Potential biomarkers of an exaggerated response to endotoxemia

Serial plasma protein analysis was used to study the acute plasma proteome response to endotoxemia (presence of toxic bacterial products called endotoxins in the blood stream). Plasma samples from healthy volunteers before and multiple time points up to 24 h following administration of low-dose endotoxin were evaluated. Plasma protein profiles were obtained by rapid extraction of whole plasma followed by analysis with matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The profiles were unique to each individual and stable over the time of the experiment. Administration of low-dose endotoxin caused profound change in six of 18 individuals. At 8 h many proteins showed quantitative oxidation, in addition to the appearance of new components and disappearance of common baseline components. An exceptionally intense new component at 4154 mass units was identified as the activation peptide of C1 esterase inhibitor. While recovery of baseline protein structure was nearly complete by 24 h, serum amyloid A, an acute-phase reactant, was still increasing and minor profile changes persisted. Clinical features did not distinguish these extreme responders from others, suggesting that plasma proteome changes offered unique insights into and potential biomarkers of subclinical events following endotoxin exposure.     

Molecular targets of HPV oncoproteins: Potential biomarkers for cervical carcinogenesis

Cervical cancer is the second most common cancer among women worldwide and is responsible for 275,000 deaths each year. Persistent infection with high-risk human papillomavirus (HR-HPV) is an essential factor for the development of cervical cancer. Although the process is not fully understood, molecular mechanisms caused by HPV infection are necessary for its development and reveal a large number of potential biomarkers for diagnosis and prognosis. These molecules are host genes and/or proteins, and cellular microRNAs involved in cell cycle regulation that result from disturbed expression of HR-HPV E5, E6 and E7 oncoproteins. One of the current challenges in medicine is to discover potent biomarkers that can correctly diagnose cervical premalignant lesions and standardize clinical management. Currently, studies are showing that some of these molecules are potential biomarkers of cervical carcinogenesis, and it is possible to carry out a more accurate diagnosis and provide more appropriate follow-up treatment for women with cervical dysplasia. In this paper, we review recent research studies on cell cycle molecules deregulated by HPV infections, as well as their potential use for cervical cancer screening.     

Proteins are potent biomarkers to detect colon cancer progression

Colon cancer is the most common type of cancer and major cause of death worldwide. The detection of colon cancer is difficult in early stages. However, the secretory proteins have been used as ideal biomarker for the detection of colon cancer progress in cancer patients. Serum/tissue protein expression could help general practitioners to identify colon cancer at earlier stages. By this way, we use the biomarkers to evaluate the anticancer drugs and their response to therapy in cancer models. Recently, the biomarker discovery is important in cancer biology and disease management. Also, many measurable specific molecular components have been studied in colon cancer therapeutics. The biomolecules are mainly DNA, RNA, metabolites, enzymes, mRNA, aptamers and proteins. Thus, in this review we demonstrate the important protein biomarker in colon cancer development and molecular identification of protein biomarker discovery.     

Potential of sperm small non-coding RNAs as biomarkers of testicular toxicity in a doxorubicin-induced mouse model

Testicular toxicity is a major concern in cancer chemotherapy and drug development as it can result in infertility; however, there are no effective biomarkers for this adverse effect. To identify new biomarkers, we investigated the expression of small non-coding RNAs (sncRNAs) in a mouse model of doxorubicin (DXR)-induced testicular toxicity. First, we performed small RNA-seq analysis of sperm from DXR-treated or control mice and observed differential expression of many genome-derived sequences. We then performed real-time RT-PCR validation of these sequences and discovered that sncRNA detected by one primers, dxRN_3, showed similar differential expression as that seen in the RNA-seq experiment. These findings suggest that the sncRNAs present in sperm have potential as clinically acceptable biomarkers for testicular toxicity.     

