Cell Surface Transporters and Novel Drug Developments.

Despite the numerous scientific and technological advances made within the last decade the attrition rates for new drug discovery remain as high as 95% for anticancer drugs. Recent drug development has been in part guided by Lipinski's Rule of 5 (Ro5) even though many approved drugs do not comply to these rules. With Covid-19 vaccine development strategy dramatically accelerating drug development perhaps it is timely to question the generic drug development process itself to find a more efficient, cost effective, and successful approach. It is widely believed that drugs permeate cells via two methods: phospholipid bilayer diffusion and carrier mediated transporters. However, emerging evidence suggests that carrier mediated transport may be the primary mechanism of drug uptake and not diffusion as long believed. Computational biology increasingly assists drug design to achieve desirable absorption, distribution, metabolism, elimination and toxicity (ADMET) properties. Perfecting drug entry into target cells as a prerequisite to intracellular drug action is a logical and compelling route and is expected to reduce drug attrition rates, particularly gaining favour amongst chronic lifelong therapeutics. Novel drug development is rapidly expanding from the utilisation of beyond the rule of five (bRo5) to pulsatile drug delivery systems and fragment based drug design. Utilising transporters as drug targets and advocating bRo5 molecules may be the solution to increasing drug specificity, reducing dosage and toxicity and thus revolutionising drug development. This review explores the development of cell surface transporter exploitation in drug development and the relationship with improved therapeutic index.

An Analysis of Mechanisms for Cellular Uptake of miRNAs to Enhance Drug Delivery and Efficacy in Cancer Chemoresistance.

Up until recently, it was believed that pharmaceutical drugs and their metabolites enter into the cell to gain access to their targets via simple diffusion across the hydrophobic lipid cellular membrane, at a rate which is based on their lipophilicity. An increasing amount of evidence indicates that the phospholipid bilayer-mediated drug diffusion is in fact negligible, and that drugs pass through cell membranes via proteinaceous membrane transporters or carriers which are normally used for the transportation of nutrients and intermediate metabolites. Drugs can be targeted to specific cells and tissues which express the relevant transporters, leading to the design of safe and efficacious treatments. Furthermore, transporter expression levels can be manipulated, systematically and in a high-throughput manner, allowing for considerable progress in determining which transporters are used by specific drugs. The ever-expanding field of miRNA therapeutics is not without its challenges, with the most notable one being the safe and effective delivery of the miRNA mimic/antagonist safely to the target cell cytoplasm for attaining the desired clinical outcome, particularly in miRNA-based cancer therapeutics, due to the poor efficiency of neo-vascular systems revolting around the tumour site, brought about by tumour-induced angiogenesis. This acquisition of resistance to several types of anticancer drugs can be as a result of an upregulation of efflux transporters expression, which eject drugs from cells, hence lowering drug efficacy, resulting in multidrug resistance. In this article, the latest available data on human microRNAs has been reviewed, together with the most recently described mechanisms for miRNA uptake in cells, for future therapeutic enhancements against cancer chemoresistance.

Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth.

Rapid changes in the number and flow cytometric behaviour of cells of E. coli taken from a stationary phase and inoculated into rich medium.Cells of E. coli were grown in LB medium, taken from a stationary phase of 2-4 h, and re-inoculated into fresh media at a concentration (105 ml-1 or lower) characteristic of bacteriuria. Flow cytometry was used to assess how quickly we could detect changes in cell size, number, membrane energization (using a carbocyanine dye) and DNA distribution. It transpired that while the lag phase observable macroscopically via bulk OD measurements could be as long as 4 h, the true lag phase could be less than 15-20 min, and was accompanied by many observable biochemical changes. Antibiotics to which the cells were sensitive affected these changes within 20 min of re-inoculation, providing the possibility of a very rapid antibiotic susceptibility test on a timescale compatible with a visit to a GP clinic. The strategy was applied successfully to genuine potential urinary tract infection (UTI) samples taken from a doctor's surgery. The methods developed could prove of considerable value in ensuring the correct prescription and thereby lowering the spread of antimicrobial resistance.

Endoplasmic reticulum chaperone prolyl 4-hydroxylase, beta polypeptide (P4HB) promotes malignant phenotypes in glioma via MAPK signaling.

