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Cell-penetrating peptides (CPPs), also known as protein transduction domains, were first identified 25 years ago. They are small, ~6-30 amino acid long, synthetic, or naturally occurring peptides, able to carry a variety of cargoes across the cellular membranes in an intact, functional form. These cargoes can range from other small peptides, full-length proteins, nucleic acids including RNA and DNA, nanoparticles, and viral particles as well as radioisotopes and other fluorescent probes for imaging purposes. However, this ability to enter all cell types indiscriminately, and even cross the blood-brain barrier, hinders their development into viable vectors. Hence, researchers have adopted various strategies ranging from pH activatable cargoes to using phage display to identify tissue-specific CPPs. Use of this phage display strategy has led to an ever-expanding number of tissue-specific CPPs. Using phage display, we identified a 12-amino acid, non-naturally occurring peptide that targets the heart with peak uptake at 15 min after a peripheral intravenous injection, that we termed Cardiac Targeting Peptide (CTP). In this chapter, we use CTP as an example to describe techniques for validation of cell-specific transduction as well as provide details on a technology to identify binding partner(s) for these ever-increasing plethora of tissue-specific peptides. Given the myriad cargoes CTP can deliver, as well as rapid uptake after an intravenous injection, it can be applied to deliver radioisotopes, miRNA, siRNA, peptides, and proteins of therapeutic potential for acute cardiac conditions like myocardial infarction, where the window of opportunity for salvaging at-risk myocardium is limited to 6 hrs.

Objective: The advent of ligand-based receptor capture methodologies, allows the identification of unknown receptor candidates for orphan extracellular ligands. However, further target validation can be tedious, laborious and time-consuming. Here, we present a methodology that provides a fast and cost-efficient alternative for candidate target verification on living cells., Results: In the described methodology a ligand of interest (e.g. transferrin, epidermal growth factor or insulin) was conjugated to a linker (TriCEPS) that carries a biotin. To confirm ligand/receptor interactions, the ligand-TriCEPS conjugates were first added onto living cells and cells were subsequently labeled with a streptavidin-fluorophore and analyzed by flow cytometry (thus referred as Flow-TriCEPS). Flow-TriCEPS was also used to validate identified receptor candidates when combined with a siRNA knock down approach (i.e. reduction of expression levels). This approach is versatile as it can be applied for different classes of ligands (proteins, peptides, antibodies) and different cell lines. Moreover, the method is time-efficient since it takes advantage of the large variety of commercially available (and certified) siRNAs.

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 (β-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 β-Crosslaps and the β-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 β-Crosslaps may reflect a beneficial response.

Neurodegenerative diseases are highly debilitating conditions characterised primarily by progressive neuronal loss and impairment of the nervous system. Parkinson's disease (PD) is one of the most common of these disorders, affecting 1-2% of the population above the age of 65. Although the underlying mechanisms of PD have been extensively studied, we still lack a full understanding of the molecular underpinnings of the disease. Thus, the in vitro and in vivo models currently used are able to only partially recapitulate the typical phenotypes of the disease. Here, we review various cell culture models currently used to study the molecular basis of PD, with a focus on alpha-synuclein-associated molecular pathologies. We also discuss how different cell models may constitute powerful tools for high-throughput screening of molecules capable of modulating alpha-synuclein toxicity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Alpha-synuclein is an abundant neuronal protein which has been associated with physiological processes like synaptic function, neurogenesis, and neuronal differentiation but also with pathological neurodegeneration. Indeed, alpha-synuclein (snca) is one of the major genes implicated in Parkinson's disease (PD). However, little is known about the regulation of alpha-synuclein expression. Unveiling the mechanisms that control its regulation is of high importance, as it will enable to further investigate and comprehend the physiological role of alpha-synuclein as well as its potential contribution in the aetiology of PD. Previously, we have shown that the protein TRIM32 regulates fate specification of neural stem cells. Here, we investigated the impact of TRIM32 on snca expression regulation in vitro and in vivo in neural stem cells and neurons. We demonstrated that TRIM32 is positively influencing snca expression in a neuronal cell line, while the absence of TRIM32 is causing deregulated levels of snca transcripts. Finally, we provided evidence that TRIM32 binds to the promoter region of snca, suggesting a novel mechanism of its transcriptional regulation. On the one hand, the presented data link the PD-associated gene alpha-synuclein to the neuronal cell fate determinant TRIM32 and thereby support the concept that PD is a neurodevelopmental disorder. On the other hand, they imply that defects in olfactory bulb adult neurogenesis might contribute to early PD-associated non-motor symptoms like hyposmia.

