BACKGROUND: Glioblastoma multiforme (GBM) is a highly heterogeneous tumor of the central nervous system (CNS) that exhibits considerable variation in its clinical course. Recently, the World Health Organization (WHO) published a classification system for tumors of the CNS that combines histological features with molecular parameters to determine tumor grade. The incorporation of molecular biomarkers that carry both prognostic and predictive value adds another level of objectivity to the glioma grading system and will help guide clinical decision. As such, the assessment of biomarkers has become an integral part of tumor evaluation in neuro-oncology. This curriculum will discuss the clinical relevance of the most recently studied biomarkers with prognostic and predictive value in the evaluation of GBM. Biomarkers regularly used for the assessment of GBM include the IDH mutations, MGMT methylation status, and EGFRvIII. Furthermore, this review will offer a perspective on experimental approaches currently under investigation for treatment of GBM. LITERATURE REVIEW FINDINGS: MGMT methylation of the promoter region is associated with better treatment response from temozolomide (TMZ), an alkylating therapeutic. Treatment benefit was most prominent in the elderly population and therapy should be individualized for that age group. Patients with GBM characterized by IDH1/IDH2 mutations carry a better overall prognosis primarily due to their higher sensitivity to chemo- and radiotherapy. The prognostic value of EGFRvIII remains controversial, although it may be associated with a worse prognosis. Nonetheless, EGFRvIII provides an ideal target for targeted molecular therapies as it is only found on tumor cells. PROPOSED METHODS: A curriculum aimed at educating primary care providers (PCPs) about the most clinically significant biomarkers in GBM will be developed. The curriculum will be in a PowerPoint format, and the hour-long lecture will be presented at continuing medical education national conferences. A pre- and post-test consisting of the same 10 multiple- choice questions will be administered on a voluntary basis to help evaluate knowledge acquisition from the curriculum. Results will be evaluated with a paired t-test analysis. The tests will be will be administered through Poll Everywhere, a smartphone survey application. CONCLUSION: There is increasing evidence to suggest that therapies should be individualized according to specific biomarkers with predictive value. PCPs are in a position where they are often the first providers to suspect the diagnosis of a brain tumor. Therefore, it is imperative for PCPs to be aware of the latest development in the field of neuro-oncology so that they may appropriately counsel patients.

There is currently no clinically useful screening test available to identify nulliparous women at high risk of developing pre-eclampsia. This study aimed to identify novel biomarkers using hypothesis generating proteomic methods applied to plasma samples obtained prior to clinical diagnosis of pre-eclampsia. Plasma samples taken at 15 weeks gestation from women who subsequently developed late pre-eclampsia (> 34 weeks), early pre-eclampsia (< 34 weeks) and two distinct groups of women with uncomplicated pregnancies (each n=12) were pooled. Pooled plasma was immunodepleted, labelled using iTRAQ-8 plex reagent and separated into fractions using high pH reverse phase chromatography. Fractions were analysed by LC-MS/MS and data interrogated using ProteinPilot 3.0. The merits of two immunodepletion systems were compared; the Seppro® IgY 14 -SuperMix LC column system removes up to 100 highly abundant plasma proteins and the Multiple Affinity Removal LC column depletes 14 highly abundant plasma proteins. Removal of more high abundance proteins allowed identification of more, potentially interesting, low abundance proteins, but was less reproducible than removing fewer proteins. Two methods of LC-MS/MS analysis were assessed; the QStar XL qTOF and 5800 MALDI-TOF-TOF. The protein identifications and the quantification data acquired by each method was comparable and complementary and increased the total number of proteins identified. A total of 502 proteins were identified. A stringent two stage analysis was developed to identify candidate proteins which changed in abundance in plasma from women who later developed pre-eclampsia compared to women with uncomplicated pregnancies. Analysis identified a total of 113 proteins which were both reproducibly quantified and changed by more than the expected range of biological variation. Six candidate proteins changed in abundance in the plasma taken from women who subsequently developed early pre-eclampsia were selected for further validation. A high throughput, low cost, method of multiple reaction monitoring which allows relative quantitation without the use of costly isotopically labelled peptides was developed to validate candidate proteins. Candidate proteins were also assessed by western blot and ELISA. Only one candidate protein; platelet basic protein, was validated by all three methods and demonstrated similar increases in the abundance. This investigation suggests that measurement of platelet basic protein at 15 weeks gestation is a novel candidate predictive marker for pre-eclampsia. Validation of platelet basic protein in a large, independent, sample set is required to confirm changes in protein expression and to evaluate potential, alongside other factors, to identify nulliparous women at high risk of developing pre-eclampsia later in pregnancy.