Haemoglobin adducts as biomarkers of exposure to tobacco-related nitrosamines

A sensitive gas chromatography/mass spectrometry (GC/MS) method was developed to measure nitrosamine-haemoglobin adducts (HPB-Hb) (4-hydroxy-3-pyridinyl-1-butanone) at trace levels in red blood cells of smoking and non-smoking mothers and their newborn babies. GC/MS methods with chemical ionization (CI) of methane reagent gas in both positive and negative ion mode as well as electron ionization (EI) were studied to determine differences in sensitivity among the various ionization methods. Detection limits using both positive and negative chemical ionization modes were found to be 30 fmol HPB, whereas detection using electron impact modes yielded a detection limit of 80 fmol HBP. In order to apply the various methods of detection to tobacco-exposed samples from human populations, we characterized adduct levels in maternal as well as paired fetal samples obtained from mothers exposed to tobacco smoke during pregnancy. Maternal samples were characterized using serum cotinine levels and were classified as non-smokers, passively smoke-exposed women, less than one pack per day smokers and greater than one pack per day smokers. Paired maternal and fetal blood samples were obtained at delivery for qualitative and qualitative analysis of nitrosamine adducts. Comparative derivatization of HPB released under alkaline hydrolysis conditions was performed using O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) and 2,3,4,5,6-pentafluorobenzoylchloride (PFBC). Both negative CI and positive CI modes of analysis were compared to the more widely accepted EI modes of mass spectrometric analysis. These results suggest that both NICI and PICI modes of detection offer a greater sensitivity of adduct characterization when compared with EI ionization techniques and that either NICI or PICI modes are preferably applicable towards the detection of human biomarker assessment of tobacco-related nitrosamines.     

Increase in transglutaminase and its extracellular products in response to an inflammatory stimulus by lipopolysaccharide

Transglutaminase (TGase) activities were measured in rat tissues 1-7 days after intraperitoneal injection of saline or lipopolysaccharide (LPS) and in the cells and media from pre-confluent human fibroblasts cultured for two days in the presence or absence of LPS. (-glutamyl)lysine and [3H]putrescine-labelled -glutamyl derivatives in extracellular and cellular fibroblast proteins were also measured. Three effects of LPS were observed. Firstly, total TGase activity is greater in the tissues from the LPS-injected animals, with the maximum increase occurring at 1 day in dermis, epidermis and liver, at 5 days in the aorta and, after a decrease at 2-5 days, at 7 days in the panniculus muscle. Secondly, the fraction of the total activity which is buffer-extractable is greater on days 1 and/or 2 in all the tissues from the LPS-injected rats. Thirdly, in cultures of human fibroblasts, LPS increases that fraction of bound [3H]putrescine and of TGase and its -glutamylamine products which occurs in the extracellular medium. In addition, a higher concentration of TGase-derived crosslinks was found in extracellular as opposed to intracellular proteins. In conjunction with previous findings in skin wound healing and in atherosclerosis these results support the concept of an extracellular function for tissue TGase and indicate that there is a widespread association of increases in TGase and its extracellular products with inflammation and the healing or fibrotic processes which follow it.     

Identification of cereal alkylresorcinol metabolites in human urine-potential biomarkers of wholegrain wheat and rye intake

Alkylresorcinols, phenolic lipids present in high amounts in wholegrain wheat and rye, are of interest as potential biomarkers of the intake of these cereals. Alkylresorcinols are known to be absorbed by humans and animals, but little is known about their metabolism or resulting metabolites. A preliminary human study was carried out to identify alkylresorcinol metabolites in human urine. Urine samples, collected before and after a wheat-bran based meal, were deconjugated with beta-glucuronidase/sulphatase and then extracted with ethyl acetate. Extracts were separated by thin-layer chromatography, and fractions containing alkylresorcinols and possible metabolites were identified by retention on the plate compared to standard compounds, and staining with fast blue B. These fractions were further analysed by gas chromatography-mass spectrometry. Deconjugated human urine after the wheat-bran based meal contained two alkylresorcinol metabolites, 3,5-dihydroxybenzoic acid and 3-(3,5-dihydroxyphenyl)-1-propanoic acid, as well as smaller amounts of unchanged alkylresorcinols, confirming the hypothesis that alkylresorcinols are metabolised in humans via beta-oxidation of their alkyl chain.     