Endoplasmic reticulum (ER) chaperone Prolyl 4-hydroxylase, beta polypeptide (P4HB) has previously been identified as a novel target for chemoresistance in glioblastoma multiforme (GBM). Yet its functional roles in glioma carcinogenesis remain elusive. In clinical analysis using human glioma specimens and Gene Expression Omnibus (GEO) profiles, we found that aberrant expression of P4HB was correlated with high-grade malignancy and an angiogenic phenotype in glioma. Furthermore, P4HB upregulation conferred malignant characteristics including proliferation, invasion, migration and angiogenesis in vitro, and increased tumor growth in vivo via the mitogen-activated protein kinase (MAPK) signaling pathway. Pathway analysis suggested genetic and pharmacologic inhibition of P4HB suppressed MAPK expression and its downstream targets were involved in angiogenesis and invasion. This is the first study that demonstrates the oncogenic roles of P4HB and its underlying mechanism in glioma. Since tumor invasion and Vascularisation are typical hallmarks in malignant glioma, our findings uncover a promising anti-glioma mechanism through P4HB-mediated retardation of MAPK signal transduction.

Microfluidic channel-assisted screening of hematopoietic malignancies.

A simple microfluidic fluorescence in situ hybridization (FISH) device allowing accurate analysis of interphase nuclei in 1 hr in narrow channels is presented. Photolithography and fluorosilicic acid etching were used to fabricate microfluidic channels (referred to as FISHing lines) that allowed analysis of 10 samples on a glass microscope slide 0.2 µl of sample volume was used to fill a micro-channel, which resembled a 250-fold reduction compared to conventional FISH. FISH signals were comparable to conventional FISH, with 50-fold less probe consumption and 10-fold less time. Cells were immobilized in single file in channels just exceeding the diameter of the cells, and were used for minimal residual disease (MRD) analysis. To test the micro-channels for application in FISH, MRD was simulated by mixing K562 cells (an established chronic myeloid leukemia cell line) carrying the BCR/ABL fusion gene across 1:1 to 1:1,000 Jurkat cells (an established acute lymphoblastic leukemia cell line). The limit of detection was seen to be 1:100 cells and 1:1,000 cells for FISHing lines and conventional FISH, respectively; however, the conventional method seemed to over-score the presence of K562 cells. This may in part be attributed to FISHing lines practically eliminating the chance of duplicate screening of cells and hastened the time of screening, enhancing scoring of all cells within the channels. This was compared to 1 in 500 cells on the slide being analyzed with the conventional FISH.

Single-cell analysis of K562 cells: an imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein.

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

Cytokeratin 15 marks basal epithelia in developing ureters and is upregulated in a subset of urothelial cell carcinomas.

The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules have been defined which regulate ureteric bud initiation and drive the differentiation of ureteric mesenchyme into peristaltic smooth muscle. Less is known about mechanisms underlying the developmental patterning of the multilayered epithelium characterising the mature ureter. In skin, which also contains a multilayered epithelium, cytokeratin 15 (CK15), an acidic intermediate filament protein, marks cells whose progeny contribute to epidermal regeneration following wounding. Moreover, CK15+ precursor cells in skin can give rise to basal cell carcinomas. In the current study, using transcriptome microarrays of embryonic wild type mouse ureters, Krt15, coding for CK15, was detected. Quantitative polymerase chain reaction analyses confirmed the initial finding and demonstrated that Krt15 levels increased during the fetal period when the ureteric epithelium becomes multilayered. CK15 protein was undetectable in the ureteric bud, the rudiment from which the ureter grows. Nevertheless, later in fetal development, CK15 was immunodetected in a subset of basal urothelial cells in the ureteric stalk. Superficial epithelial cells, including those positive for the differentiation marker uroplakin III, were CK15-. Transformation-related protein 63 (P63) has been implicated in epithelial differentiation in murine fetal urinary bladders. In wild type fetal ureters, CK15+ cells were positive for P63, and p63 homozygous null mutant ureters lacked CK15+ cells. In these mutant ureters, sections of the urothelium were monolayered versus the uniform multilayering found in wild type littermates. Human urothelial cell carcinomas account for considerable morbidity and mortality. CK15 was upregulated in a subset of invasive ureteric and urinary bladder cancers. Thus, in ureter development, the absence of CK15 is associated with a structurally simplified urothelium whereas, postnatally, increased CK15 levels feature in malignant urothelial overgrowth. CK15 may be a novel marker for urinary tract epithelial precursor cells.

