally or theoretically As input, LigMerge accepts two three-dimensional, PDB-formatted compound models. PDB files are the only supported input format. SDF or MOL files must be converted to the PDB format before using LigMerge. These models are processed in three steps. First, the maximum (largest) common substructure of the two models is identified . Second,Exhaustive lists of atom indices/element types for all heavy atoms in the two structures are first generated . HydrogeHaving identified candidate common substructures, the next step is to test for identical geometries . To faciWithout further consideration, the above protocol ignores questions of symmetry. For example, consider two models whose greatest common substructure is a toluene. Two symmetry-related superimpositions exist . The -all_symmetry_relations command-line parameter can be used to specify whether the algorithm should consider all symmetry assignments when generating merged compound models or whether it should randomly choose a single assignment from those identified . The -alIt is important to note that LigMerge ignores ligand flexibility when performing geometric comparisons. It is therefore prudent to use ligand models of compounds in docked or crystallographic poses or to choose ligand models with inflexible segments . As the number of publicly available crystal structures is ever increasing and inflexible segments are common in bioactive molecules, we expect that these two limitations will not be too problematic.All possible substructure assignments of maximum length and identical geometry are subsequently considered. For each, a transformation matrix is identified that minimizes the RMSD between substructures. Although only the common substructures are considered in generating this matrix, entire molecules are subjected to the transformation, essentially positioning the two models so that their maximum common substructures are superimposed . When idMoieties from each model, comprised of fragments with atoms bound to those of the common substructure, are next identified. The common-substructure atoms to which these moieties are bound are designated ‘handle atoms’. If the command-line parameter -all_substituent_combinations is set to false, a random fragment is selected for each handle atom, and a single merged compound is generated by combining the common substructure and the selected fragments. Special consideration is given to ‘multiple-handle fragments’, that is, fragments that externally connect to two or more handle atoms. If fragments containing more than one handle atom are selected, these fragments essentially determine the selection at multiple handle–atom locations. If the command-line parameter -all_substituent_combinations is set to true, multiple merged structures with all possible combinations of fragments are generated and saved to separate PDB files . If a spTo demonstrate the utility of the LigMerge algorithm, compounds generated by applying LigMerge to a variety of known binders were docked into three receptors: peroxisome proliferator–activated receptor (PPAR) gamma, HIV reverse transcriptase (RT), and dihydrofolate reductase (DHFR).Crystal structures of peroxisome proliferator–activated receptor (PPAR) gamma in complex with ligand 570 (PDB ID: 1FM9 ), HIV re50 values, respectively, were obtained from PubChem , logP, and polar surface area (PSA) were calculated using obprop . The setFor each of the LigMerge-generated compound sets, an in-house script was used to generate a decoy set equal in size and chemical properties. MW, logP, and PSA statistics were calculated for each of the approximately 11 000 000 compounds of the ZINC ‘All Clean’ data set  using obLigMerge is an open-source, easy-to-use tool for generating novel compounds with structural features similar to those of known ligands. Compounds derived from known ligands are more likely to be true binders themselves. Once generated, LigMerge-derived compounds can be docked into receptor structures to identify likely inhibitors for subsequent synthesis and experimental validation.To demonstrate the utility of the LigMerge algorithm, three protein drug targets with many known inhibitors were chosen as test systems: peroxisome proliferator–activated receptor (PPAR) gamma, HIV reverse transcriptase (RT), and dihydrofolate reductase (DHFR). For each of these systems, novel compounds were generated using LigMerge by combining features of known inhibitors. Predicted binding affinities were then assessed by computer docking. To demonstrate that LigMerge can generate a set of compounds enriched for high-affinity binders above and beyond screens of chemically similar molecules chosen at random, we also dock appropriate decoy databases into each of the three receptors studied to facilitate comparison.1, A total of 896 LigMerge-generated compounds were derived from the top fifteen experimentally verified PPAR binders listed in the BindingDB database  as of Oc1) is shown in 1 positions benzene and anisole substructures coincident with those of the known inhibitor. Additionally, the trifluoromethyl group of compound 1 is predicted to be proximal to a sulfonamide group of the known compound, and the trifluoromethyl group of the known inhibitor is positioned proximal to the predicted location of a compound-1 carboxylate group. Others have suggested that fluorinated methyl groups might be bioisosteres of the carboxylate group (compare PDB structures 3AEB and 3AE6) and the sulfonamide group (compare PDB structures 2XBV and 2XBX (The best-scoring binding pose of the top-ranked LigMerge molecule (compound and 2XBX ). The si2, A total of 3959 LigMerge-generated compounds were derived from the top ten experimentally verified RT binders listed in the BindingDB database  as of Oc2) is shown in 2 docked at the same location as the analogous 3-chlorobenzonitrile