Proteomics approach to identify biomarkers for upper gastrointestinal cancer

Introduction: The prognosis for patients with upper gastrointestinal cancers remains dismal despite the development of multimodality therapies that incorporate surgery, chemotherapy, and radiotherapy. Early diagnosis and personalized treatment should lead to better prognosis. Given the advances in proteomic technologies over the past decades, proteomics promises to be the most effective technique to identify novel diagnostics and therapeutic targets.

Areas covered: For this review, keywords were searched in combination with ‘proteomics’ and ‘gastric cancer’ or ‘esophageal cancer’ in PubMed. Studies that evaluated proteomics associated with upper gastrointestinal cancer were identified through reading, with several studies quoted at second hand. We summarize the proteomics involved in upper gastrointestinal cancer and discuss potential biomarkers and therapeutic targets.

Expert commentary: In particular, the development of mass spectrometry has enabled detection of multiple proteins and peptides in more biological samples over a shorter time period and at lower cost than was previously possible. In addition, more sophisticated protein databases have allowed a wider variety of proteins in samples to be quantified. Novel biomarkers that have been identified by new proteomic technologies should be applied in a clinical setting.     

Discovery of biomarkers for oxidative stress based on cellular metabolomics

Oxidative stress has a close relationship with various pathologic physiology phenomena and the potential biomarkers of oxidative stress may provide evidence for clinical diagnosis or disease prevention. Metabolomics was employed to identify the potential biomarkers of oxidative stress. High-performance liquid chromatography–diode array detector, mass spectrometry and partial least squares discriminate analysis were used in this study. The 10, 15 and 13 metabolites were considered to discriminate the model group, vitamin E-treated group and l-glutathione-treated group, respectively. Some of them have been identified, namely, malic acid, vitamin C, reduced glutathione and tryptophan. Identification of other potential biomarkers should be conducted and their physiological significance also needs to be elaborated.     

Evaluation of biogenic and anthropogenic inputs of aliphatic hydrocarbons to Lake Taihu sediments using biomarkers

Surficial sediments from 13 sites throughout Lake Taihu, one of the largest urbanized freshwater lake systems in China, were analyzed for biomarkers (e.g., n-alkanes and hopanes) to track the origin of organic inputs (biogenic or anthropogenic), and, thus, to identify any ‘hot spots’ of hydrocarbon contamination. A distinct spatial distribution of aliphatic hydrocarbons in sediments was observed in Lake Taihu. At the northern tip of the lake (i.e., Meiliang and Wuli Bays), the highest mean aliphatic hydrocarbon concentration, with a significant contribution of an unresolved complex mixture (UCM), was found, indicating possible anthropogenic petroleum contamination (mainly from untreated and partially treated industrial and domestic sewage from Wuxi, Changzhou and other cities). This was supported by the n-alkane indices (e.g., small Carbon Preference Index and odd-to-even values) and a high degree of maturity of the hopane biomarkers. However, hydrocarbons from East Taihu were mainly biogenic, with the lowest mean concentrations, negligible or no contribution of UCM, abundance of vascular plant C₂₃–C₃₃ n-alkanes with a high odd-to-even predominance, and the presence of biogenic hopanes (e.g., 17β(H), 21β(H)-hopanes and hopenes). In the other areas of the lake, however, the predominance of biogenic in combination with petrogenic hydrocarbons was indicated by the biomarkers.     