Inhibition of prolyl 4-hydroxylase, beta polypeptide (P4HB) attenuates temozolomide resistance in malignant glioma via the endoplasmic reticulum stress response (ERSR) pathways.

BACKGROUND: Glioblastoma multiforme (GBM), the most aggressive malignant primary brain tumor of the central nervous system, is characterized by a relentless disease recurrence despite continued advancement in surgery, radiotherapy, and chemotherapy. Resistance to temozolomide (TMZ), a standard chemotherapeutic agent for GBM, remains a major challenge. Understanding the mechanisms behind TMZ resistance can direct the development of novel strategies for the prevention, monitoring, and treatment of tumor relapse. METHODS AND RESULTS: Our research platform, based on the establishment of 2 pairs of TMZ-sensitive/resistant GBM cells (D54-S and D54-R; U87-S and U87-R), has successfully identified prolyl 4-hydroxylase, beta polypeptide (P4HB) over-expression to be associated with an increased IC50 of TMZ. Elevated P4HB expression was verified using in vivo xenografts developed from U87-R cells. Clinically, we found that P4HB was relatively up-regulated in the recurrent GBM specimens that were initially responsive to TMZ but later developed acquired resistance, when compared with treatment-naive tumors. Functionally, P4HB inhibition by RNAi knockdown and bacitracin inhibition could sensitize D54-R and U87-R cells to TMZ in vitro and in vivo, whereas over-expression of P4HB in vitro conferred resistance to TMZ in both D54-S and U87-S cells. Moreover, targeting P4HB blocked its protective function and sensitized glioma cells to TMZ through the PERK arm of the endoplasmic reticulum stress response. CONCLUSIONS: Our study identified a novel target together with its functional pathway in the development of TMZ resistance. P4HB inhibition may be used alone or in combination with TMZ for the treatment of TMZ-resistant GBM.

Shortened telomere length in patients with systemic lupus erythematosus.

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have a higher rate of premature death compared to the general population, suggesting a phenotype of premature senescence in SLE. Telomere length can be used to assess overall biologic aging. This study was undertaken to address the hypothesis that patients with SLE have reduced telomere length. METHODS: Telomere length was measured cross-sectionally in whole blood from SLE patients and age-matched healthy female controls, using real-time quantitative polymerase chain reaction. SLE-related and cardiovascular risk factors were assessed. RESULTS: We compared telomere length in 63 SLE patients and 63 matched controls with a median age of 50.8 years (interquartile range [IQR] 37-59 years) and 49.9 years (IQR 32-60 years), respectively. The median relative telomere length in SLE patients was 0.97 (IQR 0.47-1.57), compared to 1.53 (IQR 0.82-2.29) in controls (P = 0.0008). We then extended our cohort to measure telomere length in 164 SLE patients. Shorter telomere length was associated with Ro antibodies (ß ± SE -0.36 ± 0.16; P = 0.023), and longer telomere length was associated with steroid therapy (0.29 ± 0.14; P = 0.046). We also noted an association of longer telomere length with increasing body mass index (ß ± SE 0.07 ± 0.01; P < 0.0001) and tobacco smoking (0.64 ± 0.26; P = 0.016), as well as with the presence of carotid plaque (0.203 ± 0.177; P = 0.032). CONCLUSION: Telomere length is shortened in SLE patients compared to controls and does not appear to be a reflection of disease activity or immune cell turnover. Subsets of patients such as those positive for Ro antibodies may be particularly susceptible to premature biologic aging. The predictive value of telomere length as a biomarker of future risk of damage/mortality in SLE requires longitudinal evaluation.

Mathematical modelling of miRNA mediated BCR.ABL protein regulation in chronic myeloid leukaemia vis-a-vis therapeutic strategies.

