Quantitative proteomic analysis by iTRAQ® for the identification of candidate biomarkers in ovarian cancer serum

<p>Abstract</p> <p>Background</p> <p>Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ^®.</p> <p>Results</p> <p>Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ^®to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified.</p> <p>Conclusions</p> <p>This study provides the first analysis of immunodepleted serum in combination with iTRAQ^®to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.</p

What the geological past can tell us about the future of the ocean’s twilight zone

Paleontological reconstructions of plankton community structure during warm periods of the Cenozoic (last 66 million years) reveal that deep-dwelling ‘twilight zone’ (200–1000 m) plankton were less abundant and diverse, and lived much closer to the surface, than in colder, more recent climates. We suggest that this is a consequence of temperature’s role in controlling the rate that sinking organic matter is broken down and metabolized by bacteria, a process that occurs faster at warmer temperatures. In a warmer ocean, a smaller fraction of organic matter reaches the ocean interior, affecting food supply and dissolved oxygen availability at depth. Using an Earth system model that has been evaluated against paleo observations, we illustrate how anthropogenic warming may impact future carbon cycling and twilight zone ecology. Our findings suggest that significant changes are already underway, and without strong emissions mitigation, widespread ecological disruption in the twilight zone is likely by 2100, with effects spanning millennia thereafter

Development of an invasively monitored porcine model of acetaminophen-induced acute liver failure

Background: The development of effective therapies for acute liver failure (ALF) is limited by our knowledge of the pathophysiology of this condition, and the lack of suitable large animal models of acetaminophen toxicity. Our aim was to develop a reproducible invasively-monitored porcine model of acetaminophen-induced ALF. Method: 35kg pigs were maintained under general anaesthesia and invasively monitored. Control pigs received a saline infusion, whereas ALF pigs received acetaminophen intravenously for 12 hours to maintain blood concentrations between 200-300 mg/l. Animals surviving 28 hours were euthanased. Results: Cytochrome p450 levels in phenobarbital pre-treated animals were significantly higher than non pre-treated animals (300 vs 100 pmol/mg protein). Control pigs (n=4) survived 28-hour anaesthesia without incident. Of nine pigs that received acetaminophen, four survived 20 hours and two survived 28 hours. Injured animals developed hypotension (mean arterial pressure; 40.8+/-5.9 vs 59+/-2.0 mmHg), increased cardiac output (7.26+/-1.86 vs 3.30+/-0.40 l/min) and decreased systemic vascular resistance (8.48+/-2.75 vs 16.2+/-1.76 mPa/s/m3). Dyspnoea developed as liver injury progressed and the increased pulmonary vascular resistance (636+/-95 vs 301+/-26.9 mPa/s/m3) observed may reflect the development of respiratory distress syndrome. Liver damage was confirmed by deterioration in pH (7.23+/-0.05 vs 7.45+/-0.02) and prothrombin time (36+/-2 vs 8.9+/-0.3 seconds) compared with controls. Factor V and VII levels were reduced to 9.3 and 15.5% of starting values in injured animals. A marked increase in serum AST (471.5+/-210 vs 42+/-8.14) coincided with a marked reduction in serum albumin (11.5+/-1.71 vs 25+/-1 g/dL) in injured animals. Animals displayed evidence of renal impairment; mean creatinine levels 280.2+/-36.5 vs 131.6+/-9.33 mumol/l. Liver histology revealed evidence of severe centrilobular necrosis with coagulative necrosis. Marked renal tubular necrosis was also seen. Methaemoglobin levels did not rise >5%. Intracranial hypertension was not seen (ICP monitoring), but there was biochemical evidence of encephalopathy by the reduction of Fischer's ratio from 5.6 +/- 1.1 to 0.45 +/- 0.06. Conclusion: We have developed a reproducible large animal model of acetaminophen-induced liver failure, which allows in-depth investigation of the pathophysiological basis of this condition. Furthermore, this represents an important large animal model for testing artificial liver support systems

All in the Name: A Review of Current Standards and the Evolution of Histopathological Nomenclature for Laboratory Animals