Microwave characterization of low-molecular-weight antioxidant specific biomarkers

Antioxidants play a crucial role in the life sciences, as the regulators of biochemical reactions. We studied the dielectric properties of the low-molecular weight antioxidant specific biomarkers sodium ascorbate and glutathione in solutions of different concentrations. The biomarkers are multifunctional metabolites relevant to the reactive oxygen species (ROS) scavenging system of cells. The newly developed high-Q microwave whispering-gallery-mode (WGM) dielectric resonator based technique was applied. The technique allows investigation of liquids of nanoliter volumes filled in microfluidic channel within several milliseconds. The revealed peculiarities in the dependence of permittivity on concentrations of the sodium ascorbate and glutathione solutions are explained by differences in relaxation times and loses introduced by molecules of different shapes. We suggest that this novel approach offers the potential for the detection and characterization of ROS-relevant biomarkers with millisecond-time resolution.     

The potential biomarkers of drug addiction: proteomic and metabolomics challenges

Drug addiction places a significant burden on society and individuals. Proteomics and metabolomics approaches pave the road for searching potential biomarkers to assist the diagnosis and treatment. This review summarized putative drug addiction-related biomarkers in proteomics and metabolomics studies and discussed challenges and prospects in future studies. Alterations of several hundred proteins and metabolites were reported when exposure to abused drug, which enriched in energy metabolism, oxidative stress response, protein modification and degradation, synaptic function and neurotrasmission, etc. Hsp70, peroxiredoxin-6 and α- and β-synuclein, as well as n-methylserotonin and purine metabolites, were promising as potential biomarker for drug addiction.     

内毒素血症时大鼠心肌细胞凋亡与血管紧张素受体表达关系的研究

目的:对脂多糖诱导大鼠内毒素血症时心肌细胞凋亡与血管紧张素II受体AT1、AT2表达变化进行分析。方法:Wistar大鼠随机分为3组(n=10):空白组、脂多糖(LPS)干预2 h(L2)组、LPS干预6 h(L6)组。根据分组情况经腹腔注射LPS(10 mg/kg)制备内毒素血症模型,对照组给予等体积生理盐水腹腔注射。应用免疫组织化学法检测凋亡相关因子Bcl-2和Bax蛋白表达,并通过TUNEL法检测心肌细胞凋亡变化,并采用免疫组织化学、RT-PCR方法观察了大鼠心肌AT1、AT2受体表达变化,电镜观察心肌组织病理变化。结果:LPS干预2 h后Bcl-2阳性细胞表达开始明显上调,之后开始下降,但仍高于对照组(P<0.05)。Bax的表达随着干预时间的延长不断增加,6 h后表达与对照组相比,有显著的统计学差异(P<0.01)。Bcl-2/Bax的比率在干预开始2 h时升高,随后开始下降,心肌细胞凋亡增加。随着LPS干预的时间延长,AT1受体、AT2受体蛋白、mRNA表达水平呈下降趋势,6 h后表达与对照组相比,有显著的统计学差异(P<0.01)。结论:大鼠经LPS干预后随时间延长Bcl-2/Bax的比率呈下降趋势,心肌细胞凋亡增加,与AT1受体、AT2受体表达水平下降呈负相关,说明血管紧张素受体表达下调可能与内毒素血症时大鼠心脏功能损伤有关,为临床预防及治疗全身炎症反应所致心功能损伤提供一条新的思路以及实验室理论基础。     

The discovery and early validation of novel plasma biomarkers in mild-to-moderate Alzheimer's disease patients responding to treatment with rosiglitazone

