Receptors of the EGF receptor or ErbB family of growth factor receptor tyrosine kinases are frequently overexpressed in a variety of solid tumours, and the aberrant activation of their tyrosine kinase activities is thought to contribute to tumour growth and progression. Much effort has been put into developing inhibitors of ErbB receptors, and both antibody and small-molecule approaches have exhibited clinical success. Recently, a number of endogenous negative regulatory proteins have been identified that suppress the signalling activity of ErbB receptors in cells. These include intracellular RING finger E3 ubiquitin ligases such as cbl and Nrdp1 that mediate ErbB receptor degradation, and may include a wide variety of secreted and transmembrane proteins that suppress receptor activation by growth factor ligands. It will be of interest to determine the extent to which tumour cells suppress these pathways to promote their progression, and whether restoration of endogenous receptor-negative regulatory pathways may be exploited for therapeutic benefit. The aberrant activation of ErbB receptors in tumours can occur via mutational activation, receptor overexpression, or through tumour production of autocrine growth factor ligands.The ErbB family of receptor tyrosine kinases, consisting of the EGF receptor, ErbB2, ErbB3 and ErbB4, directs a broad range of developmental events and contributes to the malignancy of a number of tumour types. Numerous Owing to the widespread role of ErbB receptor activation in tumour growth and progression, the development of ErbB inhibitors has been a subject of intense interest. Indeed, neutralising antibodies directed towards the EGF receptor or ErbB2 are either in clinical trials or have received FDA approval for the treatment of some tumours . MoreoveMembers of the mammalian ErbB family are acted upon by EGF-like growth factor ligands, which bind to receptor extracellular domains to stimulate tyrosine kinase activity. The EGF-like domain, a 40–60 amino-acid domain characterised by six cysteine residues forming three disulphide bonds, is a very common component of the extracellular regions of a variety of proteins encompassing a wide range of functions. Only a small subset of EGF-like proteins function as activating ligands for ErbB receptors. Despite a high degree of sequence similarity, each of the ErbB receptors appears to possess distinct biochemical and biological properties. Ligand specificity is determined by the receptor extracellular domains, and EGF receptor, ErbB3 and ErbB4 each bind subsets of the known ligands. No diffusible ligand has been demonstrated to interact with ErbB2. On the other hand, although all four receptors undergo ligand-stimulated homo- and heterodimerisation events as part of their signalling mechanism, ErbB2 appears to be the preferred heterodimerisation partner for the other family members. ErbB3 is unique in that it lacks an intrinsic kinase activity and must necessarily heterodimerise with other family members to propagate signals. Pertinent to a discussion of negative regulatory mechanisms, it has been reported that only EGF receptor undergoes significant ligand-stimulated receptor internalisation and degradation . The marOne of the primary mechanisms by which cells negatively regulate growth factor receptor activity is through receptor degradation. For a quarter century, the EGF receptor has served as a prominent model for understanding how cell surface receptors undergo ligand-stimulated downregulation and degradation. In general, ligand stimulation results in receptor localisation to plasma membrane clathrin-coated pits, internalisation of coated pits and delivery to endosomes, and sorting of endosomal receptors for trafficking back to the cell surface or to lysosomes for degradation.Recent years have seen marked advances in our understanding of the molecular mechanisms underlying these events . A key pReceptor stimulation by EGF results in the phosphorylation of several tyrosine residues of the receptor intracellular region, including tyrosine 1045 that acts as a docking site for cbl. Alternatively, cbl may be recruited in complex with the adaptor protein Grb2 through a distinct phosphotyrosine residue (The cellular site(s) of cbl action toward the EGF receptor and its role in internalisation are a point of controversy. While evidence suggests that cbl is capable of interacting with the receptor prior to localisation in coated pits  protein kekkon-1 (Kek1). Kek1 was identified in a screen for downstream targets of fly EGF receptor activation (in vivo and in vitro (in vivo to suppress receptor signalling (A second EGF receptor feedback negative regulator identified in tivation , and wasThese results also beg the question of whether or not a human Kek1 homologue exists. Owing to its ability to inhibit ErbB activity, such a protein could be the product of a tumour-suppressor gene whose function is lost in some tumours. The search for a mammalian Kek1 homologue focuses on proteins containing leucine-rich repeats in their extracellular regions. This encompasses a wide variety of proteins such as growth factor receptors, growth factor-binding proteins and proteoglycans, among others. A subset of roughly a dozen of these contains both an LRR region and one or more Ig domains. Given that the Ig domain of Kek1 is dispensable for both EGF receptor binding and suppression of signalling in flies (Lig-1 was originally identified as the product of a gene whose expression is induced upon retinoic acid-induced differentiation of P19 neuronal embryonic carcinoma cells (Drosophila EGF receptor to inhibit its activity. Echinoid, a Drosophila transmembrane protein that contains seven Ig domains and two fibronectin type III domains in its extracellular region, was originally identified as a negative regulator of EGF receptor-mediated eye development in flies (Finally, recent evidence suggests that a putative L1-type cell adhesion molecule may interact directly with the in flies . In contDrosophila offer novel candidates for negative regulators, and newly developed rapid protein–protein interaction screening techniques (It is becoming increasingly clear that cells employ a variety of mechanisms to prevent oversignalling by the ErbB growth factor receptors. Degradation of receptors, mediated by E3 ubiquitin ligases associated with intracellular sorting compartments, may play a central role in suppressing signalling following ligand activation or in maintaining a narrow window of receptors at the cell surface, which are available for ligand activation. Transmembrane and secreted modulator proteins can physically interact with receptors to antagonise ligand binding or to interfere with the receptor activation mechanism. Studies from chniques  could faWhile preliminary work has been carried out on some of these negative regulators, the extent to which most of these pathways are suppressed in tumours remains to be explored. It is likely that the function of pathway components, such as E3 ligases associated with receptor expression levels, is lost in tumours associated with ErbB overexpression. The restoration of these pathways, particularly those involving proteins with extracellular regions that interact with ErbB receptors, could offer a novel approach to suppressing the growth properties of ErbB-dependent tumours.


Bone morphogenetic proteins (BMPs) contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts.In all three proteins, a bipartite nuclear localization signal (NLS) was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins  containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle.The observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell. Bone morphogenetic proteins (BMPs) were first identified nearly 20 years ago as components of a protein extract derived from bone that could direct cartilage and bone formation ,2. The Bdecapentaplegic, 60A and screw in Drosophila, and Daf-7 in Caenorhabditis elegans methionine employing the TNT Coupled Wheat Germ Extract System  according to manufacturer's instructions using an expression vector containing the rat Bmp2 cDNA. Ten units of furin  or furin plus 2 μM α1-PDX  were preincubated for 30 min at room temperature. The labeled Bmp2 proprotein was then added, and the reaction was allowed to proceed for 1 hour or 3 hours before products were separated by SDS-PAGE and visualized by autoradiography.Bmp2 protein was synthesized with the incorporation of [Nuclear and cytoplasmic proteins were separated and isolated using the Cellytic Nuclear Extraction Kit  according to manufacturer's instructions. Western blotting was performed on extracted nuclear and cytoplasmic proteins with the following primary antibodies: anti-HA antibody , anti-Bmp2  or anti-Golgi 58K . This was followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibody.JEF participated in design of the study, carried out the NLS and start codon mutagenesis, helped with the furin studies, and drafted the manuscript. JLM participated in design of the study, performed the nBmp2/GFP immunohistochemistry experiments and the furin studies, and helped draft the manuscript. TJL performed all the nBmp4 studies, JDJ and DKS performed the nGdf5 studies. SLG documented the localization of endogenous nBmps during mitosis. CAM and JBM initially identified Bmp2 in nuclear extracts from cultured cells. LCB conceived of the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.


