The pathogenesis of HIV infection, and in particular the development of immunodeficiency, remains incompletely understood. Whichever intricate molecular mechanisms are at play between HIV and the host, it is evident that the organism is incapable of restricting and eradicating the invading pathogen. Both innate and adaptive immune responses are raised, but they appear to be insufficient or too late to eliminate the virus. Moreover, the picture is complicated by the fact that the very same cells and responses aimed at eliminating the virus seem to play deleterious roles by driving ongoing immune activation and progressive immunodeficiency. Whereas much knowledge exists on the role of adaptive immunity during HIV infection, it has only recently been appreciated that the innate immune response also plays an important part in HIV pathogenesis. In this review, we present current knowledge on innate immune recognition and activation during HIV infection based on studies in cell culture, non-human primates, and HIV-infected individuals, and discuss the implications for the understanding of HIV immunopathogenesis. The natural history of HIV infection is characterized by an acute phase with very high circulating levels of virus and a rapid decline in CD4+ T cells ,2. DespiAcute or primary HIV infection is defined as the first period of infection from the detection of HIV RNA until the formation of HIV-specific antibodies 3-4 weeks after infection . FollowiStudies in SIV models and HIV-infected individuals have documented that acute SIV/HIV infection is accompanied by a massive depletion of CD4+ memory T cells, primarily in mucosal tissue, which may be explained by the high expression of the viral co-receptor CCR5 and the relatively activated state of mucosal CD4+ T cells -19. In lPlasma viraemia increases to reach a peak after 21-28 days of infection together with depressed peripheral CD4+ T cell numbers. Whereas the amount of circulating T cells subsequently return close to normal, CD4+ T cell numbers in the GALT remain severely reduced ,22. ThusAt the time of peak viraemia, patients may develop symptoms of the acute retroviral syndrome, including influenza-like illness with fever, sore throat, lymphadenopathy, and exanthema . HoweverDespite the return of circulating CD4+ T cells to near normal levels and the infection being largely asymptomatic for extended time periods in the majority of patients, it is now well established that massive immune activation and an accelerated cell turnover takes place during chronic HIV infection ,35 Figu. This apThe profound immunological damage to the gastrointestinal tract leads to breaks in the mucosal barrier allowing translocation of microbial products, including bacterial lipopolysaccharide (LPS), into the circulation. A seminal study by Brenchley and colleagues demonstrated bacterial translocation during HIV infection and correlated plasma LPS levels with immune activation . BacteriFinally, important questions regarding the immunopathogenesis of HIV infection may be learned from the study of infection in natural hosts, or potentially from HIV-infected humanized mouse models . IntriguSince one of the fundamental characteristics of HIV pathogenesis is the failure of the immune system to recognize, control, and eliminate the virus, much focus has been on early events following viral infection. The innate immune system constitutes the first line of defence against invading pathogens and is based on epithelial barriers, the complement system, and cells with phagocytotic and antigen presenting properties, such as granulocytes, macrophages, and DCs respectively ,50.Pattern recognition receptors (PRR)s have been assigned a central role in innate immune defences due to their ability to recognize evolutionarily conserved structures on pathogens, termed pathogen-associated molecular patterns (PAMP)s. A limited number of germ-line encoded receptors are responsible for triggering an innate immune response following the encounter with PAMPs, which are characterized by being invariant among entire classes of pathogens, essential for survival of the pathogen, and distinguishable from self . Among PSince microbial material is not exclusively present extracellularly or within endosomes, alternative cytosolic PRRs exist. The retinoid acid-inducible gene (RIG)-like receptors (RLR)s, RIG-I and MDA5, are RNA helicases that play a pivotal role in sensing of cytoplasmic RNA ,63. StudOverall, ligand engagement of PRRs leads to activation of a proinflammatory and antimicrobial response by triggering signal transduction pathways involving the transcription factors nuclear factor (NF)-κB and IFN regulatory factors (IRF) 3/7 as well as mitogen-activated protein kinase (MAPK) pathways, ultimately resulting in the production of cytokines, chemokines, cell adhesion molecules and antiviral type I IFN. This is depicted in Figure When considering how HIV may possibly be recognized by the innate immune system, it seems logical to contemplate the possible PAMPs that are part of the HIV particle or generated during different phases of the viral life cycle. Being a member of the retroviridae family (lentivirus subfamily), HIV is a spherical enveloped RNA virus with a diameter of roughly 100 nm. The envelope contains viral glycoproteins and encloses a cone-shaped capsid containing two identical copies of the positive ssRNA genome of 10 kilobases together with several copies of reverse transcriptase (RT), integrase, additional viral proteins and two cellular tRNAs . The virAs illustrated in Figure RT is an RNA-dependent DNA polymerase, which uses the viral positive ssRNA genome as template and the virion tRNA as primer for the synthesis of a negative-strand DNA copy , thus foSynthesis of new progeny virus is accomplished in a highly regulated manner utilizing host cell enzymes and dependent on cellular or microbial inflammatory or mitotic signals, including the HIV transactivator Tat . IntegraBased on the observation that initiation of HAART leads to an almost immediate decline in immune activation, which can be correlated to significant reduction in HIV viraemia, a direct contribution of HIV itself to immune activation has been proposed -87. The Further support for a role of TLR7/8 in HIV immune activation was provided by findings of HIV RNA rendering human lymphoid tissue of tonsillar origin or peripheral blood mononuclear cells (PBMC)s less permissive to HIV replication . In anotActivation of innate immune receptors during HIV infection does not only involve PAMPs derived from HIV but also applies to PAMPs originating from opportunistic pathogens and translocated bacteria ,96. ConsAlmost ten years ago, it was reported that bacterial LPS activates the HIV LTR through TLR4 . This isA correlation between bacterial DNA as a measure of bacterial translocation and immune activation in HIV-infected individuals has been demonstrated, and such bacterial DNA may also stimulate innate immune activation through TLR9 or cytosolic DNA receptors . HoweverOne important aspect necessary to address when describing interactions between HIV and the innate immune system, is the extensive difference observed between various cell types. Such differences add further complexity to the overall picture, since HIV targets several different cell types, including T cell subsets, monocytes, macrophages, and DCs. Therefore, entirely different recognition mechanisms and immune strategies may exist depending on the cell and tissue involved. Whereas there is solid evidence for TLR7-mediated activation of pDCs, resulting in type I IFN production , other cDespite TLRs being mainly expressed on cells of the innate immune system, mRNAs encoding TLR 1, 2, 3, 4, 5, 7, and 9 have also been detected in human primary CD4+ T cells, and engagement of specific TLRs trigger secretion of Th1 and Th17 cytokine profiles, suggesting that a subset of TLR ligands can activate resting CD4+ T cells -113. IntThe elegant mechanism, by which HIV is capable of exploiting NF-κB to its own advantage to promote viral replication, is a clear example of the ingenuity of HIV. Early studies unravelled that NF-κB perpetuates HIV enhancer activity in infected monocytes, and that κB sites in the HIV LTR are responsible for this phenomenon ,115. MorInnate immune recognition may play a central role in ongoing immune activation through PRR activation, hence resulting in the production of a range of cytokines and chemokines ,120. FurEven prior to the identification of HIV as a human retrovirus causing AIDS, the study of human retroviruses was tightly linked to IFN research ,124. OneA pertinent question is whether IFN has any antiviral activities during HIV infection. Several studies have demonstrated that type I IFN does inhibit the replication of HIV in vitro -129. In In humans, early studies reported a depletion of circulating type I IFN-producing cells in HIV-infected AIDS patients  and reduRecently, some of the molecular mechanisms, by which IFN-α may cause undesirable immune activation and contribute to disease progression have been delineated. For example, one study demonstrated IFN-α-mediated upregulation of CCR5 on T cell progenitor cells, paradoxically expanding the tropism of CCR5 trophic HIV and potentially accelerating disease progression . Type I A more complex picture of the effects of IFN may reconcile the contrasting results in the field. In particular it is important to consider the effects of IFN in the context of infection, including cell type, host, and stage of infection. With increasing insight into the interplay between HIV and IFN, it has become evident that TLR7/8 activation and IFN production may exert opposing effects depending on the stage of infection. Whereas antiviral and antiproliferative