MassIVE.quant: a community resource of quantitative mass spectrometry-based proteomics datasets.

MassIVE.quant is a repository infrastructure and data resource for reproducible quantitative mass spectrometry-based proteomics, which is compatible with all mass spectrometry data acquisition types and computational analysis tools. A branch structure enables MassIVE.quant to systematically store raw experimental data, metadata of the experimental design, scripts of the quantitative analysis workflow, intermediate input and output files, as well as alternative reanalyses of the same dataset.

Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein.

Alpha-synuclein (aSyn) is a central player in Parkinson's disease (PD) but the precise molecular mechanisms underlying its pathogenicity remain unclear. It has recently been suggested that nuclear aSyn may modulate gene expression, possibly via interactions with DNA. However, the biological behavior of aSyn in the nucleus and the factors affecting its transcriptional role are not known. Here, we investigated the mechanisms underlying aSyn-mediated transcription deregulation by assessing its effects in the nucleus and the impact of phosphorylation in these dynamics. We found that aSyn induced severe transcriptional deregulation, including the downregulation of important cell cycle-related genes. Importantly, transcriptional deregulation was concomitant with reduced binding of aSyn to DNA. By forcing the nuclear presence of aSyn in the nucleus (aSyn-NLS), we found the accumulation of high molecular weight aSyn species altered gene expression and reduced toxicity when compared with the wild-type or exclusively cytosolic protein. Interestingly, nuclear localization of aSyn, and the effect on gene expression and cytotoxicity, was also modulated by phosphorylation on serine 129. Thus, we hypothesize that the role of aSyn on gene expression and, ultimately, toxicity, may be modulated by the phosphorylation status and nuclear presence of different aSyn species. Our findings shed new light onto the subcellular dynamics of aSyn and unveil an intricate interplay between subcellular location, phosphorylation and toxicity, opening novel avenues for the design of future strategies for therapeutic intervention in PD and other synucleinopathies.

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion.

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

A Proteogenomic Resource Enabling Integrated Analysis of Listeria Genotype-Proteotype-Phenotype Relationships.

Listeria monocytogenes is an opportunistic foodborne pathogen responsible for listeriosis, a potentially fatal foodborne disease. Many different Listeria strains and serotypes exist, but a proteogenomic resource that bridges the gap in our molecular understanding of the relationships between the Listeria genotypes and phenotypes via proteotypes is still missing. Here, we devised a next-generation proteogenomics strategy that enables the community to rapidly proteotype Listeria strains and relate this information back to the genotype. Based on sequencing and de novo assembly of the two most commonly used Listeria model strains, EGD-e and ScottA, we established two comprehensive Listeria proteogenomic databases. A genome comparison established core- and strain-specific genes potentially responsible for virulence differences. Next, we established a DIA/SWATH-based proteotyping strategy, including a new and robust sample preparation workflow, that enables the reproducible, sensitive, and relative quantitative measurement of Listeria proteotypes. This reusable and publicly available DIA/SWATH library covers 70% of open reading frames of Listeria and represents the most extensive spectral library for Listeria proteotype analysis to date. We used these two new resources to investigate the Listeria proteotype in states mimicking the upper gastrointestinal passage. Exposure of Listeria to bile salts at 37 °C, which simulates conditions encountered in the duodenum, showed significant proteotype perturbations including an increase of FlaA, the structural protein of flagella. Given that Listeria is known to lose its flagella above 30 °C, this was an unexpected finding. The formation of flagella, which might have implications on infectivity, was validated by parallel reaction monitoring and light and scanning electron microscopy. flaA transcript levels did not change significantly upon exposure to bile salts at 37 °C, suggesting regulation at the post-transcriptional level. Together, these analyses provide a comprehensive proteogenomic resource and toolbox for the Listeria community enabling the analysis of Listeria genotype-proteotype-phenotype relationships.

A2A R-induced transcriptional deregulation in astrocytes: An in vitro study.