The spliceosome is a ribonucleoprotein complex involved in RNA splicing, that is, the removal of non-coding introns from precursor messenger RNA. (Alternative) Splicing events may play an essential role in tumourigenesis. The recent discovery that the spliceosome is a target for novel compounds with anticancer activity opens up new therapeutic avenues. Therefore, it is conceivable that inhibiting the spliceosome could serve as a novel target for anticancer drug development . In a process called splicing, the intron sequences are removed from the pre-mRNA and the exons fused together resulting in the formation of the mature messenger RNA (mRNA), which is subsequently capped at its 5′ end and polyadenylated at its 3′ end, and transported out of the nucleus to be translated into protein in the cytoplasm. In general, most genes give rise to multiple spliced transcripts by alternative splicing. These transcripts contain different combinations of exons, and sometimes introns or part thereof, leading to different mRNA variants and the synthesis of alternative proteins . The merTo direct correct processing of pre-mRNA, the intron sequences contain a number of core splicing signals, notably the conserved splice-site sequences at their extreme 5′ and 3′ ends and a conserved branch point region. The branch point sequence and the branch point itself, usually an adenosine, are located about 20–40 nucleotides upstream of the 3′ splice site. The critical process of recognising splice sites and the removal of introns and/or exons is the task of the spliceosome . The splFour regulatory sequence elements have been identified; two stimulative ones, splicing enhancers located in introns (ISE) or exons (ESE), and two suppressive ones, splicing silencers located in introns (ISS) or exons (ESS), respectively .In conclusion, the removal of introns and/or exons has to be precise and complete, otherwise incorrectly or differently joined exons yield no mature and functional mRNA, or result in altered genetic messages with the potential of initiating malignant behaviour in previous normal cells.Recent research has revealed that alternatively spliced products can be linked to various (genetic) diseases and also may play a role in cancer development . A systeBRCA1, APC, p53, FHIT and LKB1 and the prostate cancer susceptibility gene, KLF6 , SPN1ΔN (a deletion mutant of snRNP-specific nuclear import adapter snurportin 1), RTVP-1  and CD44v6  all favour tumourous behaviour in cells  mutations or single-nucleotide-polymorphisms (SNPs) occurring in splice sites. These changed splice-site signals may give rise to alternative splice variants of tumour suppressor genes such as Currently, three different approaches to target  splicing in cancer are explored. First, one may be able to modulate alternative splicing by identifying specific splice sites and directly or indirectly block their use. Alternatively, one may identify and characterise splice variant products  that contribute to the malignant phenotype and determine how they differ from their normal protein counterparts. Structural differences may then be used to develop antibodies or small molecules that specifically target these proteins. A second approach may be the manipulation of splice modulators, for example, hnRNPs or SR proteins, hereby indirectly influencing splicing activity. A third approach is based on the simple notion that malignant cells in general have higher metabolic rates than normal cells, and consequently have increased splicing rates and are more prone to splice inhibitors or modulators. Targeting the spliceosome directly might therefore cause tumour inhibition at drug levels that do not affect normal cells.The three strategies such as (i) splice-site modulation and targeting of variant proteins; (ii) targeting of splice modulators and (iii) spliceosome inhibitors are reviewed in detail below.DMD gene, encoding dystrophin, causing disruption of the open reading frame (ORF). The induction of exon skipping, circumventing the mutated exon through intramuscular injection of carefully designed antisense oligonucleotides, corrects the ORF of dystrophin in in vitro cell lines, animal models and humans  may fully restore SMN2 exon 7 inclusion  prove to be of predictive value and (ii) become potential prognostic markers  splice reactions in non-malignant diseases, it is conceivable that this approach might also be applicable in malignancies; Here, splice-site modulation would be a valuable concept for intervening in the expression of various oncogene and tumour suppressor gene mutations.The SR proteins comprise a family of splice factors that modulate splice-site selection by mediating the interaction between pre-mRNA transcripts and the spliceosome. Phosphorylation of the serines in their arginine/serine (RS) domains regulates the intracellular localisations of SR proteins and their interaction with related domains of other splice factors or RNA. This consequently affects alternative splicing of pre-mRNAs. There are several kinases, for example, Clk/Sty, SRPK1, SRPK2 and topoisomerase I that are known to phosphorylate SR proteins. Several compounds have been reported that target these kinases, for example, diospyrin  cannot be ruled out  splicing may contribute to cancer, we hopefully can find ways to control and intervene with splicing, hereby preventing the expression of carcinogenic proteins. The pladienolide compounds and spliceostatin A that target the spliceosome may very well become the first representatives of a totally new class of anticancer agents.


The central survival role of FOX proteins may allow a unified view of the genetic and environmental factors that cause Parkinson disease. Parkinson disease (PD) is the most common neurodegenerative movement disorder in humans, but its cause in the majority of patients is unknown. Fewer than 10% of cases appear to be inherited, and only rarely are the known Mendelian mutations identified in patients with PD. In addition to genetic factors, environmental toxins—especially those affecting mitochondrial function—have also been implicated , demonstTo study PD, researchers have used two basic types of animal models: (1) acute lesioning of DA neurons/tracts, either by surgery or toxic insult, and (2) genetic models that rely on expression of one of the rare, dominant, and highly penetrant mutations that cause PD or knockout of a similar, recessively inherited allele . Although these models have led to huge advances in our understanding of PD—implicating mitochondrial dysfunction, oxidative damage, and protein handling systems in pathogenesis—they typically either fail to recapitulate the gradual nature of this degenerative disease or lack the defining pathology ) ,4. BecauPLoS Biology by Kittappa et al. [It is in this regard that a new paper in a et al.  warrantsa et al. ,7 As thiDopamine neurons are initially generated in a permissive region at the embryonic midbrain–hindbrain border (isthmus). This region is formed by intersecting gradients of several molecules: fibroblast growth factor-8 (FGF8), which is produced by cells at the isthmus, secreted sonic hedgehog (Shh) from the ventral neural tube and floorplate, and Wnt-1 and Wnt5a from the midbrain and rostral diencephalons.. These factors induce a proneuronal program in dividing neuroepithelial precursors that specifically favors DA differentiation and concomitantly suppresses differentiation in non-DA neurons (reviewed in[. Defining an initiating role for FoxA2 is good news for the production of DA neurons from embryonic stem (ES) cells, but previous studies left open the intriguing question of what role FoxA2 plays in the functioning of adult DA neurons.Two recent studies show that Shh and FoxA1/2 are required for differentiation of both DA neurons and the serotonergic (5HT) neurons ,10. InteThe article by Kittappa et al.  highligh+/− mice. These mice show no gross histological or behavioral abnormalities throughout most of their life, but when the mice are in late adulthood (18 mo old), the researchers observed that approximately one-third of animals begin to develop progressive muscle rigidity with asymmetric posturing and tremors, not seen in age-matched wild-type littermates. These symptoms, which are suggestive of striatal degeneration, prompted the authors to demonstrate a functional lesion of the dopaminergic system using the standard amphetamine-induced rotation assay. Affected animals showed significant, asymmetric loss of DA neurons in the SN, particularly among retinoic acid–producing neurons, but the animals showed no loss in the ventral tegmental area. In contrast, age-matched FoxA2+/− mice that displayed a normal behavioral phenotype had no attendant loss of DA neurons. Interestingly, affected animals exhibited bilateral activation of astrocytes in multiple midbrain areas, suggesting that some effects of haploinsufficiency may be more diffuse.Because FoxA2-null mutants die prenatally, Kittappa et al. explored the effects of missing just one copy of FoxA2 (haploinsufficiency) in aging heterozygous FoxA2sine qua non for the disease. Many patients, including patients harboring PARK2 (Parkin) mutations, lack extensive Lewy body pathology on autopsy yet exhibit all the clinical features of PD [Together, these findings demonstrate that the authors have generated a new animal model of PD that captures both the late onset and selectivity of dopaminergic degeneration, including asymmetric motor dysfunction, which is a hallmark of the disorder in humans. However, one cardinal pathologic feature of PD not observed in this model is the presence of Lewy bodies . Althouges of PD ,14.The present study raises a host of interesting questions. First, why does FoxA2 haploinsufficiency lead to progressive loss of DA neurons and their projections to the striatum? Although detailed studies will be required to properly answer this question, one can hypothesize that loss of FoxA2 may be required for proper target innervation; when these projections recede, target-derived trophic support is lost  EvidenceCan these exciting findings be extrapolated to better our understanding of human PD or prove useful to developing ways of treating this disease? The fidelity with which this animal model seems to parallel several key aspects of PD in humans tempts one to speculate that some forms of PD may arise through dysfunction of FoxA2. Thus, FoxA2 and its targets are candidates for genetic association studies in PD in humans. Additionally, given the chronicity and late onset observed in the heterozygous mice, as well as the incomplete penetrance at 18 months, these mice provide an exciting opportunity to investigate gene-gene and gene-environment interactions that may affect DA neuron survival in vivo.Based on these findings, one may also propose that overexpression of FoxA2 could be used to drive DA differentiation of implanted neural stem cells for use in neural repair paradigms in humans with PD. It will also be worthwhile to explore whether increasing FoxA2 protein dosage, by vector-mediated overexpression of FoxA2, is neuroprotective against other genetic or environmental lesions of the nigral DA neuron population. At the same time, we need to recognize that these proposals rely on the assumption that FoxA2 will have the same subset of critical targets in human tissue as it does in mouse. Fraenkel and colleagues  analyzed


A double staining technique was developed for the simultaneous measurement of tissue hypoxia and the concentration of non-protein sulphydryls (NPSH), based on the fluorinated nitroimidazole EF5 and the fluorescent histochemical NPSH stain 1-(4-chloromercuriphenoylazo)-naphthol-2 (mercury orange). Cryostat sections of tumour tissue were examined by fluorescence image analysis, using a computer-controlled microscope stage to generate large tiled field images of the cut tumour surface. This method was applied to the human cervical squamous cell carcinoma lines ME180 and SiHa, grown as xenografts in severe combined immunodeficient (SCID) mice, in order to determine if there is a systematic relationship between tissue hypoxia and NPSH levels. Hypoxic regions of the tumours, defined by EF5 labelling, were found to show greater NPSH concentrations relative to better oxygenated regions. This is probably due to increases in glutathione, since the ME180 and SiHa xenografts contained low levels of cysteine and metallothionein; the other major cellular thiols that can bind to mercury orange. Because the effects of glutathione on radiation and chemotherapy resistance are likely to be greater under hypoxic conditions, these results have potentially important implications for the study of resistance mechanisms in solid tumours. © 1999 Cancer Research Campaign