effects may be beneficial during acute infection at the expense of a certain degree of immune activation, innate immune activation may be deleterious later during chronic infection. The central question here seems to be, whether the net effect of IFN production is beneficial or harmful to the host, i.e whether positive antiviral effects outweighs the negative consequences of IFN-induced inflammation. In this respect, IFN seems to be a double-edged sword for the organism. Many questions remain to be answered before the full picture of the role of IFN during HIV infection has been clarified.DCs are of pivotal importance, not only because they are among the earliest targets of HIV, but also due to their ability to capture antigens and initiate T cell responses . Based oSince pDCs are the major producers of type I IFN, it has been suggested that abnormal migration and localization patterns of this important cell type may be a key in understanding the interplay between HIV and type IFN ,156. Beset al., who provided evidence that HIV is capable of infecting Th17 cells in vivo and also demonstrated a significant loss of Th17 cells in the gastrointestinal tract of HIV-infected patients [Within the T cell compartment, much interest has been focused on an altered balance between proinflammatory Th17 cells and regulatory T cells (Treg)s in HIV infection. Th17 cells are CD4+ cells that produce IL-17 and play a central role in immune responses to extracellular bacteria . Initialpatients . These fpatients . Studyinpatients . These fpatients . Interespatients . Thus, sTregs is a small subpopulation of T cells involved in preventing or inhibiting autoimmune and inflammatory disorders , but mucHIV recognition by PRRs seems to be rather limited, which may indicate that HIV is particularly successful in preventing intimate encounter with the innate immune system. Accumulating evidence suggest that this virus actively avoids recognition by PRRs in order to prevent activation of a proinflammatory and antiviral responses. On a theoretical basis, it can be hypothesized that HIV is in possession of specific strategies to shield or modify its PAMPs within infected cells, for instance by hiding its RNA/DNA in the viral capsid throughout most of the viral life cycle, or by altering its nucleic acids in order not to appear foreign to the host. Below, some of the strategies, by which HIV evades innate immune activation, are described.Despite the importance of NF-κB for transcription of the viral genome, it may be advantageous for HIV to prevent NF-κB activation in certain situations. In a study addressing the mechanisms of cellular innate immune responses, HIV infection of primary monocyte-derived macrophages did not activate NF-κB , indicatet al. who observed depletion of IRF3 in HIV-infected CD4+ T cells [Evidence is also accumulating with regards to HIV interference with the IFN system. First, direct interaction of HIV gp120 with pDCs inhibits TLR9-mediated responses, including pDC activation, IFN-α secretion, and cytolytic activity of NK cells . Within  T cells . IRF3-de T cells . As to t T cells . Intrigu T cells .In addition to interfering with the production of IFN, HIV also counteracts the action of IFNs or IFN-inducible proteins. Mammalian cells harbour intrinsic cell-autonomous activities, which can act to suppress viral replication and collectively are referred to as host restriction factors. These host restriction factors are naturally connected to the innate immune response by virtue of their IFN inducibility . InteresTRIM5α belongs to a large family of proteins, several of which are involved in control of viral infections . The actMore recently, an IFN-induced restriction factor that prevents retrovirus release from the plasma membrane was identified and named tetherin ,184. TetAn alternative way to gain understanding of the role of innate immune components in the antiviral response and immune activation during HIV infection is through epidemiological studies of genetic polymorphisms in human populations. One of the first studies addressing this question was the description of almost complete protection from HIV infection conferred by homozygosity of a 32 base deletion in CCR5 . MoreoveIn the case of TLRs, somewhat more insight into polymorphisms and HIV-induced inflammation exists. One study focused on HAART naïve HIV positive patients from the Swiss HIV cohort, in which Bochud and co-workers reported an association between two single nucleotide polymorphisms (SNP)s in TLR9 and rapid HIV progression as measured by CD4+ T cell decline , althougThe interactions between HIV and the innate immune system have only recently caught the attention of HIV researchers, and as a consequence remain poorly described. The present picture is that, unlike most other pathogens, innate immune recognition of this virus may not be very elaborate. However, it is still not very well understood, how HIV evades innate immune recognition. This interesting issue points back to the central questions in HIV pathogenesis, as to why the host is unable to recognize and respond adequately to acute HIV infection to prevent the virus from establishing latent viral reservoirs and thereby lifelong chronic infection. Recent insight into this subject indicates that some of the answers should indeed be sought in the interactions between HIV and the innate immune system . The faiAs described is this review, surprisingly few innate immune receptors have been implicated in HIV recognition. Eventually, this may be explained by the fact that HIV PAMPs and their respective PRRs still await identification. Alternatively, understanding the mechanisms by which HIV avoids immune recognition by PRRs may provide insight into pivotal aspects of HIV virology and possibly identify molecular targets for therapeutical interference with the viral life cycle. Clearly, the search for innate immune receptors for HIV is still at an early stage, and this interesting subject is likely to lead to answers to central questions in HIV immunopathogenesis. Therefore, an integration of knowledge on the interactions between HIV and both innate and adaptive immunity is a prerequisite for gaining a more profound understanding of HIV immunopathogenesis, and ultimately for applying this knowledge into the development of novel treatment and vaccination strategies to clinical benefit for patients.The authors declare that they have no competing interests.THM was responsible for drafting the manuscript. THM and SRP were responsible for creating Figures


E. coli; superoxide anion generation after PMA; leukotriene B4 (LTB4) release from whole blood and isolated neutrophtls, before and after different stimuli) were evaluated during an acute exacerbation of asthma, and after 14 – 54 days of treatment with systemic glucocorticosteroids (GCS). During acute exacerbation, superoxide anion generation was higher in asthmatics than in eleven normal subjects ; there was a significant correlation between FEV1 (% of predicted) and neutrophil chemotaxis . After treatment, there was no significant change in all neutrophil functions, except for a decrease in neutrophil chemotaxis in subjects who showed an FEV1 increase > 20% after GCS treatment . Chemokinesis sicantly decreased in all subjects, and the changes significantly correlated with an arbitrary score of the total administered dose of GCS . These data suggest that neutrophil activation plays a minor role in asthma, and that treatment with GCS is not able to modify most functions of peripheral neutrophils in asthmatic subjects; chemotaxis seems to be related only to the severity of the asthma and it could reflect the improvement of the disease.In 21 asthmatic subjects, several functions of isolated peripheral neutrophils (chemokinesis and chemotaxis toward 10%


In this mini-review, we provide an overview of the histone deacetylase superfamily, highlight the positive results of deacetylase inhibitors in cancer clinical trials, and comment on the prospects for the next generation of such inhibitors.Histone deacetylase inhibitors represent a promising new class of compounds for the treatment of cancer. Inhibitors of this kind currently under clinical evaluation mainly target the classical (Rpd3/Hda1) family of histone deacetylases. Of particular note, the U.S. Food and Drug Administration recently approved the first histone deacetylase inhibitor  for the treatment of cutaneous T-cell lymphoma. Dozens of such inhibitors are now in phase  As the name implies, these enzymes remove acetyl groups from the side chain of specific lysine residues located mainly at the N-terminal flexible tails of core histones, thereby increasing the affinity of histone octamers for the dna that is wrapped around them , α-tubulin, heat shock protein 90 (Hsp90: an important anticancer drug target), and various signalling proteins  used even for enzymes that deacetylate non-histone proteins. Herein, we therefore use the term hdac to refer to lysine deacetylase enzymes exclusively.In addition to histones, proteins  3–5. Thehdac activity may play a causative role in both tumorigenesis and metastasis (The following five lines of evidence (among others) suggest that altered tastasis  6: First, histone hypoacetylation is characteristic of most cancers hdacs are often aberrantly recruited to gene promoters by fusion proteins derived from chromosomal translocations  Second, in leukemia, hdacs occurs in several cancer types, and the growth of cell lines derived from these tumours is suppressed by small interfering rna (sirna)–mediated knockdown of the overexpressed hdac  Third, overexpression of specific hdacs may block the function of tumour suppressor proteins such as p53  Fourth, elevated expression of hdacs target oncogenic regulators