Molecular Biomarkers & Diagnosis

Background: Leptin is important for the regulation of energy metabolism. Fatty acids are essential components of all biological membranes and represent an important form of energy storage in both animals and plants. In addition to leptin’s central effects on appetite control and energy expenditure, it has been shown to have a strong influence on fatty acid metabolism.

Approach: This review summarizes recent knowledge on leptin and fatty acids and their roles in biochemistry and clinical chemistry

Background: Human TOX3 (TOX high mobility group box family member 3) regulates Ca2+ dependent transcription in neurons and has been associated with breast cancer susceptibility. Aim of the study was to investigate the expression of TOX3 in bladder cancer tissue samples and to identify genes and pathways altered upon TOX3 dysregulation using a cell line model. Methods: We performed microarray transcript profiling of biopsies and validated the data with RT-qPCR. We used cell line models for over expression and siRNA mediated knockdown of TOX3. Pathway analysis was applied for target gene identification and immunoprecipitation studies were used for DNA binding studies. Results: Microarray transcript profiling of 89 bladder biopsies showed a significant up-regulation of TOX3 (p<10-4) in non-muscle invasive (Ta-T1) bladder tumors compared to muscle-invasive (T2-T4) bladder tumors and normal urothelium. Microarray expression profiling of human bladder cancer cells over expressing TOX3 followed by Pathway analysis showed that TOX3 over expression mainly affected the Interferon Signaling Pathway. TOX3 up regulation induced the expression of several genes with a gamma interferon activation site (GAS), e.g. STAT1. In vitro functional studies showed that TOX3 was able to bind to the GAS-sequence located at the STAT1 promoter. siRNA mediated knockdown of TOX3 in RT4 bladder cancer cells decreased STAT1 expression suggesting a direct impact of TOX3 on STAT1. Immunoprecipitation of TOX3 over expressing cell extracts with an artificial “GAS”- DNA element resulted in an enrichment of the GAS containing DNA-sequence, providing evidence for a potential interaction of TOX3 with the GAS-sequence of STAT1. Conclusions: These results provide evidence for an alternative activation of the downstream interferon targets, independent of the initial interferon-receptor interaction, and consequently a biological role for TOX3.

Background: Rapid diagnostic test using rk39 antigen is widely used for visceral leishmaniasis. However it detects anti-rk39 antibodies in 20-32% of endemic healthy individuals. In search for a better biomarker of infection, we identified a protein of molecular weight 70 kDa (BHUP1), specifically recognized by sera of visceral leishmaniasis (VL) patients. Methods: The protein was cloned as His-tagged fusion protein and purified. We evaluated the sensitivity and specificity of this protein in an enzyme linked immunosorbant assay (ELISA) format in comparison to the rk39 antigen using sera collected from various groups of individuals. Results: The sensitivity of rBHUP1 was 96.5% compared to 98.8% with rk39. For healthy controls from non endemic and endemic regions, the specificity of rBHUP1 was 100% and 95.6% compared to 100% and 84.9% for rk39, respectively. For other infectious diseases such as malaria, tuberculosis, viral fever, etc., specificity of rBHUP1 was as low as 74.5% when compared to 94% of rk39. At six month and one year follow-up, 74% and 22.5% patients tested positive with rBHUP1, respectively, compared to 97% and 77.4% with rk39 antigen. Conclusion: Though the high sensitivity and specificity of rBHUP1 antigen for VL and healthy controls would have made it a good diagnostic biomarkers, however, its non-specific reaction with other infectious diseases limit its utility.

Background: Drug-induced hepatotoxicity is one of the major reasons for drug recall and hence it is of major concern to the FDA and consumers. Overdose of acetaminophen (APAP) can cause acute hepatic injury. The current clinical biomarkers of liver injury are insufficient in predicting the extent of injury; thus novel biomarkers are needed to integrate with the current biomarkers for better risk assessment during drug development and clinical use.

Methods: Sprague-Dawley rats were orally gavaged with a single dose of 0.5% methylcellulose (control), 100 mg APAP/kg body weight or 1250 mg APAP/kg body weight. Urine, terminal blood samples and tissues were collected at 6, 24, 72, and 168 h for clinical chemistry and histopathology analyses. Based on the clinical chemistry data and histopathology, liver injury occurred in treated animals during the first 24 h, while recovery occurred during 72 to 168 h. A systems biology investigation of APAP-induced hepatic injury was conducted to elucidate novel metabolic biomarkers using an integrated transcriptomic and metabolomic approach. Both open metabolic profiling and broad metabolic profiling were utilized to examine metabolic changes in blood and open profiling was used to evaluate changes in the urinary metabolite profiles.

Results: In total, 270 metabolites were evaluated in blood and/or urine. Metabolites involved in energy, urea and bile acid pathways were found to have strong correlations to hepatic necrosis scores and elevated alanine aminotransferase levels. The pathways associated with these metabolites were altered at the first 72 h but had generally recovered at 168 h. Changes in hepatic gene expression of the bile acid pathway supported the interpretation from the metabolomics data.