ude.dravrah.egelloc@gnufynnej / This research was supported by awards from the National Institutes of Health (NIH), the Eunice Kennedy Shriver Institute of Child Health and Human Development (R01-HD-059835) and the National Institute for Minority Health and Health Disparities (T37-MD000149). The NIH had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. The authors wish to thank the dedicated staff members of Asociacion Civil Proyectos en Salud (PROESA), Peru and Instituto Especializado Materno Perinatal, Peru for their expert technical assistance with this research. / Revisión por pares

Prostate carcinoma is one of the most frequent cancer diseases of maturated men. Diagnose of this cancer disease is based on level of prostatic specific antigen and digital exam per rectum. However, presence of tumor, grade and stage must be examined by biopsy. This method may not to be accurate. This is the reason why are new diagnostic methods investigated, to increase the accuracy of diagnostic prostate cancer. Amino acid sarcosine is currently on of the most widely discussed biomarker, that can serve as a diagnostic biomarker for early stage detection in prostate cancer. The present work deals with the study of sarcosine metabolism in prostate cancer cell lines, mainly, but also by amino acid metabolism in cells in general. Understanding to importance of single amino acids in malignant and non-malignant cell pathways can significantly contribute to effective targeted therapy without side effect on healthy tissue. Prostate cancer cells in presence of sarcosine increase their migration potential, malignancy by increased doubling time, and concentration of sarcosine N-demethylation enzymes, sarcosine dehydrogenase and sarcosine oxidase. Through this biochemical pathway is increased synthesis of main methylation donor S-adenosilmethyonine (SAM) ad cellular methylation potential.

Patients on haemodialysis are highly susceptible to different forms of heart failure. To date, the benefit of Mineralocorticoid-receptor antagonist (MRA) administration in haemodialysis patients remains subject to discussion. Biomarkers play an important role in therapy guidance and pose a promising tool to detect pathological processes of heart failure in an earlier stage. The randomised-controlled Mineralocorticoid-Receptor Antagonists in End-Stage Renal Disease (MiREnDa) trial was set up to investigate the effect of 50 mg of spironolactone once daily on left ventricular mass index in haemodialysis patients and several secondary endpoints. This dissertation reports findings from the MiREnDa trial on (a) the efficacy of spironolactone to influence serum levels of biomarkers of heart failure, fibrosis and inflammation and electrolytes and (b) the ability of N-terminal pro-B-type natriuretic peptide (NT-proBNP), Galectin-3 and soluble source of tumorigenicity 2 (sST2) to reflect left ventricular hypertrophy and diastolic dysfunction assessed by imaging characteristics. Treatment of spironolactone over a 40-week period did not alter serum levels of biomarkers of heart failure, fibrosis and inflammation including NT-proBNP, Galectin-3 and sST2. A small but significant effect on serum sodium but not potassium was observed. NT-proBNP