Physiologic changes in pregnancy induce profound alterations to the pharmacokinetic properties of many medications. These changes affect distribution, absorption, metabolism, and excretion of drugs, and thus may impact their pharmacodynamic properties during pregnancy. Pregnant women undergo several adaptations in many organ systems. Some adaptations are secondary to hormonal changes in pregnancy, while others occur to support the gravid woman and her developing fetus. Some of the changes in maternal physiology during pregnancy include, for example, increased maternal fat and total body water, decreased plasma protein concentrations, especially albumin, increased maternal blood volume, cardiac output, and blood flow to the kidneys and uteroplacental unit, and decreased blood pressure. The maternal blood volume expansion occurs at a larger proportion than the increase in red blood cell mass, which results in physiologic anemia and hemodilution. Other physiologic changes include increased tidal volume, partially compensated respiratory alkalosis, delayed gastric emptying and gastrointestinal motility, and altered activity of hepatic drug metabolizing enzymes. Understating these changes and their profound impact on the pharmacokinetic properties of drugs in pregnancy is essential to optimize maternal and fetal health. Prescription and over-the-counter medications use is common in pregnancy, with the average pregnant patient in the US and Canada using more than two drugs during the course of their pregnancy . One reaFigure 1; Table 1).Pregnancy is associated with significant anatomic and physiologic remodeling of the cardiovascular system. Ventricular wall mass, myocardial contractility, and cardiac compliance increase . Both heStarting at 6-8 weeks of gestation and peaking at 32 weeks, maternal blood volume increases by 40–50% above non-pregnant volumes . This, cThe increase in total body water, blood volume, and capillary hydrostatic pressure increase significantly the volume of distribution of hydrophilic substrates. Clinically, a larger volume of distribution could necessitate a higher initial and maintenance dose of hydrophilic drugs to obtain therapeutic plasma concentrations. Additionally, because of the decrease in serum albumin concentrations and other drug-binding proteins during pregnancy; drugs, that are highly protein bound, may display higher free levels due to decreased protein binding availability, and thus higher bioactivity. For example, if a drug is highly (99%) bound to albumin in non-pregnant patients, a small drop in protein binding to 98% in pregnancy translates into doubling of the drug’s active fraction in pregnancy. Digoxin, midazolam, and phenytoin are examples of medications primarily bound to albumin .Due to the increase in estrogen concentrations in pregnancy, the respiratory system undergoes anatomic changes leading to increased vascularity and edema of the upper respiratory mucosa . This maPregnancy is associated with increase in tidal volume by 30–50%, which starts early in the first trimester. While the respiratory rate is not different compared to non-pregnant state, minute ventilation  is significantly increased, similarly, by 30–50%. These changes are mainly driven by the increase in progesterone concentrations in pregnancy . In addiWhen evaluating blood gases in pregnancy, it is important to note that the arterial partial pressure of oxygen (PaO2) is normally increased to 101–105 mmHg and that of carbon dioxide (PaCO2) decreased to 28–31 mmHg. These changes are mainly driven by the increase in minute ventilation described above. The drop of PaCO2 in the maternal circulation creates a gradient between the PaCO2 of the mother and fetus, which allows CO2 to diffuse freely from the fetus, through the placenta, and into the mother, where it can be eliminated through the maternal lungs . In addiThe effects of progesterone and relaxin on smooth muscles are also seen in the urinary system leading to dilation of the urinary collecting system with consequent urinary stasis, predisposing pregnant women to urinary tract infections . This isBoth renal blood flow and glomerular filtration rate (GFR) increase by 50%, as early as 14 weeks of pregnancy . The mecThe kidneys are also mainly involved in water and sodium osmoregulation. Vasodilatory prostaglandins, atrial natriuretic factor, and progesterone favor natriuresis; whereas aldosterone and estrogen favor sodium retention . AlthougIn pregnancy, the rise in progesterone leads to delayed gastric emptying and prolonged small bowel transit time, by ~30–50%. Increased gastric pressure, caused by delayed emptying as well as compression from the gravid uterus, along with reduced resting muscle tone of the lower esophageal sphincter, sets the stage for gastro-esophageal reflux during pregnancy . In addiDrug absorption is also decreased by nausea and vomiting early in pregnancy. This results in lower plasma drug concentrations. For this reason, patients with nausea and vomiting of pregnancy (NVP) are routinely advised to take their medications when nausea is minimal. Moreover, the increased prevalence of constipation and the use of opiate medications to ease pain during labor slow gastrointestinal motility, and delay small intestine drug absorption. This may lead to elevated plasma drug levels postpartum . The incThe increase in estrogen in pregnancy leads to increase in serum concentrations of cholesterol, ceruloplasmin, thyroid binding globulin, and cortisol binding globulin, fibrinogen and many other clotting factors . Serum aDrug metabolism is also altered in pregnancy in part secondary to elevated sex hormones and changes in drug metabolizing enzymes including those involved in phase I  or phase II  metabolism . CytochrWhite (WBC) and red blood cell (RBC) counts increase during pregnancy. The first is thought to be secondary to bone marrow granulopoiesis; whereas the 30% increase in RBC mass (250–450 mL) is mainly driven by the increase in erythropoietin production. The higher WBC count can sometimes make diagnosis of infection challenging; however normally the increase in WBC is not associated with significant increase in bands or other immature WBC forms . DespitePregnancy is a hypercoagulable state secondary to blood stasis as well as changes in the coagulation and fibrinolytic pathway such as increased plasma levels of clotting factors , fibrinogen, and von Willebrand factor. Fibrinogen increases starting in the first trimester and peaks during the third trimester in anticipation of delivery. Prothrombin and factor V levels remain the same during pregnancy. Whereas, protein S decreases in pregnancy, protein C does not usually change and thus can be assayed if needed in pregnancy. Free antigen levels of the protein S above 30% in the second trimester and 24% in the third trimester are considered normal during pregnancy . Anti-thPlasma iodide concentration decreases in pregnancy because of fetal use and increase in maternal clearance of iodide. This predisposes the thyroid gland to increase in size and volume in almost 15% of women. In addition to anatomic changes, the thyroid gland increases production of thyroid hormones during pregnancy. This is due to the up-regulation of thyroid binding globulin, which is the major thyroid hormone binding protein, by almost 150% from a pre-pregnancy concentration of 15–16 mg/L to 30–40 mg/L in mid-gestation. This massive increase is driven by the hyper-estrogenic milieu in pregnancy and reduced hepatic clearance. The net result is increase in total tetra-iodothyronin and tri-iodothyronin hormones (TT4 and TT3) in pregnancy. Despite the increase in total T4 and T3, the free forms of the hormones (fT4 and fT3) remain relatively stable or slightly decreased but remain within normal values and these patients are clinically euthyroid . The incThyroid stimulating hormone (TSH) decreases during the first half of pregnancy due to negative feedback from peripheral T3 and T4 secondary to thyroid gland stimulation by human chorionic gonadotropin (hCG). During the first half of pregnancy, a normal value of TSH is between 0.5–2.5 mIU/L . Other factors that affect thyroid hormones metabolism and levels in pregnancy include: (1) the increase in maternal renal iodine excretion (secondary to increase in GFR), (2) the higher maternal metabolic demands and rate during pregnancy, (3) the thyrotropic action of hCG which shares a similar α subunit with the TSH receptor and has a weak thyroid stimulating activity, (4) the increase in thyroid hormones transplacental transport to the fetus early in pregnancy, and (5) the increase in activity of placental type III 5-deiodinase (the enzymes that converts T4 to the inactive reverse T3; Profound physiologic and anatomic changes occur in virtually every organ system during pregnancy. These have significant consequences on the pharmacokinetic and pharmacodynamic properties of various medications when used by pregnant women. Data are lacking on the implications of these changes on variety of therapeutic agents, and future research is desperately needed.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hlx was first detected via is expression in hematopoietic cells of the myelomonocytic and B lymphocyte lineages and further studies indicated a potential role for Hlx in hematopoietic differentiation [HLX expression correlates with poor overall survival. These findings, combined with the observation that HLX knockdown results in an inhibition of growth and clonogenicity of leukemia cells, suggest that HLX may be a novel promising prognostic and therapeutic target in AML.Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor clinical outcome in most subtypes of the disease. Less than one third of patients achieve durable remission with current treatment regimens, and prognostication and risk stratification are challenging. We recently reported that a new non-clustered homeobox gene, H2.0-like homeobox (HLX), regulates early hematopoiesis and promotes AML in mice and humans . Hlx wasntiation . We now Δ/Δ model of AML [Hlx expression, suggesting that Hlx overexpression is an early event in leukemogenesis and Hlx is involved in malignant transformation [Recent experimental evidence has shown that acute myeloid leukemia (AML) arises from transformed immature hematopoietic cells following the accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells (HSC) and progenitors . In ordel of AML . Using tormation .in vivo we utilized a competitive, congenic transplantation model. Experimental elevation of HLX in HSC resulted in near complete depletion of early HSPC populations  and 16-fold enrichment of myeloid progenitors with a surface phenotype slightly past the GMP stage (CD45+Kit−CD34−CD44highCD49bhighCD11bmid). Overexpression of HLX in HSPC in vitro led to a myeloid differentiation block and to formation of phenotypically similar, aberrant myeloid progenitors with unlimited serial clonogenicity. These results indicate that HLX suppresses the function of normal immature HSC and progenitors but leads to a differentiation block and confers unlimited clonogenic capacity at the level of phenotypically more mature progenitors. Because the loss of HSC did not seem to be mediated by induction of apoptosis or necrosis, we speculate that HLX exerts this dual role by triggering initial differentiation of HSC followed by suppression of terminal differentiation at a more committed progenitor level. Genetic in vivo modeling using an inducible system will be required to definitively clarify the role of HLX elevation at different stages of hematopoietic stem and progenitor cell differentiation in vivo.To assess the effects of HLX overexpression in wild-type HSPC Hlx may function in concert with cofactors that could confer cell type specificity to the effects of HLX overexpression and contribute to leukemic transformation. Combinatorial genetic modeling with other disease alleles in AML will be instrumental to uncover the exact role of Hlx in AML pathogenesis.Of note, although overexpression of HLX leads to the formation of immortalized myeloid progenitors with unlimited serial clonogenicity, we so far have not observed development of overt leukemia upon transplantation, suggesting that HLX elevation alone is not sufficient for full transformation. Like other homeobox genes, HLX is significantly overexpressed (2 to 16 fold) in more than 80% of patients with AML, and this occurs across all major disease subtypes and is independent of blast counts. Higher levels of HLX are associated with poor overall survival in 3 different, large cohorts of AML patients . Importantly, the prognostic value of HLX holds up in a multivariate analysis, indicating HLX overexpression and prognostic value is an independent phenomenon across several molecular subsets of patients. Notably, high HLX expression status seems to overrule several mutations which have been associated with favorable prognosis. Patients with CEBPA or NPM1 mutations for instance only have favorable overall survival if HLX is low, but a poor prognosis if HLX is expressed at high levels. While this needs to be confirmed in independent and, ideally, prospective studies, these observations suggest broad importance of HLX for the disease biology of AML, and a role for determining HLX expression levels in the prognostication of AML patients.Several HOX genes are expressed at high levels in subtypes of AML. Important roles in leukemic transformation have been demonstrated for specific members of the HOX-A and HOX-B cluster, and the nonclustered homeobox gene CDX2 was recently reported to be implicated in leukemogenesis -10. HoweHLX overexpression in both pre-leukemic and bulk AML cells, we studied the effects of HLX reduction in both murine and human AML cells. ShRNA-mediated reduction of HLX significantly inhibits leukemic growth and clonogenic capacity, and overcomes the differentiation block of AML cells. We found that HLX regulates a set of genes which likely mediate its leukemia-promoting functions, including candidates BTG1 (B-cell translocation gene 1) and PAK1 (p21 protein-activated kinase 1). Intersecting the gene expression data from our in vitro overexpression and inhibition studies with data from genome-wide expression analysis of a large cohort of human AML patients, we identified an HLX-dependent molecular signature of genes, which again included PAK1, with prognostic value for AML patients.Based on our findings of the significance of In summary, our studies have identified HLX as a novel key transcription factor involved in the regulation of early hematopoiesis and AML pathogenesis, and suggest HLX as a promising new therapeutic target in AML. Additional mechanistic studies will be required to identify the transcriptional targets of HLX in HSPC and to define its functionally critical downstream pathways, which will improve our understanding of the precise role of HLX in HSPC biology and malignant transformation, and may offer additional opportunities for targeted therapeutic intervention.


