Histone deacetylase (HDAC) inhibitors are powerful epigenetic regulators that have enormous therapeutic potential and have pleiotropic effects at the cellular and systemic levels. To date, HDAC inhibitors are used clinically for a wide variety of disorders ranging from hematopoietic malignancies to psychiatric disorders, are known to have anti-inflammatory properties, and are in clinical trials for several other diseases. In addition to influencing gene expression, HDAC enzymes also function as part of large, multisubunit complexes which have many nonhistone targets, alter signaling at the cellular and systemic levels, and result in divergent and cell-type specific effects. Thus, the effects of HDAC inhibitor treatment are too intricate to completely understand with current knowledge but the ability of HDAC inhibitors to modulate the immune system presents intriguing therapeutic possibilities. This review will explore the complexity of HDAC inhibitor treatment at the cellular and systemic levels and suggest strategies for effective use of HDAC inhibitors in biomedical research, focusing on the ability of HDAC inhibitors to modulate the immune system. The possibility of combining the documented anticancer effects and newly emerging immunomodulatory effects of HDAC inhibitors represents a promising new combinatorial therapeutic approach for HDAC inhibitor treatments. Within a eukaryotic cell, DNA associates with histone and nonhistone proteins to form chromatin. The degree to which DNA is wound around histone proteins affects transcription: the more tightly wounded the DNA, the more condensed the DNA, and gene expression is repressed. The N-terminal regions of histone proteins are substrates for a variety of enzymes that result in posttranslational modifications of histone proteins, including phosphorylation, methylation, ubiquitination, and acetylation. Combined, these posttranslational modifications epigenetically regulate the extent of gene transcription. Of these known epigenetic factors, histone acetylation has garnered much attention in the last decade as one widely recognized factor regulating gene expression. Acetylation of histone proteins is a balance between the activities of both histone acetyltransferases (HATs) and HDACs with histone acetylation being generally associated with an increase in gene transcription while deacetylation results in decreased gene transcription.Although it is eminently comprehensible, this simplified view leads to a vast underestimate of the effects of HDAC inhibitor treatment on chromatin structure. HDAC inhibitor treatment rapidly leads to compensating changes in histone methylation and changes in expression of histone modulators so that the effects of HDAC inhibitor treatment, even at the level of chromatin structure, are not fully delineated. In addition, as the majority of acetylation occurs on nonhistone proteins, the consequences of inhibiting HDACs using available HDAC inhibitors have profound effects on many processes independent of chromatin structure. HDAC inhibitor treatment alters gene expression at many levels including transcription factor activity, miRNA expression, and signal transduction pathways. While the most commonly reported effect of HDAC inhibitors on tumor cells is as an inducer of apoptosis, they have also been shown to interfere with cellular growth –3 and diIt is exciting that the clinical utility of HDAC inhibitors has been extended far beyond treatments for cancer, as they have now been investigated for their therapeutic potential in all top 10 leading causes of death in the US. For example, valproic acid has been used for decades for the treatment of depressive disorders with the intent to prevent suicidal behaviors (number 10 on the list) , 8. MoreIn this review, we focus on the use of HDAC inhibitors in basic biomedical research and their impact on the immune system for several reasons. First, although four HDAC inhibitors are currently FDA-approved for the treatment of hematological malignancies, clinical trials using HDAC inhibitors against other cancers, particularly solid tumors, have failed. Our premise is that a greater understanding of the complex effects of these inhibitors on HDAC activities and cellular immunomodulatory processes is necessary in order to aid in the design of the next generation of these important drugs. Second, a more complete knowledge of HDAC enzymes and inhibitors is likely to reveal additional, still unrecognized, cellular processes which could potentially impact the pathophysiology of disease processes and must be taken into account in experimental design and in data analysis and interpretation. Finally, already proven clinically useful as anticancer treatments, HDAC inhibitors have a newly delineated role in modulating the activity of the immune system. Given the emerging importance of immunotherapy in cancer treatment, HDAC inhibitors may be useful in the development of new anticancer therapeutic strategies, including combination therapy. This review will summarize what is known about the cellular and systemic effects of HDAC inhibitors, with special emphasis as to how they relate to the immune system. But in order to achieve their full therapeutic potential, HDAC inhibitors need to be understood at all levels.+-dependent. Class I HDACs, consisting of HDAC1, HDAC2, HDAC3, and HDAC8, are ubiquitously expressed in the nucleus in all tissues and are homologous to the yeast HDAC RDP3. HDAC1 and HDAC2 are primarily nuclear while HDAC3 and HDAC8 can shuttle in and out of the nucleus [Currently, 18 HDAC enzymes have been identified in mammalian cells, which are subdivided into four main classes based on their homology to yeast HDACs. Three of the four classes  are zinc-dependent enzymes while Class III HDACs are NAD nucleus  and many nucleus , 12. Cla nucleus ) and has nucleus  and expr nucleus . Class I nucleus ). Sirtui nucleus , 17. Theε-N-acetyl lysine amino acid. Although the name HDAC implies some specificity for histones, HDACs deacetylate a wide range of nonhistone proteins (see [Histone deacetylases (HDACs) are a superfamily of enzymes originally named because they remove an acetyl group from ins  and in vitro or are in current clinical trials and have been recently reviewed elsewhere [ in vitro using standard assays with recombinant HDAC proteins has been hindered due to protein misfolding, lack of enzymatic activity, and most importantly, the inappropriate assessment of isolated HDACs that exist as multiprotein complexes in physiologic conditions.”Histone deacetylase (HDAC) inhibitors are a family of natural and synthetic compounds that differ in their target specificities and activities, both in the clinical setting and in laboratory studies . HDAC inlsewhere , 29–32. lsewhere  point ou in vitro, in cell-based assays to determine cellular effects for broader impacts, and in animal/human studies to delineate any systemic effects revealed by preclinical and clinical trials. This portion of the review will consider the role of HDAC inhibitors at each of these levels.Complete understanding of the activities of compounds in the clinical setting requires an understanding of the inhibitor effects at multiple levels, including the enzymes which may be targeted by the inhibitor, as assayedIn vitro assays, usually used to definitively ascertain the mode of action of an inhibitor, have proved problematic in the case of HDAC inhibitors as the values quoted between similar assays are extremely variable. For instance, Hu et al. [50 values of 0.1–0.3 μM. On the other hand, Khan et al. [50 values in the nanomolar range, with TSA having lower efficacy towards HDAC8 by two orders of magnitude compared with HDAC1 and HDAC3. Similar differences were seen with MS-275 between the two studies. Although both studies expressed recombinant HDAC enzymes using the baculovirus expression system, they used different substrates. Hu et al. used purified, labeled histones while Khan et al. used a commercial fluorogenic peptide substrate. However, little difference between these two substrates was observed when HDAC enzymes purified from liver were used. Indeed, the IC50 values for TSA measured using isolated HDAC enzymes are similar and, more specifically, relatively similar Km and Vmax values for each reaction have been reported for both purified histone and peptide substrates [50 values truly are for any given HDAC inhibitor as there are fundamental disagreements in the conclusions which can be drawn from each set of assays.u et al.  determinn et al.  report Ibstrates , 35. AltKm of inhibitor binding to each HDAC and identify possible off-target interactions. Interestingly, a similar variation in values was seen between the two assays. Marks et al. [μM range. Only values for SAHA were measured in both assays and the values for this inhibitor range from 1.8 ± 0.3 nM in the FRET binding to HDAC6 assay and 1.0 μM using the reciprocal binding constant in the FRET competition assay [An alternate approach to determine specificity of HDAC inhibitors uses fluorescence resonance energy transfer (FRET) assays for measuring binding of an inhibitor to HDAC enzymes. FRET assays either directly measure the inhibitor-enzyme binding  or use as et al.  report bs et al.  report von assay ; these a50 of HDAC inhibitors is dependent on enzyme preparation and is also assay dependent. Without a clear understanding of the enzyme targets and potency of HDAC inhibitors, interpretation of in vivo work becomes more difficult and the uncertainty in the specificity, targets, and efficacy of HDAC inhibitors from in vitro studies must frame the interpretation of in vivo assays.The aggregate conclusion from these studies is that the IC50 derived from in vivo and in vitro measurements is expected but this variability is exacerbated by the large number of cell-based assays used. As the effects of HDAC inhibition are wide-ranging, a variety of cell-based assays are needed to assess activity of HDACs  [ in vitro [Further investigation of HDAC inhibitor activity using cell-based assays is essential to understanding the activity of HDAC inhibitors tested)  and has in vitro  and in cin vitro . Cellulain vitro . Howeverin vitro  due at lin vitro , cellula ras oncogene [ in vitro recombinant assay [Unfortunately, many commercially available HDAC inhibitors have not been sufficiently well characterized to support use in the manner for which they are marketed. For instance, depudecin is a natural product which was first identified as a compound that reversed transformation by theoncogene  but was oncogene . Althougnt assay  so that nt assay . in vitro assays of Class I HDAC inhibitors focus on histone targets and as Class I HDACs are localized to the nucleus, this seems to be a reasonable strategy. However, recent advances in mass spectrometry techniques have revealed that this ancient form of protein regulation is intimately linked to metabolism and that acetylation may affect gene expression at multiple levels  (reviewed in [Manyiewed in ). The coiewed in ). Howeveiewed in , 64, addiewed in ). Thus, In highlighting the role of HDAC inhibitors as master epigenetic regulators, it is remarkable how cells treated with HDAC inhibitors are capable of self-regulation, ensuring that global changes are not lethal. Cells, and even entire organisms, can somehow tolerate extensive hyperacetylation of core histones and other proteins that occur following treatment. Recent work has demonstrated that cells respond to HDAC inhibitor treatment by rapidly increasing H3K27me3 at the transcription start sites of genes capable of slowing growth, as well as minimizing protein hyperacetylation until gene expression patterns can be restored . Once ceRegulation of histone acetylation by the activity of histone acetylases and histone deacetylases is a fundamental step in the epigenetic control of gene expression. Surprisingly, in spite of this seemingly generic global control of gene expression, only about 5–10% of gene transcription has been shown to be influenced by HDAC inhibitor treatment, with approximately half of the affected genes being upregulated and half being downregulated. However, HDAC inhibitors also mediate changes in gene expression via a direct effect on the activity of transcription factors and on multiple modes of epigenetic control; it is this level of reversible epigenetic regulation that makes HDAC inhibitors attractive and potentially effective manipulators of cellular and immunological outcomes.Understanding the pleiotropic effects of HDAC inhibitors at the cellular level is complicated by the fact that HDACs can form complexes. Intriguingly, though SAHA and BML-210 are described as pan-HDAC inhibitors, the aminobenzamide BML-210 demonstrates a higher selectivity for the HDAC3-NCoR complex than its hydroxymate counterpart, which demonstrated higher levels of selectivity for HDAC1 and HDAC2 . This woα stability and transcriptional activity [Preventing or reducing transcription factor deacetylation represents another mechanism by which HDAC inhibitors have been shown to influence gene expression. For example, HDAC inhibitor-promoted acetylation stimulates the DNA-binding activity of p53, resulting in enhanced proapoptotic gene transcription . Convers(1) Chromatin Remodeling Enzymes. HDAC inhibitor treatment results in differential expression of epigenetic regulators such as chromatin remodeling enzymes. For example, enhancer of zeste homolog 2 (EZH2), a component of the polycomb-repressive complex involved in gene silencing and H3K27me3, is increased in aggressive tumors, but it is inhibited by HDAC inhibitor treatment [ in vitro work has demonstrated that while reexpression of these proteins does slow cancer cell growth, it also promotes metastatic traits [reatment . Decreasreatment . Similarreatment –94. Howec traits .