Adenosine A2A receptors (A2A R) are modulators of various physiological processes essential for brain homeostasis and fine synaptic tuning. In certain neurodegenerative conditions, notably Alzheimer's disease (AD), A2A Rs are pathologically upregulated in neurons but also in astrocytes. In that context, the use of A2A Rs inhibitors, normalizing impaired receptor function, is seen as a potential therapeutic strategy. However, the impact of A2A R alterations, particularly in astrocytes, is not fully understood. Here, we investigated the effect of A2A R overexpression on transcriptional deregulation in primary astrocytic cultures. By performing whole transcriptome analysis, we found that A2A R overexpression promotes robust transcriptional changes, mostly affecting immune response, angiogenesis, and cell activation-related genes. Importantly, we observed that treatment with SCH58261, a selective A2A R antagonist, restored the expression levels of several inflammatory and astrocytic activation-related genes, such as Interleukin-1beta and vimentin. This supports the notion that A2A R blockade could restore some astrocytic dysfunctions associated with abnormal A2A R expression, further arguing for a potential beneficial impact of receptor antagonists in A2A R-induced transcriptional deregulation, inflammation, and astrogliosis. Overall, our findings provide novel insights into the putative impact of A2A R overexpression on transcriptional deregulation in astrocytes, thereby opening novel avenues for the use of A2A R antagonists as potential therapeutic strategy in neurodegenerative diseases.

Sodium butyrate rescues dopaminergic cells from alpha-synuclein-induced transcriptional deregulation and DNA damage.

Alpha-synuclein (aSyn) is considered a major culprit in Parkinson's disease (PD) pathophysiology. However, the precise molecular function of the protein remains elusive. Recent evidence suggests that aSyn may play a role on transcription regulation, possibly by modulating the acetylation status of histones. Our study aimed at evaluating the impact of wild-type (WT) and mutant A30P aSyn on gene expression, in a dopaminergic neuronal cell model, and decipher potential mechanisms underlying aSyn-mediated transcriptional deregulation. We performed gene expression analysis using RNA-sequencing in Lund Human Mesencephalic (LUHMES) cells expressing endogenous (control) or increased levels of WT or A30P aSyn. Compared to control cells, cells expressing both aSyn variants exhibited robust changes in the expression of several genes, including downregulation of major genes involved in DNA repair. WT aSyn, unlike A30P aSyn, promoted DNA damage and increased levels of phosphorylated p53. In dopaminergic neuronal cells, increased aSyn expression led to reduced levels of acetylated histone 3. Importantly, treatment with sodium butyrate, a histone deacetylase inhibitor (HDACi), rescued WT aSyn-induced DNA damage, possibly via upregulation of genes involved in DNA repair. Overall, our findings provide novel and compelling insight into the mechanisms associated with aSyn neurotoxicity in dopaminergic cells, which could be ameliorated with an HDACi. Future studies will be crucial to further validate these findings and to define novel possible targets for intervention in PD.

The yin and yang of α-synuclein-associated epigenetics in Parkinson's disease.

Parkinson's disease is the second most prevalent neurodegenerative disorder. The main neuropathological hallmarks of the disease are the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of protein inclusions known as Lewy bodies. Recently, great attention has been given to the study of genes associated with both familial and sporadic forms of Parkinson's disease. Among them, the α-synuclein gene is believed to play a central role in the disease and is, therefore, one of the most studied genes. Parkinson's disease is a complex disorder and, as such, derives from the interaction between genetic and environmental factors. Here, we offer an update on the landscape of epigenetic-mediated regulation of gene expression that has been linked with α-synuclein and associated with Parkinson's disease. We also provide an overview of how epigenetic modifications can influence the transcription and/or translation of the α-synuclein gene and, on the other hand, how α-synuclein function/dysfunction can, per se, affect the epigenetic landscape. Finally, we discuss how a deeper understanding of the epigenetic profile of α-synuclein may enable the development of novel therapeutic approaches for Parkinson's disease and other synucleinopathies.

The Abrams geriatric self-neglect scale: introduction, validation and psychometric properties.

