Histone deacetylase (HDAC) inhibitors represent a potential new class of antitumor agents. Vorinostat (suberoylanilide hydroxamic acid or SAHA) is a potent inhibitor of HDAC activity and has undergone initial evaluation in several Phase I and II clinical trials. HDACs are enzymes that catalyse the removal of the acetyl moiety from the lysine residues of proteins, including the core nucleosomal histones. Together with histone acetyltransferases (HATs), HDACs regulate the level of protein acetylation. Alterations in both HAT and HDAC activity have been reported to occur in cancer. HAT activity has been found to be disrupted by translocation, amplification, overexpression or mutation in a variety of cancers, including those of haematological or epithelial origin. HDACs have been found to be overexpressed or associated with oncogenic transcription factors. Vorinostat induces growth arrest, differentiation or apoptosis in a variety of transformed cells. The antiproliferative effects of vorinostat are believed to be due to drug-induced accumulation of acetylated proteins, including the core nucleosomal histones and other proteins . Phase I and II trials have been conducted for the oral formulations of vorinostat, and results show that vorinostat inhibits its target enzyme (HDAC) in peripheral mononuclear cells and tumour tissue at doses that are well tolerated. Antitumour activity has been seen in patients with both haematological and solid tumours. Although a key characteristic of neoplastic transformation is unregulated cellular proliferation, cancer cells nevertheless retain their ability to undergo differentiation or apoptosis under certain circumstances  of action of vorinostat that may contribute to its antitumour activity.The HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid or SAHA) blocks cancer cell proliferation both ε-amino group of the lysine side chain of histones H2A, H2B, H3 and H4, thereby reconstituting the positive charge on the lysine residues.Nucleosomes comprise the repeating unit of chromatin and serve to organise and compress the DNA in the nucleus. They are composed of the octamer of core histones  spanning approximately 200 bp of DNA. The acetylation status of histones plays an important role in regulating gene expression by altering the structure of chromatin  is fused with the promyelocytic leukaemia (PML) gene on chromosome 15 forming a complex (PML-RARα) that results in HDAC recruitment and transcriptional repression  is a nanomolar inhibitor of HDAC activity that has undergone initial evaluation in multiple Phase I and II clinical trials. Vorinostat is a small molecular weight (<300) linear hydroxamic acid compound that inhibits HDAC activity thereby inducing the accumulation of acetylated histones as well as nonhistone proteins, blocks the proliferation of cultured cells, and inhibits tumour growth in a variety of animal models. Vorinostat is a broad inhibitor of HDAC activity and inhibits both classes I and II enzymes . As withCrystallographic studies have revealed that vorinostat inhibits HDAC activity by binding in the active site of the enzyme , the hydin vitro, including lymphoma, myeloma, leukaemia, and non-small cell lung carcinoma with concentrations that inhibit growth by 50% compared to no treatment ranging from approximately 0.5 to 10 μM  and SV40 large T antigen transformed WI-38 (VA-13 cells)  in nude mice  while others are repressed   of action of vorinostat as well as delineation of its clinical utility in various cancer types should help formulate rational combinations with other chemotherapeutic agents that may provide synergistic or additive antitumour efficacy. Based on a mechanistic rationale, vorinostat has the potential to be combined with several different types of anticancer therapies, including radiation, anthracyclines, cisplatin, taxanes, 5-fluorouracil (5-FU), flavopiridol, bevacizumab and trastuzumab . PrelimiM, induces histone and protein acetylation and alters gene expression. Vorinostat blocks growth promoting signal transduction pathways and the proliferation of a broad spectrum of cultured cancer cells. Parenteral and oral administration of vorinostat at doses producing little or no toxicity to normal cells results in growth arrest in rodent models of several solid tumours and haematological malignancies, including prostate cancer, leukaemia, breast cancer, colon cancer and lung cancer. The mechanism underlying the antitumour action of vorinostat is not yet clear but may involve changes in the expression of specific genes via acetylation of histones and transcription factors as well as nontranscriptional effects such as inhibition of mitosis. Further research to delineate the mechanism(s) of action of vorinostat and other HDAC inhibitors may pave the way to developing rational combinations with other chemotherapeutic agents and perhaps ultimately to optimising chemotherapy regimens for cancer patients.Vorinostat, a potent inhibitor of Classes I and II HDAC activity with an IC50 <86 n


Gradient-recalled echo magnetic resonance imaging (GRE MRI), which gives information on blood flow and oxygenation changes , was used to observe the responses of six rodent tumour models to carbogen breathing. In one transplanted rat tumour, the Morris hepatoma 9618a, and a chemically induced rat tumour, the MNU-induced mammary adenocarcinoma, there were marked image intensity increases, similar to those previously observed in the rat GH3 prolactinoma. In contrast, the rat Walker carcinosarcoma showed no response. In two mouse tumours, the RIF-1 fibrosarcoma and the human xenograft HT29, carbogen breathing induced a transient fall in signal intensity that reversed spontaneously within a few minutes. The rat GH3 prolactinoma was xenografted into nude mice, and an increase in image intensity was found in response to carbogen, suggesting that any effects that carbogen may have had on the host were not significant determinants of the tumour response. The increases in GRE image intensity of the MNU, H9618a and GH3 tumours during carbogen breathing are consistent with increases in tumour oxygenation and blood flow, whereas the responses of the RIF-1 and HT29 tumours may be the result of a transient steal effect followed by homeostatic correction.


Ependymomas are tumours that arise throughout the central nervous system. Little is known regarding the aberrant cellular and molecular processes that generate these tumours. This lack of knowledge has hampered efforts to reduce the significant mortality and morbidity that are associated with ependymoma. Here, we review recent data that suggest that radial glia are cells of origin of ependymoma, and discuss the processes that might transform these neural progenitors into ependymoma cancer stem cells. Ependymomas are central nervous system (CNS) tumours that originate from the wall of the ventricular system along the entire craniospinal axis  and geneCancer researchers have invested a great deal of effort in characterising the genetic alterations that accumulate in end-stage tumours. Although these studies can provide lists of the genetic events that contribute to the expansion of malignant clones, they do not inform us regarding the chronology or relative importance of each of these alterations in the cancer process. A more comprehensive understanding of cancer that spans the life of the disease from the birth of the first malignant cell to clinical presentation, would be invaluable in the hunt for more effective treatments of cancer. With this in mind, considerable excitement has surrounded the recent discovery of cancer stem cells (CSC) . Cancer +/CD38− immunophenotype of haematopoietic stem cells (HSC)  . Thus, tComparative studies of normal and malignant HSC have been facilitated by the availability of robust assays for all stages of haematopoiesis (HOX) family members that coordinate antero-posterior tissue patterning and development of the spine  – that are neural progenitor cells (see below) – and those observed in human ependymomas from the corresponding region of the CNS. The great majority of intracranial tumours in our study arose in children; whereas the spinal tumours were obtained from adult patients. Therefore, our data suggest that RG are a likely source of ependymomas independent of patient age. It is noteworthy that astroglial cells with functional and molecular characteristics of RG persist in the SVZ of the lateral ventricles and possibly the spinal cord, suggesting that some RG give rise to adult neural stem cells  + immunophenotype of RG; and are both required and sufficient to generate tumours To determine whether ependymoma CSC arise from RG, and how this malignant transformation may occur, it is important to understand the processes that regulate the generation and fate of RG in the CNS. In vertebrates, neural stem cells first appear as a layer of pseudostratified epithelium that lines the neural plate and neural tube before the onset of neurogenesis. These neuroepithelial cells (NEC) are highly polarised along their apical–basal axis . Of partAs neurogenesis begins, NEC give rise to RG. RG retain the highly polarised features of NEC and express CD133, Nestin and RC2, but they also express astrocyte-specific proteins that include glutamate transporter and BLBP . PreviouNeuroepithelial cells and RG each possess the capacity for symmetric and asymmetric cell division . SymmetrEmx2 induces symmetric divisions that can lead to expansion of RG cell numbers, whereas Pax6 activates neurogenic, asymmetric division (EMX2, but not PAX6, is markedly upregulated in supratentorial ependymomas compared to ependymomas from other regions of the CNS (37-fold expression difference P<0.0001; EMX2 within embyonic cortical RG might contribute to increased symmetric cell division and the formation of CSC of pediatric supratentorial ependymoma (Emx2 can operate as a negative regulator of symmetric cell division in adult neural stem cells (The apical plasma membrane appears to play an important role in determining the fate of NEC and RG daughter cells . Daughtedivision . Interesendymoma . One impαE-Catenin from neural progenitor cells in mice results in severe disruption of the apical cell junctions, loss of cell polarity, increased stem cell proliferation and the formation of tumour-like masses in the brain (Neuroepithelial cell and RG each contain concentrations of adherens junctions that are located immediately beneath the apical plasma membrane . Evidenche brain .Lethal Giant Lavae-1 from dividing neural stem cells of mice, prevents the asymmetric localisation the Notch inhibitor Numb to daughter cells, resulting in failure of asymmetric cell divisions and the formation of tumour-like masses within the brain (ERBB2 oncogene that is expressed to high levels in ependymoma, is an important target of Notch signalling in RG (Certain cell signal pathways that control RG self-renewal are deregulated in ependymoma. Most notable among these is the NOTCH cell signal pathway. Upregulation of the Notch ligand Jagged1 maintains the self-renewal and multipotency of adult neural stem cells, whereas deletion of Notch1 depletes the neural stem cell fraction (ng in RG . Downregng in RG .Arf and p16Ink4a that are encoded by the Ink4a/Arf locus, are two additional regulators of neural stem cell proliferation. In this regard, Bmi1 promotes the self-renewal of neural stem cells by repressing transcription at the Ink4a/arf locus; whereas deletion of Ink4a significantly expands the neural progenitor cell population (INK4A/ARF affect the great majority of supratentorial ependymomas. Specifically, using array comparative genomic hybridisation and fluorescence in situ hybridisation, we have shown that INK4A/ARF is selectively deleted from >90% of tumour cell nuclei of supratentorial ependymomas but is rarely deleted from tumours arising in other regions of the CNS (INK4A/ARF.p19Drosophila tumour suppressor protein Brat is normally distributed asymmetrically to one of the daughters of dividing neural stem cells, fating that cell to differentiate, whereas the remaining daughter cell self-renews (Drosophila brat mutants both daughter cells self-renew, resulting in expansion of the stem cell pool and the formation of larval brain tumours.Further studies will be required to characterise fully the factors that contribute to the formation of ependymoma CSC. These efforts are likely to be assisted by ongoing studies of flies that have identified already tumour suppressor genes that regulate the proliferation of neural stem cells. For example, the If the CSC hypothesis proves correct and brain tumours, including ependymoma, arise from rare fractions of stem-like cancer cells, then these findings will lead to a paradigm shift in the way we treat CNS tumours. In particular, we should begin to develop classification systems and targeted treatment strategies that focus on the eradication of CSC. This strategy may prove particularly effective in tumours such as ependymoma that include developmentally and molecularly distinct subgroups that are unlikely to respond uniformly to all treatments.γ-secretase, are currently underway among patients with leukaemia, and plans to trial these agents among children with ependymoma are in advanced stages within the US Pediatric Brain Tumor Consortium. NOTCH signalling is activated following γ-secretase mediated cleavage of the NOTCH receptor (γ-secretase might be effective treatments of supratentorial ependymomas.At least two approaches might be adopted in the development of anti-CSC therapies. First, as normal stem cells are protected from environmental insults by both cell intrinsic and extrinsic factors, then CSC might be inherently resistant to conventional chemo- and radiotherapies . Thus, areceptor . Thus, iThe success of anti-CSC therapies will require not only that these drugs kill or disable CSC, but also that they spare normal stem cells. This issue is especially important when considering the treatment of children with brain tumours whose nervous system is still developing. Thus understanding further the origins of CSC in the brain and how best to target these in the clinic will likely require the ongoing collaboration of developmental biologists, cancer biologists and clinicians.