Evidence for the causal contribution of genotype to inherited epitypes and epigenetic risk comes primarily from two different kinds of studies discussed herein. The first and direct method of research proceeds by the examination of the transgenerational inheritance of epitype and the penetrance of phenotype among genetically related individuals. The second approach identifies epitypes that are duplicated (as DNA sequences are duplicated) and evolutionarily conserved among repeated patterns in the DNA sequence. The body of this article summarizes particularly robust examples of these studies from humans, mice, Arabidopsis, and other organisms. The bulk of the data from both areas of research support the hypothesis that genotypes predispose the likelihood of displaying various epitypes, but for only a few classes of epitype. This analysis suggests that renewed efforts are needed in identifying polymorphic DNA sequences that determine variable nucleosome positioning and DNA methylation as the primary cause of inherited epigenome-induced pathologies. By contrast, there is very little evidence that DNA sequence directly determines the inherited positioning of numerous and diverse post-translational modifications of histone side chains within nucleosomes. We discuss the medical and scientific implications of these observations on future research and on the development of solutions to epigenetically induced disorders.Clear cause-and-effect relationships are commonly established between genotype and the inherited risk of acquiring human and plant diseases and aberrant phenotypes. By contrast, few such cause-and-effect relationships are established linking a chromatin structure  with the transgenerational risk of acquiring a disease or abnormal phenotype. It is not entirely clear how epitypes are inherited from parent to offspring as populations evolve, even though epigenetics is proposed to be fundamental to evolution and the likelihood of acquiring many diseases. This article explores the hypothesis that, for transgenerationally inherited chromatin structures, “ The inheritance of numerous genetic risk factors for human and plant diseases as well as biotic and abiotic stress susceptibility phenotypes are well established -6. Partiskeletal-muscle ryanodine-receptor gene (RYR1) that causes congenital myopathies and the MutL Homolog 1 gene (MLH1) that causes increased risk of colorectal or endometrial tumors, which are discussed in the following section.Epigenetics is cited as contributing to the risk of acquiring numerous diseases and aberrant phenotypes in human and plant populations based primarily on correlations between changes in chromatin structure and penetrance of the undesired phenotype -10. TherInherited risk epitypes should evolve in populations in ways similar to the evolution of genotypes . The proThis study explores the cause-and-effect relationships among genotype, epitype and phenotype, where the epitype of a single gene or an entire genome is defined as its various cis-linked chromatin structures Figure  19]. Th. Th19]. A working hypothesis that emerged from a preliminary examination of the inheritance and evolution of various epitypes  is that It will be useful at this point to make the distinction between the transgenerational epigenetic inheritance among parents and offspring and the somatic inheritance between mother and daughter cells within developing tissues and organs ,26,41-45To test this hypothesis our discussion is focused on finding evidence for gene-specific epitypes that supports or rejects cause-and-effect relationships between genotype, epitype and phenotype. Some of the strongest evidence we found, for or against our hypothesis, is summarized in Table The second and less direct approach (B) searches out epitypes that are duplicated, as DNA sequences are duplicated, and examines multiple copies of DNA sequence and epitype that have been evolutionarily co-conserved. This approach establishes an unambiguous, and in many cases a statistically significant, correlation of a particular epitype with highly reiterated DNA sequence motifs, and/or examines the conservation of an epitype among duplicated gene sequences. With this strategy, the evolutionary conservation of epitypes among conserved sequences is used as a filter to identify epitypes that were transgenerationally inherited ,51. In oRYR1 and MLH1 genes, resulting in muscle myopathies and cancer, respectively. However, the complexity of the data on these two systems highlights the problems that arise when trying to establish such cause-and-effect epigenetic relationships, particularly in humans.Only a handful of studies have succeeded in fully demonstrating that the transgenerational transmission of an epitype produces changes in known target gene expression, which results in a disease or its risk of penetrance . Two of the best examples from humans concern chromatin structure at the RYR1: Genetic mutations causing a loss of expression of RYR1 function are associated with susceptibility to malignant hyperthermia and congenital myopathies  [ryr1 allele [RYR1 allele appears to account for the loss of functional RYR1 protein expression. For example, among a sampling of 11 patients with the disease, six patients showed tissue-specific silencing of the maternally inherited functional RYR1 allele, which apparently resulted from cytosine hypermethylation of that allele [RYR1 silencing and onset of an epigenetically determined form of the disease  disease) -54. Howe1 allele . The epit allele . Treatine Figure . However disease)MLH1: The human MLH1 gene encodes a homologue of the bacterial mismatch DNA repair protein MutL and, hence, MLH1 is classified as a tumor suppressor. Hypermethylation of DNA cytosine residues and silencing of a particular functional MLH1 alleles ) [MLH1 allele and its transcriptional silencing is found in most organs and tissues of individuals who also have hypermethylation of this MLH1 allele in their tumors. Hence, one might expect that this heritable epimutation resulted from the transgenerational inheritance of this epitype. However, studies of the children of these individuals generally show loss of hypermethylation and loss of silencing of this MLH1 allele in the first generation of transmission. Out of several individuals examined, only in one case was the epitype of hypermethylation and silencing inherited through the male parent to the individual with the disease. The MHL1 silencing phenotype in females with colorectal cancer was associated with a particular CG island centered at −93 bp from the start of transcription in a particular MHL1 allele containing a SNP, -93 SNP, in this region as illustrated for the more general case in Figure MHL1 epitypes of aberrant nucleosome position and cytosine methylation appear to be dependent upon the genotype of the epigenetically silenced MHL1 allele  m (SNP)) , when pam (SNP)) ,58. The m (SNP)) . The hypfor MHL1 .. A few of the best cases with solid supporting evidence for a relationship between epitype and phenotype will be summarized.There are considerably more robust examples of the transgenerational epigenetic inheritance from model genetic organisms and wild plants, where it is easier to analyze aberrant epitypes and associated phenotypes through multiple generationsAGOUTI: In mice, the secreted AGOUTI peptide functions normally as a paracrine regulator of pigmentation. However, the dominant constitutive expression of the AGOUTI gene also targets changes in the hypothalamus and adipose tissues and this aberrant expression causes obesity. Hypomethylated, transcriptionally active dominant epialleles of the agouti gene may be maternally inherited through meiosis. Variation in the penetrance of different active epialleles generates a distribution of offspring from abnormal yellow (agouti) obese mice to darker mice with normal amounts of fat [agouti  that are associated with a high penetrance of the yellow coat color and obesity phenotypes have promoter-containing retrotransposons positioned just upstream of the natural Agouti promoter [agouti and constitutive AGOUTI protein expression. However, a recent detailed examination of the DNA methylation profiles of active and silent alleles suggest that hypomethylation alone may not fully account for the complex ectopic expression of Agouti [genotype predisposes epitype and aberrant phenotype. It would not be surprising to find a shift in promoter nucleosome position resulting from the various retrotransposon insertions contributing to the causative epitype.(3) s of fat -65. Sevepromoter ,67. For f Agouti . NonetheAXIN1-FUSED: Axin1 is an inhibitor of Wnt (a hybrid of the names for Wingless and Integration1) signaling that regulates embryonic axis formation in deuterostome animals. In mice, Axin1 is the product of the mouse Fused locus. Some murine alleles of Axin1-fused (Axin1Fu) show variable and stochastic expression levels, where high expression of a hypomethylated allele correlates with an abnormal kinked tail. Highly penetrant Axin1Fu alleles contain an upstream retrotransposon or retrotransposon-mediated DNA rearrangement that alters chromatin structure and contributes to dominant transcript expression [Axin1Fu expression and risk of abnormal tail development [Axin1Fu expression and halved the incidence of kinked tails. Conversely, treatment of mice with the histone hyperacetylation agent Trichostatin A increased Axin1Fu expression and the frequency of a kinked tail phenotype [Axin1Fu/+ embryos shows that dimethylation of lysine-4 on histone H3 (H3K4Me2) as well as acetylation of lysine-9 on histone H3 (H3K9Ac) correlate with penetrant alleles [Axin1Fu gene expression and the kinked tail phenotype. By this view, genotype determines the nucleosomal epitype, which produces other aberrant hypomethylation and histone PTM epitypes, leading to increased gene expression and the novel kinked tail phenotype  pression ,69. An aelopment -72. The CNR: The tomato colorless non-ripening gene CNR encodes a homolog of the animal SQUAMOSA promoter binding protein (SPB box protein). CNR is essential to normal carotenoid biosynthesis and fruit ripening in the tomato and provides one of the best examples of a stable transgenerationally inherited epitype producing an abnormal phenotype. The natural epialleles of CNR in the tomato Lycopersicon esculentum contain 18 methylated cytosine residues  in a 286 bp contiguous region [CNR gene leads to colorless tomatoes low in carotenoids  s region . HypermeCYCLOIDEA: The perennial plant in which CYCLOIDEA was first identified, Linaria vulgaris , normally produces yellow and orange asymmetric flowers composed of three petals of different morphologies. “Mutant” plants are found in wild populations with aberrant abnormally symmetrical “peloric” flowers that are comprised of five evenly arrayed petals of similar morphology. Plants with these aberrant flowers were first characterized by Carl Linnaeus 260 years ago and collected as herbarium specimens [cyc epialalele is relatively stable, follows Mendelian segregation and, hence, appeared upon initial investigation to be a normal mutant allele. However, gene silencing always maps to a DNA polymorphic cyc308G allele with a single nucleotide polymorphism in an unmethylated region 308 nt downstream of the stop codon and never to the