moiety of the co-crystallized inhibitor. Additionally, the aromatic imidazopyridazine moiety of compound 2 docks at the same location, and in the same plane, as a crystallographic benzene moiety of the known inhibitor. Again, the similarities of these binding modes are not likely the result of mere chance; LigMerge-generated compounds likely have improved docking scores because they are based on known inhibitors rather than chosen at random.The best-scoring binding pose of the top-ranked LigMerge molecule , deep wHistograms showing the Vina score distributions of the 30 known DHFR inhibitors, as well as the LigMerge-derived compound models, are shown in We propose that the LigMerge-generated compound set included compounds with improved predicted affinities over those of known inhibitors because LigMerge generates novel compounds from known inhibitors in an intelligent and systematic way. However, it could be that these improved compounds were identified simply because the set of LigMerge-generated compounds was much larger than the set of known inhibitors, making it statistically more likely that a high-affinity predicted ligand would be found. To rule out this possibility, we compared the docking performance of the three LigMerge-generated compound sets to that of decoy libraries similar in size and average chemical properties.http://www.nbcr.net/ligmerge/.We here present an algorithm called LigMerge that considers two three-dimensional models of known or suspected small-molecule inhibitors and forms derivative models with similar chemical features. In the process of merging models, LigMerge first identifies the maximum common substructure (MCS). The MCS, which can be saved for later examination, may itself be a valuable tool for ligand evaluation. Next, the program aligns the two molecules by their mutual MCS, so that they are partially superimposed. Finally, the chemical moieties attached to each superimposed atom of the maximum common substructure are recombined, producing composite molecules similar to known or suspected inhibitors, but with potentially higher affinities. LigMerge is freely available through the National Biomedical Computation Resource (NBCR) and can be downloaded at


Pancreatic cancer exhibits an extraordinarily high level of resistance to almost any kind of systemic therapy evaluated in clinical trials so far. Therefore, the identification of novel therapeutic targets is urgently required. High-throughput screens have emerged as an important tool to identify putative targets for diagnosis and therapy in an unbiased manner. More than a decade ago, microarray technology was introduced to identify differentially expressed genes in pancreatic cancer as compared to normal pancreas, chronic pancreatitis and other cancer types located in close proximity to the pancreas. In addition, proteomic screens have facilitated the identification of differentially secreted proteins in body fluids of pancreatic cancer patients, serving as possible biomarkers. Recently, RNA interference-based loss-of-function screens have been used to identify functionally relevant genes, whose knock-down has impact on pancreatic cancer cell viability, thereby representing potential new targets for therapeutic intervention. This review summarizes recent results of transcriptional, proteomic and functional screens in pancreatic cancer and discusses potentials and limitations of the respective technologies as well as their impact on future therapeutic developments. It is the fourth leading cause of cancer death in the U.S. with approximately 36,800 deaths attributable to pancreatic cancer in 2010 [KRAS2 gene, and 85–98% have mutations, deletions, or hypermethylation in the CDKN2 (p16) gene. Furthermore, 50% of the tumors show inactivating mutations of p53 and about 55% have homozygous deletions or mutations of SMAD4 [KRAS2 gene mutations and telomere shortening. During PanIN progression, p16/CDKN2A gets frequently inactivated [During the last decades, the genetic alterations underlying pancreatic cancer have been well characterized: 80–95% of pancreatic ductal adenocarcinomas carry activating mutations in the The identification and characterization of these cancer-related genes have increased our understanding of the genetic basis of pancreatic cancer development, but unfortunately this knowledge has not translated into clinical practice, since survival of patients with this disease has not improved significantly over the past two decades. It appears that a multitude of transcriptional and posttranslational events regulate the expression and function of oncogenic and tumor-suppressive genes. To identify new therapeutically exploitable targets differentially expressed in pancreatic cancer, coordinated screening efforts not only at the DNA level, but also at the RNA and protein level are necessary. In addition, screening of proteins for their functional impact on cardinal hallmarks of cancer such as survival, invasiveness and proliferation, e.g., by loss-of-function screens, are mandatory to identify novel functionally relevant targets in an unbiased manner.2.Owing to the fact that hybridization reactions between complementary nucleic acid strands are relatively uniform and well-predictable in their behavior, transcriptome analyses were the natural choice for the development of the first high-throughput screening approaches in molecular biology. To this day, they represent the most widely available and most commonly employed “-omics” technology.Expression profiling by cDNA-array technology has been introduced almost two decades ago. Originally developed with nylon arrays comprising a few hundred genes in the 1990s, the technology has rapidly advanced and offers now the possibility to perform genome-wide screens on various technological platforms . DNA micet al. as early as 1996 [A