Linking aberrant glycosylation of plasma glycoproteins with progression of myelodysplastic syndromes: a study based on plasmonic biosensor and lectin array.

Aberrant glycosylation of glycoproteins has been linked with various pathologies. Therefore, understanding the relationship between aberrant glycosylation patterns and the onset and progression of the disease is an important research goal that may provide insights into cancer diagnosis and new therapy development. In this study, we use a surface plasmon resonance imaging biosensor and a lectin array to investigate aberrant glycosylation patterns associated with oncohematological disease-myelodysplastic syndromes (MDS). In particular, we detected the interaction between the lectins and glycoproteins present in the blood plasma of patients (three MDS subgroups with different risks of progression to acute myeloid leukemia (AML) and AML patients) and healthy controls. The interaction with lectins from Aleuria aurantia (AAL) and Erythrina cristagalli was more pronounced for plasma samples of the MDS and AML patients, and there was a significant difference between the sensor response to the interaction of AAL with blood plasma from low and medium-risk MDS patients and healthy controls. Our data also suggest that progression from MDS to AML is accompanied by sialylation of glycoproteins and increased levels of truncated O-glycans and that the number of lectins that allow discriminating different stages of disease increases as the disease progresses.

Targeted mass spectrometry for monitoring of neural differentiation.

Human multipotent neural stem cells could effectively be used for the treatment of a variety of neurological disorders. However, a defining signature of neural stem cell lines that would be expandable, non-tumorigenic, and differentiate into desirable neuronal/glial phenotype after in vivo grafting is not yet defined. Employing a mass spectrometry approach, based on selected reaction monitoring, we tested a panel of well-described culture conditions, and measured levels of protein markers routinely used to probe neural differentiation, i.e. POU5F1 (OCT4), SOX2, NES, DCX, TUBB3, MAP2, S100B, GFAP, GALC, and OLIG1. Our multiplexed assay enabled us to simultaneously identify the presence of pluripotent, multipotent, and lineage-committed neural cells, thus representing a powerful tool to optimize novel and highly specific propagation and differentiation protocols. The multiplexing capacity of this method permits the addition of other newly identified cell type-specific markers to further increase the specificity and quantitative accuracy in detecting targeted cell populations. Such an expandable assay may gain the advantage over traditional antibody-based assays, and represents a method of choice for quality control of neural stem cell lines intended for clinical use.

Anaerobic microbial corrosion of carbon steel under conditions relevant for deep geological repository of nuclear waste.

We examined microbial corrosion of carbon steel in synthetic bentonite pore water inoculated with natural underground water containing microorganisms over a period of 780-days under sterile and anaerobic conditions. Corrosion behaviour was determined using the mass loss method, SEM-EDS analysis and Raman spectroscopy, while qualitative and quantitative changes in the microbial community were analysed using molecular-biological tools (16S rDNA amplicon sequencing and qPCR analysis, respectively). Corrosion rates were significantly higher in the biotic environment (compared with an abiotic environment), with significant localisation of corrosion attacks of up to 1 mm arising within 12-months. Nitrate reducing bacteria, such as Pseudomonas, Brevundimonas and Methyloversatilis, dominated the microbial consortium, the high abundance of Methyloversatilis correlating with periods of highest localised corrosion penetrations, suggesting that this bacterium plays an important role in microbially influenced corrosion. Our results indicate that nitrate-reducing bacteria could represent a potential threat to waste canisters under nuclear repository conditions.

Power of proteomics and progress in precision medicine - 13th central and eastern European proteomic conference (CEEPC), Ustron, Poland.

INTRODUCTION: Central and Eastern European Proteomic Conference (CEEPC) provides a platform for researchers to discuss multi-disciplinary integrated approaches to address a range of challenges from present day viral pandemic to on-going progress in Precision Medicine. CEEPC brings together various multi-omics entwined with novel enabling technologies, thus facilitating conceptual advances from cell to society for the benefit of mankind. AREAS COVERED: Proteomic methodologies, databases and software has revolutionized our ability to assess protein interactions and cellular changes, allowing the establishment of biological connections and identification of important cellular regulatory proteins and pathways previously unknown or not fully understood. Additionally, Mass spectrometry (MS) remains a major driving force in the field of 'multi-omics' and a powerful technology for the structural characterization of biomolecules and for analysis of proteins and small molecules such as lipids, sugars and metabolites. Combination of measurements from proteomics, genomics, epigenomics, transcriptomics and metabolomics, present a powerful decision-making format allowing deeper interpretation of a disease scenario in Precision medicine. EXPERT COMMENTARY: Precision Medicine offers novel and promising ways to identify and treat a wide range of diseases. The future success of these therapies will be underpinned by novel proteo-genomic approaches linked to sophisticated databases to evaluate and predict drug-patient interactions.

Serum proteomic analysis of melanoma patients with immunohistochemical profiling of primary melanomas and cultured cells: Pilot study.

