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The Central and Eastern European Proteomic Conference (CEEPC), has reached a special milestone as it celebrates its 10th anniversary. Today, an expansive network of proteomics in Central and Eastern Europe stands established to facilitate scientific interactions and collaborations in and around Central and Eastern Europe, as well as with international research institutions worldwide. Currently, when many conferences are struggling to attract participants, CEEPC is thriving in its status and stature as well as expanding by attracting newer member countries. CEEPC's success is driven by mutual respect between scientists sharing interest in proteomics and its applications in multidisciplinary research areas related to biological systems. This effort when interwoven with exciting ambience steeped with culture, and tradition is also a reason why participants enjoy it. CEEPC's careful balance between excellence and cohesion holds the key to its success. It is evident that CEEPC is ready for the next decade of excitement and expectations of multifaceted proteomics in Central and Eastern Europe. Additionally, in the era of emerging personalized medicine where treatment selection for each patient is becoming individualized, CEEPC and proteomics is expected to play a significant role moving forward for the benefit of mankind.

As Romanians prepared to celebrate 100 years of the '‘Great Unification of 1918ʹ' which united all provinces into one Romania, the 12th Central and Eastern European Proteomic Conference (CE...

Studies on Huntington's disease (HD) demonstrated altered immune response in HD gene carriers. Using multiplexing immunoassay, we simultaneously investigated seven cytokines in secretomes of microglia and blood monocytes, cerebrospinal fluid (CSF) and serum collected from transgenic HD minipigs at pre-symptomatic disease stage. Decline in IFNα and IL-10 was observed in CSF and secretome of microglia whilst elevated IL-8 and IL-1β levels were secreted by microglia. Additionally, IL-8 was increased in serum. The proportion of mutant huntingtin in microglia may have causative impact on cytokine production. IFNα, IL-10, IL-8 and IL-1β represent promising biomarkers reflecting immuno-pathological mechanisms in porcine HD model.

Pluripotent stem cell-derived committed neural precursors are an important source of cells to treat neurodegenerative diseases including spinal cord injury. There remains an urgency to identify markers for monitoring of neural progenitor specificity, estimation of neural fate and follow-up correlation with therapeutic effect in preclinical studies using animal disease models. Cell surface capture technology was used to uncover the cell surface exposed N-glycoproteome of neural precursor cells upon neuronal differentiation as well as post-mitotic mature hNT neurons. The data presented depict an extensive study of surfaceome during neuronal differentiation, confirming glycosylation at a particular predicted site of many of the identified proteins. Quantitative changes detected in cell surface protein levels reveal a set of proteins that highlight the complexity of the neuronal differentiation process. Several of these proteins including the cell adhesion molecules ICAM1, CHL1, and astrotactin1 as well as LAMP1 were validated by SRM. Combination of immunofluorescence staining of ICAM1 and flow cytometry indicated a possible direction for future scrutiny of such proteins as targets for enrichment of the neuronal subpopulation from mixed cultures after differentiation of neural precursor cells. These surface proteins hold an important key for development of safe strategies in cell-replacement therapies of neuronal disorders. Biological significance Neural stem and/or precursor cells have a great potential for cell-replacement therapies of neuronal diseases. Availability of well characterised and expandable neural cell lineage specific populations is critical for addressing such a challenge. In our study we identified and relatively quantified several hundred surface N-glycoproteins in the course of neuronal differentiation. We further confirmed the abundant changes for several cell adhesion proteins by SRM and outlined a strategy for utilisation of such N-glycoproteins in antibody based cell sorting. The comprehensive dataset presented here demonstrates the molecular background of neuronal differentiation highly useful for development of new plasma membrane markers to identify and select neuronal subpopulation from mixed neural cell cultures.

Neurodegenerative diseases are devastating disorders and the demands on their treatment are set to rise in connection with higher disease incidence. Knowledge of the spatiotemporal profile of cellular protein expression during neural differentiation and definition of a set of markers highly specific for targeted neural populations is a key challenge. Intracellular proteins may be utilized as a readout for follow-up transplantation and cell surface proteins may facilitate isolation of the cell subpopulations, while secreted proteins could help unravel intercellular communication and immunomodulation. This review summarizes the potential of proteomics in revealing molecular mechanisms underlying neural differentiation of stem cells and presents novel candidate proteins of neural subpopulations, where understanding of their functionality may accelerate transition to cell replacement therapies.