OBJECTIVE: Self-neglect is an imprecisely defined entity with multiple clinical expressions and adverse health consequences, especially in the elderly. However, research has been limited by the absence of a measurement instrument that is both inclusive and specific. Our goal was to establish the psychometric properties of a quantitative instrument, the Abrams Geriatric Self-Neglect Scale (AGSS). METHODS: We analyzed data from a 2007 case-control study of 71 cognitively intact community-dwelling older self-neglectors that had used the AGSS. The AGSS was validated against two "gold standards": a categorical definition of self-neglect developed by expert consensus; and the clinical judgment of a geriatric psychiatrist using chart review. Frequencies were examined for the six scale domains by source (Subject, Observer, and Overall Impression). Internal consistency was estimated for each source, and associations among the sources were evaluated. RESULTS: Internal consistency estimates for the AGSS were rated as "good," with the Subject responses having the lowest alpha and omega (0.681 and 0.692) and the Observer responses the highest (0.758 and 0.765). Subject and Observer scores had the lowest association (0.578, p < 0.001). Using expert consensus criteria as the primary "gold standard," the Observer and Overall Impression subscales were "good" at classifying self-neglect, while the Subject subscale was "fair." CONCLUSIONS: The AGSS correctly classified and quantified self-neglect against two "gold standards." Sufficient correlations among multiple sources of information allow investigators and clinicians to choose flexibly from Subject, Observer, or Overall Impression. The lower internal consistency estimates for Subject responses are consistent with self-neglectors' propensity to disavow symptoms. Copyright © 2017 John Wiley & Sons, Ltd.

Does idiopathic hypercalciuria affect bone metabolism during childhood? A prospective case-control study.

BACKGROUND: A limited number of studies have evaluated biochemical bone metabolism markers in children with idiopathic hypercalciuria, which in adults has been linked with osteopenia. Our aim was to investigate in children with idiopathic hypercalciuria biochemical markers of bone formation and resorption and the osteoprotegerin (OPG) and soluble receptor activator of nuclear factor kB ligand (sRANKL) system which is involved in the osteoclastogenesis process. METHODS: A prospective study was conducted on 50 children with idiopathic hypercalciuria and 50 healthy age-, sex-, and Tanner stage-matched control subjects. Following the diagnosis, patients were requested to follow a 3-month dietary recommendation for idiopathic hypercalciuria. In patients, at diagnosis and at 3 months of follow-up, and in controls, bone-related hormones and serum/urine biochemical parameters were studied. The bone formation markers (total ALP and osteocalcin) and the bone resorption markers (ß-Crosslaps) and the OPG and sRANKL levels were determined. RESULTS: No differences were found in the bone formation markers or OPG and sRANKL between the children with idiopathic hypercalciuria and controls. The ß-Crosslaps and the ß-Crosslaps/osteocalcin ratio were higher in the patients at diagnosis than in controls (p = 0.019 and p = 0.029, respectively), with a trend to decrease after the 3-month dietary intervention. The initially increased 24-h urinary Ca in the patients decreased after the 3-month dietary intervention (p = 0.002). CONCLUSIONS: Children with idiopathic hypercalciuria had biochemical markers compatible with normal bone formation but increased bone resorption. After a 3-month dietary intervention, the trend observed towards decrease in the serum ß-Crosslaps may reflect a beneficial response.

Dynamics of Protein Expression Reveals Primary Targets and Secondary Messengers of Estrogen Receptor Alpha Signaling in MCF-7 Breast Cancer Cells.