such as Hsp90 that are important for cancer development  Fifth and finally, in addition to affecting chromatin structure and regulating gene expression and genomic stability, hdac inhibitors (hdacis) revealed their selective toxicity toward cancer cells, a property that positioned them as promising anticancer therapeutics. In the past decade or so, various hdacis have entered the clinical trial stage for a variety of cancers hdac superfamily, describe recent success in clinical trials for hdacis, and discuss the potential of class- and isotype-selective hdaci.With the benefit of hindsight, it is not surprising that early studies on hdacs are grouped into two families and four classes that differ from each other in terms of structure and biologic activity i , class iia , class iib (hdac 6 and hdac10), and class iv (hdac11). The class iii hdacs (sirt1 through sirt7) comprise the sirtuin (Sir2-related protein) family (The 18 known human ) family . The famhdaci is often used to describe compounds that inhibit the classical hdacs of classes i, ii, and iv—compounds to which sirtuins are insensitive (hdacs. Compounds targeting sirtuins are briefly discussed toward the end of this paper.The term ensitive . Owing ti hdacs (excluding hdac8) are subunits of multi-protein chromatin-modifying complexes iia hdacs shuttle between the nucleus and the cytoplasm in response to extracellular signals; they are unique among hdacs for apparently weak deacetylase activity of vertebrate homologs iib member, hdac6, is found mostly in the cytoplasm, where it deacetylates cytoskeletal proteins such as α-tubulin and cortactin hdacs play roles in many cellular processes, including proliferation, differentiation, apoptosis, autophagy, cell motility, and stress responses Class hdacis represent a broad family of chemical compounds that comprises four main structural classes:The vpa)] Short-chain fatty acids  Hydroxamates  Cyclic tetrapeptides hdacs. Hydroxamates are the only known pan-hdacis, with the ability to target all classical hdacs. In contrast, butyrate, vpa, and trapoxin A target class i and iia hdacs. Other compounds are more specific: SNDX275 inhibits class i hdacs (but not hdac8), romidepsin preferentially targets hdac1 and hdac2, and tubacin selectively inhibits hdac6 hdacis have been extensively reviewed elsewhere Not only do these compounds vary in structure, they also have distinct affinities for various hdacis as chemotherapeutic agents. The hdac inhibitory activity of the naturally occurring compounds butyrate and trichostatin A (tsa) was found to be responsible for their ability to cause differentiation and cell cycle arrest of transformed cells saha), which were known to induce differentiation or apoptosis of cancer cell lines (or both), were also identified as hdacis saha was approved by the U.S. Food and Drug Administration in October 2006 under the trade name Zolinza , for the treatment of cutaneous T-cell lymphoma hdacis are achieving successful results in phase ii and iii clinical trials for a wide range of cancers In the late 1970s and then in the early 1990s, a small number of studies ushered in the era of hdacs as canonical transcriptional co-repressors, the global transcription changes elicited by hdacis are somewhat surprising. It is estimated that the expression level of approximately 2%–10% of genes is altered by hdaci treatment, with as many genes downregulated as upregulated CDKN1A, which encodes the cell cycle inhibitor kinase p21. Although the re-expression of this gene explains the antiproliferative effect of hdaci, cell-cycle arrest is not crucial for hdaci-mediated tumour suppression because hdacis cause cell-cycle arrest in normal and cancer cells alike, and unlike many chemotherapeutic drugs, hdacis can kill proliferating cancer cellsBecause of the function of hdacis induce cancer cell cytotoxicity is apoptosis (programmed cell death). Apoptosis can occur through the extrinsic (death-receptor) or the intrinsic (characterized by cytochrome C release from mitochondria) pathway hdaci-induced apoptosis. Instead, the pro-apoptotic Bax and BH3-only proteins Bid or Bim are necessary for this effect L abrogates apoptosis induction by hdaci As for the method of this tumour cell killing, there are a plethora of routes through which it can occur. The main process through which hdaci also depends on elevations in reactive oxygen species (ros) caused by hdaci treatment. This phenomenon likely reflects the inability of tumour cells to upregulate thioredoxin, an antioxidant protein, during hdaci-induced ros accumulation Apoptosis induced by hdacis can also induce genomic instability by impairing dna damage repair hdacis can still induce cancer cell death by an autophagy-dependent process hdacis may still be effective against cancers with apoptosis defects The hdacis also extend to more systemic effects involving the tumour blood supply, metastatic potential, and host immune response. The hdacis inhibit angiogenesis by repressing the expression of pro-angiogenic factors such as vascular endothelial growth factor and by reducing the sensitivity of endothelial cells for angiogenic stimuli hdacis also inhibit the expression of matrix metalloproteinase enzymes, which degrade the basement membrane surrounding tumours, an important initial step in metastasis hdacis can sensitize tumour cells for destruction by the immune system hdacis can induce diverse cellular and physiologic processes that act in concert to prevent the initiation and progression of cancer.The antitumorigenic properties of i-selective hdacis  possess antitumorigenic capacities strongly suggests that inhibition of class i hdacs may be sufficient for chemotherapy. In support of this hypothesis, genetic deletion and sirna-mediated knockdown studies support the non-redundant role of these hdacs in controlling cell proliferation and survival HDAC1 knockout mice die during embryogenesis because of a lack of cell proliferation HDAC2 reduces tumour growth in a tumour-prone mouse model HDAC3 causes embryonic lethality associated with decreased proliferation, dna damage, and apoptosis hdacs is seen in several cancer types The fact that class i hdacs, the class iib member hdac6 is likely also an important target of hdaci-mediated cancer suppression because of its ability to control cell migration through deacetylation of α-tubulin and cortactin, and through its ability to deacetylate Hsp90 and to disrupt that protein’s function as a chaperone for oncogene products In addition to these class iia hdacs in tumorigenesis. In fact, inhibition of class iia hdacs  could lead to a host of unwanted side effects, as inferred from the role of class iia hdacs in limiting the activation of myocyte enhancer factor 2 (Mef2) target genes. Genetic deletion of each class iia member causes unrestrained MEF2 activation, leading to skeletal defects, pathologic cardiac hypertrophy, or collapse of the vascular system i-selective hdacis with recipients of pan-hdacis to see if patients in the latter group selectively suffer from these types of side effects.In contrast, there is much less evidence implicating class i–selective hdacis elicit fewer side effects than pan-hdacis do, it is clear that hdacis in general are associated with certain toxicities, including fatigue, nausea, diarrhea, and vomiting hdaci toxicity must be addressed if drugs with a broad range of clinical utilities in diverse patients are to be developed. Currently, only one truly specific hdaci has been developed. Tubacin selectively targets hdac6, and it can induce apoptosis of multiple myeloma cells in vitro hdacis will reduce toxic side effects will remain unknown until such agents are developed and tested. Yet, even if the next generation of hdacis lack better toxicity profiles, they will nevertheless be instrumental in elucidating the function or functions of individual hdacs involved in various types and stages of cancer and in other human diseases.Although it has not yet been established whether class hdaci used for specific cases may also be of critical importance independent of the hdacs it inhibits. A recent study demonstrated that specific hdacis can have dramatically different effects depending on the molecular profile of the tumour. In a mouse model of B cell lymphoma, Bcl2 overexpression blocks the ability of the hydroxamates, but not of the cyclic tetrapeptide romidepsin to prevent lymphoma hdacis are equal in terms of biologic activity. This result also provides a glimpse into a future, when therapies are tailored to the individual gene expression profiles of tumours in patients hdaci.The specific hdacis is highly likely to be in combination with other anticancer therapies. Several studies have shown that hdacis can synergize with a large set of other chemotherapeutic drugs such as cisplatin, etoposide, bortezomib, and gemcitabine. Other therapeutics that exhibit an additive or synergistic effect when used with hdacis include differentiation agents such as retinoic acid (ra) and all-trans retinoic acid (atra) for the treatment of leukemic cells; imatinib, an inhibitor of the Bcr-Abl tyrosine kinase expressed by the Philadelphia chromosome in chronic myelogenous leukemia; the breast cancer drug trastuzumab, a monoclonal antibody against the her2/neu (ErbB2) receptor; and 17AAG, an inhibitor of the oncogenic protein chaperone Hsp90 in vitro, these drugs cooperate with hdacis to enhance apoptosis—or, in the case of ra and atra, differentiation—of cancer cells. Further, hdacis can induce differentiation or apoptosis, respectively, of ra- or imatinib-resistant cells. In addition, hdacis enhance the radiosensitivity of gamma- or ultraviolet-irradiated tumours and can bolster the effect of the trail (tumour necrosis factor–related apoptosis-inducing