Intravascular or intracardiac stenoses occur in many forms of congenital heart disease (CHD). Therefore, the implantation of stents has become an accepted interventional procedure for stenotic lesions in pediatric cardiology. Furthermore, stents are know to be used to exclude vessel aneurysm or to ensure patency of existing or newly created intracardiac communications. With the further refinement of the first generation of devices, a variety of “modern” stents with different design characteristics have evolved. Despite the tremendous technical improvement over the last 20 years, the “ideal stent” has not yet been developed. Therefore, the pediatric interventionalist has to decide which stent is suitable for each lesion. On this basis, currently available stents are discussed in regard to their advantages and disadvantages for common application in CHD. New concepts and designs developed to overcome some of the existing problems, like the failure of adaptation to somatic growth, are presented. Thus, in the future, biodegradable or growth stents might replace the currently used generation of stents. This might truly lead to widening indications for the use of stents in the treatment of CHD. Since the first description of an interventional procedure in the field of congenital heart disease (CHD) by Rashkind in 1966, which laAlthough intravascular stents still do not have the Food and Drug Authority (FDA) approval for use in congenital lesions and pediatric patients, since 1996, their use for congenital vascular lesions has been accepted as the standard of care by all centers and professional medical societies associated with the treatment of pediatric and CHD. This review will address some key questions in the common practice of the use of stents in the CHD population. Advantages and disadvantages of the different types of stents and stent designs currently used in CHD are presented. Furthermore, recent developments in stent technology intended to overcome some of the present problems in stent therapy that might expand the spectrum of indications for stenting are addressed. As this paper focuses on stent therapy within the heart and the central great vessels, the use of stents in peripheral arteries and veins is not discussed.Common indications for stent deployment in CHD include treatment of obstructive lesions of the (1) branch and peripheral pulmonary arteries, (2) systemic and pulmonary veins, (3) aorta and branches, (4) right ventricular outflow tract (RVOT) conduits, (5) maintenance of patency of the arterial duct in duct-dependent circulation, (6) maintaining patency of stenosed aortopulmonary collateral vessels or surgically created but obstructed shunts and (7) maintaining patency of intracardiac communications.Stents are classified on the basis of the material of which they are made, the target region, their configuration and their size. Additional features used in classifying them include coverage, special surface treatment and coatings and drug-eluting properties. But, the most common classification is based on their delivery mechanism: Balloon-expandable versus self-expandable stents.Balloon-expandable stents (BES) are mounted on balloons, positioned across the site of obstruction and are implanted by inflating the balloon. The size of the inflated balloon determines the expanded diameter of the stent. BES were introduced for the treatment of congenital heart lesions in 1987,5 with thDesirable features for an ideal stent design in pediatric cardiology are: (1) low stent profile combined with (2) high trackability and (3) flexibility to negotiate steep curves, (4) good radio-opacity and visibility for precise placement, (5) compatibility with magnetic resonance imaging (MRI) with no artifacts, (6) predictable expansion with minimal foreshortening, (7) sufficient radial strength, (8) low rigidity with no material fatigue over time, (9) full biocompatibility with resistance to thrombus formation and corrosion, (10) prevention of plaque protrusion, (11) avoidance of neointimal proliferation, (12) round and soft edges for avoidance of intimal damage, (13) possibility of redilation with patients' growth, (14) wide struts to maintain blood flow to jailed vessel branches and (15) retrievability and possibility of repositioning if needed. Obviously, there is no stent yet available that combines all of these requirements so that selecting the right stent for the appropriate condition is one of the most difficult challenges encountered by pediatric interventional cardiologist.Technically speaking, stent performance is related to the material characteristics, form, fabrication mode and geometry.The most widely used material for BES is still stainless steel, typically 316 L. It is particularly corrosion resistant and in its fully annealed condition, easily deformable. Alternative materials for BES used in CHD are platinum alloys , tantalum  and cobalt alloys . Cobalt chromium alloys are very interesting for use in pediatric cardiology as they allow lower crimping profiles with high radial strength. Recently, biodegradable stents have been introduced that are composed of polymeric substances, magnesium or biocorrodible iron. This new class of stents is discussed in the section “New developments in stent design and future perspectives” at the end of this article.The vast majority of coronary and peripheral vascular stents are produced by laser cutting from tubing, the so-called slotted tube design. Intricate patterns can be produced using tube sizes from 0.5 mm diameter. BES are cut in crimped or near-crimped conditions, allowing a low profile. In slotted tube stents, those with the more conservative closed cell design need to be distinguished from those with the newer open cell design.The traditional closed cell design describes a sequential ring construction wherein bridging elements connect all internal infliction points of the structural members . This isThus, older Palmaz stents such as the Palmaz 8 series  and others have been replaced by the Genesis series. The Genesis stents represent the latest evolutionary step of the Palmaz “biliary” stent series. They are similar to the Corinthian stent , except for the new sigmoidal hinge. This element adds greater flexibility without compromising radial strength. They are available as unmounted and pre-mounted, the latter for only the smaller diameters – paradoxically named Genesis Medium. The Genesis Medium stents can easily be dilated to 5–8 mm, but overdilation up to 12 mm is possible. Genesis Large  are labeled as 5–10 mm, but may be dilatable to 12–15 mm. The Genesis XD  can be dilated optimally to 10–12 mm but can easily be overexpanded to 18 mm. Some other commonly used stents with the closed cell design are listed in The primary advantage of the closed cell design is optimal scaffolding and a uniform surface, regardless of the degree of bending. However, these advantages result in a structure that is typically less flexible than with a similar open cell design.In open cell design, geometry does not connect consistently throughout the stent, forming incomplete and non-bridged cells. With expansion, the individual cells merge to form larger open areas; thus, there is no consistent shape of cells . Example6In contrast to stents with the slotted tube design, the Cheatham platinum (CP) stent has been developed using a welded tube. It is manufactured from a wire, which is bent and welded to a cylindrical meshwork, forming the stent. This leads to more adjustable flexibility and a wide range of size and length, but radial strength is usually less than the ones with slotted tubes.The most commonly used stent in pediatric cardiology is the CP stent, which is made from 90% platinum and 10% iridium. Each row of zigs is laser welded with the addition of gold soldering (since December 2002) at each welded spot to increase the total strength of the weld. The number of zigs affects the final diameter and the degree of shortening as well as the profile of the stent. While it is available in 6 and 8 rows, it is mostly used in the 8-zig configuration, as this shortens much less than the 6 zig at the equivalent diameters and can be dilated up to 28 mm (instead of 18 mm for 6 zig), with foreshortening of about 22–28%; however, dilatation to larger diameters is possible.[99CP stents are available in a covered version, with an outer polytetrafluoroethylene (PTFE) membrane. The covering is initially approximately 7 mm in diameter and will stretch over the range of diameters of expansion  and will always be taut over the stent when expanded. To date, the covered CP stent is the most widely used covered stent in patients with CHD.11–26A very practice-orientated classification for the use of BES in CHD is based on the maximum expandable size of stents. It is important to note that, due to the lack of official recognition for use in pediatric and CHD patients, nearly all stent manufacturers test and label their stents with the maximum diameters for use in approved smaller diameter biliary system and peripheral vessels, rather than their true potential maximum diameter. Thus, stents can be categorized into four different sizes by their “true” maximal expandable size: Small (3–6 mm), medium (10–12 mm), large up to 18 mm) and extralarge (up to 25 mm).[ mm and eIn The large majority of balloons currently used for stent deployment are still adapted from the adult vascular angioplasty and “biliary” applications. These large- diameter single-balloon catheters tend to expand first at their ends and thereby evert the stent ends such that they protrude radially from the stent center. Deploying a stent in this orientation can cause injury to the vessel wall and may be a risk factor for the development of aneurysm or dissection. One of the most important developments in equipment for the delivery of large-diameter stents has been the Balloon-in-Balloon  catheter, the first balloon specifically designed for stent delivery in the CHD population. These catheters have an inner balloon and a longer outer balloon that is double the diameter of the inner balloon. The BIB catheters are available in outer balloon sizes of 8–24 mm. The BIB catheters offer the important advantage of opening the stent more uniformly along its length but require a larger arterial sheath for introduction. With a stent hand crimped onto the balloon, it is necessary to upsize the long sheath by 1F, greater than is necessary for the BIB catheter alone. Therefore, with hand-mounted stents, BIB catheters with outer balloon diameters of 8–14 mm require a 9F sheath, 16 mm catheters a 10F sheath, 18–20 mm catheters an 11F sheath and 24 mm balloons a 12F sheath. Thus, while BIB catheters prevent stent flare and offer more precise control over stent placement, single-balloon catheters are still sometimes preferable in smaller patients to reduce risk of injury to the femoral artery at the access site.The development of ultra high pressure (UHP) balloons like the Conquest (5–12 mm diameter) and the Atlas  may facilitate successful treatment of stent-associated stenoses that are resistant to conventional high-pressure dilation. These balloons, originally developed and approved for treatment of stenotic hemodialysis fistulas, contain For lesions resistant to conventional high-pressure angioplasty, evidenced by a persistent waist, cutting balloons may be used before stenting. A Cutting Balloon™  has three or four longitudinal