Patients diagnosed with lung adenocarcinoma (AD) and squamous cell carcinoma (SCC), two major histologic subtypes of lung cancer, currently receive similar standard treatments, but resistance to adjuvant chemotherapy is prevalent. Identification of differentially expressed genes marking AD and SCC may prove to be of diagnostic value and help unravel molecular basis of their histogenesis and biologies, and deliver more effective and specific systemic therapy.MiRNA target genes were predicted by union of miRanda, TargetScan, and PicTar, followed by screening for matched gene symbols in NCBI human sequences and Gene Ontology (GO) terms using the PANTHER database that was also used for analyzing the significance of biological processes and pathways within each ontology term. Microarray data were extracted from Gene Expression Omnibus repository, and tumor subtype prediction by gene expression used Prediction Analysis of Microarrays.Computationally predicted target genes of three microRNAs, miR-34b/34c/449, that were detected in human lung, testis, and fallopian tubes but not in other normal tissues, were filtered by representation of GO terms and their ability to classify lung cancer subtypes, followed by a meta-analysis of microarray data to classify AD and SCC. Expression of a minimal set of 17 predicted miR-34b/34c/449 target genes derived from the developmental process GO category was identified from a training set to classify 41 AD and 17 SCC, and correctly predicted in average 87% of 354 AD and 82% of 282 SCC specimens from total 9 independent published datasets. The accuracy of prediction still remains comparable when classifying 103 AD and 79 SCC samples from another 4 published datasets that have only 14 to 16 of the 17 genes available for prediction . Expression of this signature in two published datasets of epithelial cells obtained at bronchoscopy from cigarette smokers, if combined with cytopathology of the cells, yielded 89–90% sensitivity of lung cancer detection and 87–90% negative predictive value to non-cancer patients.This study focuses on predicted targets of three lung-enriched miRNAs, compares their expression patterns in lung cancer by their GO terms, and identifies a minimal set of genes differentially expressed in AD and SCC, followed by validating this gene signature in multiple published datasets. Expression of this gene signature in bronchial epithelial cells of cigarette smokers also has a great sensitivity to predict the patients having lung cancer if combined with cytopathology of the cells. Lung cancer is the most common cause of cancer mortality in the world , and oveLate diagnosis of extensive diseases is the main reason for treatment failure in lung cancer. Although a recent large randomized study suggested that computed tomography screening may not decrease number of diagnosis of advanced lung cancer and death from the tumor , early dMicroRNAs (miRNAs) are a group of small non-protein coding RNAs that often demonstrate temporally or spatially restricted expression patterns, and have great potential to mark the cytological and histological origins of tissues with less clear differentiated phenotypes, such as poorly differentiated tumors . In our Current identification of miRNA targets mainly follows computational prediction, and only a handful of them have been experimentally verified. Many investigations presumed expression of miRNA is opposite to that of their targets, based on observations in which transfecting miRNAs into human cells down-regulated large number of target mRNAs . A majorIn this study, expression of all predicted target genes of three lung-enriched miRNAs was examined in different subtypes of lung cancer without relating to matched miRNA expression. After a series of data filtering by classification of tumor subtypes, a minimal gene signature was identified to correctly predict the majority of AD and SCC cases from multiple published datasets. Expression of the same signature was also examined in two airway epithelial cell gene expression datasets to evaluate its diagnostic value for early detection of lung cancer in cigarette smokers.In general, the union of combining different prediction algorithms gives much greater number of predicted miRNA target genes, and therefore less false negatives and more false positives, than individual algorithms or the intersection of combining different algorithms. The strategy used in this study was to obtain maximally possible number of candidates, followed by a series of data reduction to filter out genes. MiRNA target genes were predicted by union of miRBase Target v4 (powered by miRanda), TargetScan 4.0, and PicTar, followed by screening for availability of gene symbols in NCBI human sequences and Gene Ontology (GO) terms using the PANTHER database v6 . Genes wst, 2007 were reviewed. Only one dataset, GSE7339, was removed from further analysis due to too many genes missing from the 17-gene signature for prediction analysis. The 14th dataset GSE2514 in Table Microarray data were extracted from Gene Expression Omnibus repository, and all databases used in this study were summarized in Table Clustering of gene expression profiles used Gene Cluster 3.0, and the parameters used are the following: after removing genes that had good data in less than 80% of specimens examined, data were adjusted by gene centering only (no array centering), and the hierarchical clustering used uncentered correlation as the similarity metric and average linkage as the clustering method. Classification of lung cancer specimens by miRNA target genes was compared to the clustering profiles of mRNA expression in the original article publishing the Database 1 . PredictBecause microarray datasets use different platforms and the data are presented in different formats, the microarray data were adjusted to similar scale and distribution patterns before class prediction based on four guidelines  that had approximately a thousand copies or less each cell in testes, fallopian tubes, lung, and trachea, while the rest of tissues examined had no or barely detectable levels of expression , while tTwo independent datasets were used to test the hypothesis whether these miRNAs (miR-449 was not available in these two databases) had reduced expression in lung cancer. One is from our previous published miRNA expression profiles in the NCI-60 panel of cell lines derived from human cancers that used real-time PCR for quantitation , and theIt has been shown that the union of miRNA target genes predicted by three computational algorithms  is one of the strategies that give the highest sensitivity , which pThe developmental process genes with eligible data points in the Database 1 were re-analyzed by PAM as a training set. After cross-validation, the error plot shows the lowest error rate between threshold 0 and 1.6, indicating that the genes represented by the threshold 1.6 is the minimal subset of "core genes" that can best separate the 41 AD and 17 SCC in this dataset (which outputs the 17 genes). SAM algorithm also showed that these 17 genes have the lowest false detection rate to separate AD from SCC . Examining the predicted target sites of miR-34b/34c/449 in these 17 genes showed that most of the genes have target sites predicted by at least 2 of the 3 algorithms and at least 2 target sites predicted by at least one of the algorithms Table . ExpressExpression of the 17 core genes in another 8 validation datasets ,30-35 waThe Database 14 contains 44 specimens derived from a urethane-induced mouse lung tumor model in which the neoplastic tissues exhibit histological appearance and molecular changes similar to human lung AD . This daThe pathway ontology terms of the 17 genes shows that only the TGF-beta signaling pathway is overrepresented after Bonferroni correction , while tumors with a more differentiated phenotype resemble bronchioalveolar carcinoma ("bronchioid") . Stagingin vivo might be subject to regulation by these miRNAs, and that delivery of these miRNAs might be able to modulate tumorigenic phenotypes, such as TGF-beta pathways that is over-represented in the identified gene signature. Secondly, identifying differentially expressed genes often follows data filtering, which only hones in the most variable expression between the groups, and apparently will discount genes whose differential expression is under the threshold but might be better candidate markers. In fact, several genes in the signature were also identified as differentially expressed between AD and SCC previously .Click here for fileSummary of gene lists generated from random controls (predicted targets of miR-141/146b/216 and first and second sets of 17 random genes) and 11 TGF-beta pathway genes from predicted targets of miR-34b/34c/449.Click here for fileDiagnosis and prediction of the bronchoscopic specimens from the training set.Click here for fileDiagnosis and prediction of the bronchoscopic specimens from the validation set.Click here for file


A highly significant correlation was found between agar clonogenicity and patient survival after biopsy. However, problems related to inherent tumour heterogeneity, quality of sample and tissue disaggregation indicate that this technique may have limited applicability in the routine assessment of patients. Only 41 biopsy specimens (36%) from 31 patients (44.3%) complied with the prerequisite criteria for agar clonogenic assessment, namely: (a) the confirmed presence of malignant cells in the biopsy, (b) the ability to prepare a single-cell suspension, and (c) adequate viable cell numbers for assay. Furthermore, although the dominant patterns of agar clonogenic growth could be identified and correlated with stage of disease, the heterogeneity in both initial clonogenic capacity and "self-renewal" capacity assessed by the ability of primary clones to propagate in liquid culture and reclone in agar was too inconsistent for the assay to be used as a prognostic index for the individual patient.