The need for international collaboration in rodent pathology has evolved since the 1970s, and was initially driven by the new field of toxicologic pathology. First initiated by the World Health Organization’s International Agency for Research on Cancer for rodents, it has evolved to include pathology of the major species (rats, mice, guinea pigs, nonhuman primates, pigs, dogs, fish, rabbits) used in medical research, safety assessment and mouse pathology. The collaborative effort today is driven by the needs of the regulatory agencies in multiple countries, and by needs of research involving genetically engineered animals, for “basic” research, and for more translational preclinical models of human disease. These efforts led to the establishment of an international rodent pathology nomenclature program. Since that time, multiple collaborations for standardization of laboratory animal pathology nomenclature and diagnostic criteria have been developed, and just a few are described herein. Recently, approaches to a nomenclature that is amenable to sophisticated computation have been made available and implemented for large-scale programs in functional genomics and ageing. Most terminologies continue to evolve as the science of human and veterinary pathology continues to develop, but standardization and successful implementation remain as critical for scientific communication, now as ever in the history of veterinary nosology.Government of Canada through Genome Canada and Ontario Genomics (OGI-051), Commission of the European Community QLRI-1999-CT-0320, the Ellison Medical Foundation, and the National Institutes of Health (CA34196, CA089713, and AG038070-05

Reasoning deficits among illicit drug users are associated with aspects of cannabis use

Background. Deficits in deductive reasoning have been observed among ecstasy/polydrug users. The present study seeks to investigate dose-related effects of specific drugs and whether these vary with the cognitive demands of the task. Methods. One hundred and five participants (mean age 21.33, S.D. 3.14; 77 females, 28 males) attempted to generate solutions for eight one-model syllogisms and one syllogism for which there was no valid conclusion (NVC). All of the one model syllogisms generated at least one valid conclusion and six generated two valid conclusions. In these six cases one of the conclusions was classified as common and the other as non-common. Results. The number of valid common inferences was negatively associated with aspects of short term cannabis use and with measures of IQ. The outcomes observed were more than simple post intoxication effects since cannabis use in the 10 days immediately before testing was unrelated to reasoning performance. Following adjustment for multiple comparisons, the number of non-common valid inferences was not significantly associated with any of the drug use measures. Conclusions. Recent cannabis use appears to impair the processes associated with generating valid common inferences while not affecting the production of non-common inferences. It is possible, therefore, that the two types of inference may recruit different executive resources which may differ in their susceptibility to cannabis-related effects

Risk factors for presentation to hospital with severe anaemia in Tanzanian children: a case-control study.

In malaria endemic areas anaemia is a usually silent condition that nevertheless places a considerable burden on health services. Cases of severe anaemia often require hospitalization and blood transfusions. The objective of this study was to assess risk factors for admission with anaemia to facilitate the design of anaemia control programmes. We conducted a prospective case-control study of children aged 2-59 months admitted to a district hospital in southern Tanzania. There were 216 cases of severe anaemia [packed cell volume (PCV) < 25%] and 234 age-matched controls (PCV > or = 25%). Most cases [55.6% (n = 120)] were < 1 year of age. Anaemia was significantly associated with the educational level of parents, type of accommodation, health-seeking behaviour, the child's nutritional status and recent and current medical history. Of these, the single most important factor was Plasmodium falciparum parasitaemia [OR 4.3, 95% confidence interval (CI) 2.9-6.5, P < 0.001]. Multivariate analysis showed that increased recent health expenditure [OR 2.2 (95% CI 1.3-3.9), P = 0.005], malnutrition [OR 2.4 (95%CI 1.3-4.3), P < 0.001], living > 10 km from the hospital [OR 3.0 (95% CI 1.9-4.9), P < 0.001], a history of previous blood transfusion [OR 3.8 (95% CI 1.7-9.1), P < 0.001] and P. falciparum parasitaemia [OR 9.5 (95% CI 4.3-21.3), P < 0.001] were independently related to risk of being admitted with anaemia. These findings are considered in terms of the pathophysiological pathway leading to anaemia. The concentration of anaemia in infants and problems of access to health services and adequate case management underline the need for targeted preventive strategies for anaemia control

A peptide mimic of the chemotaxis inhibitory protein of Staphylococcus aureus: towards the development of novel anti-inflammatory compounds

Complement factor C5a is one of the most powerful pro-inflammatory agents involved in recruitment of leukocytes, activation of phagocytes and other inflammatory responses. C5a triggers inflammatory responses by binding to its G-protein-coupled C5a-receptor (C5aR). Excessive or erroneous activation of the C5aR has been implicated in numerous inflammatory diseases. The C5aR is therefore a key target in the development of specific anti-inflammatory compounds. A very potent natural inhibitor of the C5aR is the 121-residue chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Although CHIPS effectively blocks C5aR activation by binding tightly to its extra-cellular N terminus, it is not suitable as a potential anti-inflammatory drug due to its immunogenic properties. As a first step in the development of an improved CHIPS mimic, we designed and synthesized a substantially shorter 50-residue adapted peptide, designated CHOPS. This peptide included all residues important for receptor binding as based on the recent structure of CHIPS in complex with the C5aR N terminus. Using isothermal titration calorimetry we demonstrate that CHOPS has micromolar affinity for a model peptide comprising residues 7–28 of the C5aR N terminus including two O-sulfated tyrosine residues at positions 11 and 14. CD and NMR spectroscopy showed that CHOPS is unstructured free in solution. Upon addition of the doubly sulfated model peptide, however, the NMR and CD spectra reveal the formation of structural elements in CHOPS reminiscent of native CHIPS