more common wild type CYC308A allele. Peloric individuals are homozygous recessive for the cyc308G allele with both copies being hypermethylated and completely silenced for RNA expression. Thus, it is reasonable to conclude that genotype predisposes epitype, gene silencing, and the peloric phenotype (Table (6) pecimens . The pelpecimens . Inheritpe Table C,D.Histone H3K4Me2 demethylase erases epigenetic memory in each generation: A number of histone PTMs such as H3K4Me2 are acquired during transcription and are associated with active genes [Caenorhabditis elegans  are found throughout the four eukaryotic kingdoms and a number of these genes are known to be essential for normal organismal development [(7) ve genes . These es Figure F. Removagermline ,78. Withelopment -81. The Inheritance of quantitative epigenetic trait loci. Two separate genome-wide epigenetic studies demonstrate that multi-generational inheritance of complex traits such as flowering-time, plant height, biomass, and bacterial pathogen resistance behave as quantitative epigenetic trait loci in Arabidopsis thaliana [ddm1/ddm1 that is highly compromised in a number of phenotypic traits due to DNA hypomethylation. DECREASED DNA METHYLATION1 (DDM1) is a Swi2/Snf2-like DNA-dependent ATPase chromatin remodeler required for most DNA cytosine methylation. The ddm1/ddm1 line was backcrossed to wild type, and this heterozygous F1 ddm1/DDM1 was backcrossed to wild type again and screened to obtain hundreds of separate DDM1/DDM1 lines. These lines were selfed to establish hundreds of epiallelic recombinant inbred plant lines (epiRILs) [DDM1/DDM1 epiRILs displayed aberrant morphological phenotypes affecting flowering time and plant height, among other phenotypes. They assayed 22 epiRILs for the methylation of 11 candidate genes that are normally cytosine hypermethylated, but are hypomethylated in ddm1. Six alleles showed partial remethylation and five alleles were completely remethylated producing the identical complex epitype for this later gene set to wild type. Control genes that were previously unmethylated remained unmethylated.(8) thaliana ,82,83. TepiRILs) . For sevFWA gene, for which the ectopic expression of the hypomethylated epiallele in ddm1 parental plants produces strong late flowering phenotypes [FWA and expressed high levels of FWA transcripts, accounting for their phenotype. Hence, out of hundreds, only a few of the epiRILs escaped from the remethylation of FWA, when DDM1 was restored.In one particular example, Johannes and colleagues  followedenotypes . All of de novo remethylation of these completely unmethylated loci is encoded in the Arabidopsis genome and remethylation did not require hemi-methylated DNA templates to be newly inherited. These data suggest that genotype predisposes this global cytosine methylation epitype.In summary, aberrant DNA methylation epitypes at many loci and the resulting changes in downstream molecular and developmental phenotypes appear to be transgenerationally inherited. Most genes regain wild type methylation patterns and phenotypes within a few generations and the restoration appears to be sequence specific. Hence, the genetic machinery necessary for the Reprogramming of DNA cytosine methylation by double stranded dsRNAs. The 5´-methylation of DNA cytosine residues occurs in three sequence contexts: 5MeCG, 5MeCHG and 5MeCHH  H Figure C. A numbde novo DNA cytosine methylation. If pure epialleles are truly independent of genotype, then they stand as strong evidence against our hypothesis. The well studied hypermethylation and silencing of wild-type CNR and RYR1 alleles fit the definition of pure epialelles. Schmitz and colleagues [Epialleles in heterochromatic DNA that display obligate DNA cytosine methylation always remain methylated due to the presence of large numbers of transposable elements in various orientations producing dsRNA that promote a strong RNA interference response and adjacent target gene remethylation . Genes wlleagues  examinedOne relevant question for this discussion is the following: are ostensibly pure epialleles truly independent of genotype, or are they simply facilitative epialleles for which we have not yet identified the associated cis- or trans-acting genes making dsRNAs that program inherited CG, CHG and CHH methylation epitypes? There is recent evidence supporting the latter interpretation that we now summarize.ddm1/ddm1 plant backcrossed to wild type. Those loci that were remethylated after a few generations in the epiRILs contained cytosine rich gene sequences that were highly complementary to the sequence of siRNAs. Those loci with similar cytosine rich composition for which they could not identify complementary siRNAs remained hypomethylated. siRNAs attract RNA interference (RNAi) and DNA methylation machinery to complementary DNA sequences and thereby template sequence-related DNA methylation [de novo methylation described by Schmitz and colleagues [Despite being generated through slightly different mechanisms, many classes of small RNAs  are known to template the remethylation of cytosine in different sequence-specific contexts Figure C for thehylation . This shhylation  raises fhylation ,89,93. Fhylation -98. The lleagues . ApproprReprogramming epitype during somatic cell nuclear transfer. In most of the above examples, genotype determines the likelihood, but not the certainty, of particular epitypes and phenotypes being displayed, because the same DNA sequence may be flexibly reprogrammed into many different chromatin conformations. It is fundamental to epigenetics that as cell types differentiate the same DNA sequence may display multiple epitypes and some epitypes may be more or less stable than others. An interesting example of a variety of epitypes descending from one genotype comes from research using somatic cell nuclear transfer (SCNT) to produce identical or genetically modified laboratory and farm animals. SCNT is achieved by transplanting a somatic cell nucleus into a functional embryonic cell capable of forming a viable organism. This technology has met with modest success, generating cloned mice, rabbits, pigs, sheep, cows and more, but the efficiency of obtaining viable healthy offspring is low. Even if genetically modified embryos are established in surrogate mothers, developmental abnormalities and spontaneous abortions are common. A major limitation to obtaining relatively normal full-term development appears to be variations in epigenetic reprogramming of the transplanted nucleus [10)  nucleus -102. The nucleus ,104. Wit0  nucleu nucleus -109. HowIf genotype pedisposes epitype then a reasonable corollary is that some transgenerationally inherited chromatin structures should align with particular DNA sequence motifs and be passed on to duplicate gene copies. In this model, the range of possible epitypes for a sequence would evolve by gene duplication and mutation in parallel with genotype ,51. RapiShort DNA sequence repeats such as RRRRRYYYYY determine the bending and positioning of DNA around the nucleosome. More than 30 years ago, Trifonov and his colleagues [RRRRRYYYYY)  lleagues ,111 pres lleaguesGCCCCC-G ,113. Thethe time . Becausethe time . These dThe geneome-wide positioning of H2AZ nucleosomes. The histone variant H2AZ and likely other histone variants are inserted into assembled nucleosomes by histone variant exchange complexes (HVE) such as SWR1  Arabidopsis, and yeast. These three genes are estimated to have diverged from a common gene ancestry in the eudicot plant lineage in the last 100 million years and stand alone in their own distinct clade, among more than 100 other MADS box genes in Arabidopsis that do not have a bimodal distribution of H2AZ nucleosomes. These data are consistent with the bimodal distribution of H2AZ being inherited following gene sequence duplication from an ancestral MADS gene [(2b) Further support for the concept that DNA sequence positions H2AZ nucleosomes comes from a comparison of duplicated genes in Arabidopsis. A single peak of H2AZ enriched nucleosome(s) is found at the 5´ end of nearly half of all plant, animal, and fungal genes that have been examined ,119-121.The genome-wide positioning of CENH3 centromeric nucleosomes. Recent experimental evidence demonstrates that CENH3 enriched centromeric nucleosome positions are determined by DNA sequence. Animal and plant centromeres are composed of a diverse variety of retroelements and repetitive satellites that generally appear unrelated in their DNA sequences. Numerous earlier studies of centromere and neocentromere sequences concluded that a distinct conserved DNA sequence was not essential to centromere activity. However, a very recent analysis of 100,000 centromeric histone CENH3 enriched nucleosomal DNA fragments from maize suggests that a 10 bp repeat of AA or TT dinucleotides contributes to determining the positioning of centromeric nucleosomes [(3) leosomes . The CENCytosine methylation in the human plasminogen gene family. In an attempt to show that epitypes and associated phenotypes can evolve by gene duplication and divergence, Cortese and colleagues [PLG) precursor gene family, encoding blood-clotting factors found only in hominids. Cytosine DNA methylation patterns are well conserved among seven CG sites located −171 to −378 nucleotides upstream from the start of transcription within all four PLG gene promoters  lleagues  comparedlleagues  also comHistone side chain modifications in human segmental sequence duplications. Barski and colleagues [visa versa. These data imply that histone PTM epitypes may not be the direct transgenerationally inherited “cause” of the phenotypes with which they are associated. Thus, these data on histone PTM epitypes at SDs do not support our working hypothesis. If these results are supported by more experimental studies, it will not mean that histone modifications are not useful epitypes for predicting risk, but that they may be further from the inherited cause of epigenome-induced pathologies than other epitypes such as nucleosome position and cytosine methylation. Histone PTMs are indeed important to somatic inheritance and development [(5) lleagues  publishelleagues  examinedelopment ,126.Nucleosome positioning and H3K4Me2 modifications in the HOXD cluster. There are six genes at the HOXD gene cluster  covering approximately 100,000 bp on human Chromosome 2. In human sperm, there are one or two spikes of general nucleosome occupancy and H3K4Me2-enriched nucleosome occupancy within each of the promoters of these genes, whereas the approximate 100,000 bp of 5´ flanking region is relatively free of nucleosomes [HOXD promoters cannot be determined from these data or compared to the results from Barski and colleagues [HOXD promoters in human sperm are similar to those for H2AZ-enriched nucleosomes among the FLC-related MADS genes in Arabidopsis shoot tissue [(6) leosomes   inibands -130. Forinibands . These sinibands ,132. Theinibands -135. HenAn examination of several examples of the direct transgenerational inheritance of epitype and the epitypes of duplicated and/or conserved DNA sequences revealed the complexities of determining cause-and-effect relationships among genotype, epitype and phenotype. However, in balance, there are robust experimental data supporting the hypothesis that “genotype