(2) Methylation. HDAC inhibitor treatment leads to a rapid change in histone and DNA methylation. In pulmonary fibroblasts, hypermethylation of the Thy-1 promoter region results in decreased Thy-1 expression and enhanced fibrosis. Treatment of Thy-1 (−) rat fibroblasts with the HDAC inhibitor TSA significantly decreased DNA methylation while coordinately increasing H3K4me3 in the promoter region of Thy-1, resulting in restored Thy-1 expression [ in vivo using mice with Huntington's disease phenotype, the researchers also observed that these changes in methylation status were associated with improved motor function and cognition in mice treated with an HDAC inhibitor compared to mice treated with a vehicle control [pression . Howeverpression . Followi control .(3) miRNA. The regulation, and dysregulation, of miRNA in human health and disease also demonstrates the utility of HDAC inhibitors in the epigenetic control of cellular gene expression as miRNA expression is also altered by HDAC inhibitor treatment. miRNAs are small noncoding RNA molecules that posttranscriptionally repress target gene expression. HDAC inhibition can rapidly alter miRNA levels and in turn indirectly mediate miRNA target gene expression. For example, treatment of breast cancer cells with a combination of sodium butyrate and panobinostat increased the expression of miR-31 which was accompanied by a subsequent repression of its target protein BIM1 that resulted in the induction of cellular senescence [nescence . A studynescence .HDAC inhibitors also have the potential to decrease miRNA abundance on a much broader scale as HDAC1 has been shown to enhance miRNA processing by acetylating the miRNA processing protein DGCR8, which increases the production of mature miRNA species . HoweverThe first clinical success regarding use of HDAC inhibitors was seen in the treatment of certain forms of cancer, most notably cutaneous and peripheral T cell lymphomas. Development of these Class I-specific HDAC inhibitors focused on their ability to change gene expression, which subsequently led to inhibition of cell growth and apoptosis. This early success was not recapitulated in clinical trials using HDAC inhibitors for the treatment of solid tumors –102 whicAs delineated above, HDAC inhibitor treatment leads to diverse effects via multiple mechanisms to influence pathways which affect immune function. In aggregate, these signals may result in modulation of immune function and impact the role of the immune system in a variety of diseases, including cancer. Knowing that many of the underlying immune mechanisms that are activated to fight tumor cells are also important in regulating immune responses, it comes as no surprise that HDAC inhibitors have been studied for their utility in treating chronic inflammatory disorders .γ. What is interesting is that decreased mRNA expression of IL-10, which is a major anti-inflammatory cytokine, was also noted [γ was also seen [α. Since this initial work, numerous studies from both human and animal models of inflammatory or autoimmune diseases have documented the anti-inflammatory properties of several HDAC inhibitors [μM) than lower doses (50–200 nM) that resulted in a reduction in the production of the proinflammatory cytokines TNF-α, IL-1β, IFN-γ, and IL-12. As reviewed in [Early studies examining the influence of HDAC inhibitors on the immune response led to mixed results. In a mouse model of systemic lupus erythematosus (SLE), treatment with TSA resulted in decreased mRNA expression of several inflammatory cytokines, including IL-6, IL-12, and IFN-so noted . Earlierlso seen . Two othlso seen , or FK22lso seen  resultedhibitors , 119–124hibitors  documentiewed in , severaliewed in  showed t in vitro study using pancreatic beta cells, both TSA and SAHA reduced cytokine-mediated cellular destruction in an NF-κB dependent manner, indicative of a reduction in the inflammatory pathology [In recent years, the use of HDAC inhibitors for the treatment of chronic inflammatory diseases has gained considerable attention. Two HDAC inhibitors (TSA and SAHA) have shown promise in future treatment for type 1 diabetes, a metabolic disease which has a substantial inflammatory component. In anathology . In otheathology –128. Howathology . Thus, aα and subsequently treated with the novel Class I HDAC1 inhibitor NW-21 decreased synthesis of the proinflammatory cytokines MIP-1α and MCP-1, suggesting that HDAC inhibitor treatment may help reduce synovial inflammation and be potentially useful in the management of rheumatoid arthritis [α and IL-1β, were not affected [ in vitro and in vivo models for inflammatory bowel disease (IBD), both valproic acid and SAHA drastically reduced TNF-α and IFN-γ levels, suggesting that HDAC inhibitors may prove fruitful as a novel therapy for the treatment of IBD  or TNF-rthritis . It is iaffected  when treiewed in ).Taken together, these studies reveal that a greater understanding of the specific cellular and molecular effects of HDAC inhibitors in mediating inflammation is sorely needed and will enhance their utility in the treatment of inflammatory diseases. These required future studies include the delineation of the pathways involved in immune modulation, either pro- or anti-inflammatory, of HDAC inhibitor treatment in a variety of experimental systems.The reversible nature of epigenetic aberrations contributing to human diseases makes them desirable therapeutic targets. As such, HDAC inhibitors, which can act as epigenetic rewriters, are particularly attractive therapeutic tools. However, epigenetic processes are susceptible to both inherited and environmental conditions and are often cell-type specific, making their interpretation difficult for both researchers and clinicians alike. Furthermore, though there is certainly evidence to suggest that epigenetic aberrations may underlie many disease pathologies, there is also evidence to suggest that epigenetic changes may arise from subclinical influences  or may be secondary to other diseases processes , leading to the question of causality. Some illustrations of the challenges associated with the use of HDAC inhibitors as epigenetic regulators are highlighted below.For example, HDAC inhibitor treatment of two colon cancer cell types, HCT116 and HT29, revealed differential changes in gene expression profiles as assessed by microarray analysis . SimilarInterestingly, the Myc-mediated control of miRNA in lymphoma cells was dependent upon cellular transformation. In untransformed cells, Myc transcriptionally activates the miR-15 and let-7a families leading to the repression of miRNA targets Bcl-2 and Bcl-xL, triggering apoptosis, while this pathway is inactive in transformed cells . HoweverHowever, while apoptosis and growth inhibition are almost ubiquitously observed in all cancer cells when treated with HDAC inhibitors, other studies have shown that many cell types will also develop metastatic properties. For example, Lin et al. reported that HDAC inhibitors enhanced metastatic properties in 13 of 30 human cancer cell lines tested . These cIn addition to cancer therapeutics, the successful use of HDAC inhibitors to target epigenetic dysregulation in psychiatric disorders and findings that HDAC inhibitors can function as potent anti-inflammatory agents have recently sparked much intrigue as to the possibility of using HDAC inhibitors to treat or even prevent neurodegenerative disorders as well. Additionally, evidence suggests that cognitive aging deficits are a result of disrupted epigenetic regulation and a decreased capacity to deal with inflammatory events , 135. SoThis type of scenario brings up another significant challenge in the field of epigenetic therapeutics, which is the identification of definitive epigenetic biomarkers and therapeutic targets. For example, the use of epigenome studies has shown that DNA methylation status is associated with CVD risk, but so are things like age, obesity, air pollution, and smoking, which in turn can also influence DNA methylation status . WithoutHematologic malignancies have been successfully treated with HDAC inhibitors, albeit with a relatively low therapeutic efficacy. Yet, HDAC inhibitors have generally been less effective against solid tumors, with cancer progression being observed in solid tumors following HDAC inhibition. In a study to investigate why this might occur, Lin et al. observed that HDAC inhibition resulted in the development of a metastatic phenotype in 43% of the 30 human cancer cell lines tested . As 90% HDAC inhibitors have dual actions in the treatment of melanoma. Several groups have shown impaired proliferation characteristic of HDAC inhibitor treatment in melanoma cell lines , 141 andHowever, as with other examples of the clinical uses of HDAC inhibitors, the effects of HDAC inhibition may result in opposing effects. Although abrogation of HDAC6 in macrophages and dendritic cells results in diminished production of IL-10 and induction of inflammatory APCs that activate naïve T cells , inhibitThis review has highlighted just some of the tremendous sources of variability one must consider during experimental design and selection of an HDAC inhibitor for experimental use. Typical of any inhibitor-based study, it is important to consider not just the desired class of HDAC targets, but also the concentration of the inhibitor and the type of inhibitor being investigated. However, because of the variable functions of HDAC inhibitors on multiple pathways, responses to treatment with HDAC inhibitors, including alterations in gene expression, are likely to vary not only from individual to individual, but also from cell type to cell type within an individual. Therefore, each individual patient might respond differently to treatment for a number of reasons, and the continued investigation into molecular mechanisms underlying HDAC inhibitor function is of utmost importance as the therapeutic potential of HDAC inhibitors appears to be almost unlimited. in vitro, in vivo, and in clinical settings to treat cancer, neurodegenerative diseases, and immune disorders, and even as combinatorial approaches for antiviral therapy. However, due to the pleiotropic nature of HDAC inhibitors, modes of action, and their interactions with large numbers of proteins, it is almost certain that a single mechanism of action for a given HDAC inhibitor will never be described. Furthermore, the gene signature of the cell, as well as the cellular environment, can greatly affect the response to HDAC inhibitor treatment. Thus, as we continue to investigate the therapeutic potentials of HDAC inhibitors, it will likely be helpful to first perform comprehensive chemoproteomic profiling as the action of HDAC inhibitors should be considered in the context of the multiprotein complexes they target and not as purified catalytic subunits. Not only does this methodology allow for the identification of off-target effects, but also it supports our understanding of the function of individual HDAC inhibitors in a greater biological context in order to better predict clinical efficacy.In summary, HDAC inhibitors have been used with varying degrees of success