Hepatitis C virus (HCV) is a major cause of chronic liver disease, with about 170 million people infected worldwide. Up to 70% of patients will have persistent infection after inoculation, making this disease a significant cause of morbidity and mortality.The severity of disease varies widely, from asymptomatic chronic infection to cirrhosis and hepatocellular carcinoma. Since the discovery of HCV, the treatment of hepatitis C has considerably improved. Recently, combination of pegylated interferons with ribavirin gives a response rate of about 55%. Treatment is indicated in patients with moderate or severe fibrosis. The tolerability of combination treatment is relatively poor, with a frequent flu-like syndrome and an impaired quality of life.In addition to viral and environmental behavioural factors, host genetic diversity is believed to contribute to the spectrum of clinical outcomes in HCV infection. The sequencing of the human genome, together with the development of high-throughput technologies that measure the function of the genome, have afforded unique opportunities to develop profiles that can distinguish, identify and classify discrete subsets of disease, predict the disease outcome or predict the response to treatment. This paper reviews the published literature on gene expression associated with HCV infection , and also according to response to treatment. HCV infection may activate host signalling pathways that induce type I IFNs.14It should be noted that HCV RNA encodes specific proteins that may inhibit the induction of type I IFNs. An example is the NS3-4A protease of HCV, which blocks dsRNA-induced IFN production by interfering with IRF-3 phosphorylation.HCV-related effects may also attenuate IFN signalling. Proteins called suppressor of cytokine signalling (SOCS) are known to inhibit cytokine signalling via Jak–STAT. The HCV core protein has been shown to induce the expression of SOCS3, which can suppress Jak–STAT signalling events and block the IFN-induced formation of ISGF3.The natural history of hepatitis C virus infection is influenced by both genetic and environmental factors. T lymphocytes, which are important in terms of both viral clearance and hepatotoxicity, are stimulated in their response after presentation of foreign material by antigen-presenting cells in association with major histocompatibility complex (MHC) molecules. Several studies, predominantly in Caucasians, have found associations of the human leucocyte antigen (HLA) class II alleles DQB1 0301 and DRB111 with self-limiting infection populations. Also a high quasispecies population is believed to be associated with chronicity.–33The major factors known to be associated with fibrosis progression are male gender, older age at infection and excessive alcohol consumption. Interestingly, viral factors such as viral load and genotype do not seem to influence the progression rate significantly. Progression of fibrosis is more rapid in immunocompromised patients. Insulin resistance may also contribute to more rapid progression of fibrosis.31–HCV is a member of the Flaviviridae family, genus Hepacivirus. Error-prone replication of HCV, resulting in a complex quasispecies population within each infected individual, enables rapid adaptation to changing environments. Six HCV genotypes and a large number of subtypes have been identified.8Together, these findings suggest that HCV modulation of IFN induction and signalling limits the expression of IFN-stimulated genes, allowing HCV to evade the antiviral actions of the host response.MicroRNAs (miRNAs) are a class of small non-coding RNA molecules of 20–22 nucleotides that control gene expression by targeting mRNAs for translational repression or cleavage.Silencing of the RNAase III Dicer has been shown to inhibit HCV replication by ∼7-fold.The recent development of efficient tools for large-scale analysis of gene expression has provided new insights into the role of gene networks and regulatory pathways in various tumour processes.18STAT1, STAT2, ISGF3G/IRF9, IFI27, G1P3, G1P2, OAS2 and MX1) and IFNγ-inducible genes . Dysregulation of these genes was mainly HCV specific (no upregulation in hepatitis B virus (HBV) infection).Little is known about the molecular mechanisms associated with HCV infection in humans. Liver gene expression was studied, by large-scale real-time RT-PCR, in patients with untreated chronic hepatitis C and mild fibrosis compared with histologically normal controls.Interestingly, similar results were found in a recent DNA microarray analysis in nine individuals with chronic hepatitis C compared with non-diseased liver controls.Liver fibrosis is the accumulation in excess of extracellular matrix proteins including collagen.et al analysed genomic DNA from a well-characterised German cohort of 329 patients with chronic hepatitis C and found that previously described and novel keratin 8 (K8) variants are present and collectively associate with the progression of fibrosis.35In a recent study, Strnad Until recently, almost all association studies performed to identify genetic variants associated with HCV clearance, the progression of fibrosis or the response to antiviral treatment have focused on variations in one or a few candidate genes, chosen on the basis either of their known function or from animal models. Recent advances in high-throughput genotyping technology have now made large-scale association studies a reality, and the number of candidate genes investigated in each study is rapidly increasing.Genome-wide association studies (GWAS) are now ongoing thanks to the rapidly decreasing cost of genotyping, massively multiplexed genotyping technologies, large-scale single nucleotide polymorphism (SNP) discovery as well as the genotyping efforts of the SNP Consortium and the HapMap project. GWAS use dense maps of genetic markers that cover the human genome to look for allele frequency differences between cases and controls. A significant frequency difference suggests that the corresponding region of the genome contains functional DNA variants that influence the trait of interest. The candidate gene strategy has the advantage of focusing resources on a manageable number of genes and polymorphisms that are likely to be important. The strength of the genome-wide screening is its ability to reveal not only genes that would be expected to play a significant role, but also genes that would not, potentially adding new insight into pathophysiology or pharmacology.DDX5) gene which was significantly associated with an increased risk of advanced fibrosis in both the discovery and replication cohorts . The second one was a missense SNP in the carnitine palmitoyltransferase 1A (CPT1A) gene associated with a decreased risk of advanced fibrosis in both the discovery and replication cohorts . These two novel markers could play a useful role in predicting risk of fibrosis in HCV patients. After the initial findings of two replicated SNPs, the confirmation of all significant SNPs from their genomic scan was completed and 361 SNPs were selected for signature building.38A genome-wide scan with putative functional SNPs was carried out on a group of 433 HCV patients (discovery cohort); those markers associated with advanced fibrosis were validated in a separate group of 483 patients (replication cohort).In chronic hepatitis C, the transition from mild to moderate fibrosis seems to be a major prognostic step. Several consensus conferences have proposed that patients with mild fibrosis might not be treated because their prognosis is good.39KRT19 and SCG10), growth factors/cytokines , growth factor receptors , in extracellular matrix production , in extracellular matrix remodelling  and in the cell junction (ITGA2 and CLDN4) specimens and compared with F1 (mild fibrosis) specimens.nd CLDN4).A stellate cell is a key cell in fibrogenesis and fibrolysis. Many pathways are involved in hepatic stellate cell activation. These genes encode proteins involved in extracellular remodelling, oxidative stress, signal transduction pathways, cell cycle control, apoptosis, angiogenesis, interferon signalling, the immune response, and so forth.Interestingly, a recent study has demonstrated that histologically normal liver tissue obtained in two different ways  has different gene expression patterns although all specimens are histologically normal.Finding molecular markers of the progression of fibrosis has several clinical implications. First, many of the genes found to be upregulated between mild and moderate fibrosis encode molecules secreted in the serum (cytokines). Therefore, looking for genes dysregulated in the liver can constitute a logical functional approach for the discovery of serum markers of the progression of fibrosis. For example, serum levels of cytokines have been associated with changes in the severity of chronic hepatitis C. Secondly, since a primary goal in the treatment of HCV infection is eradication of the virus and another is to stop the progression of fibrosis, molecular markers of the progression of fibrosis could help define new endpoints during antiviral treatment. Therefore, gene changes could be new markers of the progression of fibrosis during antiviral treatment. Thirdly, many of the upregulated genes identified in this gene expression study are potential molecular targets for the development of antifibrotic drugs.An altered pattern of epigenetic modifications is central to many common human diseases, including cancerHuman tumours undergo a massive overall loss of DNA methylation, but also acquire specific patterns of hypermethylation at certain promoters. In addition, these DNA methylation changes are linked with the presence of an aberrant pattern of histone modification. Unlike genetic alterations such as single-base mutations or deletions, epigenetic changes are potentially reversible.DNA methylation changes are the most widely epigenetic modifications studied in humans. DNA methylation, the addition of a methyl group to the fifth carbon position of the cytosine residue, occurs in the CpG dinucleotides; 3–6% of all cytosines are methylated in normal human DNA. Potentially methylatable CpG dinucleotides are not randomly distributed in the human genome; instead, CpG-rich regions known as CpG islands, which span the 5′ end region  of many genes, are usually unmethylated in normal cells. This unmethylated status corresponds with the ability of CpG island-containing genes to be transcribed in the presence of the necessary transcription factors. In cancer cells, the transcriptional silencing of tumour suppressor genes by CpG island promoter hypermethylation is key to the tumourigenic process.DNA methylation occurs in a complex chromatin network and is influenced by the modification of histone structure. Histones are no longer considered