predisposes epitype,” for some epitypes Table . In partcis-acting CG, CHG, and CHH sequences in the target gene and the sequence of trans-acting small RNAs. However, while histone PTM may be strongly correlated with epigenetically controlled phenotype, there is no evidence that any histone PTM is causal to transgenerationally inherited risk. Histone PTM epitypes may represent the effect of other epigenetic and genetic controls and may be principally important to somatic inheritance of epigenetic controls. The clear relationship between novel genotypes and many of the most robustly characterized inherited epitypes of nucleosome position and cytosine methylation is a recurrent theme in the literature of the most thoroughly studied genes under epigenetic control. This suggests that human and animal therapeutic treatments or plant and animal genetic breeding strategies that address harmful meiotically inherited epitypes should consider the possibility that there are genotypic causes predisposing these epitypes. If, for example, the environment of a developing somatic tissue  is influencing RNA sequence directed cytosine remethylation and gene silencing, drugs targeting downstream histone PTM epitypes of that gene may be less effective than ones addressing remethylation. Strategies directed at controlling gene expression by altering histone PTM epitypes may be useful if they target the gene or genes producing the disease’s phenotype. Finally, the undeniable influence of genotype on epigenetic controls leading to deleterious phenotypes has to be taken into account in a consideration of epigenetic risk, even if it confounds many current, working definitions of epigenetics.Based on this analysis, it is worth ranking the utility of various classes of epitype in estimating epigenetic risk. A risk pyramid linking the relationships of genotype and epitype with epigenetic risk phenotype is shown in Figure the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” [the study of mitotically and/or meiotically inherited changes in gene function that cannot be explained by the classical central dogma of molecular genetics”  allow us to quantitatively determine the precise genome-wide sequence-specific positioning of every nucleosome, methylated cytosine residue, and dozens of distinct histone PTMs in a genome. These epitypes may be correlated with the risk of cancer, behavioral disorders, pathogen susceptibility, or the role of aging and environmental factors on risk, as examples. The lower costs of genome-wide approaches is enabling the epitypes of larger populations of humans, laboratory animals, and plants to be examined in order to identify the epigenetic causes of complex diseases such as obesity, lupus, or pathogen susceptibility -140. Sec culture  and to e culture ,151. UnlThere is substantial evidence that altered epigenetic controls contribute to a variety of diseases ranging from cancer and developmental malformations to susceptibility to various forms of biotic and abiotic stress. We reviewed experimental genetic, epigenetic, cell biological, and biochemical data surrounding the transgenerational inheritance of several examples of well studied epigenome-induced pathologies and the contribution of conserved DNA sequence motifs to epitype. The preponderance of evidence suggests that genotypes predispose epitypes for most chromatin structures that are transgenerationally inherited and this relationship contributes to the penetrance of epigenetically controlled diseases. Genotypes influencing inherited epigenetic risk are often obscurely encoded in DNA sequence and small RNAs. Furthermore, the remethylation of DNA cytosine residues may only be reprogrammed at particular times in development and only in particular tissues such that a special effort may be required to identify and characterize these mechanisms. Some of the best characterized examples that were discussed herein suggest we are only just beginning to understand the molecular biology behind inherited epigenome-induced disorders. Finally, the paths to effective therapeutic development or to lowering epigenetic risk will be easier to trace out once we understand the mechanisms by which genotype predisposes epitype for a particular disease.aTrifinov did not have nucleosome specific DNA sequence data available 30 years ago.CNR, Colorless non-ripening; CENH3, Centromeric histone H3; CYC, CYCLOIDEA; DDM1, DECREASED DNA METHYLATION1; DMT, DNA methyl-transferase; dsRNA, Double stranded RNA; epiRIL, Epiallelic recombinant inbred plant line; FACS, Fluorescent activated cell sorting; FWA, FLOWERING WAGENINGEN; H3K4Me2, Histone H3 dimethylated at lysine4; H3K9Ac, Histone H3 acethylated at lysine-9; HVE, Histone variant exchange complexes; INTACT, Isolation of nuclei tagged in specific cell types; ISW, Imitation switch; MLH1, MutL Homolog 1 gene; piRNA, Piwi-interacting RNA; PLG, Plasminogen; PTM, Post-translational modification; R, Purine; RIL, Recombinant inbred line; RNAi, RNA interference; RYR1, Skeletal-muscle ryanodine-receptor gene; SCNT, Somatic cell nuclear transfer; SD, Segmental chromosome duplication; siRNA, Small inhibitory RNA; SMP, Single methylation polymorphism; SNP, Single nucleotide polymorphism; SPB box protein, SQUAMOSA promoter binding protein; SWI/SNF, Switch/sucrose nonfermentable; TBX, T Box; Wnt, Is a hybrid of the names for Wingless and Integration1; Y, Pyrimidine.The authors declare that they have no competing interests.RBM proposed the hypothesis that drove the content of the manuscript and wrote most of the manuscript. KJM helped with the conception of the manuscript and topics, designed the epigenetic risk pyramid, and contributed to the writing and fact checking. Both authors read and approved the final manuscript.


Brain aging is characterized by progressive loss of neurophysiological functions that is often accompanied by age-associated neurodegeneration. Calorie restriction has been linked to extension of lifespan and reduction of the risk of neurodegenerative diseases in experimental model systems. Several signaling pathways have been indicated to underlie the beneficial effects of calorie restriction, among which the sirtuin family has been suggested to play a central role. In mammals, it has been established that sirtuins regulate physiological responses to metabolism and stress, two key factors that affect the process of aging. Sirtuins represent a promising new class of conserved deacetylases that play an important role in regulating metabolism and aging. This review focuses on current understanding of the relation between metabolic pathways involving sirtuins and the brain aging process, with focus on SIRT1 and SIRT3. Identification of therapeutic agents capable of modulating the expression and/or activity of sirtuins is expected to provide promising strategies for ameliorating neurodegeneration. Future investigations regarding the concerted interplay of the different sirtuins will help us understand more about the aging process, and potentially lead to the development of therapeutic approaches for the treatment of age-related neurodegenerative diseases and promotion of successful aging. The brain, similar to other organs, undergoes a gradual decline in energy metabolism during aging . Since nSaccharomyces cerevisiae, and named silent information regulator 2 (Sir2) in lower organisms  which are found in different subcellular locations, including the nucleus , cytosol (SIRT2), and mitochondria . Most of the studies have described a key role for SIRT1 in regulating the metabolic response to CR , a dietaIn this review, we focus on the effects of SIRT1 and SIRT3 on metabolic regulation and their anti-aging activity in brain, and further discuss potential pharmacological approaches to remedy and prevent age-associated neurological disorders by targeting sirtuins.During mouse embryogenesis, SIRT1 is highly expressed in the brain, spinal cord, and dorsal root ganglion, with the peak expression at E4.5 . SIRT1 iSIRT1 contains 747 amino acids in humans, with a predicted molecular weight of 81 kDa and a measured one of 120 kDa. In addition to histones, SIRT1 also deacetylates a number of non-histone substrates, including p53 and peroxFigure 1).The effects of SIRT1 on longevity rely on its enzymatic activity of deacetylation of histone and non-histone substrates. While the deacetylation of histones leads to their interaction with DNA and consequent gene silencing , the deaThese metabolic changes and the cytoprotective involvement of CR are generally considered to occur in non-neural organs, such as the liver, pancreas, muscle, and fat tissues . HoweverSIRT1 has been found to delay aging and promote longevity by regulating the activity of key cellular proteins like p53, FOXO and Ku70 that are involved in either apoptotic processes or cellular repair mechanisms. SIRT1 may thus promote health and longevity partly by either decelerating cell death and/or by boosting repair mechanisms in the cells .It has become increasingly evident that the salutary effects of the CR, are in part due to the promotion of sirtuins . The expSIRT1 interacts with and modulates other key factors involved in mammalian aging, such as NF-κB that controls a low-grade systemic inflammation along with human aging process-inflamm-aging , mammaliAging process is also regulated by autophagy. It has been identified the signaling pathways that regulate autophagic degradation and SIRT1 is a potent regulator of autophagic degradation , SIRT1 c+, respectively, AMPK signaling is involved in the regulation of energy metabolic homeostasis , Parkinson’s disease (PD), Wallerian neurodegeneration, Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS). The pathomechanisms involved in these disorders involve common biochemical pathways and processes, including protein misfolding, oligomerization, and aggregation, proteolysis, post-translational modifications, mitochondrial dysfunction, abnormal metabolic processes, and proinflammatory and proapoptotic responses that we discuss in the next section.Wallerian degeneration refers to axonal death and degradation after focal injury, followed by breakdown of myelin sheath. The neuroprotective effect of SIRT1 against Wallerian degeneration was first discovered in wlds transgenic mice . These mAlzheimer’s disease is a terminal neurodegenerative disease, causing neuronal death and brain atrophy. The pathological hallmarks of AD are the intracellular tangles and extracellular plaques in brain. The tangles, also known as neurofibrillary tangles, are formed by accumulation of insoluble tau proteins, and the plaques are deposits of β-amyloid (Aβ) peptides, typically consisting of 40–42 amino acid residues.The protective effect of SIRT1 against AD was initially observed in CR studies, where CR reduced Aβ and plaque generation in the brains of transgenic AD mice . Similarin vitro and in vivo  in neurodegenerative diseases relies overwhelmingly on developing new strategies and designing molecules based on the sirtuin chemistry and molecular pathways activated by SIRT1. A very recent study performed by Figure 2).This section summarizes the studies on the role of mitochondrial SIRT3 in energy metabolism and protection against oxidative stress and age-associated dysfunction  are generated and the intrinsic apoptotic signaling pathway is