microtome blades bonded to a non-compliant balloon. Folded between the material of the balloon, the blades are exposed when the balloon is slowly inflated, achieving a profile of 0.127 mm from the surface. Their microblades are intended to incise only the intima and part of the media in a vessel, resulting in a more controlled dissection of the vascular intima. On deflation, the balloons refold very smoothly and the blades recess back to their original configuration in the surface of the balloon. As they are only available in sizes up to 8 mm, this limits their use in larger vessels. Currently, they have been shown to be very effective in pulmonary branch artery stenosis,30 major 32Only a minority of BES suitable for CHD are pre-mounted, like the Genesis medium stent (Cordis) that is available on a Slalom™, OptaPro™ and Aviator™ Balloon (Cordis). The pre-mounting represents a unique incorporation of the stent into the wall of the balloon. This fixes the stent very securely on the balloon and allows delivery of the stents to very circuitous locations without the need for a long sheath and with no displacement during delivery – although using a short sheath access may not always be advantageous, as monitoring of placement with contrast medium injection is more difficult. Currently, most stents for CHD come unmounted and have to be hand crimped over the desired balloon, which also has its own advantages. This offers more flexibility, as stocking is easier because multiple stent–balloon combinations are possible. We sometimes use a customized crimping device  to obtain a very low profile and tight stent/balloon assembly. Thus, it has been possible to deliver a Mega LD stent (EV3) on a 6 mm Powerflex P3™ (Cordis) balloon through a 6 Fr-long sheath .Materials for self-expanding stents should exhibit large elastic strains. The most widely used material is nitinol, a nickel-titanium alloy that can be recovered from elastic deformations of up to 10%. This unusually large elastic range, commonly known as superelasticity, is the result of a thermoelastic martensitic transformation. The limited elastic range of more conventional materials, such as stainless steel (Cook “Z Stent”) or certain cobalt-based alloys , offer limited design options. While the WallStent offers excellent wall coverage and flexibility, its shortcoming is its length change during deployment of up to 53.8%, making the newer design  more supin vitro study.[Self-expandable stents, constrained within a covering delivery sheath, are delivered to the site of stenosis. Withdrawal of this sheath uncovers the stent, which then reassumes its original shape (the chosen diameter) to dilate the stenotic lesion. As these stents do not require a balloon for expansion, they can be delivered through a lower-profile delivery system. Compared with BES, they are more flexible and thus can be passed through very tortuous vessels and lesions, but have significantly lower radial strength. They are made from nitinol  and are therefore fully MRI compatible. Because of the unique memory effect of nitinol, they show a delayed 10–20% additional expansion within the first month following implantation. This might partially compensate for the development of more neointimal hyperplasia than witro study.Traditionally, they have been used for peripheral vascular applications, where external compression can permanently deform a stainless steel stent, but not the nitinol thermoelastic stent. Their main disadvantage is that they cannot be further dilated to accommodate vessel growth. Therefore, use of this type of a stent in growing children is limited–37 unlesSelf-expandable stents currently used in CHD are listed in PA obstructions occur in both congenital and acquired disease and they are found as isolated or multiple lesions, discrete stenosis or diffuse hypoplasia. Genetic syndromes such as Williams or Allagille are associated with such obstructions. They may be part of simple or complex cardiac malformations, such as ventricular septal defect, pulmonary valve stenosis, tetralogy of Fallot (TOF), pulmonary atresia and others. The majority of the acquired lesions are remnants after surgical procedures in complex heart defects. Thus, stenoses may develop as a result of extensive scarring at a previous PA repair site or at the anastomosis between the PA and a conduit or along the aortopulmonary shunt. Sometimes stretching of vascular structures may contribute to formation of stenosis, as seen following the LeCompte maneuver for repair of d-TGA. Such examples indicate that the underlying etiology and thus the pathomechanism of the obstruction differ from case to case. Therefore, optimal therapeutic strategies, based on case characteristics, should be adapted. Universally accepted indications for intervention include symptoms, right ventricular systolic pressure of more than 50% of systemic systolic pressure, right ventricular dysfunction and significant differential pulmonary perfusion in unilateral stenosis. Non-invasive imaging, such as MRI or echocardiography, are usefThe disappointing results of surgical management of branch PA stenoses led to the introduction of balloon angioplasty in the early 1980s.40 With s4144414850Indications and use of stents for dealing with PA stenosis have increased in recent times. Stents are mainly used to deal with origin stenosis occurring naturally or after previous surgery, kinking or tenting of the branch PA, external compression of the branch PA, elastic recoil, intimal tear after balloon angioplasty and recanalization of totally occluded vessels. In infants with prior implantation of an RV–PA conduit, stenting is used as a bridge to delay conduit replacement. Although stents are commonly placed after previous balloon angioplasty, there are indications where primary stenting should be considered. These include long-segment stenoses, where balloon angioplasty shows unsatisfactory results, subatretThere are numerous published papers demonstrating excellent mid- and long-term results after st50575752The incidence of significant restenosis of single-vessel PA stents has been reported to be 1.5–7% in large pediatric series.5055 Two 505159Interventional treatment of bifurcating stenoses in the PA involving either the proximal branch or lobar branch vessels can be technically challenging. Standard balloon angioplasty alone is often inadequate and placement of stents is frequently required to relieve obstruction. In such bifurcating pulmonary stenoses, a simultaneous approach (simultaneous stent placement in two or more adjacent pulmonary arteries) is in our view mandatory and has been described previously.62 The adDistal PA stenoses are usually multiple and represent complex lesions that are not accessible to the surgeon. The smalFailure of a bioprosthetic right ventricle-to-pulmonary artery conduit has been well documented and may require repeat surgical conduit replacement. Pathomechanisms involved are external compression , calcification, kinking, development of fibrotic intimal peel and aneurysm. In addition, conduit diameters are limited and young patients may outgrow the diameters. Balloon dilatation of stenotic valves has shown poor results, with minimal extension of the life span of the conduit.66 Thus, 4767Potential complications with PA stent deployment occur in 4–5% and include stent migration (2.4%), stent malposition, intimal flap obstruction, jailing of side branches, stent fracture, dissection, aneurysm, vessel rupture, hemoptysis (1.5%), thrombosis of stent, ipsilateral pulmonary edema (1.5%) and death (1.5%). In a retBalloon dilation of aortic recoarctation (Re-CoA) was introduced in 1982 as treatment for post-operative restenosis after surgical repair with subsequent hypertension.–72 Intim7074741074881026768183878181838686818181818181818181819981et al. 2007; Riede, Schneider et al. 2007; Jhang, Chang et al. 2008; Wong, Yoo et al. 2008) or systemic administration of anti-proliferative drugs such as everolimus may theoretically overcome some of these problems,[In general, CoA redilatation demonstrates good intermediate follow-up results.94 Aneury868694problems, but no sproblems,In 1999, the first covered stent was used to treat coexistent CoA and aneurysm of the aorta in a young man. Aneurysm9111217182225Aneurysm formation may occur even after redilatation of a previously implanted uncovered stent; the sten88111191113171817182111182226915One obvious limitation of covered stents is their potential to cover side branch vessels during deployment, usually the subclavian artery ostium. Jailing of subclavian artery is usually well tolerated without functional deficit but an intact vertebrobasilar system should be documented to avoid subclavian steal.92 To cirAlthough covered stents are primarily used for CoA treatment, they might be applied to other vessels. Thus, the use of a covered 8-zig CP stent for treatment of post-operative RVOT has been reported Baffle l15et al. described an improvement to more than 90% of the “normal” adjacent aortic arch.[7With increasing experience in CoA stenting, the concept has been extended to the treatment of complex stenoses of the aortic arch and the descending aorta.17110 It 17ic arch.7Several conditions may lead to stenosis of systemic veins, including superior and inferior caval vein obstruction occurring after atrial switch operation or cardiac transplantation, uni- or bidirectional Glenn anastomosis and total cavopulmonary connection, correction of anomalous pulmonary venous connections and the presence of transvenous pacing electrodes or central venous catheters.–113 As b1437117119Pulmonary venous stenosis may be congenital or acquired after cardiac surgery. It is reported after repair of anomalous pulmonary venous return in about 10% of the patients. Suturele1321321355252132138For some complex congenital heart defects, an unrestrictive ASD is essential for adequate blood mixing at the atrial level or relief of right or left atrial hypertension to achieve an adequate cardiac output and/or systemic saturation. Transcatheter creation or enlargement of an ASD is usually achieved employing techniques including transeptal puncture or radiofrequency perforation (in the case of intact septum), balloon or blades septostomy and static balloon dilatation of the interatrial septum (IAS). However, due to the increased thickness of the IAS in infants beyond the neonatal period or in neonates with hypoplastic left heart syndrome, the use of those techniques may be ineffective to achieve and maintain an adequately sized communication. Therefore, atrial septal stent implantation has been successfully applied to promote a durable unrestrictive ASD.33141–145333233In duct-dependent CHD, ensuring patency of the arterial duct may be a life-saving procedure. Stenting the arterial duct in defects such as pulmonary atresia, right ventricular hypoplasia, critical pulmonary stenosis, TOF and other complex lesions with reduced pulmonary blood flow may avoid (emergency) surgical shunt. The concept of stenting the ductus arteriosus to increase pulmonary blood flow in cyanotic neonates began with the arrival of stents designed for coronary arteries in the early 1990s. Unfortun158In severe forms of TOF with PA hypoplasia, critical RVOT obstruction and the presence of MAPCAs major aortopulmonary collateral arteries (MAPCA), primary repair may not be possible. For such a two-staged repair, early surgical options include a central aortopulmonary anastomosis, a modified Blalock–Taussig anastomosis, an RV outflow patch or ductal stenting as discussed in the previous section. In selected cases, RVOT stent implantation may be another alternative to surgical palliative treatment in order to achieve sufficient oxygen delivery and to induce PA development through pulsatile flow.161 RVOT 57164Stenosis of aortopulmonary collateral arteries is common and frequently progressive in patients with unrepaired pulmonary atresia and ventricular septal defect, leading to severe hypoxemia and limitation of exercise capacity in up to 58–68% of the patients. Surgical31There are some recent developments in technology intended to overcome some of the disadvantages of currently used stents.As scaffolding is necessary to overcome late vessel remodeling only for a limited period – approximately the first 6 months following stent implantation – the concept of biodegradable stents has emerged. In addition to other advantages, this concept might solve one of the main problems in pediatric stenting – the adaption to growth. These systems should deliver a temporary longitudinal and radial straightening effect, offer better physiologic repair, allow reconstitution of local vascular compliance and should not restrict surgical or interventional revascularization thus allowing the possibility of growth and late positive remodeling. The ideal characteristics of a biodegradable stent have been defined as (1) sufficient radial strength to prevent vascular recoil, (2) minimal thrombotic and inflammatory response, (3) avoidance of intimal proliferation, (4) reabsorption of stent components within months, (5) no release of toxic products or embolic material during breakdown and (6) easy processing and sterilization. Based on these considerations, two different concepts have evolved: Absorbable polymer stents and absorbable metal stents.There are several polymeric degradable stents in development but just a few merit mention at this stage on the basis of clinical data. The BVS stent  is made from poly-L-lactic acid (PLLA) and results from clinical trials were recently published.