Huntington's disease (HD) is the most common inherited neurodegenerative disorder among polyglutamine (polyQ) diseases caused by cytosine-adenine-guanine repeat expansion in exon 1 of the huntingtin gene whose translation results in polyQ stretch in the N-terminus of the huntingtin protein (HD protein). This mutation significantly affects huntingtin conformation, proteolysis, PTMs, as well as its ability to bind interacting proteins. As a consequence, a variety of cellular mechanisms such as transcription, mitochondrial energy metabolism, axonal transport, neuronal vulnerability to oxidative stress, neurotransmission, and immune response are altered and involved in the pathogenesis of HD. Promising candidate molecular biomarkers of HD have emerged from proteomic studies. Recent analyses focused on HD protein itself, its PTM, and interacting proteins, which are of great importance for disease course. Furthermore, brain, body fluids, and immune system are intensively studied in order to search for additional proteins with a view to their use as a biomarker(s) or set of biomarkers in clinical trials in HD translational research.

Drug resistance is the major obstacle to successful cancer therapy. Our study focuses on resistance to Aurora kinase inhibitors tested as anti-cancer drugs in clinical trials. We have used 2D electrophoresis in the pH ranges of 4-7 and 6-11 followed by protein identification using MALDI-TOF/TOF to compare the protein composition of HCT116 colon cancer cells either sensitive to CYC116 and ZM447439 inhibitors or resistant toward these drugs. The analysis also included p53(+/+) and p53(-/-) phenotypes of HCT116 cells. Our findings demonstrate that platelet-activating factor acetylhydrolase and GTP-binding nuclear protein Ran contribute to the development of resistance to ZM447439 only where resistance is related to p53. On the other hand, serine hydroxymethyltransferase was found to promote the tumor growth in cells resistant to CYC116 without the influence of p53. Computer modeling of interaction networks highlighted a direct link of the p53-independent mechanism of resistance to CYC116 with autophagy. Importantly, serine hydroxymethyltransferase, serpin B5, and calretinin represent the target proteins that may help overcome resistance in combination therapies. In addition, serpin B5 and calretinin appear to be candidate biomarkers that may be accessible in patients for monitoring of cancer therapy with ease.

A comprehensive proteome map of T-lymphoblastic leukemia cells and its alterations after daunorubicin, doxorubicin and mitoxantrone treatments was monitored and evaluated either by paired comparison with relevant untreated control and using multivariate classification of treated and untreated samples. With the main focus on early time intervals when the influence of apoptosis is minimized, we found significantly different levels of proteins, which corresponded to 1%–2% of the total amount of protein spots detected. According to Gene Ontology classification of biological processes, the highest representation of identified proteins for all three drugs belong to metabolic processes of proteins and nucleic acids and cellular processes, mainly cytoskeleton organisation and ubiquitin-proteasome pathway. Importantly, we observed significant proportion of changes in proteins involved in the generation of precursor metabolites and energy typical for daunorubicin, transport proteins participating in response to doxorubicin and a group of proteins of immune system characterising response to mitoxantrone. Both a paired comparison and the multivariate evaluation of quantitative data revealed daunorubicin as a distinct member of the group of anthracycline/anthracenedione drugs. A combination of identified drug specific protein changes, which may help to explain anti-cancer activity, together with the benefit of blocking activation of adaptive cancer pathways, presents important approaches to improving treatment outcomes in cancer.

Resistance to anti-cancer drugs is a well recognized problem and very often it is responsible for failure of the cancer treatment. In this study, the proteome alterations associated with the development of acquired resistance to cyclin-depedent kinases inhibitor bohemine, a promising anti-cancer drug, were analyzed with the primary aim of identifying potential targets of resistance within the cell that could pave a way to selective elimination of specific resistant cell types. A model of parental susceptible CEM T-lymphoblastic leukemia cells and its resistant counterpart CEM-BOH was used and advanced 2-D liquid chromatography was applied to fractionate cellular proteins. Differentially expressed identified proteins were further verified using immunoblotting and immunohistochemistry. Our study has revealed that Rho GDP-dissociation inhibitor 2, Y-box binding protein 1, and the HSP70/90 organizing protein have a critical role to play in resistance to cyclin-depedent kinases inhibitor. The results indicated not only that quantitative protein changes play an important role in drug-resistance, but also that there are various other parameters such as truncation, post-translational modification(s), and subcellular localization of selected proteins. Furthermore, these proteins were validated for their roles in drug resistance using different cell lines resistant to diverse representatives of anti-cancer drugs such as vincristine and daunorubicin.

The isolation of neural stem cells from fetal and adult mammalian CNS and the demonstration of functional neurogenesis in adult CNS have offered perspectives for treatment of many devastating hereditary and acquired neurological diseases. Due to this enormous potential, neural stem cells are a subject of extensive molecular profiling studies with a search for new markers and regulatory pathways governing their self-renewal as opposed to differentiation. Several in-depth proteomic studies have been conducted on primary or immortalized cultures of neural stem cells and neural progenitor cells, and yet more remains to be done. Additionally, neurons and glial cells have been obtained from embryonic stem cells and mesenchymal stem cells, and proteins associated with the differentiation process have been characterized to a certain degree with a view to further investigations. This review summarizes recent findings relevant to the proteomics of neural stem cells and discusses major proteins significantly regulated during neural stem cell differentiation with a view to their future use in cell-based regenerative and reparative therapy.