initiated . The funl matrix . Since ml matrix . Althougl matrix .in vitro. It is proteolytically processed in mitochondria to a mature 28 kD catalytically active deacetylase  is a key enzyme that breaks down fatty acids and generates acetyl-CoA, stimulating β-oxidation. In SIRT3 knockout mice, LCAD is hyperacetylated at Lys42, leading to decreases in enzymatic activity, β-oxidation, and ATP level . Interesrogenase , facilitGlucose is the major energy source for cells. When its availability is limited, however, alternative fuels become increasingly important for cell survival. The first step of glycolysis is the conversion of glucose to glucose-6-phosphate, a reaction catalyzed by hexokinases. It is reported that SIRT3 deacetylates cyclophilin D , which lAcetate derived from acetic acid and alcohol is also used as a mitochondrial fuel, although this only occurs in extreme circumstances of nutrient depletion. In the initial step acetate is converted to acetyl-CoA catalyzed by acetyl-CoA synthetases. Acetyl-CoA synthetase 2 is the mitochondrial form of the enzyme. SIRT3 deacetylates acetyl-CoA synthetase 2 and enhances its activity, leading to increased production of acetyl-CoA . The aceNADH dehydrogenase 1 alpha subcomplex subunit 9 (NDUFA9) is an enzyme of mitochondrial complex I that is acetylated at Lys370 . SIRT3 pSIRT3 is also reported to bind the alpha subunit 1 of the F1 particle of ATP synthase , but theMitochondria are also the major sites for the generation of the ROS, superoxide, and also where the superoxide is dismuted by mitochondrial MnSOD. Recent reports show that SIRT3 deacetylates MnSOD at Lys122 and increases its activity, reducing oxidative and radiation stress in mice . OverexpIn future studies, it will be of interest to define the mechanism whereby acetylation of electron transport chain subunits affects generation of ATP. It is also important to elucidate why it might be desirable under some physiologic conditions to downregulate electron transport chain activity by increased acetylation. As an added wrinkle, SIRT3 negatively regulates translation within mitochondria by deacetylating the ribosomal protein MRPL10, a function proposed to reduce respiration .SIRT3 levels are increased in adipose tissue, skeletal muscle, and liver during CR , and conThe role of SIRT3 in aging is of considerable interest because it appears to suppress ROS, one of the causes contributing to the process of aging. In addition to elucidating its roles in regulating specific biochemical pathways in mitochondria, there is great interest in testing whether SIRT3 might modulate age-associated phenotypes, or indeed lifespan itself. In this regard, some studies have linked polymorphisms in the SIRT3 genomic locus to human longevity, although others have failed to demonstrate this association . A polym+. NADPH in turn allows regeneration of reduced glutathione to promote mitochondrial oxidative defense. In response to CR, wild-type mice, but not SIRT3-deficient animals, show increased NADPH levels, increased reduced glutathione in mitochondria, and decreased DNA damage in the cochlea and in other tissues. In tissue culture cells, overexpression of SIRT3 or IDH2 protects against oxidative stress-induced cell death, and the two proteins together have a synergistic pro-survival effect. These results do not rule out the possibility that SIRT3 might modify other substrates in addition to IDH2 to prevent AHRL during CR. Similarly, Age-related hearing loss (ARHL) is a common problem in the elderly, occurring secondary to cell loss and other degenerative changes in the cochlea. An elegant study has firmly established a role for SIRT3 in preventing ARHL . One mecAcetylation of mitochondrial proteins plays a major role in regulating functions of this organelle. Despite the rapid progress in this area, there are still many outstanding questions remain that will no doubt provide fruitful avenues for research for years to come. In particular, how mitochondrial proteins are acetylated in the first place is currently unknown. The identity of putative mitochondrial acetyltransferases remains elusive; identification of such proteins would represent a major step forward in this field. Alternatively, or in addition to enzymatic acetylation within mitochondria, mitochondrial proteins could in principle be acetylated outside this organelle, before or concomitant with mitochondrial import or even be acetylated non-enzymatically. These latter models would not permit rapid cycles of acetylation/deacetylation of mitochondrial proteins to regulate target protein function in response to varied environmental challenges. Instead, after deacetylation, restoration of acetylation status would require new protein synthesis. Such models could be distinguished through pulse-chase experiments assessing acetylation of newly synthesized mitochondrial proteins before and after mitochondrial import.+, and therefore mitochondrial NAD+ levels play a critically important role in governing mitochondrial SIRT3 function. Increased NADH generation from NAD+ that occurs with HFD and leads to reduced SIRT3 function may explain the increased global mitochondrial protein acetylation observed during this diet, as could increase levels of acetyl-CoA, the substrate for acetyltransferases (+ levels, such as post-translational modification or interactions with regulatory proteins.Similarly, how SIRT3 activity is regulated in the mitochondria is incompletely understood. SIRT3 requires NADsferases . This ovIt is currently unclear whether interventions that have an impact on acetylation of many mitochondrial proteins – SIRT3 deficiency, such as CR, HFD – lead to modification of common sets of proteins on the same lysine sites, or whether this response is tailored to different environmental perturbations. Similarly, it remains unclear whether the mitochondrial sirtuins share common targets and/or functions in common. This question could be addressed in mice or cells with mitochondrial sirtuin deficiencies or knockdowns. Given that SIRT3 deacetylates many proteins in mitochondria as well as suppresses some age-associated phenotypes, it will be of interest to test whether acetylation of mitochondrial proteins changes with age, either individually or globally, and whether prevention of this effect might have a beneficial effect on health span or even lifespan.In addition, whereas the functional impact of acetylation on a few individual protein targets is clear, a global understanding of how SIRT3 affects the activity of metabolic pathways in mitochondria, cells, tissues, and the organism overall is still lacking. Answers to these and related questions involving SIRT3 and mitochondrial protein acetylation will no doubt reveal novel aspects of mitochondrial biology, and perhaps ultimately provide the basis for novel therapeutic strategies for a variety of disorders.In the past decade, the function of mammalian sirtuins has been investigated in greater detail than ever before, and we now have a much better molecular understanding of the multiple roles this unique family of enzymes plays in seemingly every biological process. There is little doubt that sirtuins have emerged as critical modulators of metabolic adaptive responses, and their activities have been linked to metabolic abnormalities as well as age-associated neurodegeneration. Yet, key questions will keep investigators busy in the coming years. We still have poor understanding of the molecular mechanisms regulating expression and activity of the sirtuins, and of the precise stimuli that regulate these proteins, and whether the activities of different sirtuins are regulated in a coordinated fashion. In other words, is there cross-talk between sirtuins? Will future studies cement the argument that sirtuins are, indeed, critical modulators of lifespan? This review has focused on SIRT1 and SIRT3. Even less is known about other sirtuins. Further investigation into the targets and functions of this unique family of sirtuins will help develop new strategies for protection against and recovery from common aging-related neurological diseases and promote successful aging.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thrombocytopenia is a well recognized complication of infections, including those from hepatotropic viruses. Thrombocytopenia may actually be the only manifestation of vital hepatitis, which should therefore be considered in the differential diagnosis of primary immune thrombocytopenia (ITP). The mechanisms of thrombocytopenia associated with viral hepatitis vary widely depending on the specific infectious agent and the severity of liver disease. Most of the studies have described thrombocytopenia in association with chronic hepatitis C virus (HCV) infection, the most common cause of chronic infection worldwide. Studies have shown that treatment of HCV infection often results in substantial improvement or complete recovery of the thrombocytopenia. In patients with thrombocytopenia associated with HCV-related chronic liver disease, the use of eltrombopag, a thrombopoietin receptor agonist, normalizes platelet levels thereby permitting the initiation of antiviral therapy. Thrombocytopenia, either alone or in combination with other hematologic abnormalities, is commonly associated with infectious diseasesThe onset of the thrombocytopenia is rarely abrupt and severe during acute viral hepatitis A (HAV)9/l) was observed in 17.7% of 219 patients with chronic active hepatitis B and 10.6% of 123 HBV inactive carriersOn the other hand, persistent thrombo-cytopenia is more typically associated with chronic infection from HBV or HCV. Despite chronic HBV infection is still highly endemic in areas such as South East Asia and AfricaHowever, most of the recent literature pertaining to thrombocytopenia is about chronic hepatitis C, which will be the focus of our review.HCV is now recognized as the most common viral infection causing chronic liver disease in humans. The 3rd National Health and Nutrition Examination Survey (NHANES III) estimated that nearly 3.2 million persons in the general population of the United States are infected with HCVWhile retrospective studiesA variety of pathogenic mechanisms are reported to be implicated in thrombocytopenia related to chronic HCV infection. These include: 1) sequestration of platelets in the enlarged spleen secondary to portal hypertension (hyper-splenism); 2) reduced hepatic production of thrombopoietin; 3) bone marrow suppression by HCV or antiviral treatment; and 4) increased platelet destruction mediated by immune mechanisms involving anti-platelet autoantibodies and platelet-associated immune complexes.