was significantly different in the presence or absence of left ventricular hypertrophy (LVH) (normal vs. LVH (median [IQR]): 2,120 [810; 5,040] vs. 6,340 [2,410; 15,360] pg/ml, p<0.01) or moderate and severe diastolic dysfunction (DD) (normal diastolic function and DD grade I vs. DD grade II and DD grade III: 2,300 [850; 6,050] vs. 12,260 [3,340; 34,830] pg/ml, p=0.02). NT-proBNP further showed a significant correlation at baseline with LVMi (Spearman’s rho=0.41, p<0.001), LAVi (Spearman’s rho=0.55, p<0.001) and septal E/e’ (Spearman’s rho=0.45, p<0.001). No correlation was observed between Galectin-3 and the investigated functional and morphological parameters. sST2 was mildly correlated to LVMi at baseline (Spearman’s rho=0.21, p=0.05) and NT-proBNP at baseline (Spearman’s rho=0.37, p<0.001). In conclusion, spironolactone did not affect the investigated parameters but NT-proBNP proved to be significantly correlated to cardiac imaging measurements. / Dialysepatienten erkranken häufig an Formen der Herzinsuffizienz. Zugleich ist der Nutzen von Mineralkortikoidrezeptorantagonisten bei Dialysepatienten bis heute umstritten. Biomarkermessungen ermöglichen es, pathologische Prozesse am Herzen in einem möglichst frühen Stadium zu erkennen. In der randomisiert-kontrollierten "Mineralocorticoid-Receptor Antagonists in End-Stage Renal Disease" (MiREnDa) Studie wurde der Effekt der täglichen Einnahme von 50 mg Spironolacton auf den linksventrikulären Massenindex bei Dialysepatienten zusammen mit verschiedenen sekundären Endpunkten untersucht. Diese Arbeit beleuchtet Ergebnisse der MiREnDa-Studie zur Wirkung von Spironolacton auf Serumspiegel von Biomarkern für Herzinsuffizienz, Fibrose und Entzündung sowie von Elektrolyten. Darüber hinaus wird der Zusammenhang zwischen N-terminalen natriuretischen Peptid Typ B (NT-proBNP), Galectin-3 und Soluble source of tumorigenicity 2 (sST2) und Veränderungen in den wichtigsten bildgebenden Merkmalen linksventrikulärer Hypertrophie und diastolischer Dysfunktion beschrieben. Die Einnahme von Spironolacton über 40 Wochen hatte keinen Effekt auf Biomarker für Herzinsuffizienz, Fibrose und Entzündung wie NT-proBNP, Galectin-3 und sST2. Lediglich die Natriumspiegel, nicht aber die Kaliumspiegel, wurden signifikant beeinflusst. NT-proBNP unterschied sich signifikant zwischen Patient*innen mit und ohne links-ventrikulärer Hypertrophie (LVH) (normal vs. LVH (Median [IQR]): 2.120 [810; 5.040] vs. 6.340 [2.410; 15.360] pg/ml, p<0,01) beziehungsweise mit und ohne relevanter diastolischer Dysfunktion (DD) (normale diastolische Funktion und DD Grad I vs. DD Grad II und DD Grad III: 2.300 [850; 6.050] vs. 12.260 [3.340; 34.830] pg/ml, p=0,02). NT-proBNP korrelierte außerdem signifikant mit LVMi (Spearman's rho=0,41, p<0,001), LAVi (Spearman's rho=0,55, p<0,001) und E/e' (Spearman's rho=0,45, p<0,001). Galectin-3 war unabhängig von allen untersuchten Parametern. sST2 korrelierte mäßig mit LVMi (Spearman's rho=0,21, p=0,05) und deutlich mit NT-proBNP (Spearman's rho=0,37, p<0,001). Zusammenfassend beeinflusste Spironolacton keinen der untersuchten Parameter relevant und lediglich NT-proBNP wies eine signifikante Korrelation zu kardialen Bildgebungsparameters auf.