to be simple “DNA-packaging” proteins; they are recognised as being dynamic regulators of gene activity that undergo many post-translational chemical modifications, including acetylation, methylation, phosphorylation, ubiquitylation and sumoylation. The status of acetylation and methylation of specific lysine residues contained within the tails of nucleosomal core histones is known to have a crucial role in regulating chromatin structure and gene expression, and is commonly disrupted in cancer cells.Recent studies indicated that epigenetic alterations might initiate the expansion of premalignant cells during the early stages of tumourigenesis. During the early steps of development of the principal tumour types, such as colon, lung and prostate, but also liver tumours, a subset of these premalignant cells undergo subsequent irreversible genetic alterations that allow them to mediate tumour growth.Tools for large-scale analysis of CpG island hypermethylation include array-based epigenomic analysis methods such as DHM  for a genome-wide screening of CpG island hypermethylation (DNA methylome) or large-scale quantitative real-time PCR-based methylation analysis methods for more accurate and quantitative analysis of DNA methylation of specific candidate genes.42et alMMP14, miR-219, CIDEA, etc.) or in the transition between tissues adjacent to HCC .There are only a few studies in the literature on array-based epigenomic analysis and the early stages of liver fibrosis associated with chronic HCV infection. Gao –et al report that aberrant methylation of a limited number of loci is common in the normal ageing liver and that these epigenetic alterations gradually progress and expand to a larger panel of methylation markers in HCC.p16, RIZ1/PRDM2, HIC-1, etc.), the Wnt/β-catenin pathway , the Ras pathway (SPROUTY and RASSF1A), the Jak–STAT pathway , the TGFα pathway (RUNX3) and metabolism of xenobiotics .Several studies using quantitative real-time PCR-based methylation analysis for DNA methylation of specific candidate genes suggest that DNA methylation is an early event preceding cirrhosis.44Although interesting biological insights and promising tools are being developed, HCV-induced liver fibrosis epigenomics is still in its infancy. The impact of the powerful epimutagen HCV on the progression of aberrant methylation must be elucidated for the different steps of liver fibrosis. For this, comprehensive epigenomic profiling must be performed at every step of liver fibrogenesis as well as hepatocarcinogenesis. Gene silencing can be viewed as an “epigenetic gatekeeper” step that creates a milieu that facilitates the selection of mutations in oncogenes and tumour suppressor genes promoting tumour progression.Understanding the molecular events that initiate and maintain epigenetic gene silencing in the early stages of HCV-associated fibrosis could help in the development of strategies to reverse the silencing process for the prevention and treatment of cirrhosis and HCC.Recent evidence has shown that miRNA mutations or misexpression are associated with various human cancers and that miRNAs can function as tumour suppressors and oncogenes.There are a few pilot studies in the literature on miRNA expression profiling in hepatocellular tumours, but few or none concerning HCV-induced liver fibrosis.–The studies in HCC tissues or experimental models of HCC have all shown that specific miRNAs are aberrantly expressed in malignant HCC cells or tissues compared with non-malignant tissues.51et al suggest that important changes in miRNA expression occur during the development of chronic viral hepatitis and cirrhosis.58Although there are few studies in the literature on miRNA genes and HCV-induced liver fibrosis, we hypothesise that miRNAs play a critical role in HCV infection, in the progression of fibrosis and in the prediction of treatment response. Jiang –63A sustained virological response (SVR) rate of ∼55% is obtained with the combination of pegylated IFNs (PEG-IFNs) and ribavirin.59Since a significant number of patients will fail to respond to treatment, or have a virological relapse or significant side effects so that treatment must be stopped, it is important to identify non-responding patients as early as possible and ideally at baseline (before treatment) both for patient welfare and for cost-effectiveness. The probability of an SVR essentially depends on genotype. Younger age, female gender, mild fibrosis and low viral load are also associated with a better response rate, but to a lesser extent. In patients with HCV genotypes 2 or 3, the SVR rates reach 80%; in genotype 1 patients the SVR rate is 50%.An early virological response is the best predictive factor of an SVR.64ADAR, CASP5, FGF1, ICSBP1, IFI44, TAP2 and TGFBRAP1) was used to construct a model by multiple logistic regression. The sensitivity and the specificity of the model were 68.2% and 60.7%, respectively. Nonetheless, there are several weaknesses in the logistic regression approach. First, it cannot detect complex gene–gene interactions since, like other traditional regression methods, it relies on the basic assumption of linear combinations only. Secondly, the rapid increase in the availability of large numbers of genetic markers increases the number of potential predictors considerably, which, when combined with the significantly smaller number of observations, creates a statistical problem called the ‘curse of dimensionality’.Hwang and colleagues have used this method to look at the genetic differences that were associated with IFN/ribavirin responses in 317 Taiwanese patients with chronic hepatitis C.–69Several “machine learning” approaches that have recently been applied to investigate potential gene–gene and gene–environment interactions in IFN treatment or in the risk of developing cirrhosis have been highly successful for modelling the relationship between combinations of polymorphisms and clinical endpoints.67et al used an artificial neural network (ANN) to address gene–gene and gene–environment interactions in antiviral treatment response for 523 patients with chronic hepatitis C who had received IFN and ribavirin combination treatment, including 350 sustained responders and 173 non-responders.ADAR, CASP5, ICSBP1, IFI44, PIK3CG and TAP2). A feedforward neural network was used to model the responsiveness of IFN, and the back-propagation algorithm was used for the learning scheme. Inputs were genetic and clinical factors including SNP markers, viral genotype, viral load, age and gender. Outputs were the IFN-responding status. The prediction accuracy of each model was estimated using a fivefold cross-validation procedure, and a permutation test was applied to measure the statistical significance of an association between predictors and drug response. All possible combinations of N factors were evaluated sequentially and the N-factor model displaying the highest prediction accuracy was retained. IFI44 was found in the significant two-locus, three-locus and four-locus gene–gene effect models as well as in the significant two-factor and three-factor gene–environment effect models. Furthermore, viral genotype remained in the best two-factor, three-factor and four-factor gene–environment models. Thus, these results strongly support the hypothesis that viral genotype and IFI44, a member of the family of IFN-inducible genes, play a role in the pharmacogenomics of IFN treatment. In addition, this approach identified a panel of 10 factors that may be more significant than the others for further investigation. Hence, theses results suggest that an ANN-based approach may be useful to analyse the complex non-linear relationship between genetic and clinical factors and the responsiveness of IFN. Similarly, a support vector machine (SVM) algorithm was applied to build a tool to predict responsiveness to IFNα–ribavirin combination treatment using host polymorphisms and viral genotype.Lin Another study genotyped eight SNPs spanning the entire IFNγ gene in two cohorts and assessed the association between those polymorphisms and treatment response or spontaneous viral clearance.Liver gene expression profiling has recently been applied to chronic hepatitis C to determine response to treatment. Knowledge of the antiviral mechanisms of IFN is crucial for the discovery of new treatment response markers . In a reIFI27 and CXCL9) signature, which accurately predicted treatment response in 79.3% (23/29) of patients from the validation set (Group B), with a predictive accuracy of 100% (9/9) and of 70% (14/20) in non-responders and sustained virological responders, respectively. The expression profiles of responder–relapsers did not differ significantly from those of non-responders and sustained virological responders, and 73% (8/11) of them were predicted as sustained virological responders with the two-gene classifier. In conclusion, non-responders and sustained virological responders have different gene expression profiles prior to treatment. The most notable changes in gene expression were mainly observed in the IFN-stimulated genes (IFI27 and CXCL9) in two independent groups of patients . Most relapsers clustered with sustained virological responders. Interestingly, the baseline liver levels of expression of IFN-stimulated genes were higher in non-responderss than in sustained virological responders. The failure to respond to exogenous PEG-IFN in non-responders could indicate a blunted response to IFN. This suggests that IFN-stimulated genes are already maximally induced in non-responderss. Genes included in the signature encode molecules secreted in the serum and provide a logical functional approach for the development of serum markers to predict response to treatment.In a recent study, a selection of genes associated with liver gene expression dysregulation during HCV infection was studied by large-scale RT-PCR assay according to response to treatment.ed genes . We wereOne study analysed gene expression patterns in peripheral blood mononuclear cells during IFN treatment and confirmed upregulation of genes thought to be IFN-stimulated genes or involved in antigen processing and presentation.Interestingly, data in HCV-infected chimpanzees indicate a predominantly defective hepatic response to IFN, which is probably mediated through the activation of SOCS3 and may explain the lack of response to IFN-based treatment in many HCV patients.The diversity of microarray platforms utilised for gene expression analysis and the variability in microarray data emphasise the need for quality assurance. High quality RNA samples are essential for gene expression analysis, and the quality of each RNA preparation must be rigorously assessed. Careful measures must be taken during all steps of the RNA extraction to prevent the RNA from