Estrogen receptor alpha (ERα)-mediated proliferation of breast cancer cells is facilitated through expression of multiple primary target genes, products of which induce a secondary response to stimulation. To differentiate between the primary and secondary target proteins of ERα signaling, we measured dynamics of protein expression induced by 17ß-estradiol in MCF-7 breast cancer cells. Measurement of the global proteomic effects of estradiol by stable isotope labeling by amino acids in cell culture (SILAC) resulted in identification of 103 estrogen-regulated proteins, with only 40 of the corresponding genes having estrogen response elements. Selected reaction monitoring (SRM) assays were used to validate the differential expression of 19 proteins and measure the dynamics of their expression within 72 h after estradiol stimulation, and in the absence or presence of 4-hydroxytamoxifen, to confirm ERα-mediated signaling. Dynamics of protein expression unambiguously revealed early and delayed response proteins and well correlated with presence or absence of estrogen response elements in the corresponding genes. Finally, we quantified dynamics of protein expression in a rarely studied network of transcription factors with a negative feedback loop (ERα-EGR3-NAB2). Because NAB2 protein is a repressor of EGR3-induced transcription, siRNA-mediated silencing of NAB2 resulted in the enhanced expression of the EGR3-induced protein ITGA2. To conclude, we provided a high-quality proteomic resource to supplement genomic and transcriptomic studies of ERα signaling.

Prox1 Is Required for Oligodendrocyte Cell Identity in Adult Neural Stem Cells of the Subventricular Zone.

Adult neural stem cells with the ability to generate neurons and glia cells are active throughout life in both the dentate gyrus (DG) and the subventricular zone (SVZ). Differentiation of adult neural stem cells is induced by cell fate determinants like the transcription factor Prox1. Evidence has been provided for a function of Prox1 as an inducer of neuronal differentiation within the DG. We now show that within the SVZ Prox1 induces differentiation into oligodendrocytes. Moreover, we find that loss of Prox1 expression in vivo reduces cell migration into the corpus callosum, where the few Prox1 deficient SVZ-derived remaining cells fail to differentiate into oligodendrocytes. Thus, our work uncovers a novel function of Prox1 as a fate determinant for oligodendrocytes in the adult mammalian brain. These data indicate that the neurogenic and oligodendrogliogenic lineages in the two adult neurogenic niches exhibit a distinct requirement for Prox1, being important for neurogenesis in the DG but being indispensable for oligodendrogliogenesis in the SVZ. Stem Cells 2016;34:2115-2129.

Epigenetics in Parkinson's Disease.

Parkinson's disease (PD) is a highly complex neurodegenerative disorder with a multifactorial origin. Although several cellular mechanisms and genes have been implicated in the onset and progression of the disease, the precise molecular underpinnings of the disease remain unclear. In this context, epigenetic modulation of gene expression by environmental factors is emerging as an important mechanism in PD and in other neurodegenerative disorders. Thus, epigenetic mechanisms, such as DNA methylation, histone modifications and altered microRNA expression, have been under intense investigation due to their possible involvement in PD. Epigenetic modulation is responsible for inducing differential gene expression, a phenomenon which is essential throughout life in order to regulate multiple cellular responses such as development, cellular fate commitment and adaptation to the environment. Disturbances of a balanced gene expression can, therefore, have detrimental effects. Environmental factors can challenge the establishment and maintenance of epigenetic modifications and could thereby fill the gap in our further understanding of origin and/or progression of neurodegenerative diseases. In this chapter, we focus on the role of epigenetics in PD.

Neural stem cells in Parkinson's disease: a role for neurogenesis defects in onset and progression.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, leading to a variety of motor and non-motor symptoms. Interestingly, non-motor symptoms often appear a decade or more before the first signs of motor symptoms. Some of these non-motor symptoms are remarkably similar to those observed in cases of impaired neurogenesis and several PD-related genes have been shown to play a role in embryonic or adult neurogenesis. Indeed, animal models deficient in Nurr1, Pitx3, SNCA and PINK1 display deregulated embryonic neurogenesis and LRRK2 and VPS35 have been implicated in neuronal development-related processes such as Wnt/ß-catenin signaling and neurite outgrowth. Moreover, adult neurogenesis is affected in both PD patients and PD animal models and is regulated by dopamine and dopaminergic (DA) receptors, by chronic neuroinflammation, such as that observed in PD, and by differential expression of wild-type or mutant forms of PD-related genes. Indeed, an increasing number of in vivo studies demonstrate a role for SNCA and LRRK2 in adult neurogenesis and in the generation and maintenance of DA neurons. Finally, the roles of PD-related genes, SNCA, LRRK2, VPS35, Parkin, PINK1 and DJ-1 have been studied in NSCs, progenitor cells and induced pluripotent stem cells, demonstrating a role for some of these genes in stem/progenitor cell proliferation and maintenance. Together, these studies strongly suggest a link between deregulated neurogenesis and the onset and progression of PD and present strong evidence that, in addition to a neurodegenerative disorder, PD can also be regarded as a developmental disorder.