Background: Respiratory tract infections are responsible for millions of deaths annually. Interferon-stimulated genes (ISGs) play a significant role in fighting off viral respiratory tract infections in the antiviral defence system. Measuring extracellular protein products of ISGs could be potential biomarkers of viral infection. Although, the feasibility and performance of ISGs as functional and robust clinical biomarkers from a non-invasive sample format remains unknown. Methods: Three ISGs, CXCL10, CXCL11, and TNFSF10, were examined in in-vivo and in-vitro gene expression datasets (RNA-sequencing and microarray) infected with common respiratory tract infections (Rhinovirus, Respiratory syncytial virus, influenza A and SARS-CoV-2) samples and compared to negative controls. Using qualitative selection criteria of 1) elevated presence in at least one dataset with viral infection, 2) secreted protein product, and 3) commercially available antibodies for detection, CXCL10, CXCL11 and TNFSF10 gene expression levels were assessed. A correlation analysis was performed with SARS-CoV-2 infection severity and gene expression kinetics. CXCL10 was subsequently validated at the protein level in saliva as a prerequisite for developing a host-response LFA. Results: CXCL10 and CXCL11 upregulation were positively correlated with RSV compared to control (p < 0.05). CXCL10/CXCL11/TNFSF10 were not different between samples collected from RV infected subjects relative to controls (p > 0.05). No significant association was found with influenza A for all three genes. CXCL10/CXCL11/TNFSF10 upregulation was positively correlated with SARS-CoV-2 infection compared to control (p < 0.001). CXCL10 expression correlated with COVID-19 viral load. CXCL10 was chosen as a lead biomarker candidate based on these analyses that included different virus infections, time-courses, and measures of severity. CXCL10 was not detected at the protein level in healthy saliva but was elevated in saliva from COVID-19 patients. A CXCL10 LFA was developed with a sensitivity of 2 ng/ml in a buffer and artificial saliva. Conclusion: We establish and validate the potential of developing rapid test techniques to examine host immune response from a bioinformatic approach to developing a prototype rapid test with capabilities to be used in point-of-care settings. / Thesis / Master of Science (MSc) / Respiratory tract infections are a leading cause of death and one of the main reasons to seek primary care. Both historically and in the present day, respiratory tract infections remain a massive socioeconomic burden. Current diagnostics fail to quickly identify a respiratory tract infection's etiology, and prognosis, leading to suboptimal patient care and the over prescription of antibiotics. Advanced tools used in academia and research, including next-generation -omics sequencing and metagenomics, have capabilities to identify all nucleic acid material in a sample - including host RNA- which offers potential to improve the diagnosing of respiratory tract infections. However, these technologies have not been integrated into routine care due to economic, technical, and logistical barriers. We explored host RNA (transcriptomics), looking at antiviral interferon-stimulated genes for their potential as a biomarker of viral infection amenable to point-of-care testing platforms from non-invasive sample types.

Die Diagnosestellung von unipolarer und bipolarer Depression basiert bis heute ausschließlich auf der Bewertung klinischer Symptome. Objektive biochemische Marker, wie sie bei zahlreichen somatischen Krankheiten zur Diagnosestellung angewendet werden, sind bisher nicht verfügbar. Da sich die beide Krankheitsbilder vor allem in der depressiven Episode stark ähneln, ist eine Unterscheidung in diesem Krankheitsstadium häufig nicht eindeutig möglich. Dies kann zu Fehldiagnosen, einer Verschlechterung des Krankheitsverlaufs, einer erhöhten Krankheitslast und höheren Gesundheitskosten führen. Periphere Biomarker wären daher wertvoll, um die klinische Diagnosestellung zu unterstützen und eine adäquate Behandlung frühzeitige zu ermöglichen. In einer vorherigen Studie der Arbeitsgruppe haben Proteom-Analysen bestimmte Proteine wie den Wachstumsfaktor PDGF-BB und das Thrombospondin TSP-1 identifiziert, die potenziell als Biomarker fungieren könnten. In der vorliegenden Studie wurde