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disease resulting from an aberrant BCR.ABL gene and protein. To predict BCR.ABL protein abundance and phosphorylation in individual cells in a population of CML cells, we modelled BCR.ABL protein regulation through associated miRNAs using a systems approach. The model rationalizes the level of BCR.ABL protein heterogeneity in CML cells in correlation with the heterogeneous BCR.ABL mRNA levels. We also measured BCR.ABL mRNA and BCR.ABLp phosphorylation in individual cells. The experimental data were consistent with the modelling results, thereby partly validating the model. Provided it is tested further, the model may be used to support effective therapeutic strategies including the combined application of a tyrosine kinase inhibitor and miRNAs targeting BCR.ABL. It appears able to predict different effects of the two types of drug on cells with different expression levels and consequently different effects on the generation of resistance.

Dupuytren's disease metabolite analyses reveals alterations following initial short-term fibroblast culturing.

Dupuytren's disease (DD) is an ill-defined fibroproliferative disorder affecting the palm of the hand, resulting in progressive and irreversible digital contracture. In view of the abnormal gene dysregulation found in DD, and its potential effect on metabolites at a functional level, we chose to examine the metabolic profile involved in DD. Using Fourier transform infrared (FT-IR) spectroscopy to generate metabolic fingerprints of cultured cells, we compared the profiles of DD cords and nodules (1) against the unaffected transverse palmar fascia (internal control), (2) against carpal ligamentous fascia (external control), and (3) against fibroblasts from fat surrounding the nodule and skin overlying the nodule (environmental control). We also determined the effects of serial passaging of the cells on DD fingerprints. Subsequently, gas chromatography-mass spectrometry (GC-MS) was employed for metabolic profiling in order to identify metabolites characteristic of the DD tissue phenotypes. We developed a robust metabolomic analysis procedure of DD using cultured fibroblasts derived from DD tissues. Our carefully controlled culture conditions, combined with assessment of metabolic phenotypes by FT-IR and GC-MS, enabled us to demonstrate metabolic differences between DD and unaffected transverse palmar fascia and between DD and healthy control tissue. In early passage (0-3) the metabolic differences were clear, but cells from subsequent passages (4-6) started to lose this distinction between diseased and non-diseased origin. The dysregulated metabolites we identified were leucine, phenylalanine, lysine, cysteine, aspartic acid, glycerol-3-phosphate and the vitamin precursor to coenzyme A. Early passage DD cells exhibit a clear metabolic profile, in which central metabolic pathways appear to be involved. Experimental conditions have been identified in which these DD data are reproducible. The experimental reproducibility will be useful in DD diagnostics and for DD systems biology.

Protein alterations associated with temozolomide resistance in subclones of human glioblastoma cell lines.

Temozolomide (TMZ) is the standard chemotherapeutic agent for human malignant glioma, but intrinsic or acquired chemoresistance represents a major obstacle to successful treatment of this highly lethal group of tumours. Obtaining better understanding of the molecular mechanisms underlying TMZ resistance in malignant glioma is important for the development of better treatment strategies. We have successfully established a passage control line (D54-C10) and resistant variants (D54-P5 and D54-P10) from the parental TMZ-sensitive malignant glioma cell line D54-C0. The resistant sub-cell lines showed alterations in cell morphology, enhanced cell adhesion, increased migration capacities, and cell cycle arrests. Proteomic analysis identified a set of proteins that showed gradual changes in expression according to their 50% inhibitory concentration (IC(50)). Successful validation was provided by transcript profiling in another malignant glioma cell line U87-MG and its resistant counterparts. Moreover, three of the identified proteins (vimentin, cathepsin D and prolyl 4-hydroxylase, beta polypeptide) were confirmed to be upregulated in high-grade glioma. Our data suggest that acquired TMZ resistance in human malignant glioma is associated with promotion of malignant phenotypes, and our reported molecular candidates may serve not only as markers of chemoresistance but also as potential therapeutic targets in the treatment of TMZ-resistant human malignant glioma, providing a platform for future investigations.

Human papilloma virus strain detection utilising custom-designed oligonucleotide microarrays.