degrading. Furthermore, improved analytical procedures and the use of large numbers of patients are needed for validation. Gene signatures will probably be used in the future for optimised and tailored treatment.Many of the genes found to be upregulated between non-responders and responders encode molecules secreted in the serum (cytokines) and are a logical functional approach for the development of serum markers as predictors of response to treatment. For instance, one study examined the levels of chemokines that bind to CXC chemokine receptor 3 (CXCR3) to determine whether they play a role in the failure of the immune system to clear HCV infection.73Characterisation of the liver and serum proteomes is the next step in the investigation of patients with liver diseases since protein signatures are a potentially powerful tool in the diagnosis and prognosis of patients with HCV infection. Although there are several studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been extensively characterised. Proteomics uses a combination of sophisticated techniques including 2D gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing and bioinformatics.et al provide a global proteome analysis of changes induced by HCV infection by multidimensional liquid chromatographic separation coupled with mass spectrometry in the full-length HCV replicon model.There are very few studies in the literature addressing proteomic changes in hepatitis C. Jacobs et al compared proteomic dynamics in in vitro and in vivo processes of HSC activation.Liver fibrosis and cirrhosis are the consequences of chronic HCV infection. They are characterised by marked modifications of proteins including synthesis of extracellular matrix proteins, suggesting that proteomics might provide new insights for diagnosis of fibrosis or cirrhosis. Several studies have been performed to characterise protein changes in tissues in experimental models of liver fibrosis. For instance, several proteins that are significantly deregulated during liver fibrosis have been identified by 2D gel electrophoresis and mass spectrometry.et al showed that expression of 59 protein spots significantly changed upon thioacetamide administration and that a protein of 3.495 kDa, sharing homology with a histidine-rich glycoprotein, was consistently decreased in sera of cirrhotic rats.et al tried to define serum protein signatures associated with various degrees of fibrosis and develop a proteomic fingerprinting model for predicting fibrosis and cirrhosis in patients with chronic hepatitis B infection.To date, few studies have focused on serum protein changes during the development of liver fibrosis. In thioacetamide-induced liver fibrosis, Xu In addition to viral and environmental behavioural factors, host genetic diversity is believed to play a role in each step of the different clinical outcomes in HCV infection . The sequencing of the human genome, together with the development of high-throughput technologies that identify gene function, have provided unique opportunities to develop profiles to distinguish, identify and classify discrete subsets of the disease, predict disease outcome or predict response to treatment. Multiplexed protein measurement (such as flow cytometric microbead assays using Luminex technology) is a rapidly advancing field that has the broadest potential technology to transform clinical diagnostic in the next 10 years.In the near future, further studies must meet several challenges: first, studies with large prospective cohorts with well phenotyped patients are needed. For instance, in the studies evaluating the severity of the disease, the stage of fibrosis  must be carefully determined, taking into account the duration of HCV infection. For response to treatment, studies must use the same definition of SVR and the same treatment regimen. Statistical analysis must take into account all factors associated with fibrosis progression or response to treatment. Secondly, technologies must be improved for routine use. For instance, the diversity of microarray platforms used for gene expression analysis and the variability in microarray data emphasise the need for quality control. Careful measures must be taken during all steps of RNA extraction to prevent the RNA from degrading. Furthermore, analytical procedures must be improved and large numbers of patients must be studied for validation. Gene signatures will probably be used in the future for personalised medicine (optimised and tailored treatment).


Neopterin is regarded as an early biomarker of the cellular immune response. This low molecular mass compound belongs to the class of pteridine and is a metabolite of guanosine triphosphate, which is produced by the activated macrophages and dendritic cells after stimulation with γ-interferon. An international group acknowledges the fact that the levels of serum neopterin can be used as a marker of the effect of exposure to silica and other occupational diseases. The determination of neopterin is an innovative tool for monitoring diseases associated with the activation of cell-mediated immunity. Neopterin [D-erythro-6--pterin][In vitro studies reveal that neopterin derivatives exhibit distinct biochemical effects, most likely via interactions with reactive oxygen or nitrogen intermediates, thereby affecting the cellular redox state.[Neopterin is pyrazino–pyrimidine compound of molecular weight 253 D belonging to the class of pteridines. Pteridine is a chemical compound of fused pyrimidine and pyrizine rings. A pteridine is a group of heterocyclic compounds containing a wide variety of substitutions in this structure. Pterins Neopterin is derived from GTP and is produced by stimulated macrophages under the influence of INF-γ of lymphocyte origin. Cleavage of GTP by GTP-cyclohydrolase I results in neopterin, yielding 7,8-dihydroneopterin triphosphate, which is a joint precursor of dihydroneopterin, neopterin, tetrahydrobiopterin, a necessary cofactor of aromatic amino acid monooxygenase, and nitric oxide synthases (NOS). Human monocytes/macrophages are the unique source to produce an excess of neopterin derivatives at the rate of 5, 6, 7, 8-tetrahydrobiopterin,8 In trypt10κB, an important modulator of inflammatory gene expression, including pro-inflammatory cytokines and endothelial cell adhesion molecules.[Recent data suggest that neopterin derivatives exhibit distinct biochemical functions. Neopterin was found to enhance the effects of toxic reactive oxygen species originating from chloramine T and hydrogen peroxide,13 sugges15olecules. The prodDC produces neopterin derivatives on stimulation, which is additionally determined by the degree of IDO-dependent tryptophan degradation. As observed, neopterin production and IDO activation were found to closely correlate in a large variety of diseases in vivo, including systemic lupus erythematosus, hepatitis immunodeficiency virus and in pregnancy.181918To activate the gene transcription by initiation of intracellular signaling via a complex mechanism, INF binds to specific cell surface receptors. The gene gets modulated by INF stimulation and effects on inhibition of viral replication, cell proliferation and immunomodulation in infected cells. An effector protein such as neopterin and 2′, 5′-oligoadeylates synthetase gets stimulated by INF. Neopterin has been used in the INF studies to demonstrate its immune activation by the INF. Neopterin has been validated in a large number of studies as a marker of INF activation. The level of neopterin increases with the INF.Occupational exposure to lead affects the neuromuscular junctions and it might cause disturbances in the locomotor activity. This study was carried out to evaluate pteridine metabolism for the synthesis of neurotransmitters in urinary neopterin, biopterin, creatinine and blood dihydropteridine reductase (DHPR) activity in battery workers and the delta-aminolevulonic acid (delta-ALA) was measured. Blood and urine lead levels were detected by an atomic absorption spectrophotometer. A significant increase in the blood and urine lead levels shows traces of urinary neopterin, biopterin and delta-ALA among exposed workers. DHPR actIt has been suggested that it is an excellent marker for the activation of the monocyte/macrophage axis in some clinical situations. Increased amounts of neopterin in body fluids are associated with a variety of diseases in which activation of the cellular immune mechanism is involved, such as certain malignancies, allograft rejection, autoimmune diseases and viral infections.23–25 Ele212The most important clinical application of the determination of neopterin is as a prognostic indicator and as a follow-up for chronic infection, immune stimulation monitoring, differential diagnosis of acute bacterial and viral infection and also as an early indication for the complication of allograft recipient.24 As the


Since some authors have stated a certain role for so-called “free fatty acids” (FFA) in the pathogenesis of AP and the subsequent systemic complications we tried to find possible correlations between FFA, pancreatitis and lung injury using a rat model. AP was induced by intraductal infusion of two different concentrations of glycodeoxycholic acid (GDOC 17 mmol and 34 mmol). An equal number of animals had only cannulation of the pancreatic duct without infusion and served as controls (GDOC-control). In another experimental model iv.-infusion of oleic acid (OA) was used to create severe lung injury comparable to human ARDS. In this model control animals received iv.-infusion of saline solution only (SAL). At 2,6,12,24 and 48 hours the animals were sacrificed and blood was collected for determination of FFA, amylase and pO2. The pancreas and lungs were removed for histologic examination and the lungs were weighed. GDOC-34 animals developed severe pancreatitis with hemorrhage and necrosis. Histology of the lungs showed edema, inflammatory infiltrates, hemorrhage and thickening of the alveolar membrane in GDOC-34 rats as well as in OA-animals. In contrast, there was only pancreatic edema until 24 hours in the GDOC 17 group and less severe histological changes in the lungs. Amylase, FFA, pO2 and lung weight were directly influenced by the different kinds of treatment. Furthermore, FFA correlated positively with the levels of amylase and lung weight and negatively with pO2. Infusion of OA alone also caused an increase in levels of amylase with pancreatic edema and focal necroses in some animals. These results show that it was possible to create different degrees of severity of AP which was in concordance with different degrees of morphologic changes and dysfunction in the lungs. FFA values correlated significantly with the clinical course as well as with increasing amylase, lung weight and decreasing pO2.