untersucht, ob sich die Konzentration von PDGF-BB und TSP-1 im Blut zwischen Patient*innen mit unipolarer bzw. bipolarer Depression signifikant unterscheidet. Es konnte gezeigt werden, dass PDGF-BB bei unipolaren Patientinnen signifikant niedriger ist als bei bipolaren Patientinnen und gesunden Kontrollpersonen. Zudem sank die PDGF-BB-Konzentration bei bipolaren Patientinnen während einer remittierten Episode im Vergleich zu einer depressiven Episode signifikant ab. Im Gegensatz dazu zeigte TSP-1 keine signifikanten Unterschiede zwischen den Patient*innengruppen und Kontrollpersonen. Die Arbeit konnte zeigen, dass PDGF-BB das Potenzial hat, als diagnostischer Biomarker für die Unterscheidung zwischen unipolarer und bipolarer Depression zu dienen, während TSP-1 in dieser Hinsicht nicht geeignet erscheint. Weitere Forschung ist jedoch notwendig, um die Rolle von PDGF-BB in der Pathogenese affektiver Erkrankungen besser zu verstehen und seinen Einsatz als Biomarker im klinischen Alltag zu validieren. / The diagnosis of unipolar and bipolar depression is still based on clinical symptoms. Objective biochemical markers, which are commonly used for diagnosing somatic diseases, are not yet available. Since both conditions, particularly during the depressive phase, exhibit very similar symptoms, distinguishing between them in this stage is often complicated. This can lead to misdiagnosis, a worsening of the disease course, increased disease burden, and higher healthcare costs. Peripheral biomarkers would therefore be of great value in supporting clinical diagnosis and enabling early and appropriate treatment. A previous study by our research group identified proteins such as the growth factor PDGF-BB and thrombospondin TSP-1 through proteomic analysis, which could potentially serve as biomarkers. In the present study, the concentrations of PDGF-BB and TSP-1 in the blood of patients with unipolar and bipolar depression were examined to determine if there were significant differences. The results showed that PDGF-BB levels were significantly lower in unipolar patients compared to bipolar patients and healthy controls. Additionally, PDGF-BB concentrations decreased significantly in bipolar patients during a remitted episode compared to a depressive episode. In contrast, TSP-1 did not show significant differences between the groups studied. The study suggests that PDGF-BB has the potential to serve as a diagnostic biomarker for differentiating between unipolar and bipolar depression, whereas TSP-1 appears less suitable in this context. However, further research is needed to better understand the role of PDGF-BB in the pathogenesis of affective disorders and to validate its clinical utility as a biomarker.

Eine große Herausforderung in der Therapie des Prostatakarzinoms liegt in seiner ausgeprägten Heterogenität, die sich durch histomorphologisch und genetisch stark variierende Tumorläsionen auszeichnet. Zuverlässige klinische Marker, die den individuellen Krankheitsverlauf a priori vorhersagen können und somit eine bessere Risikostratifizierung und Therapieplanung ermöglichen, werden als Ergänzung zu bereits bestehenden Markern benötigt. Daraus ergibt sich die Notwendigkeit, weitere potentielle Biomarker zu erforschen. Ziel dieser Arbeit war es, die Rolle der miR-301a als Prognosemarker im Hochrisiko-PCa zu untersuchen sowie mittels in vitro Experimenten Rückschlüsse auf miR-301a vermittelte molekulare Mechanismen im PCa zu ziehen. Expressionsanalysen aus PCa Nativmaterial zeigten eine signifikante Überexpression der miR-301a in LK-Metastasen. Im verwendeten PCa Hochrisiko-Kollektiv konnte jedoch keine signifikante Überexpression der miR-301a gegenüber BPH-Proben nachgewiesen werden. Dennoch war eine hohe miR-301a Expression jeweils mit PSAProgress, klinischem Progress sowie mit tumorbedingter Mortalität assoziiert, sodass eine onkogene Funktion der miR-301a aufgezeigt werden kann. Ebenfalls erwies sich die miR-301a im untersuchten Hochrisiko-Patientenkollektiv mittels uni- und multivariater Cox-Regressionsanalysen als ein geeigneter Prädiktor zur Abschätzung der Prognose. Die Ergebnisse legen nahe, dass eine miR-301a Überexpression als ein Marker für Tumorprogress sowie als potentieller Prognosemarker herangezogen werden kann. Die onkogene Funktion der miR-301a ließ sich in den durchgeführten in vitro Versuchen bestätigen, welche