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.

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 17β-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., Competing Interests: The authors declare that they have no conflict of interest., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

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/β-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.

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.

Systemic mining of cancer exoproteome/secretome has emerged as a pivotal strategy for delineation of molecular pathways with mechanistic importance in cancer development, as well as the discovery of diagnostic/prognostic biomarkers. Although major advances in diagnostic and therapeutic management of colorectal cancer have been underscored in the last decade, this cancer still remains the second leading cause of cancer-related deaths in the developed world. Despite previous studies on deciphering the colorectal cancer exoproteome, such studies lack adequate depth and robustness due to technological limitations. Here, using a well-established LC-MS/MS method on an LTQ-Orbitrap mass spectrometer, we extensively delineated the exoproteome of 12 colon cancer cell lines. In total, 2979 non-redundant proteins were identified with a minimum of two peptides, of which ~62% were extracellular or cell membrane-bound, based on prediction software. To further characterize this dataset and identify clinical opportunities, first, we investigated overrepresented molecular concepts of interest via enrichment map analysis and second, we demonstrated translational importance of certain proteins, such as olfactomedin-4 and kallikrein-related peptidases-6 and -10, by investigating their expression levels in patient tissues and/or fluids. Overall, the present study details a comprehensive colorectal cancer exoproteome dataset, and may be used as future platform for biomarker discovery, and hypothesis-generating studies., Biological Significance: This article represents one of the most extensive and comprehensive proteomic datasets regarding the secreted/extracellular proteome of colorectal cancer cell lines. The reported datasets may form a platform for a plethora of future, discovery-based or hypothesis-generating studies, attempting to either delineate putative cancer biomarkers for CRC, or elucidate questions of mechanistic importance (e.g. investigation of deregulated pathways for CRC progression)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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 (β = 0.54, p < 0.01). Accelerated postnatal growth was positively related with kidney volume (β = 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.

Mir-17-5p and mir-20a, members of the mir-17-92 family, down-regulate E2F1, which is over-expressed in myelodysplastic syndromes (MDS). Moreover, let-7a down-regulates KRAS, which is aberrantly expressed in MDS. We evaluated the expression of the aforementioned microRNAs in CD34+ cells of 43 MDS patients using real-time PCR and their target proteins (E2F1, MYC, BCL2, CCND1, and KRAS) by Western blot. Mir-17-5p and mir-20a were under expressed in high risk MDS patients, compared to low risk MDS patients. Similarly, let-7a was under expressed in patients with intermediate or high-risk karyotype. Interestingly, there was an inverse correlation between microRNA and the expression levels of their targets. Importantly, mir-17-5p and mir-20a constitute favorable prognostic factors in MDS, since their expression was associated with increased overall survival of MDS patients., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

Emerging proteomic tools and mass spectrometry play pivotal roles in protein identification, quantification and characterization, even in complex biological samples. The cancer secretome, namely the whole collection of proteins secreted by cancer cells through various secretory pathways, has only recently been shown to have significant potential for diverse applications in oncoproteomics. For example, secreted proteins might represent putative tumor biomarkers or therapeutic targets for various types of cancer. Consequently, many proteomic strategies for secretome analysis have been extensively deployed over the last few years. These efforts generated a large amount of information awaiting deeper mining, better understanding and careful interpretation. Distinct sub-fields, such as degradomics, exosome proteomics and tumor-host cell interactions have been developed, in an attempt to provide certain answers to partially elucidated mechanisms of cancer pathobiology. In this review, advances, concerns and challenges in the field of secretome analysis as well as possible clinical applications are discussed., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Cancer is a leading cause of death. Early detection is usually associated with better clinical outcomes. Recent advances in genomics and proteomics raised hopes that new biomarkers for diagnosis, prognosis or monitoring therapeutic response will soon be discovered. Proteins secreted by cancer cells, referred also as "the cancer cell secretome", is a promising source for biomarker discovery. In this review we will summarize recent advances in cancer cell secretome analysis, focusing on the five most fatal cancers (lung, breast, prostate, colorectal, and pancreatic). For each cancer type we will describe the proteomic approaches utilized for the identification of novel biomarkers. Despite progress, identification of markers that are superior to those currently used has proven to be a difficult task and very few, if any, newly discovered biomarker has entered the clinic the last 10 years., (Copyright © 2010 Elsevier B.V. All rights reserved.)