ligand) death receptor ligand on tumour cell death hdac activity and dna methylation in controlling gene expression hdacis and dna methyltransferase inhibitors also exhibit enhanced anti-neoplastic activity The optimal use of hdacis and other compounds can be recapitulated in a single hybrid compound. The first compound of this kind, termed triciferol, in which the side chain of 1,25-vitamin D3 (the active form) is replaced with the dienyl hydroxamic acid of tsa 3 (vitamin D receptor agonism) and tsa (histone hyperacetylation) and exhibits effects similar to co-treatment with the two original compounds in terms of cancer cell cytotoxicity in vitro hdacis can be combined with other drugs to form new antitumorigenic hybrid compounds.Excitingly, it appears that the principle of synergism between hdacis has paralleled the rapid expansion over the past decade of research into the molecular details of hdac activity and the biologic roles of these enzymes. The discovery of their importance to multiple stages of tumour initiation and progression provided a strong rationale for the use of compounds targeting these epigenetic regulators in cancer treatment. With current compounds exhibiting positive clinical results, and one hdaci already on the market in the United States, it is clear that hdacis will likely represent an important class of chemotherapeutic drugs in the near future. Whether newer hdacis, possibly isotype-selective versions, will ultimately supplant the current collection remains to be seen, but would not be surprising given the modest toxicity problems associated with today’s generation of hdacis. Nevertheless, the clinical utility of altering hdac activity has been unambiguously demonstrated.The flurry of activity surrounding the development of sirt1 activator resveratrol—a polyphenol famously contained in red wine—can prevent cancer by targeting multiple stages of progression in mice sirt1 and sirt2 inhibitors, increase apoptosis of cancer cell lines and delay tumour growth in mice, in part by increasing the acetylation and transcriptional activity of TP53 hdacis in terms of their progression from the lab to the pharmacy. Their continued development, alongside that of hdacis, is crucial to fully exploit the importance of protein acetylation in cancer.Although we have focused on inhibitors of the Rpd3/Hda1 family of deacetylases herein, modulators of sirtuin activity  may provhdac inhibition is always desirable, but it is important to note that it is the balance of hdac and histone acetyltransferase activities, and hence the relative acetylation levels of key proteins (which may vary in different tissues and disease states) that is of ultimate importance. For this reason, cases may occur in which too much acetylation is pathologic, and inhibitors of histone acetyltransferases may therefore be efficacious The present mini-review has dealt primarily with the idea that increasing acetylation of histones or other proteins through dna code, it is increasingly clear that cancer can result from aberrations in chromatin and chromatin-modifying proteins such as hdacs. As a result, hdacis represent one promising subgroup of compounds for epigenetic therapy. The elucidation of the mechanisms through which hdacis suppress tumours, the discovery of the particular hdacs that are involved in each stage or type of cancer, the continued development of isotype-selective hdacis, and the optimization of hdacis as a component of combination chemotherapy represent important breakthroughs that will undoubtedly occur in the near future. With continued research and development, hdacis are likely to figure prominently in cancer treatment plans in the near future.Although cancer has traditionally been viewed as a genetic disease, the perspective of cancer as an epigenetic disease is rapidly emerging. Thus, in addition to mutations in the


We have examined the expression of several myeloid cell associated antigens, some of which are involved in myelomonocyte adhesion, in seven well characterized human breast cancer cell lines, since common properties of adhesiveness and migration are found in haemopoietic cells and epithelial cancer cells. Five of these cell lines were of metastatic origin and two were derived from primary breast carcinoma. Antigenic expression was evaluated by immunofluorescence (IF), flow cytometry (FCM), radioimmunoassay on live cells (RIA) and immunoperoxidase staining. None of these cell lines expressed T or B lymphoid specific antigens. Myeloid antigens My4, MO1, and MOF11  were expressed in the 7 cell lines. Leu M1, Leu M3, My9, and MO2 antigens were expressed in some of the cell lines. Leu M2 and My7 antigens were not expressed or at very low levels. The expression of these myeloid antigens was also tested by immunoperoxidase staining, and found on frozen sections of normal mammary gland, fibroadenoma of the breast, primary breast cancer, and lymph node and skin metastases of breast tumours. This common expression in epithelial breast cells and in myeloid cells might be related to common biological functions such as interaction with extracellular matrix which precedes cell migration, a normal function of macrophages and an abnormal function expressed or amplified in human cancer epithelial cells.


In the present study, a positive training set of 30 known human imprinted gene coding regions are compared with a set of 72 randomly sampled human nonimprinted gene coding regions (negative training set) to identify genomic features common to human imprinted genes. The most important feature of the present work is its ability to use multivariate analysis to look at variation, at coding region DNA level, among imprinted and non-imprinted genes. There is a force affecting genomic parameters that appears through the use of the appropriate multivariate methods  and quadratic discriminant analysis (QDA)) to analyse quantitative genomic data. We show that variables, such as CG content, [bp]% CpG islands, [bp]% Large Tandem Repeats, and [bp]% Simple Repeats, are able to distinguish coding regions of human imprinted genes. Genomic imprinting is an epigeneticmodification of dispersed regions of the genome depending on their exposure tothe maternal or paternal germline. This results in differential expression ofonly one of the two alleles depending on the parent of origin. Allele-specificCpG methylation, histone acetylation, asynchronous DNA replication, andchromatin condensation are all associated with imprinted loci .Recently, the question of whether imprintedgenes have sequence characteristics that distinguish them from non-imprintedgenes is drawing the attention of several research groups. Such structuraldifferences may elucidate the mechanisms leading to allele-specific expressionof imprinted genes . GreallyIdentifying imprinted genes experimentallyis challenging because the monoallelic expression of an imprinted gene mayoccur only in one of possibly several isoforms, only in particular tissues, oronly at particular stages of development. Many autosomal genes are imprintedonly in specific tissues or cell types, including GRB10 , Igf2/H1Consequently, in the absence of any methodfor prioritising genes, an average of 100 genes must be examined before a newimprinted gene can be identified. Indeed, experimental identification of humanimprinted genes to date has been slow. To date, only ~60 human imprinted geneshave been identified.For this reason, the application of sequenceanalysis approaches to genome-wide screening of human genes, which can beranked to identify those with a sequence composition suggestive of imprinting, isvery useful.To date, imprinted genes are predicted usinga wide range of genomic features and sophisticated strategies and methodologies–16, but We are concerned with identifying possible candidateimprinted genes to allow their imprinting status to be determinedexperimentally. For this reason, human gene coding region features areconsidered further with a view to developing an approximation to a first-stagescreening and classifying genes into imprinted and non-imprinted candidategroups. This study uses statistical approaches for a first discriminationbetween imprinted and non-imprinted genes based on the currently available codingregion sequences.http://igc.otago.ac.nz/home.html). A negative training set of 72randomly selected control genes and a test set of 31 predicted imprinted geneswere compiled from the recent literature % sequencecharacteristics data were performed using the Minitab software .PCA analysis is a multivariate statisticaltechnique. The central idea of PCA is to reduce the dimensionality of a dataset that presents a large number of interrelated variables, while retaining asmuch as possible the variation present in the data set. PCA can search the datafor qualitative and quantitative distinctions in situations where the number ofdata available is too large.The purpose of the Quadratic Discriminant Analysisis to predict membership of a group from a set of predictor variables (thesequence characteristics). The discriminant is the quadratic combination of thepredictor variables that best predicts group membership, allowing each gene tobe classified into either imprinted or control groups on the basis of itssequence characteristics.The performance of the classification wasassessed using internal and external validation methods according to oursoftware capabilities.cross-validation % genomic sequence characteristics of GCcontent, CpG islands, simple repeats (SR), large tandem repeats (LTR) and SINEsof all imprinted and non-imprinted coding region sequences were calculated.Before applying the pattern recognitionmethods, each calculated descriptor was autoscaled. In the autoscaling method,each variable is scaled to a mean of