typical application of this type of analysis is the comparison of mRNA expression levels detected in tissue samples from malignant tumors with those detected in non-malignant samples from the same organ in order to identify differentially expressed genes which may be suitable as targets for the development of novel diagnostic or therapeutic approaches. During recent years, literally thousands of studies of this type have been performed with virtually every type of malignancy encountered in humans. In pancreatic cancer research, the first expression profile of pancreatic cancer tissues was published by Gress  as 1996 ,8.The majority of these studies have used bulk tissue as a source for mRNA extraction, which represents a potential pitfall for the interpretation of the data. Pancreatic tumors are often characterized by extensive stromal depositions referred to as the desmoplastic reaction of pancreatic cancer. The stroma may comprise up to 90% of the tumor volume and consists of fibroblasts, stellate cells, vessels and numerous inflammatory cells ). In ordet al. have recently published a study combining SNP analyses, exon sequencing and transcriptome sequencing, which led to the definition of 12 “core signaling pathways” that were altered in 67% to 100% of the pancreatic cancer cases they analyzed [Although DNA microarrays are very powerful tools which have profoundly enriched our understanding of transcriptional changes that are associated with malignant transformation of pancreatic cells, they do have a number of limitations inherent to the technology. Chief among them are the need for a-priory knowledge of the sequences one wants to detect on the microarray as well as the inability to completely exclude unspecific cross hybridization events. The advent of next generation sequencing technologies has opened up the possibility to generate expression profiles by sequencing entire cDNA populations generated from a given sample, with the added value of being able to detect unknown genes, splice variants, or mutations present in the sample. As a result, microarray analyses are increasingly being replaced by sequencing-based experimental strategies. As a prominent example, Jones analyzed .http://www.pancreasexpression.org/, allows one to comprehensively search for gene expression results across many different studies in a single query. Datasets can be filtered by any attribute desired, e.g., type of samples used in the study, mode of sample preparation, type of array or hybridization protocol used, etc. Alternatively, lists of genes can be specified for which all available information can be retrieved. The different types of queries can be freely combined in order to deliver the desired information, thus making this database an invaluable resource for pancreatic cancer researchers.Due to the large number of publications and the overwhelming amount of data that have been generated in these studies, it has become virtually impossible for individual researchers to keep an overview of all available transcriptome data and to integrate and interpret their own data in the context of other groups' results. In order to deal with this problem, Chelala and coworkers have developed a web-based resource to systematically collect, annotate and make accessible all publicly available pancreatic expression datasets ,18. This3.i.e., fractionation techniques, depletion of high abundance proteins, etc.) will greatly influence the profile of proteins detected, such that comparisons of results across different studies are very difficult. Furthermore, most of these techniques do not readily lend themselves to high throughput application; thus, the numbers of samples that are analyzed in proteomic studies are often considerably smaller than what is seen in transcriptomic studies.In contrast to nucleic acids, which, regardless of the type of gene they encode, constitute a relatively uniform class of macromolecules, proteins display an extremely broad range of physicochemical properties. It is thus considerably more challenging to quantitatively recover, accurately separate and unequivocally identify proteins from complex samples on a global scale. Accordingly, several vastly different techniques have been developed and are being employed to perform this task. Detailed descriptions of the different methods and approaches, which include two-dimensional gel electrophoresis (2DE), mass spectrometry based techniques, and antibody or protein microarrays, are given elsewhere -21. NoneNonetheless, taking a direct view at the proteins that are expressed in a given cell type or tissue, as opposed to using mRNA abundance as a rather indirect measure of gene activity, is very attractive in order to elucidate functional differences between cancer and control tissues. Moreover, proteins are potentially very useful biomarkers, since they tend to be much more stable than mRNA in body fluids such as blood, urine, pancreatic or duodenal juice, and, once identified and characterized, can often very sensitively and specifically be detected by antibody-based techniques. Accordingly, many proteome studies performed in pancreatic research have aimed at identifying new protein markers of the disease:et al. [et al. [et al. [et al. [et al. [In the analysis of tissue samples to directly assess protein expression in pancreatic cancer, 2DE has historically been the most widely used technique. Tian et al.  and Qi e [et al.  used eig [et al.  used 2DE [et al.  analyzed [et al.  used comet al., who showed that distinction between pancreatic cancer and benign controls  using a panel of plasma protein markers was possible with high precision. However, accuracy dramatically deteriorated when patients suffering from biliary duct obstruction were added to the benign control cohort [As indicated above, a considerable number of studies have