eine Zunahme der Migration sowie Proliferation in miR-301a überexprimierenden PCa-Zelllinien zeigten. Ein möglicher funktioneller Zusammenhang konnte durch die Identifikation von TSC1, einem bekannten Tumorsuppressor und Modulator des mTOR-Signalwegs, als neuer Zielstruktur der miR-301a hergestellt werden. Die miR-301a vermittelte Regulierung von TSC1 könnte eine verstärkte epithelial-mesenchymale Transition sowie Veränderungen des Aktinzytoskeletts bedingen und in Folge dessen Metastasierung im miR-301a überexprimierenden PCa fördern. Die signifikante miR-301a Überexpression in den Lymphknotenmetastasen unseres Kollektivs würde sich damit erschließen. Die posttranskriptionelle Herabregulierung der TSC1 Expression durch miR-301a bietet jedoch nicht nur eine mögliche Erklärung für ihre Funktion als Onko-miR, sondern könnte zudem mit Blick auf den Einsatz zielgerichteter Tumortherapien von Interesse sein. So konnte eine Sensitivierung gegenüber dem mTOR-Inhibitor Temsirolimus in miR-301a überexprimierenden PCa-Zelllinien beobachtet werden. Diese Beobachtung lässt vermuten, dass PCa-Patienten mit hoher miR-301a Expression im Tumorgewebe als Patienten-Subgruppe eher von einer mTOR-Inhibition profitieren könnten. Somit könnte eine hohe miR-301a Expression als möglicher prädiktiver Marker für ein Therapieansprechen stehen. Trotz der in dieser Arbeit gezeigten Rolle von miR-301a als potenziellem Prognosemarker im Hochrisiko-PCa und bereits erfolgter Forschung, welche die onkogene Funktion der miR-301a im PCa untermauert, sind zusätzliche - insbesondere prospektive - Studien notwendig, um miR-301a als möglichen Biomarker zu etablieren. Im Hinblick auf miR-301a regulierte zelluläre Signalwege leistet diese Arbeit mit der Identifikation von TSC1 als neuer Zielstruktur einen Beitrag zum besseren Verständnis der zahlreichen miR-301a regulierten Mechanismen. / A major challenge in the treatment of prostate cancer lies in its pronounced heterogeneity, which is characterized by histomorphologically and genetically highly variable tumor lesions. Reliable clinical markers that can predict the individual disease course a priori and thus enable a better risk stratification and therapy planning are required to complement existing markers. This underscores the necessity of identifying additional potential biomarkers. The aim of this study was to investigate the role of miR-301a as a prognostic marker in high-risk prostate cancer (PCa) and to elucidate miR-301a mediated molecular mechanisms in PCa through in vitro experiments. Expression analyses of native PCa tissue revealed a significant overexpression of miR-301a in lymph node metastases. However, in the entire high-risk cohort, no significant overexpression of miR-301a compared to benign prostatic hyperplasia samples was detected. Nonetheless, high miR-301a expression was associated with PSA progression, clinical progression and cancer-related death, highlighting an oncogenic role of miR-301a, particularly in aggressive PCa. Furthermore, miR-301a proved to be a suitable predictor for prognosis estimation in the examined high-risk patient cohort, as shown by univariate and multivariate Cox regression analyses. These findings suggest that miR-301a overexpression can serve as a marker for tumor progression and as a potential prognostic biomarker. The oncogenic function of miR-301a was confirmed in the in vitro experiments carried out, which showed increased migration and proliferation in PCa cell lines overexpressing miR-301a. Through the identification of TSC1 as a novel target of miR301a, a possible functional connection between miR301a and TSC1, was found. TSC1 itself is a well-known tumor suppressor and modulator of the mTOR signaling pathway. The downregulation of TSC1 expression by miR-301a overexpression provides an additional explanation for its role as an oncomiR and also highlights further therapeutic options. Notably, sensitization to the mTOR inhibitor Temsirolimus was observed in miR-301a overexpressing PCa cell lines, which presents a clinically relevant perspective. PCa patients with high miR-301a expression in tumor tissue could represent a subgroup more likely to benefit from mTOR inhibition. Thus, high miR-301a expression could serve as a potential predictive marker for therapeutic response. Moreover, the miR-301a mediated regulation of TSC1 provides an explanation for the