Use of halophytes in pilot-scale horizontal flow constructed wetland treating domestic wastewater.

Recent findings encourage the use of halophytes in constructed wetlands for domestic wastewater treatment due to their special physiological characteristics as the ability to accumulate heavy metals and salts in their tissues makes them ideal candidates for constructed wetland vegetation. In this particular study, we investigated the application of halophytic plants in a horizontal flow constructed wetland for domestic wastewater treatment purposes. The pilot plant which was situated in Crete (Greece) was planted with a polyculture of halophytes (Tamarix parviflora, Juncus acutus, Sarcocornia perrenis, and Limoniastrum monopetalum). The system's performance was monitored for a period of 11 months during which it received primary treated wastewater from the local wastewater treatment plant. Results show that halophytes developed successfully in the constructed wetland and achieved organic matter and pathogen removal efficiencies comparable to those reported for reeds in previous works (63% and 1.6 log units, respectively). In addition, boron concentration in the effluent was reduced by 40% in comparison with the influent. Salinity as expressed by electrical conductivity did not change during the treatment, indicating that the accumulation of salts in the leaves is not able to overcome electrical conductivity increasing due to evapotranspiration. The results indicate an improvement in the treatment of domestic wastewater via the use of halophyte-planted CWs.

The enhanced value of combining conventional and "omics" analyses in early assessment of drug-induced hepatobiliary injury.

The InnoMed PredTox consortium was formed to evaluate whether conventional preclinical safety assessment can be significantly enhanced by incorporation of molecular profiling ("omics") technologies. In short-term toxicological studies in rats, transcriptomics, proteomics and metabolomics data were collected and analyzed in relation to routine clinical chemistry and histopathology. Four of the sixteen hepato- and/or nephrotoxicants given to rats for 1, 3, or 14days at two dose levels induced similar histopathological effects. These were characterized by bile duct necrosis and hyperplasia and/or increased bilirubin and cholestasis, in addition to hepatocyte necrosis and regeneration, hepatocyte hypertrophy, and hepatic inflammation. Combined analysis of liver transcriptomics data from these studies revealed common gene expression changes which allowed the development of a potential sequence of events on a mechanistic level in accordance with classical endpoint observations. This included genes implicated in early stress responses, regenerative processes, inflammation with inflammatory cell immigration, fibrotic processes, and cholestasis encompassing deregulation of certain membrane transporters. Furthermore, a preliminary classification analysis using transcriptomics data suggested that prediction of cholestasis may be possible based on gene expression changes seen at earlier time-points. Targeted bile acid analysis, based on LC-MS metabonomics data demonstrating increased levels of conjugated or unconjugated bile acids in response to individual compounds, did not provide earlier detection of toxicity as compared to conventional parameters, but may allow distinction of different types of hepatobiliary toxicity. Overall, liver transcriptomics data delivered mechanistic and molecular details in addition to the classical endpoint observations which were further enhanced by targeted bile acid analysis using LC/MS metabonomics.

Potential biomarkers for Turner in maternal plasma: possibility for noninvasive prenatal diagnosis.

Turner syndrome (TS) is the most common sex chromosome abnormality in females, caused by the complete or partial absence of one X chromosome. To identify biomarkers for TS, we compared the protein composition of maternal plasma samples from pregnant women with normal and TS fetuses, using a proteomic approach consisting of 2D-E separation and MS analysis for the identification of the differentially expressed proteins. Samples were routinely obtained in the second trimester of pregnancy, stored, and used after prenatal determination of the fetal karyotype. Nine proteins (C1S, CO3, CLUS, AFAM, HABP2, IGHA1, HPT, SHBG, and CD5L) were significantly increased in the plasma of women carrying TS fetuses, whereas KNG1, IGJ, and TTHY were decreased. Identified proteins were further evaluated by immunoblot analysis while functional network association was carried out to asses significance. The identification of specific biomarkers may facilitate the development of noninvasive prenatal diagnosis and improve our understanding of the pathology of TS. Nevertheless, testing a larger cohort of pregnant women is necessary to evaluate the relevance of the reported findings.