Recent advances in clinical, pathological and neuroscience studies have identified disease-modifying therapeutic approaches for Alzheimer's disease that are now in clinical trials. This has highlighted the need for reliable and convenient biomarkers for both early disease diagnosis and a rapid signal of drug efficacy. We describe the identification and assessment of a number of candidate biomarkers in patients with Alzheimer's disease and the correlation of those biomarkers with rosiglitazone therapeutic efficacy, as represented by a change in the Alzheimer's Disease Assessment Scale-Cognitive (ADAS-Cog). Plasma from 41 patients with Alzheimer's disease were analysed by open platform proteomics at baseline and after receiving 8 mg rosiglitazone for 24 weeks. From a comparison of protein expression following treatment with rosiglitazone, 97 proteins were observed to be differentially ediffer xpressed with a p-value<0.01. From this analysis and comparison to recently published data from our laboratory, a prioritized list of 10 proteins were analysed by immunoassay and/or functional assay in a wider set of samples from the same clinical study, representing a rosiglitazone dose response, in order to verify the changes observed. A number of these proteins appeared to show a correlation with change in ADAS-Cog at the higher treatment doses compared with the placebo. Alpha-2-macroglobulin, complement C1 inhibitor, complement factor H and apolipoprotein E expression showed a correlation with ADAS-Cog score at the higher doses (4 mg and 8 mg). These results are discussed in light of the pathology and other recently published data.     

Vascular response to angiotensin II is exaggerated through an upregulation of AT1 receptor in AT2 knockout mice

Blood pressure is elevated and pressor response to angiotensin II (Ang II) is exaggerated in AT2 null mice. The purpose of the present study was to elucidate the mechanism for the increased responsiveness to Ang II in the mice. The contraction of aortic strips generated by Ang II was significantly greater in the AT2 gene-deleted mice than the control, which was completely abolished by AT1 antagonist losartan. The aortic content of AT1 receptor was significantly increased (P < 0.05, n = 5) in the AT2 null mice (212 +/- 58.2 fmol/mg protein) compared with the control (98.2 +/- 55.9 fmol/mg protein). While both AT1 and AT2 mRNAs were expressed in the aorta of the control mice, only AT1 mRNA was expressed in the AT2 knockout mice. The expression of AT1 mRNA in the AT2 knockout mice was significantly higher (1.5-fold, P < 0.05, n = 5) than that in the control. The present study clearly demonstrated that the increased vascular reactivity to Ang II in AT2 knockout mice is at least partly due to an increased vascular AT1 receptor expression and suggested that AT2 counteracts AT1-mediated vascular action of Ang II through downregulation of AT1 receptor by a crosstalk between these receptors by some as yet unknown mechanisms.     

Non-targeted metabolomics of saliva to explore potential biomarkers for gastric ulceration in pigs fed hemp

Gastric ulceration is a common disease in pig production worldwide and is associated with economic losses as well as animal health and welfare issues. The aim of this study was to explore potential salivary biomarkers for gastric ulceration in pigs. In addition, the aim was to study the effect of hemp on the incidence of gastric ulcers. Approximately 440 growing-finishing pigs in the period from 30 to 110 kg BW were allocated to four different diets: meal feed (Meal); pelleted feed (Pellets); pelleted feed added 4% hempseed cake (Hemp Cake); pelleted feed added 4% hempseed hulls (Hemp Hulls). The day before slaughter, saliva samples from each pig were collected. After slaughter, the stomachs were emptied to assess the consistency of the stomach content and examined for gastric ulceration using an index scale (0–10). Noticeable changes of the gastric mucosa (total index score ≥ 6) were observed in 291 pigs. The odds of having index scores 0–5 relative to index scores 6–8 and 9–10, respectively, were higher (P < 0.001) for pigs fed Meal compared to pigs fed Pellets. The odds of suffering from severe gastric ulcers tended (P = 0.08) to be lower in pigs fed Hemp Hulls compared to pigs fed Pellets. A non-targeted liquid chromatography mass spectrometry based metabolomics analysis was performed on saliva samples to determine any separation between pigs with healthy stomachs and those with gastric ulcers and to examine a possible correlation between gastric ulcer index and potential biomarkers. Partial least-squares discriminant analysis showed a separation between pigs with ulcers and those with healthy stomachs/hyperkeratosis (HK). Metabolites contributing to the separation between groups were identified. Levels of oxylipins deriving from linoleic acid were lower (P < 0.001) in pigs with ulcers compared to healthy/HK pigs. This may indicate a shift in the metabolic pathways towards more pro-inflammatory arachidonic acid-derived eicosanoids, which might reflect an increased inflammatory response. Thus, reduced levels of oxylipins derived from linoleic acid seemed to be associated with active gastric ulcers, and thereby they might function as biomarkers for gastric ulceration in pigs. In addition, supplementation of hempseed hulls had a beneficial effect on severe gastric ulcers, as hempseed hulls changed the consistency of the gastric content by conferring more solidness. However, it was not possible to observe any reliable separation between pigs fed pellets supplemented with hemp products and pigs fed non-supplemented pellets according to the identified salivary metabolites.     