The steadily increasing incidence of malignant melanoma (MM) and its aggressive behaviour makes this tumour an attractive cancer research topic. The tumour microenvironment is being increasingly recognised as a key factor in cancer biology, with an impact on proliferation, invasion, angiogenesis and metastatic spread, as well as acquired therapy resistance. Multiple bioactive molecules playing cooperative roles promote the chronic inflammatory milieu in tumours, making inflammation a hallmark of cancer. This specific inflammatory setting is evident in the affected tissue. However, certain mediators can leak into the systemic circulation and affect the whole organism. The present study analysed the complex inflammatory response in the sera of patients with MM of various stages. Multiplexed proteomic analysis (Luminex Corporation) of 31 serum proteins was employed. These targets were observed in immunohistochemical profiles of primary tumours from the same patients. Furthermore, these proteins were analysed in MM cell lines and the principal cell population of the melanoma microenvironment, cancer­associated fibroblasts. Growth factors such as hepatocyte growth factor, granulocyte­colony stimulating factor and vascular endothelial growth factor, chemokines RANTES and interleukin (IL)­8, and cytokines IL­6, interferon­α and IL­1 receptor antagonist significantly differed in these patients compared with the healthy controls. Taken together, the results presented here depict the inflammatory landscape that is altered in melanoma patients, and highlight potentially relevant targets for therapy improvement.

Driving Precision Medicine through Proteomics and Metabolomics - 12th Central and Eastern European Proteomic Conference (CEEPC), Bucharest, Romania.

As Romanians prepared to celebrate 100 years of the ''Great Unification of 1918'' which united all provinces into one Romania, the 12th Central and Eastern European Proteomic Conference (CEEPC) jointly with the 39th Anniversary of the Institute of Cellular Biology and Pathology ''N. Simionescu'' (ICBP-NS), held their inaugural meeting at the Romanian Academy in Bucharest - a national forum of highest scientific recognition. With an exciting theme entitled, 'Advances in Proteomics and Progress in Precision Medicine', delegates gathered to debate Precision medicine's revolution in diagnosis and treatment, which now accounts for predictive, preventative, and targeted treatment strategies with informed decisions according to individual's unique clinical, molecular and genetic profile. Proteomics has a pivotal role to play in furthering precision health and medicine for the benefit of mankind. To this end, CEEPC continues to drive advances in proteomics, metabolomics, and diseases as well as raising awareness of pressing global humanitarian and health-care issues including mental health diseases, aging, chronic diseases, global epidemics and environmental issues. Today, CEEPC is a well-recognized major annual conference with a focused vision and a highly valued ideology as it continues to propagate scientific, medical and proteomic collaborations whilst expanding as more Eastern European countries prepare to join.

AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington's Disease Minipig Model.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Previously, we showed strong huntingtin reduction and prevention of neuronal dysfunction in HD rodents using an engineered microRNA targeting human huntingtin, delivered via adeno-associated virus (AAV) serotype 5 vector with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT). One of the challenges of rodents as a model of neurodegenerative diseases is their relatively small brain, making successful translation to the HD patient difficult. This is particularly relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Here, we aimed to demonstrate the translation of huntingtin-lowering gene therapy to a large-animal brain. We investigated the feasibility, efficacy, and tolerability of one-time intracranial administration of AAV5-miHTT in the transgenic HD (tgHD) minipig model. We detected widespread dose-dependent distribution of AAV5-miHTT throughout the tgHD minipig brain that correlated with the engineered microRNA expression. Both human mutant huntingtin mRNA and protein were significantly reduced in all brain regions transduced by AAV5-miHTT. The combination of widespread vector distribution and extensive huntingtin lowering observed with AAV5-miHTT supports the translation of a huntingtin-lowering gene therapy for HD from preclinical studies into the clinic.

Pursuit of proteomic excellence and the excitement in Kosice, Slovakia, at the 11th Central and Eastern European Proteomic Conference (CEEPC).

The Central and Eastern European Proteomic Conference (CEEPC) successfully launched its second decade of proteomics in Kosice, Slovakia with a program of systems biology, cellular, clinical, veterinary and sports proteomics. Whilst many conferences are struggling to attract participants, CEEPC with its outstanding track record and unique 'family - feel' packaged with excellent ambiance is thriving and bringing together proteomics experts from academia, industry, scientific specialties, clinics and precision medicine communities interested in resolving mysteries about protein functionalities in health and disease. CEEPC is also renowned for addressing humanitarian global healthcare issues, may it be ageing, chronic diseases or global epidemics. This year CEEPC intertwined with Kosice Peace Marathon's pursuit of excellence in sports and initiatives including sports medicine and global peace.

A decade of proteomics accomplished! Central and Eastern European Proteomic Conference (CEEPC) celebrates its 10th Anniversary in Budapest, Hungary.