–172 WhilThe relative ease with which magnesium corrodes and its role as an essential element in the biological system makes it an excellent candidate for the biocorrosion concept. First data was published in 2003. As an al177in vitro experiments showed that ions released from iron stents might reduce the proliferation rate of vascular smooth muscle cells by influencing growth-related gene expression and could therefore play a beneficial role in antagonizing restenosis.[Limitations posed by biodegradable polymer and magnesium alloy stents, such as chronic inflammation and premature recoil, might be overcome by biocorrodible iron stents. The NOR-I Stent is a pure iron stent that has been implanted in an animal study, demonstrating low thrombogenicity, mild inflammatory response and no toxicity, no pronounced intimal hyperplasia and excellent patency, but a very slow degradation rate of several years.182 In adstenosis.All of the degradable technologies reviewed here have many specific challenges ahead of them, but, to date, common to all is the lack of clinical evidence demonstrating a clear advantage of this approach. Polymer systems face specific challenges in achieving adequate strength and resistance to recoil as well as a need to reduce degradation times. Magnesium systems appear to offer the most promise, with degradation times being closer to what may be required. Iron stents face big difficulties in terms of establishing that the degradation products would be acceptable.Another interesting approach to overcome the “growth” problem is the development of the so-called Growth Stent. It is ma184Electropolishing is the state-of-the-art finishing process for nitinol implants, such as stents. Several studies have shown that electropolished nitinol surfaces exhibit better corrosion resistance, biocompatibility and overall surface quality than surfaces finished by other methods.187 Thus,189Over the last 20 years, the usefulness of stents in patients with CHD has become well established. Stent implantation is now considered to be a safe and effective method in relieving a wide variety of pre- and post-operative vascular stenoses not amenable to pure balloon dilatation. Thus, stent therapy is now considered to be the standard and first-line treatment for CoA in adults or post-operative PA branch stenoses. The majority of stent applications are intended to provide a long-lasting cure or reasonable palliation to avoid high-risk repeat operation. Thus, stents may now be used to ensure patency of naturally occurring  or artificially created intercirculatory communications. Because of a tremendous improvement in stent technology, the first-generation models like the original Palmaz stent have now been replaced by “modern” designs. But still the device that meets the requirements of an ideal stent for use in CHD patients remains to be developed. Thus, concepts for stent application in very small patients that allow adaptation for somatic growth until adulthood vessel size is reached are clearly needed. Another issue to be addressed is improvement of biocompatibility to avoid intimal hyperplasia and restenosis. Until these problems are solved, the crucial question in this interventional field still remains: Which stent(s) should we use for which lesion? To clarify these questions, anecdotal data are insufficient, underlying the clear need for large prospective trials to search for objective performance criteria. As the population with CHD is limited, independent objective multinational registries are needed. With these improvements, indications for stent treatment in CHD are likely to widen in the future.


We also review articles describing positive effects obtained by reducing necroptosis, including the reduction of infarct volume and improved functional recovery in animal models. Since necroptosis is characterized by cell content leakage and subsequent inflammation, in addition to reducing cell death, inhibition of necroptosis in ischemic brain stroke also reduces some inflammatory cytokines. By comparing various approaches in inhibition of necroptosis, we analyze the achieved effects from the perspective of controlling necroptosis as a part of future therapeutic interventions in brain ischemia.Due to very limited therapeutic options, ischemic brain injury is one of the leading causes of death and lifelong disability worldwide, which imposes enormous public health burden. One of the main events occurring with ischemic brain stroke is cell death. Necroptosis is a type of cell death described as a regulated necrosis characterized by cell membrane disruption mediated by phosphorylated mixed lineage kinase like protein (MLKL). It can be triggered by activation of death receptors , which lead to receptor-interacting serine/threonine-protein kinase 3 (RIPK3) activation by RIPK1 in the absence of active caspase-8. Here, we review articles that have reported that necroptosis significantly contributes to negative events occurring with the ischemic brain stroke, and that its inhibition is protective both  Beside cancer and cardiac disease, brain stroke is one of the leading causes of death worldwide. In more than 80% of cases it is caused by blood supply obstruction leading to ischemia . Stroke in vivo and in vitro models of brain ischemia. We also focus on the role of necroptosis in inflammation following ischemia and the potential role of elements of necroptosis in the nucleus following ischemic brain injury.Necroptosis is a regulated type of cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase like protein (MLKL), which results in membrane permeabilization . VariousCell death is both a physiological and pathological event present during the development of and in the processes linked to homeostasis. It is also one of the central elements accompanying various pathological processes. For a long time, cell death has been primarily divided into the programmed cell death or apoptosis, and “accidental” cell death or necrosis, which is caused by physiochemical stress. Apoptosis is mediated by proteins, among which are caspases, and is characterized by cytoplasmic shrinkage, chromatin condensation, and plasma membrane blebbing, leading to the formation of small vesicles, which are phagocytosed by neighboring cells . On the Necroptosis is the most studied form of regulated necrosis, and its main characteristic is cell membrane disruption mediated by phosphorylated MLKL (pMLKL). Necroptosis can be triggered by the activation of death receptors , which can lead to RIPK3 activation by RIPK1 in the absence of active caspase-8  Figure . Activatin vitro and in vivo. Here, we discuss the effect of inhibition of different elements of necroptosis on infarct volume and neurological/cognitive functions of experimental animals. We focus on specific cell types that are protected by the inhibition of necroptosis following ischemic brain stroke. We also report the effects of inhibition of necroptosis in neurons and astrocytes subjected to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Finally, we describe the effects of inhibition of necroptosis on inflammation following ischemic brain injury and discuss a possible role of elements of necroptosis in the nucleus.Necroptosis significantly contributes to negative events occurring with the ischemic brain stroke, and its inhibition is proven to be protective both in vitro, which was then successfully transferred to a mouse model of ischemic brain injury, where its administration reduced infarct volume (It has been shown that chemical inhibitors of necroptosis are protective in animal models of ischemic brain injury  Figure . The firt volume . Since tt volume  demonstrt volume . In addit volume ,17.Inhibition of RIPK3 and MLKL, downstream regulators of necroptosis, has also been shown to be protective in ischemic brain stroke. The administration of RIPK3 inhibitors, GSK’872 or dabrafenib, reduced lesion size in mice subjected to ischemic brain injury ,19. In aThe protective effect of inhibition of necroptosis, seen as a reduction of infarct volume and improvement in neurological functions, is primarily achieved by the protection of the brain cells. Ni et al  demonstrInhibition of necroptosis in neurons subjected to OGD has also been shown to have a protective role. Treatment of rat cortical neurons with Nec-1 before OGD significantly reduced the percentage of necrotic cell death measured 3 h post recovery: in OGD group the percentage of necrotic cell death was 60.5 ± 5.8%, while in the OGD+Nec1 group it was 40.8 ± 4.3% . PretreaRipk1. At the same time, neural stem cells did not influence Ripk3 and Mlkl levels, which remains to be explained (In our own experiments, we analyzed how neural stem cell transplantation after stroke influenced the expression of necroptosis markers. Interestingly, we found that neural stem cell transplantation, but also enriched medium, significantly down-regulated xplained .in vitro and in vivo (Inhibitors specific for a certain element of necroptosis affect not only an isolated path but also the whole necroptosis pathway. MLKL small interfering RNA notably inhibited MLKL expression and RIPK1-RIPK3-MLKL interaction in the rat cortex and hippocampus subjected to hypoxia-ischemia . Ni et a in vivo . Nec-1 t in vivo .in vitro experiments using conditioned medium generated from WT and RIPK3−/− neurons to treat macrophages, Yang et al (Since necroptosis is characterized by cell membrane rupture and cell content leakage, one of the most obvious effects of inhibition of necroptosis is the reduction of inflammation. Inhibition of necroptosis by Nec-1 following BACS has been shown to have long term effects manifested in the down-regulation of inflammatory cytokines TNF-α, INF-γ, IL-1β, and IL-33, 15 days after treatment both on mRNA and protein levels . Althougng et al  showed tNecroptosis is a specific type of cell death mediated by RIPK1, RIPK3 and MLKL, resulting in cell membrane permeabilization by MLKL . On the Necroptosis has become one of the most studied forms of cell death because of its presence in many pathological events. Recent articles clearly showed that necroptosis is responsible for a large portion of negative effects occurring after brain ischemia. Equally important, inhibition of elements of necroptosis significantly reduces brain damage. Taken altogether, necroptosis entered the scenery of brain pathophysiology as one of its central elements. Current efforts focused on better understanding how to control necroptosis have a potential to lead to new therapeutic options for reducing damage caused by brain ischemia.