examined protein content in different body fluids in an attempt to identify novel biomarkers for non-invasive diagnostic applications. Since blood is easy to obtain and a classic source of protein biomarkers in many diseases, it is not surprising that most of these studies have been aimed at identifying differentially expressed proteins in whole blood, serum or plasma of pancreatic cancer patients and controls, using essentially all proteomic techniques available today -35. Pancl cohort .Very recently, a novel approach to pancreatic cancer biomarker discovery has been developed to enrich and reliably detect differentially expressed and secreted proteins from cell cultures, which employs stable isotope labeling with amino acids in cell cultures (SILAC) coupled with extensive multidimensional separation coupled with tandem mass spectrometry (MS/MS), which successfully identified several promising targets in pancreatic cancer cells -45.4.In order to decipher the function of cancer-relevant genes in a high-throughput manner, functional genomic screens have been successfully applied in various cellular systems. During recent years, screens which are performed as so-called forward genetic screens to discover the genes underlying a defined phenotype, have been revolutionized by the advent of RNA interference technology .C. elegans by Fire and Mello 1998 [RNA interference represents a naturally occurring mechanism for post-transcriptional suppression of gene expression using double-stranded RNA (dsRNA) . During llo 1998 , and itsllo 1998 , it evolllo 1998 . ArtificRNA interference can be introduced into mammalian cells either by transfection of double-stranded small-interfering RNAs (siRNA) or by transfection or transduction of short-hairpin RNA containing plasmids (shRNA) which are transcribed and subsequently cleaved intracellularly into effective siRNA oligonucleotides by the enzyme Dicer . For scret al. screened the human kinome for kinases whose inhibition could increase spontaneous or gemcitabine-induced apoptosis in MiaPaCa-2 pancreatic cancer cells. By screening siRNAs directed against 645 kinases, they identified a panel of kinases acting synthetically lethal with gemcitabine including AK1, GRAF, MAP3K7, CSNK2A1, and PAK7, whose inhibition led to significantly enhanced drug-induced apoptosis in several pancreatic cancer cell lines [Several RNA interference-based screens have been performed to identify novel diagnostic or therapeutic targets in pancreatic cancer ,53. Giroll lines .et al. [A second synthetic lethal screen was published recently by Azorsa et al. : They utet al. .et al. applied a high-throughput loss-of-function screen by RNA interference across this amplicon to identify functionally relevant genes whose down-regulation affected cell viability.Apart from classical synthetic lethal approaches, several combinatorial approaches utilizing RNAi libraries have been published. Microarray-based, genome-wide analysis for DNA copy number aberrations in pancreatic cancer identified several recurrent amplifications, including a 1.1 MB amplicon at 19q13. Based on these data, Kuuselo This screen revealed five genes whose knock-down affected cell viability selectively in amplified but not in non-amplified pancreatic cancer cells. Of these, the transcriptional regulator intersex-like (IXL) was consistently overexpressed in amplified cells and had the most dramatic effect on cell viability. IXL silencing also resulted in G0-G1 cell cycle arrest and increased apoptosis, suggesting that IXL is required for cancer cell survival in 19q13-amplified tumors ,55.Apart from cell viability, other features of tumor progression can be used as read-out for the screens, such as cell migration, apoptosis, cell morphology or activation of distinct signaling pathways of interest in luciferase-based approaches. Our group identified the transcription factor CUX1/CUTL1 in an shRNA library based screen for mediators of cell motility comprising several hundred genes . We coulThese reports utilizing RNAi libraries in different experimental settings demonstrate that RNAi-based loss-of-function screens have evolved as important tools to identify novel potentially relevant targets in pancreatic cancer.5.During the last decade, the advent of several new technologies for high-throughput screening has revolutionized research aiming to identify novel targets with potential diagnostic, prognostic, predictive or therapeutic impact. Ranging from RNA expression profiling, which has evolved as a tool with potential impact for therapeutic decision making and response prediction, up to functional screens aiming to decipher the functional consequences of large scale inhibition of gene expression, high- throughput screening offers a unique opportunity to identify novel targets in an unbiased manner. Based on the unprecedented wealth of data which may result from single screening experiments, bioinformatic analysis of the experimental data and target selection remains challenging, which requires careful statistical considerations while setting-up the screen conditions. Hopefully, the novel screening technologies will indeed allow the identification of genes relevant for tumor progression which can be therapeutically targeted, in order to improve the dismal prognosis of pancreatic cancer. However, it has to be emphasized that up to now, candidates identified by these screening approaches have not entered clinical application and therapeutic management has largely been unchanged over the last decades. One reason for this disappointing fact might be the lack of preclinical validation platforms for published screening targets, e.g., by the use of genetically engineered mouse models of pancreatic cancer which could be used to bridge the gap between basic science and clinical advances and drive translational approaches forward.