Within the past 15 years, the utilisation of microarray technology for the detection of specific pathogen strains has increased rapidly. Presently, it is possible to simply purchase a pre-manufactured "off the shelf " oligonucleotide microarray bearing a wide variety of known signature DNA sequences previously identified in the organism being studied. Consequently, a hybridisation analysis may be used to pinpoint which strain/s is present in any given clinical sample. However, there exists a problem if the study necessitates the identification of novel sequences which are not represented in commercially available microarray chips. Ideally, such investigations require an in situ oligonucleotide microarray platform with the capacity to synthesise microarrays bearing probe sequences designed solely by the researcher. This chapter will focus on the employment of the Combimatrix® B3 CustomArray™ for the synthesis of reusable, bespoke microarrays for the purpose of discerning multiple Human Papilloma Virus strains.

The influence of immunosuppressive drugs on vascular endothelial growth factor production in relation to VEGF -1154 g and -2578 C genotypes.

BACKGROUND: The pathogenesis of many diseases is correlated to irregularity in vascular endothelial growth factor (VEGF) expression. RESULTS from several association studies show that variation in the level of VEGF expression is related to polymorphic sequences within the VEGF gene. Additionally, there are many studies showing that some gene polymorphisms significantly influence the pharmacokinetics of immunosuppressive drugs. OBJECTIVE: The aim of this study was to determine the influence of immunosuppressive drugs on VEGF production in individuals with different VEGF genotypes. METHODS: ARMS-PCR was used to genotype VEGF polymorphisms at positions -1154 and -2578 within the promoter of VEGF gene. A VEGF-specific ELISA was used to determine the influence of immunosuppressive drugs on VEGF production in PBMCs of individuals with different VEGF genotypes. RESULTS: Suppressive effect of mycophenolic acid was observed just in individuals with GG -1154/CC -2578, GG -1154/CA -2578 and GA -1154/CC -2578 haplotypes. Additionally, VEGF was significantly suppressed in all individuals after treatment with rapamycin except those who had AA -1154/CA -2578 and AA -1154/AA -2578 VEGF genotype combinations. CONCLUSION: RESULTS of a recent study revealed that MMF treatment might be effective in preventing chronic renal rejection only in recipients with IL-10 high producer genotype. Additionally result of another study showed that CYP3A5 genotype markedly influences the pharmacokinetics of rapamycin in kidney transplant recipients. Therefore with regard to our results, different suppressive effect of mycophenolic acid and rapamycin on VEGF production might also be dependent on VEGF genotype.

Systems biology and modeling in neuroblastoma: practicalities and perspectives.

Neuroblastoma (NB) is a common pediatric malignancy characterized by clinical and biological heterogeneity. A host of prognostic markers are available, contributing to accurate risk stratification and appropriate treatment allocation. Unfortunately, outcome is still poor for many patients, indicating the need for a new approach with enhanced utilization of the available biological data. Systems biology is a holistic approach in which all components of a biological system carry equal importance. Systems biology uses mathematical modeling and simulation to investigate dynamic interactions between system components, as a means of explaining overall system behavior. Systems biology can benefit the biomedical sciences by providing a more complete understanding of human disease, enhancing the development of targeted therapeutics. Systems biology is largely contiguous with current approaches in NB, which already employ an integrative and pseudo-holistic approach to disease management. Systems modeling of NB offers an optimal method for continuing progression in this field, and conferring additional benefit to current risk stratification and management. Likewise, NB provides an opportunity for systems biology to prove its utility in the context of human disease, since the biology of NB is comprehensively characterized and, therefore, suited to modeling. The purpose of this review is to outline the benefits, challenges and fundamental workings of systems modeling in human disease, using a specific example of bottom-up modeling in NB. The intention is to demonstrate practical requirements to begin bridging the gap between biological research and applied mathematical approaches for the mutual gain of both fields, and with additional benefits for clinical management.

Proteomics of hepatocellular carcinoma: serum vimentin as a surrogate marker for small tumors (

Small hepatocellular carcinomas (HCCs) can be effectively cured by surgery with good clinical outcomes. However, the conventional AFP marker is ineffective in detecting small tumors. Here we employed a proteomic profiling approach to identify a candidate marker for HCC (

Potential risk factors associated with the use of cidofovir to treat benign human papillomavirus-related disease.