The composition was based on the PICO model and the PRISMA checklist. First author, year, subject characteristics, intervention, outcome measures & variables, results and wavelet specification were extracted. Eighteen studies included the use of wavelets to investigate electromyograms of lower extremity muscles. Three main topics were discussed: 1.) The capability of the method to correctly assign participants to a specific group (recognition rate) varied between 68.4%-100%. 2.) Patients with ankle osteoarthritis or total knee arthroplasty presented a delayed muscle activation in the early stance phase but a prolonged activation in mid stance. 3.) Atrophic muscles did not contain type II muscle fiber components but more energy in their lower frequencies. The simultaneous information of time, frequency and intensity is of high clinical relevance because it offers valuable information about pre-and reflex activation behavior on different walking and running speeds as well as spectral changes towards high or low frequencies at every time point of the gait cycle.Surface electromyography is often used to assess muscle activity and muscle function. A wavelet approach provides information about the intensity of muscle activity and motor unit recruitment strategies at every time point of the gait cycle. The aim was to review papers that employed wavelet analyses to investigate electromyograms of lower extremity muscles during walking and running. Eleven databases were searched up until June 1 Electromyograms (EMG) is a commonly used method to measure muscle activity and to assess muscle function –5. WhileP (Population): “human adults”, I (Intervention): “impact of gait, running or walking on muscle activity of the lower extremities”, C (Control): “healthy or different gait conditions” and O (Outcome): “EMG wavelet parameters”  theNext, the following data were independently extracted and analyzed by two investigators using a standardized extraction form: First author, year; subject characteristics; intervention; outcome: measures & variables; results; wavelet specification.Following “The Cochrane Collaboration`s tool for assessing risk of bias”, all studies showed a high or unclear risk of bias concerning random sequence generation, and blinding of participants and personnel. None of the studies provided information about blinding of outcome assessments. Two studies gave some information about randomization ,13, 14 sIn 18 studies, EMG activity patterns of the lower extremities were investigated: 13 investigated walking, 4 running and one different walking and running conditions. The summary of wavelet analysis of EMG walking data is depicted in t, are sometimes very difficult to assign to a certain condition by visual inspection because the signals can appear inconsistent [The recognition rate is the capability of a method to correctly assign participants to a specific group ,15. Diffnsistent . Howevernsistent , allows nsistent , betweennsistent  and malensistent . The appnsistent ,18. Eighnsistent ,14–19.The recognition rate discriminated between different running, walking or footwear conditions measured with different muscles of the leg ,15,17–19The behavior of EMG intensity and frequency over time was analyzed. The non-stationary wavelet approach was used for the evaluation of muscle timing, time shifts and early or delayed muscle activation ,16,20–23In healthy subjects walking presented a pre-activation peak 50–100 ms before heel-strike . The conElectromyographic signals have been found to be very subject-specific ,23. TherRecruitment patterns of slow type I to fast type II fibers have been investigated by means of the intensity levels at different wavelet frequency bands . IntensiDown shifted EMG spectra of tibialis anterior toward lower frequencies have been observed in patients with ankle osteoarthritis. The wavelet patterns of patients showed a trend towards less intensity production of tibialis anterior between 170 and 271Hz but more intensity between 19 and 62Hz . AtrophiMotor unit characteristics while running have been mainly investigated by Wakeling et al. and von Tscharner et al. ,13,21–23The aim of this systematic review was to demonstrate how wavelet analyses applied to EMG of lower extremity muscles have successfully differentiated between different walking and running conditions, and between subjects with and without certain orthopedic conditions.Since none of the studies reviewed were designed as randomized control trials, the issues of randomization and blinding are not relevant. The study designs of the included studies were appropriate regarding the research questions.Non-linearly scaled wavelets, as originally described by von Tscharner (2000)  have beeThe use of wavelets for processing non-stationary EMG has been shown to be suitable for evaluating muscle timing, time shifts and early or delayed muscle activation ,16,20–23This knowledge of muscular preparation and adaption to variable heel-strike events, as well as the interplay of the muscles are useful regarding training protocols in sports and in rehabilitation . It alloFast type II muscle fibers generate higher rates of force development than slow type I fibers, and are more likely to allow a fast release compared to slow fibers . During To the best of our knowledge, this is the first systematic review that summarizes the use of wavelet analyses of electromyographic recordings from lower extremity muscles during walking, running, and certain orthopedic conditions. The influence of the interdisciplinary knowledge of the team including physiotherapists, sports and movement scientists, biomechanics and engineers, all experienced in signal analysis is another strength of the review.However, the study has the limitation that in a couple of studies low numbers of participants were included. Furthermore, almost all reviewed articles applied the non-linearly scaled wavelet method developed by von Tscharner (2000) . HoweverWavelets derived from the lower extremity muscles’ EMG reflect signal components related to activities of small and large alpha-motoneurons and also muscle timing and intensity characteristics. The simultaneous information of time, frequency and intensitiy behavior is of high clinical relevance because it offers valuable information about pre- and reflex activation behavior on different walking and running speeds as well as spectral changes towards high or low frequencies at every time point of the gait cycle. Furthermore, the required information of motor unit recruitment behavior in the pre- and post-heel-strike phase as well as at heel-strike could be extracted, which sheds light on specific differences of muscle activation patterns. This knowledge allows for optimizing training and rehabilitation protocols. The optimized accuracy of discriminating between healthy people and people with orthopedic conditions wavelets provide a useful diagnostic tool to analyze group differences or pre-/post study designs.S1 Fig(DOC)Click here for additional data file.


The pregnancy complication preeclampsia represents an independent risk factor for cardiovascular disease. Our previous research shows a diminished function of fetal endothelial colony-forming cells (ECFC), a proliferative subgroup of endothelial progenitor cells (EPC) in preeclampsia. The aim of this study was to further investigate whether DNA methylation of fetal EPC is affected in preeclampsia.The genomic methylation pattern of fetal ECFC from uncomplicated and preeclamptic pregnancies was compared for 865918 CpG sites, and genes were classified into gene networks. Low and advanced cell culture passages were compared to explore whether expansion of fetal ECFC in cell culture leads to changes in global methylation status and if methylation characteristics in preeclampsia are maintained with increasing passage.A differential methylation pattern of fetal ECFC from preeclampsia compared to uncomplicated pregnancy was detected for a total of 1266 CpG sites in passage 3, and for 2362 sites in passage 5. Key features of primary networks implicated by methylation differences included cell metabolism, cell cycle and transcription and, more specifically, genes involved in cell-cell interaction and Wnt signaling. We identified an overlap between differentially regulated pathways in preeclampsia and cardiovascular system development and function. Cell culture passages 3 and 5 showed similar gene network profiles, and 1260 out of 1266 preeclampsia-associated methylation changes detected in passage 3 were confirmed in passage 5.Methylation modification caused by preeclampsia is stable and detectable even in higher cell culture passages. An epigenetically modified endothelial precursor may influence both normal morphogenesis and postnatal vascular repair capacity. Further studies on epigenetic modifications in complicated pregnancies are needed to facilitate development of EPC based therapies for cardiovascular alterations. The hypertensive pregnancy disorder preeclampsia is one of the most common causes of maternal and fetal morbidity and mortality in the developed world . ImpaireRecent studies suggest that preeclampsia not only represents an independent, cardiovascular risk factor for the mother, but that the cardiovascular system of the offspring is also adversely affected. The affected children display on average significantly higher blood pressure, higher body mass index (BMI) and increased vascular stiffness in the pulmonary and peripheral circulation compared to offspring from uncomplicated pregnancies . EpidemiEndothelial progenitor cells (EPC) play an important role in vascular homeostasis and development, including repair of injured endothelium, angiogenesis, and neovascularization . A decreStudies on epigenetic modifications in complicated pregnancies are urgently needed to develop epigenetically based therapeutics for the prevention and therapy of early cardiovascular alterations. Considering the potential of cell therapies based on EPCs, the aims of this study were to compare the DNA methylation status of fetal ECFCs at birth between uncomplicated and preeclamptic pregnancies, and to assess the effect of cell culture on DNA methylation.The Ethical Committee at Hannover Medical School and the University of Pittsburgh Institutional Review Board approved the study. Informed written consent was obtained from each study participant. Twelve healthy women with uncomplicated, normotensive pregnancies (controls) and 12 women with late-onset preeclampsia (diagnosis at >37 weeks gestation) provided umbilical cord blood samples directly after delivery. Clinical and demographic data of these women are presented in 7 cells/well) onto collagen-coated 6-well plates , containing endothelial growth medium-2  supplemented with suppliers recommended concentrations of growth factors, 10% FBS and 1% penicillin/streptomycin. Growth media were changed daily for the first seven days and then every other day. Colonies were assessed daily, and colony numbers and time of colony appearance were recorded. Colonies were then clonally expanded and banked for the studies as described below. The phenotype of each ECFC clonal line was confirmed by its “cobblestone” monolayer morphology, determination of acetylated low-density