zero and a standard deviation of unity. This method is very important because each variable is weighted equally, andthis provides a measure of the ability of a descriptor to discriminate classesof compounds . With thFirstly, we started applying the PCAtechnique. After several PCA analyses, the best separation was obtained byusing the following descriptors: GC content, [bp]% CpG islands, [bp]% SimpleRepeats and [bp]% Large TandemRepeats. This suggests that in this case, the other variables are notsignificant for the classification of the coding regions studied.The PCA results show that the firstcomponent PC1) is responsible for 49.6% of the variance of the data. Considering the first (PC1) and second (PC2) components, the accumulated varianceincreases to 72%. is respoXY axes.On theother hand, we can see that the major part of non-imprinted genes, the NO_I2group, has small values for [bp]% SR and [bp]% LTR, and the NO_I1 group has largevalues for the same both descriptors. It is clear that there are four codingregion groups, and each one is located in practically one specific quadrant ofthe coding sequence coefficient is higher in the I1 group (I1) than inI2 (0.03). Note that these results are in good agreement with the loadings ofthe PCA model.Genomic sequence characteristics of a total of 22544 bp fromthe coding sequences of 12 (I1 group) imprinted genes were compared to those of66959 bp of coding sequences of 18 (I2 group) imprinted genes  inorderWe foundan obvious functional difference between I1 and I2 groups in terms ofexpression pattern. We observed maternal expression for 67% of the I1 imprintedgenes and paternal expression for 61% of the I2 imprinted genes.Moreover,other important observation is that all the Large Tandem Repeats of the I1group genes are inside CpG islands while this fact is not observed in the I2group. These results agree with those of Meguro et al. : the CpGToidentify sequence fingerprints and similarities among Large Tandem Repeats inthe two imprinted groups, we used the Wilbur and Lipman pairwise sequencealignment method (see supplementary data for details). The I1 sequences group isquite consistent; all sequences are rich in GC content, and the similarityindex of the aligned fragments ranges from 60 to 100%. In contrast, thesequences of the I2 group are longer, more heterogenous in terms of nucleotidecomposition; in some of them, the presence of a polyA motif could be empathised. The I2 sequence repeats show a much more wide range of similarity index. Inaddition, because of some significant differences in nucleotide composition betweenmembers of I2 sequences, some I2 sequence pairs could not to be aligned. Fromthis analysis, we can conclude that these two Large Tandem Repeats: GC-motifs(in I1 group) and AT-motifs (in I2 group) are highly conserved sequence patternsacross their respective coding regions.Then, webuilt a new model using another statistical technique: the quadratic discriminantanalysis (QDA). QDA is also closely related toprincipal component analysis (PCA) in that both look for combinations ofvariables which best explain the data. QDA explicitly attempts to model thedifference between the classes of data (supervised pattern recognition). PCA, onthe other hand, does not take into account any difference in class(nonsupervised pattern recognition).After the employingof QDA and PCA methods, we proceeded to the validation of their respectiveclassification models.cross-validation approach was used tovalidate the QDA model. The total percentage of correct classification was 83%. Afterthat, we formed a test group of 31 supposed imprinted genes related tonutrigenomics in humans  and quadratic discriminantanalysis (QDA) to analyse quantitative genomic data. We show thatvariables, such as, CG content, [bp]% CpGislands, [bp]% Large TandemRepeats, and [bp]% SimpleRepeats are able to distinguish human coding region imprinted genes.We know that a conclusive assessment ofprediction methods for imprinted genes is problematic due to the small numberof affected genes, their clustering in small genomic regions, and the difficultyof experimental validation.However, we think that the application ofthis PCA sequence analysis approach to genome-wide screening of human genes,which can be ranked to identify those with a sequence composition suggestive ofimprinting, is potentially valuable for a first-stage approximation directingfollow-up laboratory work.Clearly an approach like this can be furtherrefined and the resolution improved as more imprinted genes are identified andconfirmed and the genome sequencing is completed.Supplementary material containing lists of genes used in the study: a negative training setof 72 randomly selected control genes and a test set of 31 predicted imprinted genes; atable with the relevant calculated features; results of Wilbur and Lipman pairwisesequence alignment method.Click here for additional data file.


Sepsis is currently a leading cause of death in hospital intensive care units. Previous studies suggest that the pathophysiology of sepsis involves the hyperactivation of complex proinflammatory cascades that include the activation of various immune cells and the exuberant secretion of proinflammatory cytokines by these cells. Natural killer T-cells (NKTs) are a sublineage of T cells that share characteristics of conventional T cells and NK cells and bridge innate and adaptive immunity. More recently, NKT cells have been implicated in microbial immunity, including the onset of sepsis. Moreover, apolipoprotein E (apoE), a component of triglyceride-rich lipoproteins, has been shown to be protective in endotoxemia and gram-negative infections in addition to its well-known role in lipid metabolism. Here, we will review the role of NKT cells in sepsis and septic shock, the immunoregulatory role of apoE in the host immune response to infection, and propose a mechanism for this immunoregulation. Natural killer T (NKT) cells are a heterogeneous and conserved lineage of T cells that have been implicated in tumor immunity, autoimmune diseases such as diabetes and multiple sclerosis, as well as the overall regulation of the immune system . They weβ2-microglobulin . Furthermore, it appears that the physiological concentration of apoE may be regulating a delicate balance point between sufficient systemic inflammation to clear the invading pathogen, yet insufficient to harm the host in the process of eradicating the infection.Indeed, we found that the apoE-mediated increase in septic mortality was also associated with increased splenic and hepatic iNKT cell frequencies, proliferation and cell numbers, and an exacerbated degree of liver injury . Consist of apoE  were fouα than mice expressing human apoE3 [The APOE gene codes for three main isoforms, apoE2, E3, and E4, only differ from each other by a single amino acid substitution, but possess differential binding affinities for the LDLR (E4 > E3 ⋙ E2) . Recent an apoE3 . Furtheran apoE3 . That saan apoE3 . Similaran apoE3 , promotein vivo [in vitro findings suggest that apoE-mediated antigen presentation is more prominent with endogenous ligands than with LPS, although the classic TLR4 pathway of direct LPS recognition may play a more substantial role in inducing an inflammatory response in vivo. Although the primary insult in CLP appears to be the systemic release of LPS, or other pathogen-associated molecular patterns (PAMPs), and induction of fecal peritonitis, the traumatic ligation and puncture of the cecum could result in the release of endogenous ligands or danger-associated molecular patterns (DAMPs) that can contribute significantly to inflammation as well. The contribution of DAMPs to dendritic cell and iNKT cell activation is not unprecedented, having been studied in attempts to develop immunotherapies against malignancies [Despite evidence that iNKT cells are activated in response to LPS in vivo , apoE fain vivo . Insteadin vivo . These ignancies . This evgnancies , 98 apoEDespite different experimental models, the studies reviewed here all constitute growing evidence for the large contribution of NKT cells to the dysregulated and overwhelming pro-inflammatory response in polymicrobial sepsis and endotoxic shock. Although many strong correlations have been made between septic mortality and NKT cell activation and cytokine production, researchers are still far from delineating and demonstrating the exact mechanism by which NKT cells participate in the septic immune response, or how their activity is regulated in general. In order to accomplish this, knowledge of the endogenous ligand(s) of NKT cells is needed. In spite of progress made by the identification of iGb3, the self-ligand(s) is still unknown, and investigators anxiously await the discovery of this missing piece of the puzzle. Considering the ability of NKT cells to bridge innate and adaptive immunity and their extensive immunoregulatory roles, manipulation of these cells provides a promising therapeutic strategy for sepsis and inflammation. However, because NKT cells have numerous effector functions and closely communicate with other cell types, it will be a challenge to direct the effect of these interventions to a single mechanism of action. In promoting the onset of sepsis, there is contradictory evidence of whether NKT cells are deleterious due to their pro-inflammatory or anti-inflammatory contribution. To further tailor therapies that modulate NKT cell activity, more work is needed to determine what specific factors trigger NKT cells to assume a Th1 or Th2-like phenotype during sepsis. Nonetheless, the work done in animal models so far provides insight on the contribution of NKT cells to inflammation and injury, and an important foundation upon which to build more targeted therapies.