- 1) Hypersplenism is a common finding in patients with advanced liver disease , who develop portal hypertension ultimately resulting in an enlarged spleen and subsequent platelet sequestration. The increased portal pressure causes redistribution of blood to the spleen, subsequent pooling of platelets, and the increased clearance of platelets from the circulation- 2) Reduced hepatic production of the thrombopoietin (TPO) may be one of the contributing causes of thrombocytopenia during chronic liver diseases. TPO, the growth factor that primarily regulates megakaryocyte maturation and platelet formation, is produced mainly by hepatocytes, and is normally released at a constant rate into the circulation- 3) Impaired production of platelets secondary to HCV infection is thought to be a contributing factor in the development of thrombocytopenia. In a study using reticulated platelets in peripheral blood as a marker of thrombopoiesis, patients with liver cirrhosis had low platelet production9/l) was not observed, and platelet counts less than 50 x 109/l rarely were observed. Current guidelines therefore recommend the management of mild-to-moderate thrombocytopenia with PEG dose reduction and the discontinuation of HCV therapy in cases of severe thrombocytopenia, which typically is seen only in the setting of cirrhosis.However, antiviral therapy is itself associated with thrombocytopenia. IFN administration can induce a rapid and sustained reduction in peripheral platelet count. Peck-Radosavljevic and colleagues demonstrated that the platelet count decreased by nearly 28% in subjects treated with at least one dose of standard IFN and pegylated interferon (PEG)- 4) Although there is a higher prevalence of thrombocytopenia and anti-platelet antibodies in patients with liver disease caused by HCV than in patients with hepatitis B infectionOther studies have shown that HCV-RNA can be detected in washed platelets of infected individuals, particularly if thrombocytopenicvs 49 ± 30 x 109/l, respectively; P<0.02). Conversely, in an American study carried out in the Los Angeles area9/l . However, 56 (74%) patients had a platelet count ≤50 x 109/l. Symptoms and signs of thrombocytopenia were less frequent in HCV-positive ITP, but major bleeding was more frequent . Serum cryoglobulins and anticardiolipin antibodies were more frequent in HCV-positive ITP (90% and 62% respectively), but rare in HCV-negative ITP . In the FrenchIn one study from JapanMost case series of patients with HCV infection and chronic immune thrombocytopenia have reported a greater than 50% platelet response to steroidset al noted that only a minority of HCV-positive patients received some form of treatment for thrombocytopenia [29 (38%) vs. 158 (91%) for HCV-negative ITP]Rajan In the report of Garcia-Suarez et al each of 6 HCV patients treated with IFN-α responded with a significant increase in platelet countConsidering the results of these various studies9/L in 75%, 79%, and 95% of patients treated with 30 mg, 50 mg, and 75 mg eltrombopag, respectively, compared to no response in placebo patients (P < 0.001) Research has focused on developing compounds specifically to stimulate thrombopoietin (TPO) activity in order to prevent or treat thrombocytopenia in chronic liver diseases. Eltrombopag is a small-molecule nonpeptide oral platelet growth factor that acts as an agonist to the thrombopoietin-receptorSince eltrombopag has shown remarkable activity in chronic ITP as wellBoth acute and chronic viral hepatitis may be associated with isolated thrombocytopenia and should be considered in the differential diagnosis of ITP when isolated thrombocytopenia is present. Most of the studies have described thrombocytopenia in association with chronic HCV infection, by far the most common cause of chronic hepatitis. Several potential mechanisms can contribute to the thrombocytopenia in chronic HCV infection, including accelerated platelet clearance due to immune complex disease, cross-reactivity of anti-platelet glycoprotein antibodies and viral or bacterial antibodies, defective platelet production, and splenic sequestration of platelets secondary to portal hypertension and decreased production of thrombopoietin.Serologic evaluation for HCV infection is indicated in patients with ITP because of the potential adverse effect of prolonged corticosteroid usage on the underlying infection and the utility of antiviral therapy in treating the both the underlying infection and the thrombocytopenia. Treatment with eltrombopag, a second generation thrombopoietin receptor agonist, appears to be very efficacious in elevating the platelet count and is well tolerated. Ongoing phase III studies in HCV-related chronic thrombocytopenia will better define the benefits of this agent compared to standard of care.


Given the critical role of the geniculate map in the development of visual cortex, these results provide a foundation for analyzing the temporal dynamics whereby the visual cortex itself develops.A neural model is described of how spontaneous retinal waves are formed in infant mammals, and how these waves organize activity-dependent development of a topographic map in the lateral geniculate nucleus, with connections from each eye segregated into separate anatomical layers. The model simulates the spontaneous behavior of starburst amacrine cells and retinal ganglion cells during the production of retinal waves during the first few weeks of mammalian postnatal development. It proposes how excitatory and inhibitory mechanisms within individual cells, such as Ca This article describes a neural model of how spontaneous retinal waves are formed in infant mammals, and how these waves organize activity-dependent development of a topographic map in the lateral geniculate nucleus, or LGN, with connections from each eye segregated into separate anatomical layers of the LGN . Recent 2+-activated K+ channels, cAMP, and afterhyperpolarization currents (AHPs) in controlling spatiotemporal properties of retinal waves Albeit simple, the new model goes beyond earlier modeling efforts by explicitly accounting for the role of intracellular mechanisms such as CaRetinal waves cause spontaneous bursts of action potentials in RGCs, on the order of 10–100 Hz, that move in a wave-like fashion across As noted above, the model  incorporAll model cells are point neurons whose single compartment voltage, l denotes the left eye and r the right eye. In the Poisson distribution in (4) that defines the probabilistic input term The activities 2+-activated K+ channels 2+-activated K+ channels, which have been shown to control the AHPs of SACs using in vitro whole cell patch clamp 2+ which, in turn, opens Ca2+-activated K+ channels that act to electrically shunt the cell by increasing K+ conductance on a slow timescale, decreasing the membrane resistance for tens of seconds. It also appears that these channels are voltage-independent  and can only be opened by an increase in cytosolic Ca2+2+ can be affected by the conductance of voltage-gated Ca2+ channels.Lastly, terms l) and right (r) eyes obey:The SACs send half-wave rectified signals to the RGCs, which smooth SACs activity through leaky integration. The activities The RGC outputs activate cells in the dorsal LGN (dLGN) in layers The activity-dependent release of learning-inducing neurotrophins from dLGN cells is modeled by sigmoid signals of LGN activities:instar learning law d equals A or A1, and RGC bursting . These intracellular mechanisms are assumed to control the AHP of SACs that modulate wave velocity and periodicity. The model replicates the general spatiotemporal properties of waves observed in vitro in a number of animals, including mice and ferrets. A sample wave is depicted in Retinal waves 2+-imaging data from First, model simulations were compared to CaIn all the retinal simulations, waves were detected by counting the number of active cells at each time step and comparing with a pre-determined threshold. The algorithm assumes that the beginning and end of a wave are marked by crossing the threshold of 10 cells going active. Thus, if more than ten cells (out of 576) burst, a wave begins, and the wave ends as soon as less than ten cells are bursting. Each time step in between these two events is considered part of a single wave. This algorithm eased the significant computational load of analyzing the simulation results . Hundreds of identified target waves were checked by eye to ensure the validity of the algorithm.he right . Additiohe right . Thus, m2+ concentration) of SACs should decrease wave velocity and WPM, while increasing maximum excitability  or intracellular excitatory mechanisms  should increase velocity and WPM. Additionally, these simulations show a significant relationship between velocity and WPM, which has been observed in experiments To more comprehensively understand how model parameters affect the IWI and velocity, simulations systematically explored the parameter space see  and 7. TWhereas all SAC parameters influence wave periodicity and velocity, the RGC parameters Simulations were carried out to demonstrate that waves generated within the model retina can drive the development of a retinogeniculate map whose connections from each eye are segregated into distinct LGN layers A and A1 The simulations demonstrate that retinal waves can drive the development of eye-specificity in dLGN layers  as well = q) see .First, development of the retinogeniculate pathway was simulated for 5,000 seconds (simulated time) using the standard parameters to generate retinal waves see , and eacin vitroWaves were also generated using different sets of parameters to simulate perturbations to the retina and their effect on retinogeniculate development. First, ions see  and 4. Tsee  see . When , Next, to divorce layer-specificity from topography refinement, ‘random’ waves were generated by replacing the activity of RGCs with Poisson noise , to approximate the dynamics of Ca2+-activated and CAMP-sensitive K+ channels. Godfrey and Swindale 2+ processes that dictate afterhyperpolarization periods. This is more similar to our model and is quite biophysically realistic. Our approach captures most of the dynamics present in this spiking model, yet is significantly simpler and is more amenable to embedding in a larger model of retinogeniculate development, which is not simulated by Hennig et al. More precisely, in our model, the recovery variable r simulates lumped intracellular inhibitory mechanisms, based on whole-cell patch clamp 2+-imaging r suppresses the firing rate of SACs after a prolonged period of heightened firing. This approximates the process of slow Ca2+ accumulation in the cytosol during bursting, which progressively opens K+ channels that electrically shunt a given SAC. As the channels are slow to close, the SAC remains hyperpolarized or refractory for tens of seconds during the AHP, which regulates the periodicity of retinal waves observed in vitro. Additionally, upregulating cAMP through either uncaging techniques or bath application of forskolin reduces AHPs 2+ and cAMP, which agrees with whole-cell patch clamp data This foundation enables our simulation of retinogeniculate development to use a biologically plausible dynamical system with activity-dependent refractoriness to generate retinal waves. Some models simply move the center of pre-computed distributions  to approximate the bump of activity that sweeps across the retinal sheet during waves In summary, the proposed model builds on and goes beyond previous work by being the first to include both: (1) a fully dynamical explanation and simulation of retinal waves that clarifies the functional role of intracellular AHP currents, and (2) a learning rule that exploits competition for a limited neurotrophin to induce topography refinement and the dominance of one eye over the other in each dLGN layer. Testing the model's predictions can provide additional experimental data with which to develop future model refinements see .