Integrating meta-analysis of microarray data and targeted proteomics for biomarker identification: application in breast cancer.

The development of signature biomarkers has gained considerable attention in the past decade. Although the most well-known examples of biomarker panels stem from gene expression studies, proteomic panels are becoming more relevant, with the advent of targeted mass spectrometry-based methodologies. At the same time, the development of multigene prognostic classifiers for early stage breast cancer patients has resulted in a wealth of publicly available gene expression data from thousands of breast cancer specimens. In the present study, we integrated transcriptome and proteome-based platforms to identify genes and proteins related to patient survival. Candidate biomarker proteins have been identified in a previously generated breast cancer tissue extract proteome. A mass-spectrometry-based assay was then developed for the simultaneous quantification of these 20 proteins in breast cancer tissue extracts. We quantified the relative expression levels of the 20 potential biomarkers in a cohort of 96 tissue samples from patients with early stage breast cancer. We identified two proteins, KPNA2 and CDK1, which showed potential to discriminate between estrogen receptor positive patients of high and low risk of disease recurrence. The role of these proteins in breast cancer prognosis warrants further investigation.

Growth and function in childhood of a normal solitary kidney from birth or from early infancy.

BACKGROUND: Children with a solitary kidney (SK) have an increased long-term risk of hypertension, albuminuria and glomerulosclerosis. In this study, we assessed the early signs of impaired glomerular filtration in children with a SK from birth or from early infancy. METHODS: Renal growth and function at ages 4-15.5 years were studied in 38 children with SK and 40 matched control subjects in terms of accelerated growth. RESULTS: The systolic/diastolic blood pressure Z-scores (p = 0.01/<0.05) and the resistance index (RI) of the arcuate arteries (p = 0.05) were higher in the children with SK. Creatinine clearance and 24-h protein and albumin urinary excretion showed no difference. All but seven children with SK had 99mTc diethylene-triamine pentaacetic acid glomerular filtration rate values of >80 ml/min/1.73 m(2). An independent positive correlation was found between length of the follow-up time and 24-h albumin urinary excretion (ß = 0.54, p < 0.01). Accelerated postnatal growth was positively related with kidney volume (ß = 0.35, p < 0.05). CONCLUSIONS: Among our patient cohort, renal function was well preserved at ages 4-15.5 years in children who were born with a SK. However, both their higher blood pressure and RI and the correlation of 24-h albumin urinary excretion with length of follow-up time underline the need for monitoring to detect early signs of glomerular hyperfiltration and, if necessary, implement timely intervention. SK hypertrophy was found to be correlated with postnatal growth.

Arterial hypertension during treatment with triptorelin in a child with Williams-Beuren syndrome.

BACKGROUND: Arterial hypertension (AHT) is a common finding in children with Williams-Beuren syndrome (WBS). Although cardiovascular and renal abnormalities can explain the AHT in some patients with WBS, its etiology is not fully understood and most cases are considered idiopathic. CASE-DIAGNOSIS/TREATMENT: The case is reported of a 10-year-old girl with WBS who developed severe AHT during treatment with triptorelin, a long-lasting gonadotropin-releasing hormone (GnRH) analog, administered because of early normal puberty. Comprehensive diagnostic studies ruled out other known causes of AHT associated with WBS. After discontinuation of triptorelin, the blood pressure remained within the normal range for her age and height with no antihypertensive treatment on long-term follow-up. To the best of the authors' knowledge, this is the first report of AHT associated with triptorelin administration in a child with WBS. CONCLUSIONS: Clinicians should be aware of the possibility, although rare, of AHT developing during triptorelin administration in childhood, specifically in patients at increased risk of AHT, such as those with WBS.