lipoprotein uptake and Ulex europaeus lectin binding, capillary tubule-like network formation in Matrigel assay, expression of characteristic surface markers  and the absence of hematopoietic or myeloid surface antigens , as previously described  and genomic DNA and total RNA were isolated from the cell lysates using the AllPrep DNA/RNA micro kit .DNA samples were subjected to methylation analyses using the Illumina Infinium EPIC BeadChip Kit  at the University of Kiel, Institute of Clinical Molecular Biology (ICMB). Native DNA was sent from our lab to ICMB. The samples were coded for blinded sample analysis by ICMB personnel. The assay required 200 ng of original DNA sample as input. The DNA samples were denatured and neutralized and were then isothermally amplified in an overnight step. The whole-genome amplification uniformly increased the amount of the DNA sample by several thousand-fold without significant amplification bias. A controlled enzymatic process fragmented the amplified product. The process used endpoint fragmentation to prevent overfragmentation. The fragmented DNA was collected after an isopropanol precipitation and centrifugation at 4°C. The precipitated DNA was resuspended in hybridization buffer. Samples were applied to a BeadChip and separated by an IntelliHyb seal. The loaded BeadChip was incubated overnight in an Illumina Hybridization Oven. The amplified and fragmented DNA samples annealed to locus-specific 50-mers during hybridization. Unhybridized and non-specifically hybridized DNA was washed off and the BeadChip was prepared for staining and extension. Single-base extension of the oligos on the BeadChip, using the captured DNA as a template, incorporated detectable labels on the BeadChip and determined the genotype call for the sample. XStain occurred in a capillary flow-through chamber. The Illumina HiScan System scanned the BeadChip, using a laser to excite the fluorophore of the single-base extension product on the beads. The scanner recorded high- resolution images of the light emitted from the fluorophores. Processed data were sent back to our lab for analysis.1 , with protein-protein interactions restricted to experimental evidence, and without addition of interactors at the first or second shell. Biological processes or pathways were considered significantly enriched after correction for multiple testing if the false discovery rate (FDR) was FDR < 0.05. Enrichment of GO processes was additionally validated in an independent analysis using the newest version 14.0 of the PANTHER  resource (We compared methylated genes from preeclampsia versus uncomplicated pregnancy at passage 3 or passage 5 using the database of the Gene Ontology (GO) Consortium 1  for an e1  software tool . The standard setup for network analysis provided by the IPA core analysis was employed. The “molecules per network” parameter was set at 70, because of the large amount of input genes. All genes carrying single and multiple methylated CpG dinucleotides were used for the IPA analysis.p-values above 0.01 removing 3334 probes. Overall, no completely failed measurements were identified. Using general EPIC SNP list for filtering probes with SNPs as identified by Statistical analysis of the Illumina methylation bead array data was performed separately for cell culture passage 3 and 5 with the R software  following the analysis pipeline provided by the ChAMP package ; probes We first compared the methylation patterns of cord blood derived ECFC associated with preeclampsia and normal pregnancy controls by means of the Infinium EPIC BeadChip microarray. This methylome analysis identified a number of differently regulated CpG sites, the majority of which were hypomethylated in fetal ECFCs from preeclamptic compared to control pregnancies.In cell passage 3 a total of 346 hypermethylated and 920 hypomethylated CpG dinucleotides were identified in the preeclamptic group compared to the control group. Thirty out of 346 hypermethylated and 204 out of 920 hypomethylated CpGs mapped to regions not associated with any known gene. We identified 316 out of 346 hypermethylated and 716 out of 920 hypomethylated CpG dinucleotides that mapped in or near 954 known genes . Of thesIn cell culture passage 5 we found 555 hypermethylated and 1807 hypomethylated CpG dinucleotides in the preeclamptic group compared to the control group. Here, 43 out of 555 hypermethylated and 351 out of 1807 hypomethylated CpGs mapped to regions not associated with any known gene. We identified 512 out of 555 hypermethylated and 1456 out of 1807 hypomethylated GpG mapped in or near 1719 known genes . Analogo-16), more specifically in the positive regulation of RNA metabolic processes , in cellular protein modification processes , and in the positive regulation of transcription . Consistent with this, the protein-protein interaction clusters with most nodes were observed for proteasomal function, RNA transcription and pre-mRNA splicing (-9), cellular nitrogen compound metabolism , adherens junction assembly , chromatin modification , cellular response to growth factor stimulus  and tube morphogenesis . These observations point to a broad spectrum of enriched cellular developmental processes. KEGG pathway analyses indicated enrichment for 33 biological pathways, with the most significantly enriched pathways related to cell-cell interaction , Wnt signaling  and mTOR signaling   . Estroge-9), cell surface receptor signaling , protein phosphorylation  and cell cycle   , S4. Aga × 10-7) .We also performed IPA to evaluate and classify genes influenced by significantly altered CpG regions. IPA recognized 552 out of 954 injected genes in cell culture passage 3 and 1038 out of 1719 injected genes in cell culture passage 5. For the creation of a gene network, only the known genetic associations and linkages for mammalian endothelial cells from the IPA database were used. Both the significantly altered CpGs from preeclampsia compared to the control group from cell culture passage 3 and passage 5 were read and evaluated in parallel manner.CDKN1A: hypermethylated at 1 position, EGFR: hypomethylated at 1 position, KRD: hypermethylated at 1 position, S1PR1: hypermethlyated at 1 position, SMAD3: hypomethylated at 1 position, SMAD7: hypermethylated at 1 position). The most important canonical pathways of the altered genes are summarized in The highest-ranking network for cell culture passage 3 was the “Cardiovascular System Development and Function, Organizational Development, Cellular Development” network . This neSMAD3 was significantly hypomethylated and SMAD7 hypermethylated. SMAD activity is also part of the second pathway called “Cell Cycle: G1/S Checkpoint Regulation.” For instance, preeclampsia was associated with a hypermethylation of CDKN1A, encoding the cyclin dependent kinase inhibitor p21Waf1/Cip1. Further pathways in web 1 included “Sphingosine-1-phosphate Signaling” and “ERK5 Signaling.”The canonical pathway analysis of the IPA data yielded 2 main webs . The mosThe second web was dominated by glucose metabolism, especially glucose and glucose-1-phosphate degradation and GDP-glucose biosynthesis.Patterns in cell culture passage 5 were similar to cell culture passage 3. Again, the highest-ranking network was “Cardiovascular System Development and Function, Organizational Development, Cellular Development” , comprisAmong all samples irrespective of pregnancy outcome group, a total of 1096 CpG regions were significantly altered in cell culture passage 5 compared to cell culture passage 3. Of these, 770 were gene-associated. Interestingly, only five gene-associated altered CpG regions, which were detectable in cell culture passage 3, were no longer detectable in passage 5. All other CpGs altered in passage 3 were also significantly altered in passage 5. The overlap between differentially methylated coding genes in passage 3 cells and passage 5 cells and the ratio of the coding genes in passage 3, passage 5 and passages 3 + 5 in percent is shown in The molecular and cellular dysfunctions associated with preeclampsia are still largely unknown. Here we compared the global DNA methylation profiles of fetal ECFCs from uncomplicated pregnancies and pregnancies complicated by preeclampsia. We identified 954 genes in passage 3 and 1719 genes in passage 5 with significantly differentially methylated CpG sites between the two groups. Almost all changes between the group with preeclampsia and the control group detected in cell culture passage 3 were also confirmed in cell culture passage 5 (1260 out of 1266), indicating that the methylation differences in passage 3 are stably propagated and methylation dysregulation accumulates at later passages. Several studies showed that higher cell passage numbers lead to more inconsistent production of certain biomarkers and methylation levels at least in cancer cells and endocrine cells . An analHypermethylation predominantly results in a decrease in gene expression whereas hypomethylation tends to be gene activating . InteresOur results for both GO biological processes and protein-protein interaction networks indicated that ECFCs from preeclamptic pregnancies are differentially methylated in regions corresponding to a broad range of processes regulating cell metabolism, transcription and cell cycle, with a likely role for protein networks involved in proteasomal, transcriptional and pre-mRNA splicing processes. KEGG pathway analyses pointed to differential regulation of cell-cell interaction, Wnt signaling and mTOR signaling. In regard to preeclampsia, these pathways might be expected to negatively impact the capacity for endothelial development/repair and trophoblast invasion. Our results from IPA, restricted to interactions in mammalian endothelial cells, revealed “Cardiovascular System Development and Function, Organizational Development, Cellular Development” as the highest ranking network.The methylation pattern of fetal ECFC reveals a unique epigenetic signature which may represent a close link to cardiovascular system development and function and contribute to the cardiovascular risk of offspring born of preeclamptic pregnancies. As this is a study on fetal ECFC we can imagine that epigenetic modules may act as a molecular readout of cumulative cardiovascular risk factor exposure, with implications for the improvement of clinical risk prediction in the future. However, this has never been proven and follow up studies on these children from preeclamptic pregnancies in a larger cohort are clearly necessary. Comprehensive reviews and studies also suggest that changes in DNA methylation states contribute to the regulation of biological processes underlying cardiovascular disease, such as atherosclerosis, hypertension, and inflammation  and seveStudies on the methylation of human gestational tissue, e.g., placental tissue or cells derived from umbilical cord blood are only at the beginning. Therefore, this project is novel in both the cells studied and the implications of fetal progenitor cells for pathogenesis of preeclampsia and its long-term consequences for the offspring. An epigenetically modified endothelial precursor could affect both normal feto-placental vascular development and repair capacity and might influence the subsequent