Exceptional scientific work inquires into new areas or utilizes techniques circumventing existing limitations to study. Alternatively, it may provide insights extending or complementing existing understanding, or generate hypotheses amenable to or worthy of further testing. Adeniran et al.  completeKCNH2 and KCNJ2 and therefore in repolarizing IKr  and cytoplasmic volumes, SR-Ca2+ leak and pump currents, Ca2+ current from dyadic to bulk cytoplasmic space, and background, plateau and Na+/Ca2+ exchange membrane Ca2+ current. The resulting Ca2+-troponin binding coupled this to a myofilament mechanics model chosen for its realistic basis in the cross-bridge cycling model of cardiac muscle contraction (Rice et al., 2+ and sarcomere length-Ca2+ relations. The simulations compared results from inclusion or exclusion of stretch-activated current, ISAC.At the cellular level, an established electrophysiological single cell model recapitulated human ventricular myocyte electrical and membrane channel properties as well as transmural action potential timecourse heterogeneities across the ventricular wall (Ten Tusscher et al., These cellular level findings were built into predictions for mechanical activity in two and three-dimensional human ventricular tissue models. The modeling of cardiac tissue mechanics permitted a non-linear elasticity (Marsden and Hughes, ISAC in normal as well as SQT hearts. In addition, at the theoretical level, the present modeling inevitably entails assumptions of particular implicit physical mechanisms operating within the reconstructions of physiological behavior. It did yield predictions matching previous experimental force-frequency data over frequencies including that at which the simulations were conducted. As outlined in the limitations section of the article, further validation including improved and more physiologically detailed representations of Ca2+ dynamics (Iribe et al., P, its derivative, dP/dt, as well as the timecourse and amplitude of chamber contraction and relaxation.The action potential shortening associated with SQT did appear to reduce ventricular mechanical function. Furthermore, this was rectified by an inclusion of stretch-activated channels. This prediction justifies future experimental testing as to whether such channels indeed exert functional effects on cardiac electro-mechanical coupling in SQTS1 and SQTS3. The experimental testing might then explore involvements of 2+ homeostatic and biomechanical changes with realistic descriptions of cardiac anatomy and mechanical properties. At the broader level we look forward to applications and developments of such approaches at the whole organ level in the analysis of other ion channel exemplars of abnormal cardiac physiology hitherto mainly studied at the cellular or tissue levels. These might include models for the long QT, Brugada and catecholaminergic polymorphic ventricular tachycardia, syndromes (Thomas et al., Nevertheless, the approach adopted by the authors draws attention to the potential value for modeling that integrates cellular electrophysiological, CaThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cardiovascular complications are the leading cause of mortality in patients with end-stage kidney disease. Research indicates that the Mediterranean diet is protective of cardiovascular disease in the general population. Components of this diet have been trialled in haemodialysis patients with the aim of reducing the risk of cardiovascular disease and improving associated risk factors. Components include fish, fruit and vegetables in the form of fish oil supplements and vitamin and antioxidant supplements. This narrative review provides an overview of observational studies, and interventional and randomised controlled trials examining the association of these supplements with cardiovascular outcomes in haemodialysis patients.We reviewed the relevant literature by searching English-language publications in Web of Science and references from relevant articles published since 1992. Eight-seven abstracts were reviewed and 38 relevant articles were included.The extant literature suggests that risk of mortality is reduced in patients with a higher fish intake and those with higher serum omega-3 fatty acid levels. However, the pathways by which risk of mortality is reduced have not been fully extrapolated. While only a few studies have examined the effect of vitamin B supplementation in haemodialysis patients, these studies suggest that supplementation alone does not reduce the risk of mortality. Finally, studies examining vitamin E supplementation have drawn inconsistent conclusions regarding its pro-oxidant or antioxidant effects. Differences between studies are likely due to methodological variations in regards to dose, route of administration and treatment duration.Nutritional and dietary supplementation in haemodialysis patients is an area which requires larger, more methodologically robust randomised controlled trials to determine if risk of cardiovascular outcomes can be improved. Cardiovascular complications are the most common cause of mortality in people with end-stage kidney disease (ESKD). Dietary approaches in this population have therefore focused predominantly on reducing the risk of incident cardiovascular disease (CVD) and cardiovascular events. Research indicates that healthy adults can reduce their risk of CVD by adopting a diet that is either i) low in fat, cholesterol, saturated fatty acids or sodium or ii) high in fruit and vegetables, fibre, fish, polyunsaturated or monounsaturated fatty acids, or potassium . In healThe Mediterranean style diet incorporates many of these recommendations and is now widely regarded as a beneficial approach to maintaining cardiovascular health throughout life. This dietary approach originated from the traditional diets of Greece, Italy and Spain, and promotes increased intake of high fibre foods such as fruit, vegetables and legumes; increased intake of fish; moderate intake of dairy products and wine (particularly red wine); and low intake of red meats. Previous research generally indicates a positive effect of the Mediterranean diet on traditional cardiovascular risk factors, including blood pressure, and total and LDL cholesterol levels, as well as outcomes including CVD morbidity and mortality . A recenGiven that the Mediterranean diet appears to be cardioprotective in the general population , studiesThe aim of this narrative review is to provide an overview of recently published observational studies and interventional trials examining the association of fish oil, and vitamin and antioxidant supplementation with risk of CVD morbidity and mortality in haemodialysis patients.The relevant literature was reviewed by searching English-language publications in Web of Science and references from relevant articles published from January 1992 to July 2015. Web of Science spans eight science databases, including MEDLINE via PubMed. Literature was searched from 1992 in order to examine contemporary evidence relevant to current treatment practices. Key words (including spelling variants) used in the search included terms relating to: i) haemodialysis ; ii) study design ; iii) CVD morbidity and mortality ; iv) omega-3 fatty acids  and v) vitamins and antioxidants . Eighty-seven abstracts were reviewed and 38 relevant full-text articles were subsequently included.Fish, especially oily fish, are a rich source of omega-3 polyunsaturated fatty acids. In humans, the most biologically-important omega-3 fatty acids are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Omega-3 fatty acids compete with omega-6 fatty acids, such as arachidonic acid and linoleic acid, to be incorporated into the phospholipid bilayer of the cell membrane. Once incorporated into the cell membrane, omega-3 and omega-6 fatty acids can be converted to eicosanoids, such as prostaglandins and leukotrienes . Omega-3Since 2002, six studies have examined the relationship between fish oil and risk of cardiovascular events or mortality in haemodialysis patients  . While nN = 42) in Eastern Europe erythrocyte DHA levels were negatively associated with plasma levels of IL-6 and tumour necrosis factor-α (TNFα) [N = 176) erythrocyte DHA levels correlated positively with serum LDL cholesterol levels [N = 517), with serum levels of EPA and DHA correlating positively with non-HDL cholesterol levels [The full benefits of fish oil consumption in haemodialysis patients and the mechanisms by which it may reduce risk of cardiovascular events or mortality are not yet completely understood. Studies examining the association of fish consumption with various physiological markers indicate that patients with a higher consumption of fish have higher HDL cholesterol  and albuα (TNFα) . Howeverl levels . Similarl levels . DifferiRecent intervention and randomised controlled trials examining the effect of omega-3 fatty acid supplementation on the risk of CVD mortality, morbidity and relevant physiological markers in haemodialysis patients are displayed in Table N = 200), examined the effect of omega-3 fatty acid supplementation on risk of mortality in haemodialysis patients over a 2-year follow-up. Three months of supplementation with 1700 mg of omega-3 fatty acids daily reduced the risk of myocardial infarction (MI) by 70 % , but did not reduce the risk of cardiac events and/or all-cause mortality  [Only one randomised controlled trial, conducted in Denmark (72–1.48) . A large72–1.48) .Pooled results from a recent review of the literature suggest that there may be no effect of vitamin or mineral supplementation, including vitamins A, C and E, and folic acid, on the risk of CVD or mortality in the general population . HoweverTable Only one randomised controlled trial examined outcomes for patients supplemented with α-tocopherol . In this2) or ESKD. These studies involved large sample sizes, long durations of supplementation [Table entation  or extenentation . Neitherentation . Cardioventation .Table 4RCardiovascular complications are the most common cause of mortality in people with ESKD. Given that the Mediterranean diet appears to have cardioprotective properties in the general population, it is feasible that this dietary approach may also be beneficial in this patient population. However, people living with ESKD have altered metabolic processes and fluid and electrolyte balance, causing their uraemic symptoms. Therefore, the beneficial effects of interventions in the general population may not always be true in this group. In the 4D and AURORA studies, for example, common LDL cholesterol-lowering drugs failed to reduce the risks of cardiovascular events in haemodialysis patients, despite being successful in the general population , 37.The extant research suggests that those with higher fish intakes tend to have more favourable albumin and HDL cholesterol levels, as well as a lower risk of mortality compared to those with lower fish intake. Increased levels of omega-3 fatty acids in the blood also appear to lower risk of mortality. However, randomised controlled trials of supplementation with omega-3 fatty acids have assessed different markers of inflammation and nutritional status thereby limiting conclusions. The majority of studies included in this review had small sample sizes and only a few studies were adequately powered randomised controlled trials. Intervention studies involving robust methodology are required to properly examine the physiological mechanisms of fish oil, and its associations with mortality, morbidity and other physiological markers.Only two studies have examined the effects of vitamin B supplementation on cardiovascular events and mortality in haemodialysis patients, with both showing no effect. Likewise studies examining the role of vitamin E supplementation in haemodialysis patients have had inconsistent results and small sample sizes. This is likely due to methodological differences in dose, route of administration, and treatment duration. Randomised controlled trials of vitamin supplementation should involve combinations of multiple relevant vitamins, rather than just one vitamin in isolation, to examine the synergistic complexity of the antioxidant system. Investigating the cardioprotective role of supplementation with whole foods including fish, fruit and vegetables in these patients, a group with a high medication load, may also provide an alternative avenue of research aimed at improving cardiovascular health in people with ESKD.