The ethnicity-specific association of biomarkers with the angiographic severity of coronary artery disease

Background

Risk factor burden and clinical characteristics of patients with coronary artery disease (CAD) differ among ethnic groups. We related biomarkers to CAD severity in Caucasians, Chinese, Indians and Malays.

Methods

In the Dutch-Singaporean UNICORN coronary angiography cohort (n = 2033) we compared levels of five cardiovascular biomarkers: N-terminal pro-brain natriuretic peptide (NTproBNP), high-sensitivity C-reactive protein (hsCRP), cystatin C (CysC), myeloperoxidase (MPO) and high-sensitivity troponin I (hsTnI). We assessed ethnicity-specific associations of biomarkers with CAD severity, quantified by the SYNTAX score.

Results

Adjusted for baseline differences, NTproBNP levels were significantly higher in Malays than in Chinese and Caucasians (72.1 vs. 34.4 and 41.1 pmol/l, p < 0.001 and p = 0.005, respectively). MPO levels were higher in Caucasians than in Indians (32.8 vs. 27.2 ng/ml, p = 0.026), hsTnI levels were higher in Malays than in Caucasians and Indians (33.3 vs. 16.4 and 17.8 ng/l, p < 0.001 and p = 0.029) and hsTnI levels were higher in Chinese than in Caucasians (23.3 vs. 16.4, p = 0.031). We found modifying effects of ethnicity on the association of biomarkers with SYNTAX score. NTproBNP associated more strongly with the SYNTAX score in Malays than Caucasians (β 0.132 vs. β 0.020 per 100 pmol/l increase in NTproBNP, p = 0.032). For MPO levels the association was stronger in Malays than Caucasians (β 1.146 vs. β 0.016 per 10 ng/ml increase, p = 0.017). Differing biomarker cut-off levels were found for the ethnic groups.

Conclusion

When corrected for possible confounders we observe ethnicity-specific differences in biomarker levels. Moreover, biomarkers associated differently with CAD severity, suggesting that ethnicity-specific cut-off values should be considered.     

Mining the fecal proteome: from biomarkers to personalised medicine

Introduction: Fecal proteomics has gained increased prominence in recent years. It can provide insights into the diagnosis and surveillance of many bowel diseases by both identifying potential biomarkers in stool samples and helping identify disease-related pathways. Fecal proteomics has already shown its potential for the discovery and validation of biomarkers for colorectal cancer screening, and the analysis of fecal microbiota by MALDI-MS for the diagnosis of a range of bowel diseases is gaining clinical acceptance.

Areas covered: Based on a comprehensive analysis of the current literature, we introduce the range of sensitive and specific proteomics methods which comprise the current ‘Proteomics Toolbox’, explain how the integration of fecal proteomics with data processing/bioinformatics has been used for the identification of potential biomarkers for both CRC and other gut-related pathologies and analysis of the fecal microbiome, outline some of the current fecal assays in current clinical practice and introduce the concept of personalised medicine which these technologies will help inform.

Expert commentary: Integration of fecal proteomics with other proteomics and genomics strategies as well as bioinformatics is paving the way towards personalised medicine, which will bring with it improved global healthcare.