health of the offspring. Although implications for maternal pathogenesis of preeclampsia can not be inferred by our data, there is evidence that fetal ECFCs have a role in normal placental vasculogenesis and further that these cells traverse the placenta and home to sites of vessel formation in the maternal uterine microvasculature .Our data, while in need of further exploration, suggest involvement of certain pathways in the epigenetic imprinting of preeclampsia in utero. The main canonical pathway implicated deals with embryonic stem cell pluripotency and is regulated by extrinsic and intrinsic factors. Here the regulation of TGF-beta signaling via SMAD proteins is in center. The hypo- and hypermethylation of SMAD coding genes changes in cell differentiation associated with preeclampsia Linked through SMAD activity a second important pathway for cell cycle checkpoint regulation was observed. The key components involved are the cell cycle kinases and the transcription complex. The bioactive sphingosine-1-phosphate has been linked to a wide spectrum of biological processes, including cell growth, survival and motility and forms the bridge to pathway 3. The hypermethylation of this central gene in this pathway can possibly lead to a reduction of cell survival and migration capacity via the described pathway . The ligGiven the potential utility of ECFCs in cell-based therapies, a subanalysis focused on the comparison of two different cell culture passages to assess whether the cell culture passage affects the methylation pattern. Cell culture passage 3 corresponds to the earliest time at which a sufficient number of cells can be harvested after the initial isolation. Passage 5 was chosen as a comparison group, as ECFC at this cell age are commonly used for functional studies. Although the results in cell culture passage 3 and 5 were similar in terms of both the patterns of the gene networks and the pathways, some differences were nevertheless detected. The differentially methylated genes by cell culture passages could be assigned to cellular development, function and maintenance. The canonical analysis revealed pathway differences which regulate signal transduction, cell behavior and differentiation, proliferation and apoptosis. Based on these results, it can be interpreted that the culture of ECFC leads to a change in the methylation profile primarily of cell culture specific pathways. As almost all differences between control group and preeclampsia group detected in cell culture passage 3 were also confirmed in cell culture passage 5 (1260 out of 1266), the gene modification caused by preeclampsia appeared to be largely stable through higher cell culture passages.IPA results suggest that there are similarities between epigenetic changes in preeclampsia and cardiovascular disease outside the setting of pregnancy. Cardiovascular diseases are complex multifactorial and polygenetic, with interaction of numerous genes, genetic variants, and environmental factors contributing to development . In atteAs a note of caution, the IPA results were obtained from an endothelium-restricted database of interactions, and thus the emergence of a cardiovascular pathway could be partially attributed to these presettings. However, our unrestricted STRING analyses also identified clear links to the cardiovascular system, such as the GO term “tube morphogenesis” that includes angiogenesis, or the Wnt signaling pathway known to regulate the mobilization and proliferation of cells in endothelium and epicardium in an ischemic heart , thus adThe altered methylation profile of fetal ECFC may have been present prior to the onset of clinical manifestations of preeclampsia. If already present, they could contribute to risk of preeclampsia. However, the changes could also be caused by the preeclampsia milieu, i.e., by oxidative stress and DNA oxidation .To the best of our knowledge, our study is the first to explore whether the human intrauterine environment in preeclampsia is associated with abnormal fetal ECFC epigenetic gene network programming. These data suggest that fetal ECFC methylation status differs between preeclampsia and uncomplicated pregnancy. Our pathway analysis of the genes impacted by the methylation regulation may provide insights into possible mechanisms of pathogenesis. Future studies are needed to clarify which factors impact specific methylation changes, which methylation changes are involved in various obstetric complications, and what bearing these changes might have on offspring health.LB and FvV-H conceived and designed the experiments. LB and SvH performed the experiments. LB, TZ, TD, and HF analyzed the data. FvV-H, CH, and CvK contributed reagents, materials and analysis tools. LB, FvV-H, CH, and BS-H contributed to the writing of the manuscript.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.


Epigenetics is defined as heritable changes in genomic function that occur without changes in the DNA sequence. Accumulating evidence has revealed that epigenetics is critical in understanding the basic molecular mechanisms that take place in chromatin , and epigenetics is ultimately expected to provide insights into complicated biological processes such as stem cell maintenance and development and be applied to human diseases, including cancers.Chromatin is bound by a diverse group of proteins that dynamically regulate its structure by modifying, reading and erasing its basic components—histones and DNA. In particular, DNA and histones are primarily post translationally modified through various enzymatic activities, such as phosphorylation, acetylation, methylation, ubiquitination and O-GlcNAcylation. Combinations of these epigenetic modifications regulate chromatin dynamics by preparing specific loci for activation or repression ultimately governing the global chromatin architecture with regard to gene expression during the cell cycle and development.5-cytosine and N6-adenosine in DNA and at lysine and arginine residues in histones and chromatin-binding proteins. Further, there are three forms of methylation on lysine residues—monomethyl, dimethyl, and trimethyl. For arginine, monomethylated, symmetric and asymmetric dimethylated forms appear. Each form has a specific function, rendering it difficult to determine the purpose of their combinations. Also, the position of the affected lysine (or arginine) in the amino-acid sequence of histones influences its epigenetic function because methylation at various positions is programmed to recruit cognate reading proteins for repression or activation. Thus, methylation of DNA and histones is the most advanced epigenetic marker in the regulation of chromatin dynamics.Methylation is the most complicated epigenetic modification. Unlike other changes, methylation alone affects both DNA and histones: methylation occurs primarily at the CExperimental & Molecular Medicine, we focus on the regulation of cytosine methylation on DNA and lysine methylation of histones in stem cell pluripotency and cancer development.In this special issue of Mirang Kim  and Joseph Costello  review DNA cytosine methylation as an epigenetic marker of cellular memory. 5-Methylcytosine (5 mC) was initially discovered as a minor base that was introduced into the progeny strand during DNA replication. Later, a family of DNA methyltransferases  was discovered to catalyze the methylation of the 5-carbon of cytidine in CpG dinucleotides. They also introduce an advanced method for analyzing DNA cytosine methylation. By developing DNA methylation analysis and genomewide sequencing tools, we now understand that DNA cytosine methylation is dispersed throughout chromatin and serves as an epigenetic marker of memory in the development, differentiation and maintenance of cellular identity. Further, they discuss the epigenetic function of cytosine methylation in various types of stem cells and cancer cells.In the second issue, Myunggon Ko’s group  presents a comprehensive review on DNA demethylation in stem cells and cancer. DNA cytosine methylation has been considered a stable epigenetic modification, but the pioneering discovery by Anjana Rao’s group  that ten-eleven translocation 1 (TET1) oxidizes 5 mC to 5-hydroxymethylcytosine (5 hmC) confirms that cytosine methylation is dynamically modulated during stem cell differentiation and development. Notably, three isoforms of TET can catalyze the successive oxidation of 5 mC to 5 hmC, 5-formylcytosine (5 fC) and 5-carboxylcytosine (5 caC), which differs from the reversible demethylation of methylated lysines in histones. It will be interesting to study why mammalian cells adopt a more complicated reactive pathway for 5 mC demethylation. In addition, the authors discuss the involvement of TET in cancers. Loss of TET function is common in various cancers, and thus the in-depth study of TET-mediated DNA cytosine oxidation should increase our understanding of how epigenetic dysregulation causes tumorigenesis.Histone lysine methylation is more complex than cytosine methylation in DNA. Thus, Jaehoon Kim’s group  summarizes all known enzymes that are involved in lysine methylation and demethylation and their functions in various processes. Several lysine methyltransferases catalyze methylation marks on chromatin loci, to which cognate proteins are recruited and eventually determine epigenetic function. Conversely, demethylases remove these marks to reset loci for second-round methylation or simply antagonize the methylation signal. Many methyltransferase-demethylase pairs have been identified in mammals, but no H3K79 demethylase has been discovered. The finding of an H3K79 demethylase will be necessary to understand the complete pairing system between histone lysine methylation and demethylation.Fei Lan’s group  focuses on the epigenetic function of histone demethylases in embryonic development. They first discuss how lysine demethylases regulate epigenetic states at specific genomic loci, including bivalent promoters, enhancers and repetitive elements, because the methylation state of these chromatin elements is linked to cell fate decisions. They also review the epigenetic function of histone lysine demethylases in various processes during embryonic development.In the final article, TaeSoo Kim’s group  and Stephen Buratowski  discuss the cotranscriptional mechanism by which histone methylation at H3K4 and H3K36 regulates transcription in cooperation with other histone epigenetic markers, such as acetylation and mono-ubiquitination. For instance, H3K4me3 and histone acetylation are enriched in the promoters of genes. H3K36me2/me3 are found preferentially in gene bodies, in which Eaf3-mediated recruitment of HDACs effects the deacetylation of histones during transcription. Given that cotranscriptional regulation of histone methylation and acetylation has been linked to many cancers, more detailed research in this field will help identify cancer-related genes and apply this to anticancer drug development.Experimental & Molecular Medicine, five review articles summarize the general concepts of methylation of histones and DNA and provide an overview of the current information on how methylation regulates stem cell maintenance, differentiation and cancer development.In this special issue of