KLKs) encodes proteins with diverse and pleiotropic functions in normal physiology as well as in disease states. Currently, the most widely known KLK is KLK3 or prostate-specific antigen (PSA) that has applications in clinical diagnosis and monitoring of prostate cancer. The KLK gene family encompasses the largest contiguous cluster of serine proteases in humans which is not interrupted by non-KLK genes. This exceptional and unique characteristic of KLKs makes them ideal for evolutionary studies aiming to infer the direction and timing of gene duplication events. Previous studies on the evolution of KLKs were restricted to mammals and the emergence of KLKs was suggested about 150 million years ago (mya). In order to elucidate the evolutionary history of KLKs, we performed comprehensive phylogenetic analyses of KLK homologous proteins in multiple genomes including those that have been completed recently. Interestingly, we were able to identify novel reptilian, avian and amphibian KLK members which allowed us to trace the emergence of KLKs 330 mya. We suggest that a series of duplication and mutation events gave rise to the KLK gene family. The prominent feature of the KLK family is that it consists of tandemly and uninterruptedly arrayed genes in all species under investigation. The chromosomal co-localization in a single cluster distinguishes KLKs from trypsin and other trypsin-like proteases which are spread in different genetic loci. All the defining features of the KLKs were further found to be conserved in the novel KLK protein sequences. The study of this unique family will further assist in selecting new model organisms for functional studies of proteolytic pathways involving KLKs.The gene family of human kallikrein-related peptidases ( KLK1-15) in the human genome mapped to chromosomal locus 19q13.4 KLK contiguous cluster is not interrupted by other non-KLK genes, an additional feature that makes this family unique. Collectively, all the above characteristics establish the KLK family as a family of great importance for evolutionary studies. Tissue KLKs are usually divided into two groups the “classical” and the “non-classical” KLKs. The term “classical” KLKs is referred to the first members of the human KLK family that were identified, namely KLK1, KLK2, and KLK3 (PSA), whereas the rest are often referred to as “non-classical” Human tissue kallikrein-related serine peptidases (KLKs) constitute a single family of 15 highly conserved trypsin- or chymotrypsin-like serine proteases encoded by the largest contiguous cluster of protease-encoding genes  which is implicated in stabilizing the oxyanion intermediate of the internal peptide bond during hydrolysis All currently reported KLKs are expressed in a wide variety of tissues including the pancreas, heart, lung, central nervous system, salivary glands and endocrine-regulated tissues such as thyroid, breast, testis, ovary, prostate, indicating that they participate in important biological processes Previous efforts focused on the evolutionary history of KLKs focused in the characterization of the mouse In the present study, a comprehensive phylogenetic analysis of the KLK proteins was performed employing a maximum likelihood-based method in order to unravel the evolutionary history of the KLK family. Interestingly, three reptilian, two avian and one amphibian KLK homologues were detected which allowed us to trace the evolutionary origin of KLKs earlier than it was previously thought, approximately 330 mya. Primary sequences, as well as the predicted secondary and tertiary structures of putative KLK peptide sequences were analyzed. The genomic organization of the KLK genes was further examined and it was shown that in different species these genes cluster together at syntenic loci. Collectively, we suggest that KLKs are also present in non-therian species covering an evolutionary distance from amphibia to eutheria and they cluster at a single locus.The complete or almost complete genomes of species representing major taxonomic divisions (according to the NCBI taxonomy database) 57, Asp102 and Ser195) are conserved among species. The core trypsin domain of the 260 retrieved sequences was used to reconstruct a maximum likelihood-based phylogenetic tree . This mutation renders the enzyme inactive, since Asp102 is a catalytic residue.As mentioned previously, the identified KLK homologues contain the invariant residues of the active-site catalytic triad . HoweverFurthermore, it is demonstrated that the novel KLK amino acid sequences possess the secondary structure of known KLKs KLK gene family in different species. As shown in . However, due to incomplete genomic studies, KLK genes of several species KLK genes that flank it in the prototypical sequential order (1) are detected in the same chromosome/scaffold/contig. The two KLK2 pseudogenes (KLK2-ps) found in murinae KLK2 gene.The exponential accumulation of genomic sequences allowed us to study the evolution of the , apart from the fifteen “conventional” KLK family members, three ‘orphan’ KLKs are present in anole lizard. The lizard KLK orphans appear to arise from the basal node  thescaffold . KLK6, Kscaffold . Besidesscaffold . KLK7, Kscaffold . Regardirst time . We propeudogene . Also, it genome . Both thKLK2 gene maintains the same orientation in all genomes except in perissodactyla suggesting that the direction of KLK2 transcription differs from species to species. Although, the equine KLK2 has a predicted chymotrypsin-like specificity similar to that of KLK3 KLK2 which produced a KLK3 in catarrhini. Both KLK2 and KLK3 enzymes are secreted by the prostate gland where the zymogen KLK3 was shown to be activated by KLK2 KLK9 and KLK11. Similarly, KLK4 appears to be the product of a KLK5 duplication which has occurred presumably after the marsupial-placental split -encoding genes have different splicing patterns when compared to the rest of serine protease-encoding genes prompting that the split between the serine proteases took place at that time point Inspection of the KLK protein sequences  suggestsKLK gene family ideal for evolutionary studies. For example, MMPs constitute another important gene family (consisting of 25 genes in vertebrates and 24 in humans). MMPs are widely distributed in the animal kingdom and appear to have evolved from a single domain protein which underwent successive rounds of duplication, gene fusion and exon-shuffling. However, in contrast to KLKs, the members of this family are distributed along different chromosomes The process of gene duplication is essential to the efficient generation of genes with novel or altered functions. When the duplicated gene is fixed to the genome and is functionally preserved by natural selection it may diverge either by neofunctionalization or subfunctionalization. The significance of this process is demonstrated by the widespread existence of gene families. The unique characteristic of co-localization and the large number of members is what makes the KLK genes were present in the genome of chicken, frog, or the song bird zebra finch Xenopus tropicalis) KLK gene, confirmed the absence of a KLK-like sequence in chicken , but in contrast a KLK homologous sequence in turkey  and a partial KLK gene (likely pseudogene) were revealed in zebra finch.The increased availability of fully sequenced genomes from multiple organisms enabled us to conduct a detailed phylogenetic analysis of KLKs in order to reconstruct the evolutionary history of the KLK family. Contrary to the prevailing notion, in the present investigation it was shown that putative KLKs exist in non-therian species, covering an evolutionary distance from amphibia to eutheria. Previous work In view of our findings it would also be tempting to speculate that the evolutionary origin of KLKs should be moved further back to the radiation of amphibia (330 mya). Noticeably, despite extensive database searches no piscine, ascidian or insect KLK-related proteins were detected. The importance of our findings has implications for the physiological functions, while evolution of KLKs parallels that of their well-established substrates.KLK2 and KLK3 genes appeared later in evolution of the KLK gene family. Their functional roles are mainly linked to reproduction and more specifically to liquefaction of semen in humans KLK2 gene is absent (i.e. inactivated due to missing coding exons), as also absent are the KLK2-specific substrates in the seminal clot, i.e. semenogelin I, semenogelin II, and TGM4 (prostate transglutaminase 4) that are inactivated due to premature stop codons. Lack of seminal proteins diminishes the viscosity of semen that is liquefied upon ejaculation, therefore the KLK2 enzymatic activity is not needed in this case Macaca mullata (Rhesus monkey), KLK2 has a mutation (active site Asp102Ala) that renders the enzyme inactive as previously reported . On the contrary, gorillas are monogamous in nature and there is no need for mate guarding and sperm competition, therefore the aforementioned genes have been inactivated through selection. In addition, a copulatory plug does not exist in cow which is further characterized by the absence of TGM4TGM4 has been reported in opossum and again we did not find KLK2 and KLK3 genes in opossum KLK4 showed abnormalities in teeth maturation KLK4 knockout mice although the enamel layer thickness was normal it was rapidly abraded following weaning even when they were maintained with soft chow KLK4 is important for proteolysis and degradation of the 32 kDa fragment of enamelin since this procedure provides space for apatite growth. Retention of this fragment disturbs the biomineralization process. Consistently, knockout mice for KLK4 is unnecessary in these animals; indeed we were unable to detect this gene. Consistently, we showed here that chicken genome encodes a non-functional enamelin pseudogene and no KLK4 or other KLKs KLK4 gene as we could not detected it in platypus  i.e. KLK) as they are essential for replenishment of the skin surface. It is currently not clear whether the KLK skin cascade emerged in platypus, since we were unable to identify a KLK7 orthologue in platypus but this may be due to incomplete genome sequencing.It is well established that the skin desquamation process involves a proteolytic cascade, which is initiated by activation of proKLK5 either auto-catalytically SPINK5 encodes for LEKTI that contains 15 protease inhibitory domains, mouse and rat Spink5 encode for LEKTI that contains only 14 domains and lacks domain 6 Anolis carolinensis  has low-permeability skin. While putative orthologs for desmocollin (ENSACAG00000017830) and desmoglein (ENSACAG000000 17850) are encoded in lizards, the absence of KLK5 and KLK7 is compatible with decreased skin shedding and the low permeability of their stratum corneum. Additionally, skin permeability is also decreased by expression of hard-beta keratins and high amounts of lipids that “insulate” the skin It should be noted that frog skin and the skin of amphibia, in general, is more permeable than that of mammals since it is engaged in respiration and regulation of internal water and ion loss . Interestingly, Kita et al. The loop-99 (starting at amino acid residue 99) is necessary for kininogenase activity and is present only in KLK1, KLK2 and PSA/KLK3 KLKs  are coordinately regulated in biological fluids and tissues, while they often display common patterns of aberrant expression in disease states KLK9 and KLK11 are highly expressed in esophagus, vagina, stomach, breast, salivary gland and pancreas, KLK4 and KLK5 are highly co-expressed in breast and cervix, KLK10 and KLK12 in salivary gland, esophagus, fallopian tube, and pancreas KLK tissue-specific co-expression supports the hypothesis that each KLK gene is independently regulated by conserved regulatory mechanisms of transcription. Regulatory involvement of a locus control region (LCR) is not likely as the KLK locus evolved through a series of gene duplication events. Lack of a LCR is corroborated by studies showing that in transgenic mice bearing genomic fragment combinations of 2–3 neighboring rat Klk genes, rat-tissue KLK expression patterns are preserved There is ample evidence that duplicated in vivoRecent functional studies implicate certain KLKs in various types of cancer The fact that, during the course of evolution, KLKs have survived with significant similarity in terms of sequence, gene organization and number in higher organisms (from monotremes to primates) suggests that they likely play important roles in normal physiology. Elucidating the evolutionary history of KLKs would serve in the development of model systems for the study of gene function(s) in future studies. Collectively, the biological functions of the extended KLK family are currently under investigation. Pleiotropic physiological roles of KLK enzymes are being revealed, while aberrant regulation of KLKs is implicated in diverse diseases such as hypertension, renal dysfunction, skin disorders, inflammation, neurodegeneration, and cancer In order to identify KLK orthologues, a combination of queries based on key terms and BLAST searches was employed. The names and/or accession numbers of the characterized kallikreins, including all human, mouse and rat KLKs, as well as the canine and equine prostate-specific antigen (KLK3), were used to retrieve their corresponding amino acid sequences. Then, the entire peptide sequences of those KLKs were used as probes to search the publicly available non-redundant databases, UniProt The consensus boundaries of the core trypsin domain in the sequences included in the phylogenetic analyses, were determined from full-length sequences combining the outputs of Pfam i.e. 3 out of 5 predictions) of the combined output of CDM The secondary structure of the identified putative KLK homologous proteins was predicted as a consensus Click here for additional data file.Figure S2ML phylogram of KLK homologues and related proteins. The CFD/Adipsin sequences were included in the phylogenetic analysis as well. The sequences which are subject to question are indicated by arrows. Conventions are the same as in (0.26 MB TIF)Click here for additional data file.Figure S3Rate shift analysis of KLK7, 10, and 12 subfamilies. The analysis further supports our phylogenetic analysis by demonstrating that KLK10 and KLK12 subfamilies are sister groups.(5.08 MB TIF)Click here for additional data file.Table S1Names and accession numbers of the sequences analyzed in the present study. The KLK pseudogene names are shown in italics.(0.32 MB DOC)Click here for additional data file.