It is a goal we can achieve.”Melinda Gates, co-founder,Bill & Melinda Gates FoundationWith more than 1 million child deaths annually, malaria remains the single leading killer of young children in sub-Saharan Africa In the early 1970s, an intensified effort to interrupt the transmission of malaria was conducted in a group of villages near the town of Garki in northern Nigeria There is yet another promising, low-cost and highly cost-effective, and complementary approach for potentially reducing the morbidity of malaria in sub-Saharan Africa, which builds on existing efforts and could be implemented for as little as US$0.50 per person per year or less than 10% add-on to projected malaria control costs In Africa and other tropical developing countries, the great killer and disabler that results from malaria and NTD coinfections is anemia Similarly, most of the 7.5 million pregnant women infected with hookworm likely live in areas of sub-Saharan Africa that place them at risk for malaria In sub-Saharan Africa, there are several opportunities to link malaria and NTD control programs Together, malaria and the seven most common NTDs listed above cause almost 2 million deaths and are responsible for the loss of almost 100 million disability-adjusted life years (DALYs) annually  It is likely that focusing control efforts on malaria alone will thwart global efforts to sustain malaria control, much less achieve eradication. Ultimately, by reducing anemia in sub-Saharan Africa, linking the NTDs with malaria control would have a major impact on almost all of the Millennium Development Goals


A microarray analysis provides new evidence suggesting that specific cellular processes in the mammalian CNS are coordinated at the level of alternative splicing, and that a complex splicing code underlies CNS-specific alternative splicing regulation. Alternative splicing (AS) functions to expand proteomic complexity and plays numerous important roles in gene regulation. However, the extent to which AS coordinates functions in a cell and tissue type specific manner is not known. Moreover, the sequence code that underlies cell and tissue type specific regulation of AS is poorly understood.Using quantitative AS microarray profiling, we have identified a large number of widely expressed mouse genes that contain single or coordinated pairs of alternative exons that are spliced in a tissue regulated fashion. The majority of these AS events display differential regulation in central nervous system (CNS) tissues. Approximately half of the corresponding genes have neural specific functions and operate in common processes and interconnected pathways. Differential regulation of AS in the CNS tissues correlates strongly with a set of mostly new motifs that are predominantly located in the intron and constitutive exon sequences neighboring CNS-regulated alternative exons. Different subsets of these motifs are correlated with either increased inclusion or increased exclusion of alternative exons in CNS tissues, relative to the other profiled tissues.Our findings provide new evidence that specific cellular processes in the mammalian CNS are coordinated at the level of AS, and that a complex splicing code underlies CNS specific AS regulation. This code appears to comprise many new motifs, some of which are located in the constitutive exons neighboring regulated alternative exons. These data provide a basis for understanding the molecular mechanisms by which the tissue specific functions of widely expressed genes are coordinated at the level of AS. Alternative splicing (AS) is the process by which the exon sequences of primary transcripts are differentially included in mature mRNA, and it represents an important mechanism underlying the regulation and diversification of gene function -4. CompaDrosophila AS factor Transformer-2 (Tra2). Binding of Tra2 to a specialized exonic splicing enhancer element regulates the AS of transcripts encoding the transcription factors Doublesex and Fruitless, which activate sets of genes that are involved in sex determination and courtship behavior, respectively [The extent to which AS events in different cell and tissue types are regulated in a coordinated fashion to control specific cellular functions and processes is not known. Evidence for coordination of cellular functions by AS was recently provided by a study that employed a custom microarray to profile AS in mouse tissues. It was shown that deletion of the mouse gene that encodes Nova-2  primarily affects AS events associated with genes encoding proteins that function in the synapse and in axon guidance . In the ectively ,12.cis-acting elements is known to affect splice site choice by a variety of specific mechanisms that generally result in the promotion or disruption of interactions that are required for the recruitment of core splicing components during early stages of spliceosome formation [Current evidence indicates that tissue specific AS events may be regulated in some cases by different combinations of widely expressed factors and in other cases by cell/tissue specific factors ,13,14. Icis-acting sequences bound by AS regulators were initially identified by deletion and mutagenesis studies employing model pre-mRNA reporter constructs, in conjunction with in vitro or transfection based assays that recapitulate cell or tissue specific AS patterns [cis-acting control sequences and cognate regulatory factors have only just been attempted [In several cases, patterns . In othepatterns ,20. Howettempted ,22. Suchcis-acting motifs, many of which are new, that correlate strongly with CNS associated regulation of AS. Unexpectedly, many of these new motifs are located in neighboring constitutive exons and adjacent intron sequences. Together, our results suggest a widespread role for tissue coordinated AS events and associated cis-acting regulatory elements in controlling important functions in the mouse CNS.In the present study, we used a new microarray to profile AS levels for thousands of cassette type alternative exons  across a diverse spectrum of mouse tissues. Analyses of these data resulted in the identification of genes with single or multiple alternative exons that display tissue correlated AS levels and the discovery of many new central nervous system (CNS) associated AS events that are enriched in functionally related genes. A computational search also led to the identification of Using a new AS microarray, we generated quantitative profiling data for 3,707 cassette-type AS events in 27 diverse mouse cells and tissues. These AS events were mined from expressed sequence tag (EST) and cDNA sequences represented by 3,044 UniGene clusters . The profiled tissues included whole brain, five brain subregions, spinal cord, three embryonic stages, embryonic stem cells, three muscle-based tissues , gastrointestinal and reproductive tissues, and several additional adult tissues. Quantitative, confidence-ranked estimates for percentage exclusion ('skipping') levels of each alternative exon were determined using the computational analysis tool GenASAP (Generative Model for the Alternative Splicing Array Platform) ,24. ConfIn the present study, we used our dataset to detect alternative exons that display inclusion level differences specific to groups of physiologically related tissues, as compared with all other tissues. We also considered whether pairs of alternative exons belonging to the same genes have coordinated inclusion levels across the profiled tissues. From these analyses, we investigated which AS events may be coordinated functionally and potentially form AS-regulated networks, and which sequence elements in transcripts are likely to play a role in the regulation of functionally coordinated AS events.AS events specific to groups of related tissues were initially analyzed. The use of the term 'specific' in this context, and below, refers to the detection of a statistically significant AS level difference in a group of tissues, relative to all of the other profiled tissues . We observed that about ten alternative exons displayed inclusion level differences in embryonic stem cells and the three whole embryo samples representing different stages of development, relative to the other profiled tissues. In addition, about ten alternative exons displayed pronounced inclusion level differences in the three muscle-based tissues , and five alternative exons displayed AS patterns common to both CNS and muscle tissues. Interestingly, some of the genes displaying AS differences in embryonic stem cells and embryonic samples are associated with regulation of development, and several of the genes with differential AS levels in muscle-based tissues are associated with muscle specific functions. These and other non-CNS-regulated AS events are described in Additional data file 1 and are listed in Additional data file 2. These findings suggest that AS could play an important role in coordinating gene functions in a tissue specific manner, although a larger set of tissue specific AS events is required to test this hypothesis.The largest numbers of tissue dependent AS events detected in our microarray data were associated with CNS tissues, with about 110 events displaying specific AS level differences Figure . This obP < 1 × 10-16; see Additional data file 1).The probable functional relevance of the majority of the 110 most significant CNS-associated AS events is underscored by the observation that 60% of the alternative exons in this group could be detected in aligned human EST and cDNA sequences, whereas only about 24% of the non-CNS-associated alternative exons represented on the microarray could be detected in both human and mouse cDNA/EST sequences. This finding represents a statistically significant enrichment of conserved cassette alternative exons with detected CNS-associated AS levels, while controlling for variable cDNA/EST counts . Only 8% of the CNS regulated exons have the potential to introduce a premature termination codon that could elicit nonsense mediated mRNA decay, in contrast to about 37% of the other AS events . These results are consistent with recent findings indicating that a relatively small proportion of conserved AS events introduce premature termination codons [Consistent with this observation, and with the results of previous reports ,27, we fn codons ,29, and P = 0.017, by Fisher's exact test). This difference, although based on a small sample size, is consistent with our observation that CNS regulated AS events are significantly enriched in conserved alternative exons, whereas