This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer.Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays,  Adult lung is lined by surface airway epithelium. In order to meet the need of regional functionalities of the lung, the epithelia within each domain of the conducting airway are properly composed with distinct types of epithelial cells. The integrity of epithelium is essential for maintaining normal lung functions. However, the lung continually undergoes injury during the process of respiration caused by environmental insults from inhaled air; the injury repair of re-epitheliumis thus required for the preservation of epithelial integrity. In this regard, a variety of stem/progenitor cells with functional specificity are responsible for both of the injury repair and the normal turnover at steady state throughout the airway tree –7.Similar to other adult tissues and organs, epithelial stem/progenitor cells in adult lung are a subset of undifferentiated cells that undergo asymmetric cell division during normal lung morphogenesis and possibly regeneration . With chIn terms of the potential stem cell niches in lung, studies using murine models have revealed several unique regional niches for distinct epithelial stem/progenitor cell populations along the proximal-distal axis of airway, along which the epithelial stem cells reside in their specific local niches in order to maintain tissue homeostasis during injury repair and normal turnover. In this context, the coordination of local molecular and cellular events in the microenvironment of niches play pivotal roles in maintaining the balance of stem and differentiated cells for injury repair and regeneration in lung  4, 9, 2–31. AlthThe adult lung is a highly complex organ comprised of diverse cell types, and over 40 different unique cell types with specific functions have been historically described in the lung , 36. BasThe proximal airway is lined by pseudostratified columnar epithelial cells predominantly including basal, club, ciliated, and goblet cells and interspersed with submucosal glands (SMGs) beneath the surface airway epithelium , 28, 37.The epithelial stem/progenitor cells are crucial in the development, tissue homeostasis, and injury repair in lung. To date, at least a subset of basal, secretory, and mucous cells in the SMGs of the proximal airways, variant club cells in the bronchioles, bronchoalveolar stem cells (BASCs) in BADJ, and a subset of AEC II cells in alveolar space have been suggested as region-specific epithelial stem/progenitor cell populations in the adult lung of mice and humans  29, 32,,32, 39.In the cartilaginous airway (trachea and main stem bronchi), a subset of basal cells are the dominant epithelial stem cell type responsible for the homeostasis and injury repair, which possess a capacity to generate all the major epithelial cell types found in the proximal airway, including the basal, ciliated, goblet, and granular secretory cells , 40, 41.α6  could be used in combination for isolation of human basal cells that have stem cell potentials [bright/Sca-1+/ALDH1+  subset of tracheal basal cells as region-specific stem cells that could generate their own niches in vitro and contribute tracheal epithelial maintenance and injury repair [In order to further define the subpopulation of basal stem cells with stem cell properties, cellular markers including the cytokeratin 5 (CK5), cytokeratin 14 (CK14), and Aquaporin 3 have been employed in the identification and isolation of basal cells . In thistentials . Using ay repair . These s+, CyP450-2F2−) show potentials of self-renewing and giving rise to club cells and ciliated cells, which meet the criteria of bronchiolar tissue-specific stem cells in adult lung [+ club cells was also found at the BADJ of the distal bronchioles, at which this population of cells were responsible for repopulation of the airway epithelium after the lung injury induced by depletion of club cells using naphthalene [ in vivo and an ability to differentiate into club cells and alveolar epithelial cells as determined by an ex vivo clonogenic assay [ in vivo lineage tracing analysis [+ cells [In the intralobar bronchiolar airways, a subset of variant club cells expressing Clara cell secretory protein (CCSP) but not CyP450-2F2 low cells were variant club cells resistant to naphthalene progenitors, while the AFhigh cells were sensitive to naphthalene [+/Sca-1low/CD24low cell fraction [+/Sca-1low/integrin α6β4+/CD24low which was capable of self-renewing and differentiating into the airway epithelial lineage cells, including the alveolar epithelial cells [Other surface cell markers were also employed for the isolation and identification of BASCs. For instances, a study by Teisanu et al. showed that a subpopulation of CD45hthalene . Anotheral cells . Mechanial cells . These s in vitro cell proliferation and clonogenic assays, in vivo epithelial injury repair, and lineage tracing analysis. These studies provided a solid evidence that a subset of AEC II cells are able to proliferate and restore the alveolar epithelium by giving rise to either new AEC II or the squamous AEC I cells following an injury; even AEC II cells have only shown their unipotency to generate AEC I cells [−/integrin α6β4+ cells were found to sporadically localize in alveolar epithelia and were able to regenerate SPC+ AEC II cells in the alveoli, indicating that the expression of integrin α6β4 may be a potential biomarker for the stem/progenitor cells in alveolar epithelium [ in vivo genetically SPC-labelled lineage tracing analysis and an in vitro 3D culture model. In this study, the authors showed convincing evidence that AEC II cells were stem cells for maintaining the homeostasis of alveolar epithelia during the steady state and injury repair [In the gas exchange region, a subset of AEC II cells have long been recognized as stem/progenitor cells for AEC I cells in the adult lung , 49. Thi I cells , 51. Morithelium . Importay repair , 22. Sev+ cells [ in vitro and in vivo  expressing CGRP [+/CyP450− variant club cells as putative stem/progenitor cells in the distal airway [low/CyP450−/Scgb3a2+ expressing club cells residing in the NEBs in which Notch signal and transcription factor TTF1 (Nkx2.1) played a crucial role in the induction of secretory cell fate determination in developing murine airways [In the bronchiolar airway, the calcitonin gene-related peptide (CGRP) marked NEBs have been proposed as a niche for lung stem/progenitor cells responsible for the homeostasis of bronchiolar epithelium during normal turnover and injury repair , 61. In ing CGRP , 62. Seving CGRP  and a suing CGRP , 20. In l airway , 62, Xin airways . These s airways . These s in vitro studies revealed the capacity of BASCs to differentiate into club, AEC II, and AEC I cells [ in vivo lineage tracing study using CCSP-CreER murine model showed an inability of CCSP positive cells to contribute alveolar injury repair following a naphthalene or hyperoxic acute lung injury [In the terminal bronchioles, mounting evidence has suggested that the BADJ is the niche for BASCs that are capable of regeneration of both bronchiolar and alveolar epithelial cells in response to injury , 64–67.  I cells , 21, in g injury . Neverthg injury , 65, 66.+-AEC II cells, suggesting that SPC expressing cells were not the major contributors for alveolar repair following the bleomycin-induced parenchymal injury [α6β4, but not the AEC II cell marker SPC, was progenitor cell type. This subset of AECs was able to proliferate and differentiate into many epithelial cell types, including the CCSP-expressing cells and SPC+-AEC II cells in an ex vivo kidney capsule model [ in vivo, supporting the existence of alveolar stem cell population [ in vitro differentiation analysis using a 3D culture model showed that an individual AEC II cell could give rise to self-renewing “alveolospheres” comprised of both AEC I and AEC II cells. Noteworthily, a coculture of the AEC II and primary PDGFRα+ lung stromal cells showed most readily growth and differentiation of the alveolospheres. The primary PDGFRα+ lung stromal cells include a population of lipofibroblasts (lrMSCs) that reside close to AEC II cells which may play an important role in the epithelial-mesenchymal interaction-generated alveolar lung development [In the terminal end of respiratory tree, AEC I and AEC II cells, capillaries, and lung resident mesenchymal cells (lrMSCs) comprise gas exchange area, the alveolar ducts, and alveolar sacs. The AEC II cells have been histologically suggested as the stem/progenitor cells responsible for regeneration of AEC I and AEC II cells after an alveolar injury , 22, 52.elopment  and servLung cancer is a heterogeneous and complex disease in terms of its diversity of phenotypes and anatomical sites of origins in airways. It thus can be subdivided into two major groups: small cell lung cancer (SCLC) and non-small cell lung cancers (NSCLC). The SCLC is characterized by neuroendocrine cell morphology and accounts for ~15% of lung malignancies; however the NSCLC accounts for the rest ~85% of lung malignances and can be further subdivided into three distinct histological subtypes, which are squamous cells carcinoma (SCC), adenocarcinoma, and large cell carcinoma . Such he in vitro and in vivo clonogenic assay and lineage tracing analysis, a picture of coordination between the region-specific epithelial stem cells and their distinct niches has displayed. A balance of the region-specific stem cells and their distinct residential niches is pivotal for maintaining normal functions and homeostasis of lung along the airway during both the steady state and injury repair. In this context, stem cell niches located at different regions of the lung play crucial roles in protecting stem cell from depletion, and they govern the specificity of regional stem cells for normal lung turnover and injury repair. An abnormal niche activity may result in lung diseases, including lung cancer. Therefore, defining the intrinsic and extrinsic cues between lung stem cells and their unique regional niches may ultimately provide novel insights into the progresses underpinning the pathogenesis of lung diseases involving abnormalities injury repair and lung CSCs.UsingAlthough the markers and locations of several epithelial stem/progenitor cells of airway have been interrogated in great detail, such as the BASCs at BADJ and the basal cells in proximal airways, a specific marker for the identification and isolation of lung stem cells has not been defined yet. More phenotypic markers are thus needed for the unambiguous identification and isolation of lung stem cells. In addition, other unidentified subpopulations of stem/progenitor cells may be very important which has been isolated. On the other hand, an overlap immunophenotypic profile of BASCs identified from independent groups implies that there is heterogeneity among the stem cell populations. This may suggest that a variably enriched population of stem/progenitor cells could be isolated from different locations. Hence, defining better specific surface markers is necessary for the isolation of a homogenous population of lung stem cells. Equally important is the fact that an analysis at a single cell level may also enable us to interrogate a more detailed and precise lineage relationships of stem cells.Apart from the identification of the locations (niches) of lung stem cells, the molecular signaling network of niches involved in the control of stem cell fates is mostly unclear. Therefore, the understanding of the molecular structure and composition, as well as the signaling pathways of distinct niches, will allow us to better understand behaviors of different region-specific stem cells in lung.