BACKGROUND: Cidofovir is currently being used off-licence to treat different viral infections, such as benign low-risk human papillomavirus (HPV)-related recurrent respiratory papillomatosis (RRP). There are concerns over the safety of this practice as rat studies demonstrated a high malignant transformation rate. As yet, there are no clinical reports of cidofovir-induced malignant changes in humans. METHODS: Telomerase immortalised human keratinocytes (hTert) stably expressing E6 proteins from either low-risk HPV6b or high-risk HPV16 and vector control cells were treated with either low-dose (5 microg/ml) or higher dose (30 microg/ml) cidofovir for 2 days and the effects evaluated by clonogenic survival assays. Based on these results, gene expression microarray analysis was performed on cidofovir-treated low-risk E6 and vector cells before, during and after drug treatment, and the results verified by real-time PCR. RESULTS: Both low-risk and high-risk E6-expressing cells show significantly improved long-term survival compared with vector control cells when exposed to 5 microg/ml cidofovir for 2 days, (hTert T6E6 P=0.0007, hTert T16E6 P=0.00023 and hTert vector control P=0.62). Microarray and real-time PCR analyses of low-dose cidofovir-treated low-risk E6-expressing cells revealed changes in gene expression that are known to be associated with malignant progression, which were not observed in drug-treated vector control cells. CONCLUSIONS: This is the first report that cidofovir can both increase cell survival and induce alterations in gene expression that are known to be associated with malignant transformation in cells transduced only with the E6 gene from low-risk HPV. It is our belief that these data provide cause for concern over the off-license use of this drug to treat RRP.

A protein-based set of reference markers for liver tissues and hepatocellular carcinoma.

BACKGROUND: During the last decade, investigations have focused on revealing genes or proteins that are involved in HCC carcinogenesis using either genetic or proteomic techniques. However, these studies are overshadowed by a lack of good internal reference standards. The need to identify "housekeeping" markers, whose expression is stable in various experimental and clinical conditions, is therefore of the utmost clinical relevance in quantitative studies. This is the first study employed 2-DE analysis to screen for potential reference markers and aims to correlate the abundance of these proteins with their level of transcript expression. METHODS: A Chinese cohort of 224 liver tissues samples (105 cancerous, 103 non-tumourous cirrhotic, and 16 normal) was profiled using 2-DE analysis. Expression of the potential reference markers was confirmed by western blot, immunohistochemistry and real-time quantitative PCR. geNorm algorithm was employed for gene stability measure of the identified reference markers. RESULTS: The expression levels of three protein markers beta-actin (ACTB), heat shock protein 60 (HSP60), and protein disulphide isomerase (PDI) were found to be stable using p-values (p > 0.99) as a ranking tool in all 224 human liver tissues examined by 2-DE analysis. Of high importance, ACTB and HSP 60 were successfully validated at both protein and mRNA levels in human hepatic tissues by western blot, immunohistochemistry and real-time quantitative PCR. In addition, no significant correlation of these markers with any clinicopathological features of HCC and cirrhosis was found. Gene stability measure of these two markers with other conventionally applied housekeeping genes was assessed by the geNorm algorithm, which ranked ACTB and HSP60 as the most stable genes among this cohort of clinical samples. CONCLUSION: Our findings identified 2 reference markers that exhibited stable expression across human liver tissues with different conditions thus should be regarded as reliable reference moieties for normalisation of gene and protein expression in clinical research employing human hepatic tissues.

Metabolomics of serum peptides.

Diseased-cell secreted proteins/peptides offer several leads in biomarker development. Blood is a rich and universal source of biomarkers because of its proximity to all cells in the body. However, important physiological and practical aspects need consideration before serum peptidomics is effectively applied in a clinical, and later bedside, setting.

Biomarkers for early detection of liver cancer: focus on clinical evaluation.

This review summarises the screening methods from hepatic ultrasonography to serological biomarkers for early detection of liver cancer and focuses on evaluation of biomarkers ability. The development of novel biomarkers according to the 5-phase program defined by the Early Detection Research Network (EDRN) is also outlined in this review.