AS events with the potential to disrupt conserved protein coding sequences are known to be significantly under-represented by conserved alternative exons compared with species-specific alternative exons [P = 4.6 × 10-7, by Wilcoxon-Mann-Whitney test), whereas the alternative exons of AS events predicted to result in domain disruption have longer median exon lengths than those that are not predicted to result in domain disruption (116 nucleotides versus 99 nucleotides). Thus, the shorter alternative exon lengths of the CNS specific AS events appear to account, at least in part, for the lower proportion of predicted domain disruptions resulting from this set of exons. Given that these regulated exons are often conserved in human, it is interesting to consider that they may contribute numerous important roles, such as the formation and regulation of protein-protein interactions associated with neural specific complexes and pathways.We also examined the potential impact of the CNS regulated AS events at the protein level. The CNS associated AS events have the potential to result in partial or complete domain disruption in 13% (4/31) of cases, whereas 34% (201/599) of the non-CNS AS events represented on the arrays could result in such a change  of the top 125 genes (ranked according to the significance of the CNS associated AS level difference) have a reported specific functional link with the nervous system. Nervous system specific functions of genes containing CNS regulated AS events are listed in Table Consistent with the previous observation that about 7% of AS events are differentially regulated between neocortex and thymus by the AS regulator factor Nova-2  . Using information provided by the microarray probes targeting the constitutive exons flanking each alternative exon, we identified about 200 genes that have CNS associated changes at the transcript level, as represented by statistically significant changes relative to most of the other profiled tissues . Consistent with previous findings indicating that AS and transcript level regulation control different subsets of genes in mammalian tissues ,30,31, tIn addition to the detection of individual alternative exons that display regulatory patterns associated with single tissues or groups of physiologically related tissues, we investigated whether pairs of alternative exons belonging to the same genes display tissue coordinated AS levels. Previous studies of EST/cDNA sequences identified a few cases in which different alternative exons belonging to the same genes appear to be coordinated ,33. HoweApproximately 15 of the pairs of alternative exons have significantly correlating (absolute standard Spearman correlation ≥ 0.70) inclusion levels across the tissues, with an expected false-positive detection rate of one exon pair . However, higher than expected numbers of exon pairs with correlated AS levels are observed over a wide range of lower correlation levels Figure . For exaP = 0.021, by Wilcoxon-Mann-Whitney rank sum test). Consistent with these results, exon pairs displaying positive correlation are also significantly closer to each other in terms of nucleotide length, as compared with pairs of exons with high negative correlation or without high correlation . In a few of the cases shown in Additional data file 4, pairs of alternative exons with significant positive correlation are adjacent to each other. One example is a pair of alternative exons in the gene encoding Agrin, a proteoglycan that functions in the aggregation of acetylcholine receptors in postsynaptic membranes, which is a key step in neuromuscular junction development. Consistent with our microarray data indicating that this pair of exons has increased inclusion levels in CNS tissues relative to the other profiled tissues, it has been reported that the same pair of exons can be included in nervous system tissues but are excluded in all other tissues examined [Exons with high positive correlation (at a standard Spearman correlation ≥ 0.6) are mostly within one to four exons of each other, with a median of two intervening exons . In contrast, exon pairs with high negative correlation (at a standard Spearman correlation ≤ -0.60) have a median of four intervening exons, and exon pairs that are not highly correlated (with an absolute standard Spearman correlation < 0.6) have a median of four intervening exons . The difference in intervening exon numbers between the positively correlated pairs of exons and pairs of exons that are not highly correlated is statistically significant  gene  and N-methyl-D-aspartate [Neo1 is a widely expressed cell surface receptor that is involved in axon guidance and in the regulation of neuronal survival [As in the case of the pair of the positively correlated exons in Agrin transcripts, the levels of inclusion of exons belonging to a correlated pair are generally highly similar among the various CNS tissues  terms. Enrichment was observed for terms including the following: GTPase-based signaling, cell-cell signaling, cytoskeletal organization and biogenesis, vesicular mediated transport, transmission of nerve impulse, and neurophysiologic process . Because sequence conservation reflects selection pressure acting to preserve biologic activity, it can be used as a proxy to assess the probable functional importance of motifs. Accordingly, we also analyzed the relative conservation levels of the SeedSearcher motifs in the corresponding intron and exon regions of the orthologous human genes, and the statistical significance of detected conservation was determined .The lgorithm  (see AddP < 0.05) are shown in Additional data file 5, alongside any known similar motifs. Of these motifs, 26 had at least 20 occurrences in the three groups defined above, and this number of occurrences facilitated further analysis of these motifs for statistically significant, relative conservation levels. Seventeen of the 26 motifs were found to be significantly more conserved than the surrounding regions . Finally, we also directly searched for enrichment of cis elements with a previously described link to regulation of AS in the nervous system . The results of this search, as well as the ab initio search, are described in more detail below.All 39 of the SeedSearcher motifs found to be significantly enriched in the CNS regulated AS events  exons and in the intron sequences flanking these and the alternative exons. Second, some motifs are detected when sequence regions were concatenated, indicating that they could function in a spatially flexible manner and do not have to reside within a specific exon or intron location. Third, there is a high enrichment for variations of C/U-rich motifs. These predominantly reside within the 150 nucleotide intron region immediately upstream of alternative exons, although some C/U-rich motifs are also found within the 150 nucleotide intron region downstream of alternative exons. These motifs are specific to these regions, because they are not significantly enriched in the 150 nucleotide intron regions immediately flanking the C1 and C2 exons. Moreover, none of the motifs shown in Figure Figure The C/U-rich motifs resemble binding sites for nPTB and PTB, which are known to function in the regulation of alternative exon inclusion in the nervous system (see Introduction) ,43. In pab initio search, C/U-rich motifs were also found to be the most significantly enriched (hypergeometric P about 10-6) when directly searching using subsequences of known motifs, including those shown in previous experiments to directly bind nPTB and PTB . Many of the C/U-rich motifs identified in the directed searches resemble those identified by the ab initio search. However, in both searches, the identified motifs are considerably shorter and more degenerate than those inferred previously by experimental approaches and, as such, could represent the core recognition sites for nPTB/PTB or potentially other AS regulatory factors that specifically recognize pyrimidine-rich regions to regulate neural specific splicing. The C/U-rich motifs that we detected are different from the UGYUUUC motif that Sugnet and coworkers [Consistent with the results from the oworkers  found toP ≤ 0.05; see Additional data file 1 for details) position-specific scoring matrix (PSSM) based motifs . Consistent with the results obtained with SeedSearcher, Improbizer detected enrichment of several C/U-rich motifs in the intron regions flanking the regulated alternative exons, and only one motif was detected in the regulated alternative exons, although this motif barely passed the score threshold. There are obvious similarities between many of the 20 Improbizer PSSM motifs and the 39 SeedSearcher motifs, and in some cases combinations of multiple SeedSearcher motifs appear to be similar to individual Improbizer PSSMs (but not necessarily vice versa). It is also noteworthy that although the UGYUUUC motif previously reported by Sugnet and coworkers was not detected in intron sequences flanking the CNS regulated exons in our data, Improbizer did report another motif (UUUSYUU) that matched one of the SeedSearcher motifs . Our comparative results thus indicate that both differences in the sets of AS events analyzed as well as in the motif search procedures probably account for differences between the numbers and types of motifs detected in the two studies.The differences between our findings and the results reported by Sugnet and coworkers could be due to the different sets of AS events analyzed (see above) as well as differences between the motif search algorithms implemented in the two studies. To investigate the latter possibility, we employed the Improbizer algorithm described by Sugnet and coworkers to identify and score motifs enriched in our set of CNS regulated AS events. The Improbizer searches resulted in detection of 20 statistically significant (-4) of motifs corresponding to the binding sites for Fox-1/2 and Nova-1/2 motifs. This is not surprising because, as mentioned before, Nova proteins probably regulate about 7% of neural specific AS events [cis-acting sequences associated with CNS tissue regulated AS.In addition to the detection of strong enrichment of C/U-rich motifs, the directed searches on our dataset revealed a relatively modest enrichment P about 10-4 of motS events . The detab initio searches resulted in the detection of many motifs that are more highly enriched than those found in the directed searches, and several of these motifs appear to be novel , and this implies that coordination between AS events belonging