Case-based exercises fail to improve medical students' information management skills: a controlled trial

BACKGROUND: Tomorrow's physicians must learn to access, retrieve, integrate and apply current information into ambulatory patient encounters, yet few medical schools teach 'real time' information management. METHODS: We compared two groups of clerkship students' information management skills using a standardized patient case. The intervention group participated in case-based discussions including exercises that required them to manage new information. The control group completed the same case discussions without information management exercises. RESULTS: After five weeks, there was no significant difference between the control and intervention groups' scores on the standardized patient case. However, third rotation students significantly outperformed first rotation students. CONCLUSION: Case-based exercises to teach information management failed to improve students' performance on a standardized patient case. Increased number of clinical rotations was associated with improved performance

Metastatic appendiceal adenocarcinoma presenting late as epididymo-orchitis: a case report and review of literature

BACKGROUND: Whereas testicular metastases are in themselves a rare entity, testicular secondaries from an appendiceal carcinoma have not yet been described. The case also illustrates the diagnostic dilemma of a tumour presenting as epididymo-orchitis. CASE PRESENTATION: The authors present a case of an appendiceal carcinoma that, two years after radical therapy, manifested as a secondary in the testis. It was misdiagnosed as an epididymo-orchitis and was only revealed through histology. CONCLUSIONS: Practitioners need to remember that long-standing testicular inflammation may result form secondary tumours. Even "exotic" primary tumours in the medical history of the patient must give rise to an increased suspicion threshold

Leucine-rich alpha-2-glycoprotein-1 is upregulated in sera and tumors of ovarian cancer patients

<p>Abstract</p> <p>Background</p> <p>New biomarkers that replace or are used in conjunction with the current ovarian cancer diagnostic antigen, CA125, are needed for detection of ovarian cancer in the presurgical setting, as well as for detection of disease recurrence. We previously demonstrated the upregulation of leucine-rich alpha-2-glycoprotein-1 (LRG1) in the sera of ovarian cancer patients compared to healthy women using quantitative mass spectrometry.</p> <p>Methods</p> <p>LRG1 was quantified by ELISA in serum from two relatively large cohorts of women with ovarian cancer and benign gynecological disease. The expression of LRG1 in ovarian cancer tissues and cell lines was examined by gene microarray, reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, immunocytochemistry and mass spectrometry.</p> <p>Results</p> <p>Mean serum LRG1 was higher in 58 ovarian cancer patients than in 56 healthy women (89.33 ± 77.90 vs. 42.99 ± 9.88 ug/ml; p = 0.0008) and was highest among stage III/IV patients. In a separate set of 193 pre-surgical samples, LRG1 was higher in patients with serous or clear cell ovarian cancer (145.82 ± 65.99 ug/ml) compared to patients with benign gynecological diseases (82.53 ± 76.67 ug/ml, p < 0.0001). CA125 and LRG1 levels were moderately correlated (r = 0.47, p < 0.0001). <it>LRG1 </it>mRNA levels were higher in ovarian cancer tissues and cell lines compared to their normal counterparts when analyzed by gene microarray and RT-PCR. LRG1 protein was detected in ovarian cancer tissue samples and cell lines by immunocytochemistry and Western blotting. Multiple iosforms of LRG1 were observed by Western blot and were shown to represent different glycosylation states by digestion with glycosidase. LRG1 protein was also detected in the conditioned media of ovarian cancer cell culture by ELISA, Western blotting, and mass spectrometry.</p> <p>Conclusions</p> <p>Serum LRG1 was significantly elevated in women with ovarian cancer compared to healthy women and women with benign gynecological disease, and was only moderately correlated with CA125. Ovarian cancer cells secrete LRG1 and may contribute directly to the elevated levels of LRG1 observed in the serum of ovarian cancer patients. Future studies will determine whether LRG1 may serve as a biomarker for presurgical diagnosis, disease recurrence, and/or as a target for therapy.</p