Simian virus 40 (SV40) was discovered in 1960 as a contaminant in early polio vaccines. Its discovery coincided with an explosion of knowledge in the new field of molecular biology, and SV40 was quickly adopted as a model to study eukaryotic genome structure, expression, replication, and cell growth regulation in cultured cells not sufficient, to activate bidirectional helicase activity of the Tag complex in vitro or in vivo.Assembly of Tag on the viral core origin of DNA replication (64 bp) is the first step in replication Activation of the double hexamer on origin DNA requires a unique phosphorylation state of Tag: phospho-Thr124, and unmodified Ser120 and 123 Both unphosphorylated and phosphorylated forms of Tag assemble double hexamers on duplex SV40 origin DNA . HoweverInvestigation of SV40 replication has been motivated in part by anticipation that it would provide insight into host replication proteins and mechanisms. The architecture, dimensions, and assembly of Tag and yeast Mcm2-7 double hexamers on their cognate origin DNAs are closely related Recently, fluorescence microscopy of SV40 chromatin replication in infected cells has revealed that Tag and the host proteins required for SV40 replication in vitro co-localize in prominent subnuclear foci that enlarge with time after infection in permissive cells


Immunohistochemical evaluation for a BMP-binding protein Twisted Gastrulation (TWSG1) revealed mosaic pattern of staining, with some cells, including osteoclasts, strongly stained and others exhibiting faint or no staining, thus supporting active regulation of BMP signaling within the lesion. Future investigations will determine if dysregulation of BMP signaling plays a causative role or rather reflects secondary activation of repair mechanisms and/or bone remodeling.A hereditary congenital condition characterized by a fibro-osseous lesion sharing some features with fibrous dysplasia and affecting the middle aspect of the mandible is presented. The condition was initially described as congenital monostotic fibrous dysplasia in two siblings, a male and a female. However, there is sufficient evidence that the disorder is autosomal dominant since it has been encountered in two of four children, both males, of the female propositus and one child, a boy, of the male propositus. All patients presented at birth or right after birth with enlargement of the middle part of the mandible. Radiographs from affected individuals have shown mesomandibular enlargement with irregular trabeculation akin of “ground-glass” appearance. Histologically, samples from all patients revealed woven bone proliferation in a cellular fibroblastic stroma. Interestingly, the originally described siblings, now in their 30s, have no evidence of jaw lesions either radiographically or clinically, thus indicating that the condition is self-limiting or self-resolving. An autosomal dominant mode of inheritance with apparent male predilection is favored. The molecular basis of this condition is currently unknown. However, the location of the lesions in the middle aspect of the mandible suggests dysregulation of Bone Morphogenetic Protein (BMP) signaling since BMPs regulate mandibular morphogenesis  Familiarity with their clinical, radiographic, and in some cases histopathologic characteristics is important for management that can vary, depending on the type, from periodic observation, surgical recontouring for functional and esthetic purposes, to tumor resection.Benign fibro-osseous lesions (BFOLs) encompass a group of pathologic processes characterized by replacement of normal bone by generally cellular fibrous tissue and newly formed osseous, or so-called cemento-osseous, deposits. Included in the group of BFOL of the jaws are: (1) ossifying fibroma, a neoplasm, (2) fibrous dysplasia a condition associated with known mutations to the G protein subunit α . Sections were deparaffinized in xylenes and rehydrated through a series of graded ethanol. Endogenous peroxidase activity was blocked by incubating sections in a 3% hydrogen peroxide/methanol solution for 10 min and then washed in PBS. Slides were placed in Antigen Unmasking Solution diluted to manufacturer's recommendation  and microwaved for 20 min. Once cooled, slides were washed and then blocked in PBS containing 5% goat serum  and 0.3% Triton X-100 (Fisher Scientific) for 60 min at room temperature. Sections were incubated with monoclonal anti-hTWSG1 antibody  at 1:500 dilution overnight at 4°C. Detection was accomplished using SuperPicture Kit  following manufacturer's instructions.The description of the propositus and his sibling has been previously published  did not reveal any clinical lesions. There was only transposition between the right mandibular canine and first premolar. There was no evidence of radiographic abnormality. A follow-up of the male infant was recommended. At 12 Figure  and 36 mIn order to examine if BMP binding protein TWSG1 was present in either bone cells or fibrous tissue within the lesion, sections of lesional tissue obtained from this patient were evaluated by immunohistochemistry with monoclonal antibody against human TWSG1. Immunoreactivity was observed in a mosaic pattern within the lesion with some groups of cells stained strongly and other groups exhibiting low to no staining Figures . The staIn 2009, another affected male infant was born to the female patient II-3 with mesomandibular enlargement, however, not as noticeable as his brother's Figure . The lesInherited fibro-osseous lesions of the jaws are rare and appear in the literature as examples of gigantiform cementomas  fibromas and cemento-osseous dysplasias  are the result of an autosomal “dominant” lethal gene leading to loss of the zygote  Happle, . In orde Happle, . Dependi Happle, . With ti Happle, . Fibrous Happle, .GNAS or in the gene causing cherubism (Mangion et al., Although it is well established that the mutations causing fibrous dyplasia occur postzygotically, thus excluding the possibility of the disease to be inherited, there are reported cases of familial fibro-osseous lesions, clinically and histologically, similar or even indistinguishable from fibrous dysplasia (Reitzik and Lownie, The condition affecting the family described herein was previously referred to in the literature as monostotic fibrous dysplasia (El Deeb et al., GNAS mutation (Sakamoto et al., Osteofibrous dysplasia, known also as “osteitis fibroma,” a “variant of fibrous dysplasia,” and “ossifying fibroma of long bones,” is a benign bone dysplastic process affecting primarily the cortex of the tibia and/or the fibula of skeletally immature patients (Park et al., The molecular mechanism associated with the familial fibro-osseous condition presented in this paper is unknown. However, the location of the lesions in the middle aspect of the mandible suggests dysregulation of BMP signaling. BMPs regulate mandibular morphogenesis, particularly in the medial region (Ferguson et al., Msx1 and Msx2 (Vainio et al., BMP signaling is modulated in the extracellular space by BMP-binding proteins Noggin, Chordin, Chordin-like 1, TWSG1, and others (Ross et al., In summary, we present additional information on the clinicopathologic features of the family initially reported by El Deeb et al. (El Deeb et al., 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.


Since the initial discovery that a subset of patients with cutaneous melanoma harbor BRAF mutations, substantial research has been focused on determining the pathologic consequences of BRAF mutations, optimizing diagnostic techniques to identify these mutations, and developing therapeutic interventions to inhibit the function of this target in mutation-bearing tumors. Recently, advances have been made which are revolutionizing the standard of care for patients with BRAF mutant melanoma. This paper provides an overview on the pathogenic ramifications of mutant BRAF signaling, the latest molecular testing methods to detect BRAF mutations, and the most recent clinical data of BRAF pathway inhibitors in patients with melanoma and BRAF mutations. Finally, emerging mechanisms of resistance to BRAF inhibitors and ways of overcoming this resistance are discussed. Melanoma is currently the 5th and 7th most common cancer in American men and women, respectively . In addiV600E) [More recently, preclinical discoveries have led to significant advances in the understanding of the key molecular signaling events underlying the pathogenesis of melanoma. Most notably, a high percentage of tumors of melanocytic origin have been shown to harbor activating mutations of BRAF, which lead to its constitutive activity. Approximately 70–80% of acquired melanocytic nevi and 40–60% of malignant melanoma contain a BRAF mutation, the vast majority of which result in a single amino acid change at codon 600 (BRAFV600E) , 4. The V600E) . This enV600E) –7.The interaction between a growth factor receptor and its ligand typically induces a series of events, which promote cellular growth and survival. The RAS family members are GTPases which act as critical mediators in the transduction of such signals. Though RAS plays an important role in the homeostasis of normal cell turnover, death, and survival, activating mutations in RAS family members  have been identified and associated with various human malignancies . In melaκB), and B-cell leukemia 2 (BCL-2) [Activated RAS triggers MAPK pathway activation through interactions with the RAF oncoproteins (BRAF and CRAF) leading to the initiation of a progrowth signaling cascade . It is u (BCL-2) . InteresV600E mutation in a homogenous tumor nodule, this is likely not sensitive enough to detect a few tumor cells in the background of a high percentage of stromal or lymphatic elements, infiltrating lymphocytes, or peripheral blood cells. Since the identification of activating mutations of BRAF in melanoma, the technology for detection has improved dramatically. Standard mutational testing for BRAF in tumor tissue typically utilizes techniques such as bidirectional direct fluorescent sequencing and allele-specific polymerase chain reaction which are commercially available and offer high specificity. The sensitivity of these assays, however, is limited in that they are only able to detect the mutation if the tumor cells constitute >5–10% of the specimen submitted for genetic analysis , 17. WhiOne concern regarding the utilization of mutation detection techniques with enhanced sensitivity is that a positive test might actually reflect the detection of a small subset of mutant cells. While this might have interesting scientific consequences, the clinical relevance of a tumor containing a small amount of mutant BRAF cells is none, as these patients would not be expected to benefit from BRAF inhibitors. This concern is warranted, as tumor heterogeneity has been described in primary melanomas . In addiMore advanced techniques and assays have been developed which either provide increased sensitivity or obviate the need for increased sensitivity. These next generation tests allow for more accurate testing on samples which contain only a small amount of tumor, as well as for the detection mutations in various peripheral blood components . The utility of many of these tests have been explored in samples from melanoma patients with varying results.Amplification refractory mutation systems (ARMSs) are a recently described, allele-specific technique which has enhanced sensitivity (able to detect mutation sample containing 1% mutant cells) compared to standard DNA sequencing of formalin fixed paraffin-embedded (FFPE) tissues . AnotherIn addition to new technologies (ARMS) and modifications to routine techniques which lead to a greater sensitivity of mutation detection, the application of standard assays on previously untested samples is