to the same genes may involve communication between splicing factors assembled at proximally located splicing signals.Approximately half of the genes containing single and pairs of exons that are differentially spliced in CNS tissues have known neural specific functions. The CNS associated AS events we have detected by microarray profiling are significantly enriched in genes with GO terms related to GTPase based signaling, vesicular transport and cytoskeletal functions, as well as nervous system specific GO terms. Similar to the proposed role for Nova proteins in the coordinated regulation of alternative exons belonging to genes associated with functions at the synapse ,22 see . This neNew and known motifs were identified that correlate strongly with the CNS regulated AS events. Our results thus provide a large number of new CNS regulated AS events, many of which are associated with functionally related genes, as well as detailed information on sequence motifs that are predicted to regulate these CNS-associated AS events. These motifs probably comprise part of the sequence 'code' underlying the regulation of CNS tissue specific AS. As such, the data resulting from our analyses should provide a valuable resource for establishing molecular mechanisms by which the neural specific functions of widely expressed genes are regulated and coordinated at the level of AS.The detection of AS events was performed essentially as previously described ,30.Microarray design, hybridization and data analysis for 3,707 mouse cassette AS events from 3,044 UniGene clusters  was performed essentially as described previously ,24,44. IDetection of motifs enriched in exon and intron sequences associated with CNS regulated AS events was performed using a variant of the SeedSearcher algorithm . DetailsRT-PCR reactions were carried out as described previously .ab initio searches. Additional data file ab initio motifs detected at each exonic and intronic location, and in each group. Additional data file ab initio searches. Additional data file The following additional data are available with the online version of this paper. Additional data file Provided is supplemental information on genes with microarray detected tissue specific AS levels, additional details on Materials and Methods, and additional Figures (1-5).Click here for fileProvided is information on tissue specific AS events.Click here for fileProvided is information on CNS regulated AS events.Click here for fileProvided is information on correlated pairs of AS events belonging to the same genes.Click here for fileab initio searches.Summarized are motifs associated with CNS specific AS events detected from Click here for fileSummarized are experimentally defined sequences/motifs associated with neural specific AS used for searchesClick here for fileSummarized are experimentally defined motifs/subsequences significantly enriched in exons and introns associated with CNS regulated AS events identified in the AS microarray data.Click here for fileab initio motifs detected at each exonic and intronic location, and in each group.Summarized are the number and statistical significance of Click here for fileSummarized are motifs associated with CNS specific AS events detected by searching with the Improbizer program.Click here for fileab initio searches.Shown are conservation levels of motifs associated with CNS specific AS events detected from Click here for fileProvided are GenASAP values and C1-A-C2 exon sequences for 3,707 AS events profiled in 27 mouse tissues. Additional microarray data has been deposited in GEO under accession GSE8081.Click here for file


Recent evidence suggests that several human cancers are capable of uncoupling of mitochondrial ATP generation in the presence of intact tricarboxylic acid (TCA) enzymes. The goal of the current study was to test the hypothesis that ketone bodies can inhibit cell growth in aggressive cancers and that expression of uncoupling protein 2 is a contributing factor. The proposed mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration.Seven aggressive human cancer cell lines, and three control fibroblast lines were grown in vitro in either 10 mM glucose medium (GM), or in glucose plus 10 mM acetoacetate [G+AcA]. The cells were assayed for cell growth, ATP production and expression of UCP2.There was a high correlation of cell growth with ATP concentration (r = 0.948) in a continuum across all cell lines. Controls demonstrated normal cell growth and ATP with the lowest density of mitochondrial UCP2 staining while all cancer lines demonstrated proportionally inhibited growth and ATP, and over-expression of UCP2 (p < 0.05).Seven human cancer cell lines grown in glucose plus acetoacetate medium showed tightly coupled reduction of growth and ATP concentration. The findings were not observed in control fibroblasts. The observed over-expression of UCP2 in cancer lines, but not in controls, provides a plausible molecular mechanism by which acetoacetate spares normal cells but suppresses growth in cancer lines. The results bear on the hypothesized potential for ketogenic diets as therapeutic strategies. One mitochondrial adaptation to increased ROS is over-expression of uncoupling protein 2 (UCP2) which has been reported in a number of human cancer cell lines . Un,9. Un8,9most colon cancer lines, the degree of over-expression correlating with the histologic grade. It was proposed that UCP2 over-expression was an adaptive mechanism that reduced ROS-induced apoptosis among the cancer cells.An increase in UCP2 has been reported to be associated with reduction of respiratory ATP production in several cancer lines  that havWe suggest that ketone bodies, if metabolized in cancers of this type, can inhibit glycoloysis and resultant ATP generation without compensatory mitochondrial respiratory ATP production, thereby inhibiting cell growth.et al. pointed out that the Randle cycle has only been persuasively demonstrated in in vitro preparations under conditions of euglycemic, hyperinsulinemic clamp, in which intracellular glucose availability remains high and fixed. Under these circumstances, in rat myocardium preparations, for example, variable fatty acid availability does indeed regulate the rate of glycolysis.The Randle cycle was proposed more than 30 years ago as a mechanism whereby products of lipolysis (fatty acids and ketone bodies) would inhibit glycolysis in normal cells to maintain stable ATP production . Accordiin vivo for most aggressive cancer cells whose membranes express high affinity insulin-independent glucose 1 transporters (GLUT1) [Interestingly, high fixed intracellular glucose availability is a likely condition  (GLUT1) . The Km et al. reported MAC16 tumor growth inhibition in mice on ketogenic diets [Evidence for such an inefficient Randle cycle is found in the seven cancer lines of our investigation which demonstrated parallel reductions in ATP concentration and cell growth, the greatest inhibition found in CaCO2 and SW48 lines, and the least in MCF7 Figure . In addiic diets  but failic diets . The latin vivo study to predict effects under physiologic conditions. Third, in vitro culture of primary cell lines is difficult for normal human cell types, and only fibroblasts were used as controls, so the response of other normal tissue types was not measured. Further, fibroblast lines capable of growth in cell culture are not normal; however, neither are they the equivalent to malignant cell lines and their difference from malignant lines was demonstrated in this investigation. Also, the range of tumor susceptibility to acetoacetate varies widely, plausibly due to increased levels of UCP2 expression, but other factors must be considered. For example SW48, with the greatest reduction of ATP and cell growth in acetoacetate medium, was a prominent outlier with the least UCP2 expression among the cancer cell lines (Figure There are limitations in the present study. The investigation does not prove metabolic inhibition of cancer growth and ATP production due to uncoupling. Proof requires direct demonstrations of acetoacetate metabolism as well as inhibition of the rate of ATP production, not just of steady state ATP concentrations. Second, glucose and acetoacetate were both incubated at supraphysiologic concentrations to maximize detectable effects. It is not possible without further s Figure . FinallyCancers are notorious for their ability to evade metabolic regulation  and therOn the other hand the initiation of a primary event transforming a normal cell into a pre-malignant phenotype is followed by a long period of subsequent adaptive cellular response to a local hypoxic, nutrient-deprived microenvironment. During this interval of abnormal cellular growth multiple adaptations eventually result in the cancer phenotype. A typicWe have shown that acetoacetate added to glucose medium causes variable but parallel reductions of ATP concentration and cell growth in seven human cancer cell lines. The effect lies along a continuum with control fibroblasts at one extreme in which neither cell growth is inhibited nor is ATP concentration reduced. The effect is consistent with an "inefficient" Randle cycle due to over-expression of uncoupling protein 2, seen only in the cancer lines, and is plausible in light of current models of a transformed cell's adaptive microscopic development and progression toward the cancer phenotype. More work must be done to prove metabolic inhibition by this mechanism. If proven, the results speak to the hypothesized potential for ketogenic diets as non-toxic therapeutic strategies, as adjuvants to standard therapies, and to multimodality therapies to improve the control of malignant disease.The authors have no conflicting or competing interests to declare in the conception, evolution, or execution of this research.EJF conceived the study, directed its primary design and coordination, performed much of the data analysis, and drafted and revised the manuscript; AM grew and counted the cell cultures, performed the ATP assays, performed substantial data analysis and participated in writing the Methods section of the manuscript; EVQ coordinated execution of the study, contributed to important aspects of the research direction, and made important revisions to the manuscript; JMS performed the UCP2 assay and wrote the methods for this aspect of the work; RDF coordinated execution of the study, contributed to important aspects of the research direction, and made important revisions to the manuscript. All authors read and approved the final manuscript.