Sex hormone binding globulin (SHBG) concentrations were measured by immunoradiometric assay in serum samples from 1,221 healthy female volunteers aged 34-79 who had never used oral contraceptives or hormone replacement therapy, had no history of thyroid disease or cancer, and had not used any drugs known to influence SHBG in the 14 days preceding blood sampling. There were 616 premenopausal and 605 naturally postmenopausal women. In both premenopausal and postmenopausal women, SHBG decreased with increasing weight (Quetelt's Index) and was lower in single nulliparous women than in married nulliparous women or parous women. In premenopausal women, SHBG was higher in women with late menarche, was higher in smokers than in non-smokers, and was higher in blood samples taken during the first 12 days of the luteal phase than during the rest of the menstrual cycle. In postmenopausal women, SHBG increased with years since the menopause. The possible biological importance of these findings is discussed with particular reference to risk factors for breast cancer.


High fat diets have long been associated with weight gain and obesity, and the weak satiety response elicited in response to dietary lipids is likely to play a role. Suppression of appetite and food intake has consistently been shown to be diminished with high fat relative to either high protein or carbohydrate meals. There is however some evidence that the satiating capacity of lipids may be modulated when physicochemical properties are altered, but studies investigating the effect of lipid saturation on appetite have generated inconsistent findings. This study investigated the effects of changes in fatty acid saturation on post-ingestive satiety and energy intake.ad libitum.High-fat (HF) test breakfasts (2.0 MJ) containing 26 g lipid were given to 18 healthy, lean men in a 3 treatment randomised cross-over design, each treatment separated by a washout of at least 3 days. The breakfasts were high in saturated , polyunsaturated  or monounsaturated  fatty acids, and comprised 2 savoury muffins. Participants rated appetite sensations using visual analogue scales (VAS) to assess palatability immediately following the meals, and hunger and fullness prior to the HF breakfast and throughout the day. Energy intake was measured by covert weighing of a lunch meal which was served 3.5 h after the breakfast, and from which the participants ate There was no difference in VAS ratings of pleasantness, visual appearance, smell, taste, aftertaste and overall palatability between the 3 high-fat test breakfasts. However, there was also no differential effect of the 3 treatments on ratings of hunger, fullness, satisfaction or prospective food consumption during the 3.5 h following the breakfast meal and over the full 6 h experiment. Energy and macronutrient intake at lunch also did not differ between treatments . The maximum difference in energy intake between treatments was less than 2%.There was no evidence of a difference in post-ingestion satiety between high fat meals which differed in saturation profile in this group of lean, healthy men.ACTRN12610000193077 A high intake of dietary fat has long been implicated in the development of obesity with a positive association between a high-fat (HF), high-energy dense diet and a high body mass index (BMI) ,2. Whethad libitum energy intake (EI) have been reported. Whilst there is some evidence of PUFA having the strongest and MUFA the weakest suppression of food intake, clinical studies are inconsistent and variable. A gastrointestinal infusion study showed high-PUFA Intralipid® (a fat emulsion comprising largely soybean oil) and linoleic acid (18:2) to decrease intake when compared with a no-fat saline control, whilst carbon chain length (CCL) -matched stearic acid  and oleic acid  did not , 270, 330 and 390 minutes after the participant began consumption of the test breakfast. Immediately after breakfast, participants also rated the pleasantness, visual appeal, smell, taste, aftertaste and overall palatability of the breakfast using separate 100-mm VAS [Participants rated their hunger, fullness, satisfaction and prospective consumption (how much do you think you could eat now?) using VAS . Subject0-mm VAS . These qad libitum lunch consisted of a restricted buffet-style meal with predominantly cold and one hot meal choice, along with a selection of beverages. In an attempt to avoid over-consumption the variation of meal items offered was limited. Participants were advised that they could eat as much or as little as they chose and that they were to remain in the room for a period of 45 minutes. The items presented at the ad lib buffet meal, along with their serving weight, energy and macronutrient content are shown in Table The ad lib lunch meal was analysed using univariate ANOVA . Univariate ANOVA was also used to determine any differences in EI or physical activity level on the day prior to each treatment visit (Day-1). The trial was analysed on the basis of intention to treat (ITT), hence all data from all treatment visits completed were included in the analysis. Missing data was assumed missing at random and no data imputation was performed. Statistical significance was based on 95% limits (P < 0.05). Participant characteristics are presented as mean, standard deviation . Efficacy endpoints are presented as mean, standard error of the mean .VAS data were analysed using a repeated measures Linear Mixed Model ANOVA . The participant, the dietary treatment, the intervention period, and the study day were included in the procedure, as was the treatment/time interaction which addressed whether the trajectory over time during the intervention period differed between treatments (diet*time). MANOVA was also used to generate a full model for VAS incorporating all dependent variables. Energy and macronutrient intake data from the Eighteen (18) male participants were randomized into the study, of which 17 completed all 3 treatments. One participant completed only 2 treatments  due to relocation overseas, hence a total of 53 of the scheduled 54 study visits were completed. Participant characteristics are shown in Table ad lib lunch also induced significant changes in VAS-rated measures of appetite and satiety during the afternoon. There was however no significant difference in mean ratings of hunger, fullness, satisfaction or prospective consumption between the 3 treatments when measured over the 3.5 hours between the test breakfast and ad lib lunch meal  or when measured throughout the full study day . The use of MANOVA to combine VAS measurements for all treatments was also unable to identify a significant difference between treatments either before lunch or over the full study day . Analysis of the sequential blood collects also showed there to be no difference between circulating levels of serum cholesterol, triacylglycerol (TAG), glucose or insulin between the SFA, MUFA and PUFA treatments .There was no difference in VAS ratings of pleasantness, visual appearance, smell, taste, aftertaste and overall palatability between the 3 HF test breakfasts when assessed immediately following consumption  in these trials may make changes in eating behaviour more difficult to effect than studies where an outcome meal is given only 60 or 90 minutes following the test treatment. In our current trial however the ad lib energy intake was consistent with the recorded postprandial feelings of hunger and fullness which also did not differ across lipid treatments. It is possible that the varied item buffet-style lunch may have encouraged overconsumption, decreased sensitivity and masked differential effects, but again it is notable that a multi-item buffet lunch has previously been shown to be sensitive to effects of acute lipid manipulations [There are a wide range of published methods by which short-term appetite regulation has been assessed but consensus is yet to be achieved as to best practice . The metulations -51. We hulations . PleasanThere is little consensus as to whether degree of lipid saturation may play a role in regulation of body weight and adiposity through mechanisms driving changes in either intake or oxidation. Whilst epidemiological evidence of an association between lipid saturation and adiposity in studies such as the Quebec Family Study  suggest et al (2000) in a CCL-matched feeding study which demonstrated decreased ratings of appetite and a trend, albeit non-significant, towards lower ad lib EI over the rest of the day following a high-PUFA (linoleic acid) and SFA (stearic-oleic blend) lunch meal compared with a lunch rich in MUFA (oleic acid) [et al (2005) found that mean VAS scores of appetite satisfaction were greater following a high-oleic safflower oil (high-MUFA) compared with a walnut oil (high-PUFA) preload, although it is notable that this result was not substantiated by 4 other appetite-related VAS or subsequent ad lib EI at lunch [Much of the evidence for effect of saturation on EI has been derived from acute studies of intake of similar design to our current trial and also from acute GI infusion trials, several of which show increased satiety effects of PUFA. Intestinal infusion of Intralipid  and PUFA (linoleic acid), but not SFA (stearic acid) or MUFA (oleic acid) emulsions tended to decrease EI relative to a saline control in a study of men given a restricted lunch meal . The effic acid) . A seconic acid) . A more ic acid) . Conversic acid) ,40. For at lunch . Finallyat lunch -43. It iad lib outcome meal. These have been summarized in Table Some variability in outcome may in part be due to differences in methods. Important aspects of trials assessing postprandial changes in appetite may include number and characteristics of participants; lipid dose, composition and route of administration; inter-meal interval; and composition and variety of foods offered at the ad libitum outcome lunch, and the composition of the ad lib meal have all been identified as areas which may bias outcome. In this study we administered a 26 g lipid test meal which was well matched for sensory outcomes between treatments, 3.5 hours prior to a free choice lunch meal. Whether a higher dose lipid product administered as a preload rather than test meal, i.e. closer to the lunch meal, would elicit an appetite response is unknown. Certainly there is little consensus on effects of dietary lipid composition on appetite control from previous studies. Whether appetite may be altered by longer term and sustained changes in dietary fatty acid composition also remains to be demonstrated.In this study we were unable to show differential changes in postprandial feelings of hunger or fullness, or changes in energy intake at a lunch meal when alterations were made to the fatty acid saturation of a high-fat breakfast. Lipid dose, the interval between test-breakfast and ANOVA: analysis of variance; BMI: body mass index; CCL: carbon chain length; CCK: cholecystokinin; CHO: carbohydrate; EI: energy intake; FA: fatty acid; HF: high fat; HNU: human nutrition unit; MANOVA: multivariate analysis of variance; MUFA: monounsaturated fatty acid; PEPSA: Physical Exercise Programme for Sedentary Adults; PUFA: polyunsaturated fatty acid; SD: standard deviation; SEM: standard error of the mean; SFA: saturated fatty acid; VAS: visual analogue score.SDP, ATM, FEL, CMS, AKM received salary support and funding for this trial from LactoPharma, New Zealand. Other authors declare that they have no competing interests.CM Strik was responsible for patient recruitment, screening and daily management of the trial as well as data entry and contribution to manuscript preparation; FE Lithander participated in patient recruitment, screening and oversaw trial management; AT McGill was the study physician and contributed to protocol design and regulatory procedures; AKH MacGibbon contributed to the design of the trial and provision of dietary treatments; BH McArdle was responsible for all statistical analyses; SD Poppitt was the senior author, and responsible for protocol design, data interpretation, trial supervision and regulatory procedures. All authors read and approved the final manuscript.