pronounced lymph node metastases of our cohort: the downregulation of TSC1 could enhance epithelial-mesenchymal transition and induce changes in the actin cytoskeleton, subsequently promoting metastasis in miR-301a overexpressing PCa. Despite the role of miR-301a as a potential prognostic marker in high-risk PCa as demonstrated in this study and despite prior findings supporting its oncogenic function in PCa, additional – particularly prospective – studies are needed to establish miR-301a as a biomarker. Regarding miR-301a regulated cellular signaling pathways, this study contributes to a better understanding of the numerous mechanisms regulated by miR-301a by identifying TSC1 as a novel target.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder associated with aging. AD is by far the best understood and most studied neurodegenerative disease. Substantial advances have been made over the last decade, however it is debatable how much closer we are to a clinically useful therapy. A long standing goal in the AD field has been to improve the accuracy of early detection, with the assumption that the ability to intervene earlier in the disease process will lead to a better clinical outcome. Major facets of this effort have been the continued development and improvement of AD biomarkers, with a strong focus on developing imaging modalities. AD is accompanied by two pathological hallmarks in the brain: extracellular neuritic plaques composed of the beta-amyloid peptide (Aβ) and intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein. Evidence of Aβ as the driving force behind the progression of AD (the amyloid cascade hypothesis) was first published by Hardy & Higgins in 1992, and this peptide has been the focus of therapeutic and diagnostic testing for decades. Significant technological advances in recent years now allow imaging of amyloid pathology in vivo. These methods evaluate Aβ burden in a living person, and could potentially serve as both a biomarker, and as a diagnostic tool to detect disease. Pittsburgh Compound B (PiB) is currently the best studied of these imaging agents, however, our current knowledge of the quantitative relationship between PiB binding and amyloid pathology in the brain is limited. A better understanding of how these variables relate to one another is essential for the continued development of reliable diagnostic biomarkers for AD. We analyzed increasingly insoluble pools of Aβ to quantify their relative contributions to the overall Aβ burden, and to determine if any of these measures could be used to predict disease status. We found that the amount of PiB binding in a cortical region of the brain could distinguish cases of mild cognitive impairment (MCI) when corrected to the amount of PiB binding in the cerebellum. As the Aβ peptide ages, the amino acid aspartate may spontaneously convert to an isoaspartate residue through a succinimide intermediary. The presence of iso-Asp Aβ has been used to indicate the presence of aged plaques in AD and Down syndrome cases. We sought to investigate the potential relationship between levels of ‘aged’ Aβ in the plasma as indicated by iso-Asp Aβ and disease state, as a potential biomarker for the presence of AD pathology. We found that AD cases had lower levels of all forms of Aβ in plasma when standardized to the group average, and that plasma levels of Aβ and iso-Asp Aβ were reversed between disease groups. A follow up study is required, however, these initial data are a promising step towards utilizing aged iso-Asp Aβ plasma levels as a potential biomarker to indicate disease state.

The Proximity Ligation Assay (PLA) is a relatively new method which utilizes the strength of both immunoassays and DNA detection. PLA has the capacity of high multiplexing due to the high specificity achieved with both dual protein-binding and dual primer binding during detection with Real-Time PCR. We developed a multiplexed PLA protocol that can measure 28 biomarkers in human EDTA plasma. The method was tested on 46 individuals diagnosed with colorectal cancer and 48 age matched healthy controls. The results are very promising as we re-discover the most well-known biomarkers for colorectal cancer and also find some potential new markers (significance tested with students T-test with p&lt;0.05). Further improvements of the protocol are needed to decrease the variation.