The heat shock response (HSR) is a cellular response to diverse environmental and physiological stressors resulting in the induction of genes encoding molecular chaperones, proteases, and other proteins that are essential for protection and recovery from cellular damage. Since different perturbations cause accumulation of misfolded proteins, cells frequently encounter fluctuations in the environment which alter proteostasis. Since tumor cells use their natural adaptive mechanism of coping with stress and misfolded proteins, in recent years, the proteostasis network became a promising target for anti-tumor therapy. The membrane is the first to be affected by heat shock and therefore may be the first one to sense heat shock. The membrane also connects between the extracellular and the intracellular signals. Hence, there is a “cross talk” between the HSR and the membranes since heat shock can induce changes in the fluidity of membranes, leading to membrane lipid remodeling that occurs in several diseases such as cancer. During the last decade, a new possible therapy has emerged in which an external molecule is used that could induce membrane lipid re-organization. Since at the moment there are very few substances that regulate the HSR effectively, an alternative way has been searched to modulate chaperone activities through the plasma membrane. Recently, we suggested that the use of the membrane Transient Receptor Potential Vanilloid-1 (TRPV1) modulators regulated the HSR in cancer cells. However, the primary targets of the signal transduction pathway are yet un-known. This review provides an overview of the current literature regarding the role of HSR in membrane remodeling in cancer since a deep understanding of the membrane biology in cancer and the membrane heat sensing pathway is essential to design novel efficient therapies. The heat shock response (HSR) is a highly evolutionary conserved mechanism that is initiated by environmental and physiological stressors such as heat, oxidative stress, heavy metals, toxins, and bacterial infections, and is essential for the survival in a stressful environment  proteostasis, hence, with changes in the expression of chaperones  production , as exemplified in a study done in colon cancer cells. It was previously shown that lower ER Ca2+ levels promote pre-apoptotic calreticulin exposure on the surface of cancer cells treated with chemotherapy . The mammalian HSF family consists of four members: HSF1, HSF2, HSF3, and HSF4  to p53, resulting in the “holding” of p53 in the cytoplasm. However, inhibition of GRP75 using MKT-077 (rhodacyanine), an Hsp70 inhibitor, initiates disruption of the 17-AAG-induced GRP75-p53 complex. Released p53 translocates into the nucleus and triggers transcription of apoptosis-related genes  interacts with the substrate binding domain (SBD) of Hsp70, with a promising anti cancer activity when transfected into tumor xenografts in mice Murphy, .Gudkov discovered the 2-phenylethynesulfonamide (PES), a compound that inhibits the trafficking of p53 to the mitochondria, NF-kB activation and causes ribosome pausing as well as decreasing protein translation Murphy, . Hence, PES was also proposed to bind to the SBD of heat-inducible Hsp70. PES also demonstrated moderate inhibitory effects on the chaperone action of the constitutive Hsc70 and the heat inducible Hsp70. However, it was suggested that PES may also interact with low affinity and un-specifically with the SBD of Hsp70  derivates, were found not to induce a “classical” elevation of the HSR. We and others have shown that these membrane fluidizers cause induction at non-inducing temperatures in plants and bacteria  channels are members of a large family of cationic channels that serve as major players in sensory physiology, such as transduction of chemicals, temperature, and mechanic stimuli  contains modulatory domains capable of binding calmodulin in the presence of bound CaTRPs are also sensitive to PIP2—which is an important feature of cell signaling due to its high abundance in the membrane and since it serves as a substrate for phospholipase C (PLC). However, the regulation of TRPV1 by PIP2 seems to be controversial: Although some studies have shown that activation of TRPV1 does not require PIP2, others have demonstrated that the addition of PIP2 can induce TRPV1 activation. It was also shown that oxidation of linoleic acid and LPA, another enzymatic product of phospholipids that is produced during inflammation and tissue injury, can activate TRPV1 as well. The cholesterol membrane content also plays a major role in the activity and sensitivity of several TRP channels such as TRPV1 and TRPC3.Hence, ameliorations of lipid compositions and sphingolipid cholesterol signaling cascades during stress response or heat shock were found to be correlated with Hsp production  cells, induced Hsp70 expression in some membrane areas  and, as mentioned above, in MCF-7 cells.Thus, we showed that the use of the membrane receptor blockers Capsazepine or AMG-9810, TRPV1 antagonists, and TRPV1 siRNA resulted in decreased heat shock proteins, Hsp70 and Hsp90 in cancer cells.Therefore, we believe that a possible strategy to treat various cancers could utilize substances known to regulate the HSR through a membrane receptor capable of reducing the expression of heat shock proteins such as Capsazepine or AMG-132 and TRPV1 siRNA. Currently, we are exploring the consequences or the downstream pathways involved in this phenomenon (Ito et al., 2+ after stress (Gehrmann et al., As described earlier, cancer cells or tumors frequently overexpress Hsp70 in the cytosol, but also present Hsp70 on their plasma membrane, as was found in our model as well (Vígh et al., The existence of membrane Hsp70 in tumor cells may also imply that there is a release of Hsp70 in lipid vesicles—termed exosomes (Schlegel and Williamson, Some cancer therapies such as thermo/radiotherapy could induce necrosis within the central zone. Thus, since elevated Heat shock protein levels can enhance tumor cell viability and increase resistance, the peripheral or transitional zone of sublethal hyperthermia might undergo a recovery from reversible injury, rendering this therapeutic approach inefficient.However, rodent studies have indicated that irradiation was associated with a reduction in Hsp70 serum levels (Van Oosten-Hawle et al., However, Hsp70 membrane expression, or exosomal Hsp70 on highly aggressive tumor cells could also activate NK cells (Multhoff et al., Following irradiation, there is direct killing of cancer cells which is mediated by DNA damage, although this process also initiates effects that can stimulate the T and NK cell immune responses (Bayer et al., These findings might have a significant future clinical relevance for irradiation therapy that could be improved by combining it with immunotherapy targeting membrane Hsp70.The “cross talk” between the HSR, membranes, and intracellular pathways opens a new field that may help understand the mechanisms of missfolding diseases, such as aging and cancer. The plasma membrane fluidity state and its role in the pathological conditions of the cells, such as in cancer, aging, or stress response serves as the first guard against such stressors and connects between the extracellular and the intracellular signals. “Lipid therapy” as suggested by Vigh et al. in which a molecule affects membrane lipid re-organization maybe of therapeutic potential since such membrane fluidizers were found to enhance heat shock geneexpression in diseased cells without affecting healthy cells (Multhoff et al., Lipid therapy offers a possible strategy of regulating the fluidity of the membranes and modulating Hsps activities within the membrane. Hence, lipid therapy and antagonists of membrane-bound receptors, such as TRPV1, could allow us to modulate the heat sensing route of cancer cells, hence curtailing their pro-survival mechanisms.The authors ZB and YW are equally contributing authors to this current review. ZB and YW are co-collaborators for the last 15 years of joined research.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.