The in vitro co-culture models of communication between normal fibroblasts and epithelial cells, such as keratinocytes or squamous cell carcinoma cells of FaDu line representing wound healing or cancer development, were established by non-direct contact between the cells and utilised in this study to examine epithelia-induced changes in overall fibroblast proteome patterns.We were able to select the proteins co-regulated in both models in order to evaluate possible molecular commonalities between wound healing and tumour development. Amongst the most pronounced were the proteins implemented in contractile activity and formation of actin cytoskeleton such as caldesmon, calponin-2, myosin regulatory light-chain 12A and cofilin-1, which were expressed independently of the presence of α-smooth muscle actin. Additionally, proteins altered differently highlighted functional and cellular phenotypes during transition of fibroblasts towards myofibroblasts or cancer-associated fibroblasts. Results showed coordinated regulation of cytoskeleton proteins selective for wound healing which were lost in tumourigenesis model. Vimentin bridged this group of proteins with other regulated proteins in human fibroblasts involved in protein or RNA processing and metabolic regulation.The findings provide strong support for crucial role of stromal microenvironment in wound healing and tumourigenesis. In particular, epithelia-induced protein changes in fibroblasts offer new potential targets which may lead to novel tailored cancer therapeutic strategies.

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication and potentially life-threatening condition resulting from excessive ovarian stimulation during assisted reproductive technologies. Our aim was to identify candidate proteins in follicular fluid (FF) using various proteomic approaches which may help to identify patients at risk of OHSS. We analysed the proteome alterations in FF from patients suffering from severe forms of OHSS (OHSS+) compared with a control group of women without or with only mild signs of OHSS (OHSS-). The 12 abundant proteins of FF were removed using an immunoaffinity system. Pools of remaining depleted proteins were applied to the two-dimensional (2D) electrophoresis and 2D liquid chromatography and proteins in differentially expressed protein spots/fractions were identified by mass spectrometry. Among a total of 19 candidate proteins differentially expressed (P< 0.05) between OHSS+ and OHSS- FF samples, three proteins, namely ceruloplasmin, complement C3 and kininogen-1, were found using both 2D techniques. Computer modelling highlighted the important role of kininogen-1 as an anchor for mediated interactions with other identified proteins including ferritin light chain and ceruloplasmin, hepatocyte growth factor-like protein, as well as complement C3 and gelsolin, thus linking various biological processes including inflammation and angiogenesis, iron transport and storage, blood coagulation, innate immunity, cell adhesion and actin filament polymerization. The delineation of such processes may allow the development of informed corrective therapeutic intervention in patients at risk of OHSS and a set of key proteins of the FF may be helpful as potential biomarkers for monitoring IVF therapy.

Within a mammalian organism, the interaction among cells both at short and long distances is mediated by soluble factors released by cells into the extracellular environment. The secreted proteins may involve extracellular matrix proteins, proteinases, growth factors, protein hormones, immunoregulatory cytokines, chemokines or other bioactive molecules that have a direct impact on target cell phenotype. Stem cells of mesenchymal, adipose, neural and embryonic origin, fibroblast feeder cells as well as primary isolates of astrocytes, endothelial and muscle cells have recently become targets of intensive secretome profiling with the search for proteins regulating cell survival, proliferation, differentiation or inflammatory response. Recent advances and challenges of the stem cell and primary cell secretome analysis together with the most relevant results are discussed in this review.

In vitro fertilization (IVF) is fraught with problems and currently proteomics approaches are being tried out to examine the microenvironment of the follicle in order to assess biological and immunological parameters that may affect its development. Additionally, better understanding of reproductive process may help increase IVF birth rate per embryo transfer and at the same time avoid spontaneous miscarriages or life threatening conditions such as ovarian hyperstimulation syndrome. The primary aim of this study was to search for specific differences in protein composition of human follicular fluid (HFF) and plasma in order to identify proteins that accumulate or are absent in HFF. Depletion of abundant proteins combined with multidimensional protein fractionation allowed the study of middle- and lower-abundance proteins. Paired comparison study examining HFF with plasma/serum from women undergoing successful IVF revealed important differences in the protein composition which may improve our knowledge of the follicular microenvironment and its biological role. This study showed involvement of innate immune function of complement cascade in HFF. Complement inhibition and the presence of C-terminal fragment of perlecan suggested possible links to angiogenesis which is a vital process in folliculogenesis and placental development. Differences in proteins associated with blood coagulation were also found in the follicular milieu. Several specific proteins were observed, many of which have not yet been associated with follicle/oocyte maturation. These proteins together with their regulatory pathways may play a vital role in the reproductive process.

The evolution of sexual reproduction remains a mystery as well as a fascination for many clinicians and researchers. While some of the benefits of evolutionary reproduction, such as an improved qua...