BCE) and Diodorus Siculus (1st c. BCE)], a particular translation of inscriptions on some burial monuments, and the argument that if the animals had been sacrificed so too were the humans, is that Tophets represent burial grounds reserved for sacrificial victims. An alternative hypothesis acknowledges that while the Carthaginians may have occasionally sacrificed humans, as did their contemporaries, the extreme youth of Tophet individuals suggests these cemeteries were not only for the sacrificed, but also for the very young, however they died. Here we present the first rigorous analysis of the largest sample of cremated human skeletal remains  from the Carthaginian Tophet based on tooth formation, enamel histology, cranial and postcranial metrics, and the potential effects of heat-induced bone shrinkage. Most of the sample fell within the period prenatal to 5-to-6 postnatal months, with a significant presence of prenates. Rather than indicating sacrifice as the agent of death, this age distribution is consistent with modern-day data on perinatal mortality, which at Carthage would also have been exacerbated by numerous diseases common in other major cities, such as Rome and Pompeii. Our diverse approaches to analyzing the cremated human remains from Carthage strongly support the conclusion that Tophets were cemeteries for those who died shortly before or after birth, regardless of the cause.Two types of cemeteries occur at Punic Carthage and other Carthaginian settlements: one centrally situated housing the remains of older children through adults, and another at the periphery of the settlement (the “Tophet”) yielding small urns containing the cremated skeletal remains of very young animals and humans, sometimes comingled. Although the absence of the youngest humans at the primary cemeteries is unusual and worthy of discussion, debate has focused on the significance of Tophets, especially at Carthage, as burial grounds for the young. One interpretation, based on two supposed eye-witness reports of large-scale Carthaginian infant sacrifice [Kleitarchos (3 Some biblical scholars maintain that the Carthaginians frequently and systematically practiced infant sacrifice perhaps as early as Queen Dido's founding of the Phoenician colony on the northern coast of Africa in the 9th or 8th century BCE until 146 BCE, when the Romans won the third and last Punic War Other biblical scholars In the latter 1970s, excavations at Carthage were undertaken as part of a UNESCO sponsored, multinational archaeological effort to salvage as much information as possible from the vast site before expansion of building covered everything. The Tunisian Department of Antiquities granted permission to the American Team to excavate and analyze all material–osteological or otherwise–recovered from the Tophet. Once urns were removed from the field, the processing, sorting, osteological analyses of their contents, and the presentation of the results was under the direction of JHS.Here we provide the results of the first in-depth study not only of the largest sample of the skeletal remains (348 urn contents) from the Tophet at Carthage (summer field seasons 1976 to 1979), but from any Carthaginian Tophet of [see Supporting Information Because the water table rose subsequent to use of the Carthaginian Tophet, JHS determined that each excavated urn should be placed in a water-filled bucket until he could extract its contents; otherwise dissolved calcium carbonate would solidify urn contents into a cement-like block as they dried Since damage to urns and dislodging of the clay seal made possible the loss of material from an urn as well as the intrusion of silts and even bones into the urn Age estimation was based on comparative measurements of skeletal elements  Given the periodicity of enamel deposition and the fact that prenatal enamel does not normally present accentuated lines, an NL is the first postnatal hypoplasia . It thus marks the brief period of disruption of enamel secretion (decrease in daily rates of enamel formation) that occurs immediately postpartum. The emergence of an NL most likely reflects a drop in blood serum calcium values during the first 48 to 72 hours ex-utero An NL can be identified easily in ground sections because both the difference in quality between pre- and postnatal enamel and its characteristic location is specific for each tooth class Analysis of NL presence/absence is routine in forensic investigations, which is noted not only in its increasingly prevalence in analyses of archaeological populations For this analysis, JHS and FH sent LB and RM well-preserved crowns of deciduous incisors and deciduous molars of 50 individuals, whose estimated ages bracketed the morphologically determined perinatal period and thus the period of transition from in- to ex-utero. Only specimen numbers were provided to LB and RM.Specimens were cleaned in an ultrasonic bath and embedded in epoxy resin. Longitudinal labio- (bucco-) lingually oriented ground sections were prepared with a diamond blade microtome (Leica 1600) following the protocol of Caropreso et al. At least three thin sections per specimen were produced. ∼300 µm-thick slices were subsequently reduced to 80–100 µm with a motorized grinder (Minimet 1000 Buehler), polished, mounted for routine microscopy, and then etched for few seconds with a gel of phosphoric acid in order to enhance enamel microstructure. Of the three slides per tooth, the one with the least diagenetic damage and the most clear-cut microstructure was used in the analysis Sections were scrutinized under polarized light with an optical transmitted-light microscope  and images taken with Polaroid Digital Microscope Camera (DMC 1) at 100× and 400×. Contrast enhancement convolution filters (3×3 and 5×5 kernels) produced sharper detail while change in the look-up table function increased site-specific contrasts of intensity profiles. Several partial images (from 7 to 15) were used to reconstruct the entire crown as a digital photomosaic. Because tooth enamel contains significantly less organic material than bone , it reacts differently to heat and is less prone to plastic deformation Urns could contain burned bones and teeth of humans, animals (primarily lamb or kid), or both . There cBones and teeth from the same individual were rarely uniformly charred or calcined, and many were only minimally affected by heat . This irSeventy pelvic ilia were sufficiently preserved for visual assessment of sex, for which we relied on angle and depth of the greater sciatic notch and, when preserved sufficiently to be scrutinized, curvature of the iliac crest . In SchuOnly bones and teeth and tooth crowns that were preserved sufficiently intact to provide an accurate (not estimated) measurement were used in our estimation of age. Based on skeletal measurements . Comparative estimates obtained by the same investigative methods on deciduous teeth of all morphological classes were available from 124 crowns representing 102 modern European children An NL results from perturbation in matrix deposition of enamel prisms reflecting stress in the transition from an intra- to extra-uterine environment , which dThe identification of prenatal individuals in the Carthaginian Tophet sample is consistent with current data from modern-day studies on the incidence of stillbirth and spontaneous abortion as being the primary contributors to “reproductive wastage” Infectious diseases known to lead to stillbirth include smallpox, vaccinia, and listeriosis; those resulting in prematurity and perinatal mortality include severe viral infections and malaria In sum, while the Carthaginians may occasionally have practiced human sacrifice, as did other circum-Mediterranean societies Table S1Species Identification of Skeletal Remains from Urns, Carthaginian Tophet.(0.33 MB DOC)Click here for additional data file.Table S2Demographic Profile of Human Remains, Carthaginian Tophet.(0.93 MB DOC)Click here for additional data file.Table S3Dimensions of Human Cranial Bones (in mm.), Carthaginian Tophet.(0.33 MB DOC)Click here for additional data file.Table S4Dimensions of Human Pelvic Bones (in mm.), Carthaginian Tophet.(0.12 MB DOC)Click here for additional data file.