Radiation therapy (RT) is frequently used as part of the standard of care treatment of the majority of brain tumors. The efficacy of RT is limited by radioresistance and by normal tissue radiation tolerance. This is highlighted in pediatric brain tumors where the use of radiation is limited by the excessive toxicity to the developing brain. For these reasons, radiosensitization of tumor cells would be beneficial. In this review, we focus on radioresistance mechanisms intrinsic to tumor cells. We also evaluate existing approaches to induce radiosensitization and explore future avenues of investigation. Radiation therapy is a mainstay in the treatment of the majority of primary tumors of the central nervous system (CNS). However, the efficacy of this therapeutic approach is significantly limited by resistance to tumor cell killing after exposure to ionizing radiation. This phenomenon, termed radioresistance, can be mediated by factors intrinsic to the cell or by the microenvironment. One approach to overcome radioresistance has been to alter the parameters under which radiotherapy is delivered and another has centered on understanding the molecular complexity that underlies radioresistance with the aim of developing targeted therapies. This review summarizes these efforts in the context of primary brain tumors.The inherent radiosensitivity of normal brain tissue manifests as late toxicity in the form of radionecrosis and is evident in some patients after completing CNS-directed radiotherapy. This is not to be confused with the term “pseudoprogression” which describes a radiological finding thought to represent radiation damage to the tumor itself rather than normal brain toxicity . In contModulation of radiation’s delivery is an approach that can be readily pursued through alteration of total cumulative dose delivered or adjustment of the fractionation schedule in order to exploit differences between the radiobiology of the tumor and normal tissues. Data-driven incremental dose escalation has improved local control for many different tumor types, including prostate and lung carcinomas ,13,14,15Computerized approaches to image registration, both at the time of radiotherapy planning and at the time of radiation delivery, have permitted more accurate target volume definition and more accurate treatment delivery ,28. BecaIn recent years, stereotactic radiosurgery (SRS) has been evolving to safely deliver ablative high doses of focused radiation with high precision to the tumor or post-operative tumor bed, while limiting dose to critical adjacent normal structures. This modality has been tested and found to be useless, and possibly harmful in the primary management of glioblastoma ,42,43. IAn alternative strategy to combat brain tumor resistance to radiation therapy is through the administration of tumor-specific radiosensitizing agents prior to or during radiation treatment in order to enhance the sensitivity of the tumor target, while not affecting the intrinsic tolerance of in- or near-field normal brain. A framework to characterize and understand potential interactions between traditional chemotherapeutic agents and therapeutic ionizing radiation was described by Steel and colleagues in 1979 ,53. The In the management of pediatric brain tumors, radiosensitization is desirable to accomplish dose reduction, given the inherent adverse sensitivity of the developing brain, spine and encasing osseous structures to ionizing radiation. Processes critical to neuronal development, cognition, Intelligence Quotient (IQ), and achievement of developmental milestones can be severely impacted. Although the prognosis of patients with medulloblastoma, the most common pediatric brain tumor, is in general very good, with a 5 years tumor free survival of 75% to more than 95% in some subtypes , this po2, followed by 6 cycles of adjuvant monthly TMZ at a dose of 150–200 mg/m2 for 5 consecutive days per cycle vs. adjuvant RT alone. Median and 5 year-overall survival favored the combined TMZ + radiation arm at 14.6 months vs. 12.1 months and 9.8 vs. 1.9%, respectively [An example of a radiosensitizer currently used with concurrent radiotherapy as a standard of care in the treatment of glioblastoma is temozolomide (TMZ). Through pioneering work conducted in the 1970s–1980s in the UK, the compound CCRG81045 gradually found its way into early phase I and II clinical trials, where it was shown to be safe and effective in enhancing clinical and radiologic responses in small groups of patients with GBM ,58. EORTectively . The mecectively . Screeniectively ,61,62.Identifying effective radiosensitizers requires an understanding of the underlying molecular causes of radioresistance. Much progress has been made in this area and the remainder of this review will summarize our current knowledge of the molecular causes of radioresistance.Radioresistance is caused by a variety of factors that include the intrinsic biology that arises from the aberrant genetic makeup of tumor cells, the extensive heterogeneity of brain tumors ,64 and tThe genetic diversity of some brain tumors is illustrated by several recent studies that suggest a parallel evolution of genetically distinct subclones within a tumor. Surgical multisampling and molecular analysis of glioblastoma multiforme (GBM) tissues have demonstrated the presence of multiple subclones within a single brain tumor that presumably arise through branching evolution . In anotAn additional aspect of tumor complexity is provided by the inherent plasticity of tumor cells that allows adaptation to intra- as well as extracellular changes. The recent identification of a core set of neuro-developmental transcription factors  that are sufficient to fully reprogram differentiated GBM cells to ‘‘induced’’ tumor promoting cells, puts forth the possibility of bidirectional plasticity through epigenetic reprogramming .Another interesting recent finding that sheds a new light on therapeutic resistance of aggressive brain tumors is the capacity of astrocytoma cells to form tumor microtubes (TM) that create interconnected functional networks . Osswaldin vitro and within intracerebral xenografts, Jamal and colleagues [in vivo than in vitro. Together with data on gene expression that indicated higher expression levels for genes involved in ROS metabolism and antioxidant response in intracerebral xenografts than in vitro, these authors suggested that the brain microenvironment protects GBM cells from radiation-induced DNA damage and facilitates its repair.The role of the microenvironment in sustaining stem cell-like programs in brain tumors has been explored best in the context of the perivascular niche, where nitric oxide released by endothelial cells induces a stem cell state in PDGF-induced gliomas via activation of the Notch pathway . The perlleagues  observedSeveral recent comprehensive reviews have described the properties of brain tumor CSCs, the approaches taken to study them and the implications of CSCs for the development of new therapies, as well as the role of the microenvironment in maintenance of glioma stem cells ,78,79. H+ brain tumor stem cells were in fact found to be enriched in human glioma xenografts exposed to radiation [− populations. Rates of post-irradiation apoptosis were lower in CD133+ cells compared to non-stem populations [The enhanced resistance of CSC to radiation leads to preferential enrichment of this minor subpopulation following radiation treatment. CD133adiation . These culations . Anotherulations . In suppulations . Howeverulations . AlthougThe main mechanism through which radiotherapy kills tumor cells is by causing single and double stranded DNA breaks, which, if not repaired, induce cell death. Actively dividing cells are more susceptible to damage by radiation, with cells being most sensitive while in G2-M, less so in G1 and least radiosensitive in late S phase . An impo+ GBM progenitor cells possess a robust response to DNA damage, which involves detection and repair mechanisms that render them significantly more radioresistant than their differentiated counterparts. ATM kinase inhibition was shown to lead to enhanced radiosensitization in glioblastoma CSCs [+ cells in both in vitro and in vivo [In response to double-stranded DNA damage, cells mount a complex DNA damage response . This isoma CSCs . Likewis in vivo .in vitro and in vivo radiosensitization of glioma and are currently being tested in phase I and II trials to assess treatment response in CNS tumors [Tumor cells that are genetically deficient in at least one DNA repair pathway can be sensitized to radiation by targeting other redundant pathways . This phS tumors .Although elements of either the DNA damage checkpoint response or DNA repair proteins are in principle good targets for radiosensitization, simultaneous targeting of both pathways has been shown to be more effective. Inhibition of ATM that affects both cell cycle checkpoint regulation and DNA repair, achieved greater radiosensitization compared to inhibition of CHK1, ATR or PARP alone . Thus, mAnother signaling element that confers radioresistance by activating CHK2 in response to IR is the stress activated protein MRK, also known as ZAK . AlthougIn vivo expression analysis using the Olig2-TRAP transgenic system to examine transcription and translation simultaneously on a genome-wide scale, have identified the p53 target gene cluster to be among the most robust transcriptional and translational response clusters noted in proneural GBM in response to IR. These findings provide support to the notion that p53 plays a key role in modulating the radioresistant phenotype in glioblastoma by driving transcription of apoptotic gene expression programs and that of genes that are involved in stress responses, including redox imbalances [in vivo [In response to IR, p53 is implicated as a major regulator of radiation responsive genes that are believed to contribute significantly to radioresistance. Adenovirus-mediated expression of p53 in glioblastoma cells has been shown to enhance radiosensitivity . In vivobalances . Thus, p[in vivo .in silico analysis found that WEE1 is highly expressed in high grade glioma compared to normal brain tissue and siRNA-mediated depletion of WEE1 led to abrogation of G2 checkpoint-mediated cell cycle arrest and increased cell death [In addition to the DNA damage checkpoint proteins, an avenue that has been explored to prevent cell cycle arrest after DNA damage has been the inactivation of regulators of the cyclin-CDK complex that controls cell cycle progression. One such element is WEE1, a potent inhibitory kinase that directly regulates cyclin-dependent kinase 1-mediated cell cycle progression through the G2/M phase into mitosis. In response to DNA damage, cell division is halted by WEE1 to allow time for effective DNA repair ,110. An ll death . Use of ll death ,113,114.Radiation causes DNA damage by indirect energy transfer that produces chemically reactive free radicals, including ROS. In addition to an effect on DNA, radiation may induce cell death via damage of critical cellular structures like lipids and proteins . InteresIntrinsic factors include survival signaling pathways that are deregulated through alterations in growth factors or their receptors, up-regulation