The Central and Eastern European Proteomic Conference (CEEPC) proudly celebrated its 10th Anniversary with an exciting scientific program inclusive of proteome, proteomics and systems biology in Budapest, Hungary. Since 2007, CEEPC has represented 'state-of the-art' proteomics in and around Central and Eastern Europe and these series of conferences have become a well-recognized event in the proteomic calendar. Fresher challenges and global healthcare issues such as ageing and chronic diseases are driving clinical and scientific research towards regenerative, reparative and personalized medicine. To this end, proteomics may enable diverse intertwining research fields to reach their end goals. CEEPC will endeavor to facilitate these goals.

A decade of Central and Eastern European Proteomic Conference (CEEPC): Credibility, cohesion and vision for the next decade.

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.

Revelation of the IFNα, IL-10, IL-8 and IL-1ß as promising biomarkers reflecting immuno-pathological mechanisms in porcine Huntington's disease model.

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-1ß 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-1ß represent promising biomarkers reflecting immuno-pathological mechanisms in porcine HD model.

Proteomic landscape in Central and Eastern Europe: the 9th Central and Eastern European Proteomic Conference, Poznan, Poland.

Every year since 2007, the Central and Eastern European Proteomic Conference (CEEPC) has excelled in representing state-of-the-art proteomics in and around Central and Eastern Europe, and linking it to international institutions worldwide. Its mission remains to contribute to all approaches of proteomics including traditional and often-revisited methodologies as well as the latest technological achievements in clinical, quantitative and structural proteomics with a view to systems biology of a variety of processes. The 9th CEEPC was held from June 15th to 18th, 2015, at the Institute of Bioorganic Chemistry, Polish Academy of Sciences in Poznan, Poland. The scientific program stimulated exchange of proteomic knowledge whilst the spectacular venue of the conference allowed participants to enjoy the cobblestoned historical city of Poznan.

Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation.

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.

Multiplex immunoassays for quantification of cytokines, growth factors, and other proteins in stem cell communication.

Immunoassays represent valuable and broadly used techniques for detection and quantification of proteins. Thanks to their high sensitivity, such techniques are powerful for analyzing growth factors, trophic factors, angiogenic factors, hormones, cytokines, chemokines, soluble receptors, and other proteins which play key roles in intercellular communication and operate as potent regulators of stem cell survival, proliferation, differentiation, or cell death. Multiplex immunological assays, in contrast to ELISA, offer simultaneous quantification of tens of proteins across multiple samples, and have been developed to save time, costs, and sample volumes. Among them, planar antibody microarrays and xMAP(®) bead-based assays have become particularly popular for characterization of proteins secreted by stem cells, as they are relatively easy, highly accurate, multiplex to a high degree and a broad spectrum of analytes can be measured. Here, we describe protocols for multiplex quantification of secreted proteins using Quantibody(®) microarrays (RayBiotech) and xMAP(®) assays (Luminex and its partners).

Challenges of Huntington's disease and quest for therapeutic biomarkers.

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.

Proteome-wide analysis of neural stem cell differentiation to facilitate transition to cell replacement therapies.

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.

Tremendous progress in proteomics and metabolomics in Central and Eastern Europe.

The ever expanding Central and Eastern European Proteomic Conference (CEEPC) hosted its 8th annual meeting in Vienna, Austria, in July 2014 with resounding success, highlights of which are shared in this report. Tremendous progress in proteomics over the past decade in Central and Eastern Europe continues to rapidly accelerate due to networking across borders as well as access to sophisticated technologies. As the popularity of targeted proteomics in pathogenesis grows to unravel the complexities, so does the use of advanced analytical instrumentation. In addition, development of more sensitive research methodologies and a massive integration of technologies now give optimism to gain better understanding of the structure, functions and isoforms of various proteins as well as their complex interactions in biological systems. This, together with the confidence to qualitatively and/or quantitatively interrogate proteins of interest has led to promising developments for the identification of potential new drug targets for the treatment of various diseases.

Proteomics without boundaries across Central and Eastern Europe.

The 7th Central and Eastern European Proteomic Conference (CEEPC), considered as the bedrock of proteomics in Central and Eastern Europe, was held on 13­16 October 2013 at the Max Planck Institute for Chemical Ecology in Jena, Germany. Established in 2007, CEEPC now represents a cradle of proteomic interactions in and around Central and Eastern Europe, without limitations of borders and linking it to international institutions worldwide. Its mission remains to contribute to all approaches of proteomics including clinical, quantitative and structural proteomics and with a view to identifying potential targets for therapeutic interventions. The 7th CEEPC excelled at stimulating exchange of proteomic knowledge and imbibing local hospitality offered by Jena with its limestone hills and exotic charm.

Revelation of fibroblast protein commonalities and differences and their possible roles in wound healing and tumourigenesis using co-culture models of cells.

BACKGROUND INFORMATION: 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. RESULTS: 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. CONCLUSIONS: 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.