Quantitative analysis of energy metabolic pathways in MCF-7 breast cancer cells by selected reaction monitoring assay.

To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells.

Serum Neuron-Specific Enolase as a Biomarker of Neonatal Brain Injury-New Perspectives for the Identification of Preterm Neonates at High Risk for Severe Intraventricular Hemorrhage.

Neonatal brain injury (NBI) is a critical condition for preterm neonates with potential long-term adverse neurodevelopmental outcomes. This prospective longitudinal case-control study aimed at investigating the levels and prognostic value of serum neuron-specific enolase (NSE) during the first 3 days of life in preterm neonates (<34 weeks) that later developed brain injury in the form of either periventricular leukomalacia (PVL) or intraventricular hemorrhage (IVH) during their hospitalization. Participants were recruited from one neonatal intensive care unit, and on the basis of birth weight and gestational age, we matched each case (n = 29) with a neonate who had a normal head ultrasound scan (n = 29). We report that serum NSE levels during the first three days of life do not differ significantly between control and preterm neonates with NBI. Nevertheless, subgroup analysis revealed that neonates with IVH had significantly higher concentrations of serum NSE in comparison to controls and neonates with PVL on the third day of life (p = 0.014 and p = 0.033, respectively). The same pattern on the levels of NSE on the third day of life was also observed between (a) neonates with IVH and all other neonates (PVL and control; p = 0.003), (b) neonates with II-IV degree IVH and all other neonates (p = 0.003), and (c) between control and the five (n = 5) neonates that died from the case group (p = 0.023). We conclude that NSE could be an effective and useful biomarker on the third day of life for the identification of preterm neonates at high risk of developing severe forms of IVH.

Coupling proteomics and transcriptomics in the quest of subtype-specific proteins in breast cancer.

Breast-cancer subtypes present with distinct clinical characteristics. Therefore, characterization of subtype-specific proteins may augment the development of targeted therapies and prognostic biomarkers. To address this issue, MS-based secretome analysis of eight breast cancer cell lines, corresponding to the three main breast cancer subtypes was performed. More than 5200 non-redundant proteins were identified with 23, four, and four proteins identified uniquely in basal, HER2-neu-amplified, and luminal breast cancer cells, respectively. An in silico mRNA analysis using publicly available breast cancer tissue microarray data was carried out as a preliminary verification step. In particular, the expression profiles of 15 out of 28 proteins included in the microarray (from a total of 31 in our subtype-specific signature) showed significant correlation with estrogen receptor (ER) expression. A MS-based analysis of breast cancer tissues was undertaken to verify the results at the proteome level. Eighteen out of 31 proteins were quantified in the proteomes of ER-positive and ER-negative breast cancer tissues. Survival analysis using microarray data was performed to examine the prognostic potential of these selected candidates. Three proteins correlated with ER status at both mRNA and protein levels: ABAT, PDZK1, and PTX3, with the former showing significant prognostic potential.

The long journey of cancer biomarkers from the bench to the clinic.

BACKGROUND: Protein cancer biomarkers serve multiple clinical purposes, both early and late, during disease progression. The search for new and better biomarkers has become an integral component of contemporary cancer research. However, the number of new biomarkers cleared by the US Food and Drug Administration has declined substantially over the last 10 years, raising concerns regarding the efficiency of the biomarker-development pipeline. CONTENT: We describe different clinical uses of cancer biomarkers and their performance requirements. We also present examples of protein cancer biomarkers currently in clinical use and their limitations. The major barriers that candidate biomarkers need to overcome to reach the clinic are addressed. Finally, the long and arduous journey of a protein cancer biomarker from the bench to the clinic is outlined with an example. SUMMARY: The journey of a protein biomarker from the bench to the clinic is long and challenging. Every step needs to be meticulously planned and executed to succeed. The history of clinically useful biomarkers suggests that at least a decade is required for the transition of a marker from the bench to the bedside. Therefore, it may be too early to expect that the new technological advances will catalyze the anticipated biomarker revolution any time soon.