of pro-survival proteins, such as Bcl2 family members, and changes in the DNA damage response and in metabolism. Although these pathways are usually studied in isolation and in large part are considered to be independent, they are in fact interrelated. Growth factor signaling for instance can lead to deregulated anti-apoptotic signals, as well as improved DNA damage response ,118. Thein vivo is the one generated by the pleiotropic cytokine transforming growth factor-β (TGF-β). Its inhibition by LY364947 or by LY109761, two small molecule inhibitors of TGF-β type I receptor kinase, in combination with radiotherapy, has been shown to interfere with DNA damage repair, thereby improving survival in preclinical studies [Intracellular signaling is further modulated by signals contributed by the microenvironment and conditions such as angiogenesis and hypoxia. The contribution of the microenvironment to maintenance of the stem cell phenotype has been extensively discussed in recent reviews ,119. In  studies ,121. Amo studies .Another important problem recognized for decades in radiobiological research, is that of intratumoral hypoxia and its role in radioresistance ,124,125.Although the mechanisms through which the microenvironment supports radioresistance in brain tumors largely remain to be elucidated, its importance is widely recognized and it continues to be the subject of active studies. In the following chapters, we will focus our discussion on signaling elements that directly contribute to radioresistance of cancer stem cells  release after cleavage by γ secretase-like protease. NICD subsequently translocates into the nucleus and binds to specific response elements to transactivate target genes . Notch p+ cells, and treated cells showed diminished soft agar colony formation and tumor growth, indicating that Notch signaling is necessary for the maintenance of medulloblastoma stem cells [Early studies had shown that Notch blockade using a γ secretase inhibitor in medulloblastoma cells depleted CD133em cells . Over them cells ,138. In em cells . Interesem cells , a hallmem cells . In lineem cells . We noteem cells , a discr+ stem cells, but not of CD133− cells [S) and Mcl-1L toward increased expression of the latter pro-survival protein [Importantly, the Notch pathway plays a critical role in the radioresistance of glioblastoma stem cells. Interference with Notch signaling with a γ secretase inhibitor has been shown to impair clonogenic survival of CD1333− cells . Knockdo protein . The effAnother pathway implicated in the maintenance of stem cells is the canonical Wnt/β-catenin pathway, which is an important regulator of embryonic development as well as of adult stem cells and it has been found to be altered in cancer ,146. UpoThe Wnt/β-catenin pathway is involved in radioresistance of GBM in part through maintaining the stem cell phenotype. This pathway was found to be activated within the stem cell population in GBM orthotopic mouse models that had been treated with radiation. Inhibition of the Wnt pathway led to decreased survival and clonogenicity of GBM cells, as well as reduction of the glioma stem cell subpopulation . AnotherWithin glioma, Wnt and its cofactors are highly deregulated, contributing to increased tumorigenicity. As an example, the Wnt family of embryonic lineage specifying genes, SFP1 and SFP2 (soluble Frizzled-related proteins) are often over-activated in glioma. It appears that these proteins both promote glioma tumor growth and survival via enhanced clonogenicity and survival in the absence of growth factors .Control over Wnt signaling in glioma is often mediated in an epigenetic fashion. For example, the gene promoters of Wnt pathway inhibitor genes, including soluble frizzled related proteins, Dickkopf and naked genes, were found to be heavily methylated leading to epigenetic silencing . In anotOther means of Wnt pathway deregulation in glioma involve FRAT1 (frequently rearranged in advanced T cell lymphoma) that works to inhibit β-catenin phosphorylation by GSK. FRAT1, thus, acts as a potent oncogene in the induction of Wnt signaling and positively correlates with increasing grade of glioma ,153. WntIn contrast to GBM, Wnt pathway activation in medulloblastoma is associated with favorable prognosis  and its A third signaling pathway implicated as a central mediator in the pathogenesis of glioma and glioma stem cells (GSCs) is the Sonic Hedgehog (SHH). SHH binds to its transmembrane receptor patched (PTCH) to release Smoothened (SMO) from PTCH mediated-inhibition, which in turn initiates activation and nuclear translocation of Kruppel zinc finger transcription factor Gli1 ,159,160.+ tumor initiating progenitors [Up regulation of the Hedgehog signaling has been shown in glioblastoma and medulloblastoma, and to a lesser extent, in neuroblastoma . In medugenitors ,166,167.genitors . Bmi1 hagenitors  and throgenitors .in vitro [Sonic Hedgehog pathway blockade, established through treatment of medulloblastoma with cyclopamine, resulted in neuronal differentiation and decreased stemness characteristics in vitro . In glioin vitro .Because of the prominent role of the hedgehog pathway in cancer, and despite limited success so far, several antagonists of this signaling cascade are still being tested in the clinic ,174.In addition to regulators of embryonic development like the ones described in the above sections, a number of growth factors and their receptors have been strongly implicated in stem cells maintenance and in radioresistance and are discussed below.in vitro depends on FGF-2 and that its removal favored differentiation.Fibroblast growth factor-2 belongs to the FGF superfamily that comprises 22 different genes. FGF-2 signals primarily through the fibroblast growth factor receptors (FGFRs) FGFR-1 and FGFR-2, which lead to activation of the Ras-MAPK and the PI3K-Akt pathways . The derSeveral studies have implicated fibroblast growth factor signaling in radioresistance. Early studies revealed a role for FGF in the protection of endothelial cells from apoptosis , a proceDuring normal CNS development, the receptor tyrosine kinase, epidermal growth factor receptor (EGFR) plays a major role in facilitating glial progenitor survival throughout gliogenesis. Transgenic overexpression of the receptor suggested a role for EGFR in driving glial proliferation and in promoting neural stem cell survival ,189.EGFR is frequently found to be amplified in highly proliferative tumors such as glioblastoma, where it occurs in about 50% of cases . ConstitThere is strong evidence that antagonism of EGFR leads to radiosensitization in high grade glioma. Early preclinical work using EGFR tyrosine kinase inhibitors, such as AG1478, or dominant negative constructs including EFGR-CD533, have all caused significant radiosensitization with dose enhancement ratios approaching 1.85 ,196,197.125I-mAb 425) have been conducted. Results from a large phase II trial testing this strategy were encouraging with a median survival of 20.2 months when administered with TMZ [Several clinical trials have tested various small molecule and antibody-based EGFR tyrosine kinase inhibitors (TKIs), while vaccine-based and siRNA mediated EGFR targeting trials are now underway. To take advantage of the radiosensitizing effects of EGFR attenuation seen in pre-clinical models, phase I and II clinical trials designed to test the efficacy of an anti-EGFR monoclonal antibody coupled to radioactive iodine , the key factor involved in cap-dependent protein translation. Bypassing mTOR repression, via overexpression of EIF4E, leads to enhanced radiosensitivity and selective loss of the hypoxic cell population .Despite promising pre-clinical data, recent phase I and II clinical trials using mTOR pathway inhibitors, including temsirolimus monotherapy, have failed to demonstrate improved overall survival in recurrent glioma patients, although various radiographic responses were observed . Two phaFinally, downstream of intracellular signaling elements, transcription factors are responsible for regulation of gene expression and for implementing new cell programs. Here we review the role of two crucial transcription factors in establishing radioresistance.Classically, nuclear factor-kappaB (NF-κB) functions as a transcription factor kept under tight cytoplasmic regulation by the kappa B inhibitor protein, IκB. Upon ligand binding of a cell surface receptor such as TNF-R or Toll-like receptor, IκB is targeted for proteosomal degradation via IKK complex-mediated phosphorylation, thus releasing NF-κB to translocate into the nucleus and transactivate downstream target genes .NF-κB activation was recently shown to contribute via MLK4 to the development of intratumoral heterogeneity and enhanced radioresistance of glioblastoma by inducing a transition from a proneural to mesenchymal transcriptional GBM subtype ,245. Furin vitro attempts at targeting NF-κB activity in GBM have been successful in decreasing tumor cell proliferation. One approach targeted tumors cells with poly (DL-lactic-co-glycolic acid) (PLGA) microparticle-containing NF-κB decoy oligonucleotides possessing DNA-binding sites specific for the wild-type NF-κB DNA-binding domain. Release of the decoy oligonucleotides within the cell acts to bind up free NF-κB, thus effectively inhibiting transactivation of downstream target genes, leading to inhibition of GBM cell growth and proliferation [Early feration ,250. Othferation . Additioferation ,253. HowSTAT3 functions at the confluence of several important molecular pathways involved in radioresistance of brain tumors, including Wnt, Notch, Akt and the Bcl2, Bcl-xL and Mcl-1 survival genes . STAT3 iin vitro and in vivo [STAT3 is also implicated as a key driver in the proneural to mesenchymal shift seen in GBM after exposure to RT, which contributes to a radioresistant phenotype . Phospho in vivo . Thus, bOur understanding of the molecular mechanisms that promote radioresistance has revealed a number of potential targets, inhibitors of which could be developed as radiosensitizers. A number of these inhibitors have been tested in the clinic as monotherapy and shown very disappointing results. As it is expected that their benefit as radiosensitizers depends on their concurrent use with radiation, new clinical trials that evaluate these agents in conjunction with radiotherapy will be necessary. The understanding that cancer stem cells represent a source of therapy resistance and failure has suggested that targeting this cell population within the tumor will provide increased sensitization to radiotherapy as well. Thus, together with optimized radiation delivery modalities, advances in identification of radioresistance targets and development of their inhibitors may provide better future therapeutic outcomes for brain tumors. Therapeutic success of small molecule radiosensitizers will depend also on their ability to cross the blood brain barrier, which represents an outstanding challenge in brain tumor treatments. Thus, development of novel delivery modalities that overcome this obstacle will be crucial to improving therapies for brain tumors. As several chemotherapies work by causing DNA damage, it is expected that novel radiosensitizers may be effective also to overcome chemoresistance.


AbstractMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders; they are characterized by ineffective hematopoiesis and a predilection to the development of acute myeloid leukemia (AML). For a rapid evaluation of the outcome in myelodysplastic patients non-cytogenetic prognostic scores can be used.  Aim: This study proposed to demonstrate that age and gender are important factors in the outcome of the patients diagnosed with myelodysplastic syndrome. Materials and methods: This study was conducted in the Department of Hematology of the Emergency University Hospital Bucharest during October 2008 and October 2012. Results: Male sex and age higher than 60 years are associated with high risk in the studied cases by using the Spanish prognostic score. According to Goasguen score: male sex and age, patients older than 60 years, present characteristics associated with an intermediate risk. Based on the Dusseldorf score, age over 60 years and female gender were associated with pronounced risk in the examined group. By examining the Bournemouth score in our group, we found that age > 60 years correlated with a higher frequency of risk, but no significant differences regarding the sex of patients were observed.  Conclusions: We concluded that age > 60 years and male gender are important predisposing factors in the survival.AbbreviationsMDS = myelodysplastic syndromes, AML = acute myeloid leukemia, LDH = lactate dehydrogenase, FAB = French-American-British, WHO = World Health Organization, IPSS = International Prognostic Scoring System, ALIP = abnormal localization of immature precursors, WPSS = WHO classification-based prognostic scoring system, FISH = fluorescence in situ hybridization, del = deletion. A succession of MDS classification systems has been developed to facilitate the prediction of the risk of progression to AML and overall survival [5].Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders and they are characterized by ineffective hematopoiesis, morphologic abnormalities in one or more cell lines in an usually cellular bone marrow and a predilection to the development of acute myeloid leukemia (AML) [6], Goasguen score [7], Dusseldorf score [8] and Bournemouth score [10]. For a rapid evaluation of the outcome in myelodysplastic patients, non-cytogenetic prognostic scores can be used. The following prognostic scores are not based on a cytogenetic analysis and they are reliable to evaluate recently diagnosed patients with myelodysplastic syndrome: Spanish score , bone marrow blast percentages [15], and cytogenetic abnormalities [16]. Because the vast majority of primary MDS patients are elderly, age-stratified morbidity and mortality figures are needed regarding their clinical outcome [4]. Our data are similar to those previously reported, showing that age > 60 years is an important factor associated with poor prognosis and survival. Regarding the clinical outcomes, other studies have suggested the importance of a variety of clinical features, including different numbers or types of cytopenias  [1]. When all the patients were included in the analysis, sex, age, the proportion of blasts in blood or bone marrow, or the presence of abnormal localization of immature precursors (ALIP) in the bone marrow trephine were not of prognostic significance [1]. Our study revealed that male sex is more frequently associated with severe prognostic risk compared to female gender. Both myelodysplasia and myeloproliferative diseases are uncommon in childhood, perhaps because small series of patients are the norm [2]. Unfortunately, the IPSS system underweights the clinical importance of severe (life-threatening) neutropenia and thrombocytopenia, in determining the need for therapeutic intervention. Although imperfect in its clinical utility, the IPSS has been very useful in examining and comparing the outcomes of clinical trials [11]. Since its publication, its utility has been confirmed in many institutions and refinements of the IPSS  continue to be proposed [17].  Cytogenetic findings have an established role in the diagnosis and assessment of prognosis of MDS and are emerging as an important factor in the treatment selection and monitoring response to therapy [20]. Although the established prognostic scoring systems are based on conventional cytogenetics, some studies showed that chromosomal abnormalities detected by fluorescence in situ hybridization (FISH) may provide prognostic information [22], and may be useful in supporting the clinical decision making in the selected cases, such as those with del(5q) or with del(7q) or monosomy 7 [23].In addition, cytogenetic subgroups and prognostic variables have recently been suggested as providing improved prognostic evaluation of clinical outcomes of primary MDS patients [ The diagnosis of myelodysplastic syndromes is a multi-step procedure and the evaluation of the outcome of these patients can sometimes be difficult to assess. Using simple laboratory tests, such as cells blood count, bone marrow smears and level of lactate dehydrogenase, 4 non-cytogenetic prognostic scores can be applied to obtain important information about the risk assessment of the patients. By the end of this study, we concluded that age > 60 years and male gender are important predisposing factors in survival.


Systematic, high-throughput dissection of causal post-translational regulatory dependencies, on a genome wide basis, is still one of the great challenges of biology. Due to its complexity, however, only a handful of computational algorithms have been developed for this task. Here we present CINDy , a novel algorithm for the genome-wide, context specific inference of regulatory dependencies between signaling protein and transcription factor activity, from gene expression data. The algorithm uses a novel adaptive partitioning methodology to accurately estimate the full Condition Mutual Information (CMI) between a transcription factor and its targets, given the expression of a signaling protein. We show that CMI analysis is optimally suited to dissecting post-translational dependencies. Indeed, when tested against a gold standard dataset of experimentally validated protein-protein interactions in signal transduction networks, CINDy significantly outperforms previous methods, both in terms of sensitivity and precision. Reverse engineering of gene regulatory networks using gene expression profiles has proven valuable in dissecting the logic of cellular regulation in multiple species i.e. those between TF and Tg that are conditionally dependent on the expression of M. Inference of true conditional transcriptional interactions requires.These observations provided the original rationale for the development of the Modulator Inference by Network Dynamics (MINDy) algorithm First, we tested the performance of the two algorithms (using default parameters) in inferring established modulatory interactions, using two distinct gene expression profile datasets: (a) a B-cell lymphoma dataset containing 226 samples Briefly, for the MINDy algorithm, for each candidate modulator gene, M, we tested only TFs with expression statistically independent of M, as assessed by the statistical significance of the Mutual Information of their gene expression profiles. We also discarded candidate target genes whose gene expression was highly correlated with that of the associated TF, thus restricting the number of candidate target genes in the analysis. Both of these are a requirement for using the Gold). The latter was generated by taking the union of interactions obtained from four independent databases: for generic PPIs, we combined the interactions in HPRD Figure S1). Algorithm performance was evaluated by computing recall rate, defined as the fraction of inferred interactions in the PPIGold, and precision rate, defined as 1 minus the fraction of inferred interactions not present in the PPIGold. An important point to note is that the PPIGold dataset contains only a very small fraction of all true biological PPIs. Therefore, any precision estimates represent highly underestimated values. Indeed, precision should be used only as a comparative metric here such that recall may be computed either at roughly the same or better precision and is not representative of true precision, which can only be assessed from experimental validation.To objectively assess the performance of the two algorithms, we compared the modulatory interactions they inferred to a set of validated Protein-Protein Interactions (PPIs) between TFs and candidate modulator proteins  interactions . When using identical computational environments, CINDy requires almost double the time of MINDy, mostly due to using the entire dataset rather than just the top and bottom 25%. However, its memory requirements are half of those of MINDy .Due to the resulting differences in the analysis, the computational requirements of the algorithms are different , one can consider TF targets that are either experimentally assessed from CHIP-Seq and or TF silencing assays Figure S4 and Table S1).Much of the computational requirements for the algorithms are due to the fact that every gene is considered as a candidate TF target by the analysis. To reduce both computation time and memory requirements .Finally, we assessed the performance of CINDy by varying the number of gene expression profiles, Table S2), as well as several novel predictions. Among the newly inferred MYC activity modulators, we find many signaling proteins and TFs that are associated with B-lymphoma malignancies, including ATM CINDy confirmed previous predictions of modulatory interactions, such as MYC activity modulation by the STK38, MAPK1 and CSNK2A1 proteins in B-cell lymphoma In lung adenocarcinoma, CINDy specifically highlights modulatory interactions that affect epithelial proliferation, such as the direct phosphorylation by the Epidermal Growth Factor Receptor (EGFR) of the STAT1 The most pressing challenge for Systems Biology is the development of model-based approaches for the veritable interpretation of an avalanche of new biological data. Reverse engineering algorithms provide a key approach to build regulatory models representing the molecular mechanisms that control cell behavior. These models in turn can provide critical novel knowledge about mechanistic control of physiologic processes Due to its general formulation, CINDy can identify a variety of post-translational interaction mechanisms that go beyond standard post-translational modification , such as recruitment of CREBBP to FOXM1 and consequent transcriptional activation. It is also able to generate novel testable hypotheses for intriguing dependencies, such as regulation of HMGA1 activity by CDK2 Figure 4Importantly, by adopting a theoretically rigorous formulation, CINDy does away with many of the heuristics and parameter choices of the MINDy implementation. For instance, the need to select arbitrary tails of the modulator expression, the somewhat arbitrary thresholds used to evaluate a modulator TF interaction or the statistical dependency between a TF and a candidate target gene, as well as the statistical significance of  Table 1.https://www.synapse.org/#!Synapse:syn395683), RPKM-normalized. The B-Cell dataset derives from human B-cell microarray gene expression experiments We ran the CINDy and MINDy algorithms on two independent datasets, called “Lung dataset” and “B-Cell dataset”. The Lung dataset originates from the TCGA gene expression study Table S3, containing 3,203 candidate modulators and 1,673 transcription factors. 210 of these genes fall in both categories , i.e. they have both the transcriptional as well as modulatory function, and were therefore processed in our analyses both as candidate modulators and transcription factors.Transcription factors and modulator gene lists used to run MINDy in this study were defined as in Figure S1). We excluded homodimerization interactions and peptides that could not be unambiguously mapped to any Entrez gene id.We collected human PPI interactions from HPRD release 9 , Y2H (170), Strings v9.0.5  and human phosphorylation kinase/target pairs from PhosphoSite (541), totaling 82,160 distinct modulator/TF interactions  and a Target Gene (Tg), given a putative Modulator (M) is inferred by estimating the conditional probability distribution using an adaptive partitioning approach:CMI is therefore analogous to conditional partial correlation for mutual information calculation 4 distinct  pairs and estimate 5 CMI values in each bin. Within each bin, we model the distribution of CMI as an extended exponential, To assess the statistical significance of a particular CMI, we generate a series for null models, each for different ranges of mutual information between TF and Tg. To build this null model, first we randomly select 10Figure S1Number of modulator/transcription factor associations in four independent databases, and relative intersections.(TIF)Click here for additional data file.Figure S2Comparative computational performance of MINDy and CINDy. The test was performed on the human B-cell dataset (TIF)Click here for additional data file.Figure S3CINDy performance on a single TF-Modulator pair using increasing number of target genes. The vertical black line to the left indicates the average number of targets in the dataset (97.2). For this particular dataset, on average, MINDY using all genes is almost 130 times slower than using target genes, and requires almost 28 times more RAM.(TIF)Click here for additional data file.Figure S4Benchmark of MINDy runs using a subset of target genes defined by ARACNe  . A-B Precision and recall of MINDy, CINDy, intersection and union sets in the B-cell and Lung datasets, calculated over a golden set of four databases of experimentally validated PPIs between modulators and transcription factors. C-D Precision/Recall plots for MINDy (blue points) and CINDy (red points) at different robustness thresholds (see (TIF)Click here for additional data file.Figure S5Effects of sample size on precision and recall in the B-cell dataset (226 samples). The precision/recall curves were calculated using the 100 TFs and modulators with most connections in the gold standard set . The error bars indicate the standard deviation in the estimation of precision and recall obtained by running CINDy over 100 datasets generated by subsampling.(TIF)Click here for additional data file.Table S1Raw performance information for CINDy and MINDy in the lung adenocarcinoma and B-cell lymphoma datasets, at different thresholds defined by the number of target genes affected by the modulation events.(XLSX)Click here for additional data file.Table S2Significant modulation events predicted by CINDy and MINDy at standard parameters in the B-Cell and Lung datasets. The number of significant conditional target interactions for each M (XLSX)Click here for additional data file.Table S3Gene symbols used in the current manuscript as modulator genes or transcription factors.(XLSX)Click here for additional data file.