Conclusion: The combination of the transcriptomics and metabolic profiling technologies discovered novel injury biomarkers (arginine, 2-oxoarginine, medium chain dicarboxylic acids, α-ketoglutarate and bile acids), which are involved in energy, bile acid, and arginine metabolism pathway.

The numbers of chemicals which are the most important group of environmental pollution were limited to a few thousand until the beginning of this century. But nowadays, due to intensive production and the unconscious usage, the chemical pollution is increasing throughout the food chain and is damaging all living creatures. The chemical levels of the water, the sediment and the aquatic organisms are very important to determine the level of the chemical contamination of the marine environments by biological. The aquatic organisms, such as fish, crustaceans, algae, protozoa, macrophytes, bacteria and plankton are widely used as a biomarker in determining the quality of aquatic systems for the environmental contaminants. Because of this importance the description and differences of marine pollution terms are significant. In this review, the data were collected from different literatures around the World in uses terms of monitoring the aquatic organisms for chemical pollution in aquatic system.

The development of proteomics technology over the past decade has advanced the identification and validation of new biomarkers. Clinical applications of serum biomarkers will benefit us by being cost-effective and non-invasive, as well as by allowing the simple detection of diseases. Numerous serum biomarkers for lung cancer have been identified and reported in publications. Despite extensive studies, cancer detection via a single marker remains difficult due to the low sensitivity, specificity and reproducibility of the identified lung cancer serum biomarkers. An optimal combination of multiple biomarkers, rather than a single biomarker, should be considered for the development of a lung cancer detection biomarker panel and should be validated with these combinatory markers. This review summarizes the lung cancer biomarkers that have been published within the last five years.

Abstract
Background: Smoking has been perceived to increase the levels of carcinogenic and inflammatory mediators
thereby promoting presumably malignant and proinflammatory tissue destruction via activating proteolytic enzymes
such as matrix metalloproteinases (MMPs) and their regulators. We studied the effect of smoking on the diagnostic
ability of serum MMP-8-IFMA and TIMP-1-ELISA analysis to recognize patients with acute coronary syndrome
(ACS).
Methods: The case-control population (n=605) comprised 291 patients with diagnosed acute myocardial
infarction (AMI) or unstable angina pectoris (UAP) and 314 healthy control individuals. The case and the control
group included 55 and 66 smoking subjects, respectively.
Results: Smoking increased the MMP-8, but not the TIMP-1, concentration in both the control and the AMI
group. MMP-8 concentration, and consequently MMP-8/TIMP-1, distinguished the cases from the controls accurately
in the ROC analysis, but smoking decreased the AUCs in every category. The MMP-8 concentration produced an
AUC (95% CI, p-value) for ACS of 0.771 (0.723-0.818, p<0.001) and 0.684 (0.581-0.787, p=0.001) for non-smokers
and smokers, respectively. Similarly, the multivariate logistic regression model indicated that smoking decreased the
association of MMP-8 with ACS. The OR (95% CI, p-value) of MMP-8 for ACS was 8.1 (per ng/ml log-transformed
unit increase, 5.0-13.1, p<0.001) and 2.8 (1.1-7.0, p=0.025) for non-smokers and smokers, respectively.
Conclusions: One of the most important risk factors for ACS, smoking, decreases the diagnostic ability of MMP-
8 concentration and MMP-8/TIMP-1 ratio. This must be taken account if these serum determinations are used as
biomarkers of the risk for cardiovascular diseases.

Background: It should be decided before the introduction of osteoporosis therapy whether to start the therapy with the inhibitor of resorption or with the stimulator for bone formation. The decision should also be made during the therapy with antiresorptives regarding the appropriate time to stop the therapy and start the therapy with stimulators for bone formation. Assuming that normal or reduced levels of bone metabolism is limiting factor for initiation of antiresorptive therapy, and thereafter the appropriate parameter for the decision to stop antiresorptive therapy and start with stimulators of bone metabolism, we believe that the determination of bone markers should be compulsory and an integral part of the diagnostic procedure. The aim of this study was to determine whether antiresorptive therapy leads to satisfactory or excessive suppression of bone metabolic activity depending on the initial values of bone markers. Methods: We performed a prospective longitudinal study of 178 postmenopausal women with osteoporosis. We were following the values of osteocalcin, beta-crosslaps before the introduction of bisphosphonates therapy and after three months during therapy. Results: The results speak in favor of decreased bone resorption during antiresorptive therapy and that the value of bone markers during antiresorptive therapy may be predicted depending on the initial values. If we add osteocalcin and βCTx values before the therapy to the equation: -0.041*OC-0.003*βCTx+2.983, patients having result >0, will have excessive suppression of bone resorption during the therapy. If we add values to the equation: -0.054*OC-0.001*βCTx+2.075, patients having result >0, will have excessive suppression of entire bone remodeling during the therapy. Conclusion: We suggest that osteoporosis patients who are predicted to have excessive suppression of entire bone remodeling during bisphosphonate therapy, should be mainly treated with stimulators for bone formation or possibly with medications with dual mode of action.