also changing how we approach BRAF testing. BRAF analysis on free DNA in the serum and plasma has been reported as has the detection of BRAF mutations from isolated, circulating tumor cells (CTCs) –30. WhilV600E), both in tissue or blood, it also may be worthwhile to test for other mutations and anomalies as these may indicate sensitivity to a particular treatment. For example, Sequenom MassARRAY technology is being used to query larger panels of oncogenic mutations, using a primer extension reaction followed by mass spectrometry to detect the products and identify mutations with potential clinical consequences [While the role of standard and experimental molecular diagnostics is being utilized to identify specific mutations of interest , and CRAF. In addition, inhibitors of the downstream mediators of RAF activation, namely MEK and ERK, are also being developed. In this section, only agents which have been tested clinically and reported publically are reviewed.A series of small molecule inhibitors have been developed which target, with varying selectivity, wild-type BRAF, BRAFSorafenib, a multitargeted tyrosine kinase inhibitor of BRAF, CRAF, platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR) 2, p38, and CKIT which was the first RAF-inhibitor actively studied in patients with melanoma as it was available for phase II testing in the same year in which BRAF mutations were first reported. Unfortunately, despite being evaluated in numerous phase I, II, and III studies as a single agent and in combination with chemotherapy, the clinical utility of sorafenib has been disappointing. For example, in a single agent trial of sorafenib, the median progression-free survival for patients with melanoma was 11 weeks . Six patThis study was followed by several trials of sorafenib in combination with various cytotoxic agents, though the combination which was best studied was sorafenib, carboplatin, and paclitaxel –42. InitV600E predictably has led to improved clinical activity of these agents compared to sorafenib. One major explanation proposed for the ineffectiveness of sorafenib as a single agent in patients with melanoma is its inability to completely inhibit BRAF, and in particular, BRAF containing the V600E mutation. Other inhibitors of BRAF, such as PLX-4032 and GSK2118436, have been developed and are more potent and selective inhibitors of mutant BRAF than sorafenib . This enV600E mutation who received a dose ≥240 mg twice daily in the dose escalation phase experienced tumor responses while no clinical responses were seen in the five patients with wild-type BRAF-containing tumor. In addition, 26 of 32 (81%) patients with the BRAFV600E mutant melanomas treated in an expansion cohort at the recommended phase II dose of 960 mg twice daily had a clinical response, including two patients achieving a complete response (CR). The estimated median PFS was seven months which compares favorably to previously available therapies for metastatic melanoma. Further, treatment with vemurafenib leads to a reduction in levels of phosphorylated ERK (pERK) in tumors containing the BRAFV600E mutation which is associated with clinical response [V600E cells [Vemurafenib was the first higher potency BRAF inhibitor to complete phase I testing and show significant clinical benefit . In the response , 44. Lik0E cells . These findings quickly led to the rapid accrual of both a single-agent phase II study (BRIM2) and a randomized controlled phase III trial (BRIM3). The phase II trial enrolled 132 patients with advanced melanoma who had received one prior therapy. The objective response rate (ORR) was 53% with a CR rate of 5%, and the progression-free survival was 6.7 months . In the V600E mutation treated at the two highest dose levels (150 mg twice daily and 200 mg twice daily) had a high response rate  [V600E mutations  treated at a dose of ≥100 mg twice daily, three had a partial response. Both of the patients with BRAFK601E progressed after first restaging, suggesting that only patients with BRAF mutations at the 600 position will respond to therapy.GSK2118436 is a second higher potency BRAF inhibitor which has shown substantial clinical activity. In a phase I/II trial, similar to PLX4032, patients with the BRAFts, 63%) . In the V600E mutation; as mutational status correlates strongly with response to MEK inhibition in murine melanoma xenograft models [Inhibitors of MEK, the downstream mediator of RAF activation, and the only known substrate of BRAF have shown promise in preclinical studies in melanoma and have begun to be investigated in the clinic with some encouraging results. MEK inhibitors may also be most useful in patients with BRAFt models .Two phase I trials of AZD6244 involving patients with advanced solid tumors showed this agent to be well tolerated and to possess some antitumor activity in patients with melanoma , 51. In In addition, AZD6244 has shown promising results in murine models, particularly in combination with chemotherapy, setting the stage for combination trials . BuildinV600E mutant melanoma [GSK1120212 is a reversible, selective inhibitor of MEK1/MEK2 which has been shown, in a phase I trial, to have single-agent efficacy in patients with advanced, BRAFmelanoma . SpecifiA phase I trial of PD-0325901 enrolled 48 patients with advanced melanoma, of whom 3 (6%) had a confirmed PR, 10 (21%) had stable disease for ≥4 months, and a total of 15 (31%) patients showed reduction in Ki-67 tumor staining . MutatioSimilar results have been recently reported with AS703026, which is a potent MEK1/2 inhibitor. In the phase I study, three of eight patients had a partial response with treatment in one of two treatment schedules . MutatioWhile the clinical data on MEK inhibitors is encouraging, it is quite preliminary. The true value of these agents must await phase II and phase III trials in patients with BRAF mutant melanoma. One such trial that is currently ongoing is a randomized, phase III trial of GSK1220212 compared with chemotherapy (either dacarbazine or paclitaxel) in patients with melanoma harboring BRAF mutations (NCIT01245062). V600E and a high rate of objective response early in the course of therapy. Preliminary studies suggest that resistance to PLX-4032 is not related to the development of a second mutation which impairs the binding of the treatment drug to BRAF, a resistance mechanism noted for targeted therapy in other malignancies such as nonsmall cell lung cancer, chronic myelogenous leukemia, and gastrointestinal stromal tumor [Importantly, it appears that the great majority of patients treated with single agent PLX-4032 will eventually exhibit disease progression despite successful inhibition of the BRAFal tumor –59. InstV600E-mutated cells which have been generated to exhibit acquired resistance to BRAF inhibitors, that has led to the first clues as to how BRAF-mutated cells are able to survive BRAF inhibition. It appears clear that reestablishment of MAPK signaling is the key variable in acquired resistance to BRAF inhibition [It is from in vitro studies of BRAFhibition . This cahibition , 62, acthibition , 63, acthibition , and devhibition , 65. In hibition . Of notePrimary resistance to BRAF inhibition is seen in less than 10% of patients with BRAF mutant melanoma treated with vemurafenib . While tWT) melanoma cells, the MAPK kinase pathway is activated by vemurafenib  leading to upregulation of MEK and ERK and enhanced proliferation [WT cells [WT protein and/or CRAF homodimer which is most apparent in RAS-mutated cells [WT melanoma cells (particularly those which harbor an NRAS mutation) treated with BRAF inhibitors such as vemurafenib and PLX4720, the clinical relevance of this is uncertain. Specifically, it is not thought that clinically acquired resistance to vemurafenib, for example, occurs solely due to growth of a subset of BRAFWT melanoma cells as persistence of the BRAFV600E mutation has been identified in all tumors analyzed and reported to date. In fact, it appears that specific changes in BRAFV600E-mutated cells allow for adaptations which lead to renewed growth despite continued BRAF inhibition.In BRAF wild-type  with a MEK inhibitor (GSK1120212) is underway and appears tolerable with both agents being administered at their standard single-agent doses . In addiAnother approach to enhancing the effectiveness of selective BRAF inhibitors and MEK inhibitors is the addition of agents which might augment apoptosis. One such agent is ABT-263 which is a BH3-mimetic currently in clinical development. In preclinical studies, the less bioavailable homologous BH3-mimetic ABT-737 in combination with a MEK inhibitor led to enhanced lethality compared to either agent alone . WhetherIn addition to combination therapy with molecularly targeted agents which inhibit signaling induced by selective BRAF inhibition or promote apoptosis, another promising approach to maximizing the benefit of BRAF or MEK inhibiters is to combine these agents with immunotherapy. Recently, immune checkpoint inhibitors, including the anti-CTLA-4 monoclonal antibody ipilimumab and the anti-PD monoclonal antibody MDX-1106, have shown single agent efficacy in patients with metastatic melanoma , 76. ImpIt had been hoped for many years that the growing understanding of the molecular pathways involved in melanoma development and the increasing availability of specific inhibitors of these pathways would enable the rational development of future therapies. With the emergence of vemurafenib and GSK2118436, the first molecularly targeted agents to lead to tumor responses in a large percentage of patients, a new approach to the treatment of melanoma has begun. As a result, all patients with advanced melanoma should have BRAF mutational analysis prior to commencing systemic therapy. In those patients whose tumors harbor a mutation in BRAF, every attempt should be made to treat these patients with either of the two highly potent BRAF inhibitors. Additionally, as more is learned about the resistance mechanisms to BRAF inhibitors, the development of combination trials of novel molecularly targeted therapies can be expected; further clinical improvements can only be sorted out by carefully conducted preclinical and clinical studies that include pretreatment and on-treatment biopsies. With BRAF being established as the first point of vulnerability in melanoma, it is hoped that a molecular understanding of the limits of BRAF inhibition will lead to further clinical benefit.


In the present study we analyzed the reliability for cardiac output (CO) measurement of Vigileo  and MostCare  in comparison with transthoracic Doppler echocardiography (as the reference method) in patients undergoing vascular surgery.r2) and Bland-Altman analysis.Both Vigileo and MostCare were connected to the FloTrac transducer (Edwards Lifescience) for CO calculation. The data from Vigileo and MostCare were registered (COVIG and COMC respectively) and compared with those calculated with the echocardiographic standard formulation . In every patient CO was measured twice: at baseline (T1) and after volume loading (500 ml lactate Ringer solution) (T2). Agreements between COVIG, COMC, and COECHO were evaluated by means of simple linear regression (r2, bias and limit of agreement at T1 and T2 are summarized in Table Twenty patients were enrolled in the study. Values of Vigileo did not prove to be a substitute to the reference system; pre-loaded data, necessary for vascular impedance estimation, may be one of the main limitations that made Vigileo measurements less accurate than the MostCare ones. On the contrary, MostCare, an uncalibrated totally independent system, was shown to properly estimate the vascular impedance in these hemodynamically stable patients. Further comparisons in unstable conditions are needed to confirm our observations.