Proteomic analysis of amniotic fluid in pregnancies with Klinefelter syndrome foetuses.

Klinefelter syndrome is a sex chromosomal abnormality (47, XXY karyotype), occurring approximately in 1 in 1000 male live births. In the present study proteomic analysis was performed in twelve 2nd trimester amniotic fluid samples, eight coming from pregnancies with normal males and four with Klinefelter syndrome foetuses, as shown by routine prenatal cytogenetic analysis. Samples were analysed by 2-DE, coupled with MALDI-TOF-MS analysis. Three proteins (Ceruloplasmin, Alpha-1-antitrypsin and Zinc-alpha-2-glycoprotein) were found to be up-regulated in samples obtained from pregnancies with Klinefelter syndrome foetuses, whereas four proteins (Apolipoprotein A-I, Plasma retinol-binding protein, Gelsolin, and Vitamin D-binding protein) were down regulated when compared to proteins detected in samples from normal foetuses. The differential expression of Ceruloplasmin, Apolipoprotein A-I and Plasma retinol-binding protein was further confirmed by immunoblotting. Since these proteins are likely to cross the placenta barrier and be detected in maternal plasma they could be used as biomarkers for the non-invasive prenatal diagnosis of Klinefelter syndrome.

BDNF-induced changes in the expression of the translation machinery in hippocampal neurons: protein levels and dendritic mRNA.

BDNF plays a key role in neuronal development, in short- and long-term changes in synaptic activity, and in neuronal survival. These effects are mediated, to a great extent, by changes in protein synthesis. We conducted a gel-based proteome profiling of the long-term (12 h) effects of BDNF in cultured hippocampal neurons. BDNF changed the abundance of proteins involved in (i) Nucleobase, nucleoside, nucleotide and nucleic acid metabolism, (ii) protein metabolism, (iii) carbohydrate metabolism, (iv) regulators of apoptosis, and (v) regulators of cell proliferation. A large majority of the identified proteins involved in translation activity were upregulated, but not all changes in the protein content were correlated with alterations in the corresponding mRNA. The upregulation of Seryl-aminoacyl-tRNA-synthetase and Eef2 was sensitive to the mTOR inhibitor rapamycin, as determined by Western blot. Since the mRNAs for proteins involved in translation represent a large fraction of the diversity of dendritic mRNAs, we investigated the effect of BDNF on the distribution of the transcripts in the soma versus neurite compartments. The increase in mRNA for proteins of the translation machinery in the soma was differentially coupled with the upregulation of neurite transcripts. BDNF also downregulated specific mRNAs in neurite compartments suggesting that the neurotrophin may act by regulating mRNA stability and thereby affecting the dendritic protein content.

Down-regulation of microfilamental network-associated proteins in leukocytes of breast cancer patients: potential application to predictive diagnosis.

BACKGROUND: Breast cancer is one of the most frequent tumors worldwide. Currently applied diagnostic approaches are frequently not able to recognize early stages in tumor development therefore impairing outcomes. The focus of this study is the creation of a non-invasive predictive diagnostic approach by pathology-specific blood proteome analysis. PATIENTS AND METHODS: Circulating leukocytes were isolated from fresh blood samples of breast cancer patients, benign breast pathologies and healthy controls. In patients with all kinds of breast pathologies, blood samples were taken before core needle biopsy of the lump. Comparative protein mapping was performed by 2D-PAGE followed by MALDI-TOF analysis and Western-blot quantification of differentially expressed protein spots. RESULTS: By protein mapping, 64 protein spots were identified. Pathology-specific differential expression patterns comprised microfilamental network-associated proteins: Calgranulin A (S100), LyGDI (Rho GDIbeta), RhoA and profilin 1. RhoA and profilin values discriminated between healthy controls and patients with all breast pathologies. CONCLUSION: Microfilamental network-associated proteins are involved in the regulation of a variety of central cellular processes functionally linked with each other and known to be highly relevant for all stages of tumorigenesis including precancerous lesions and metastases. Pathology-related molecular patterns are currently considered for the creation of a novel highly sensitive minimally-invasive approach for predictive diagnosis of breast cancer.

Improved enrichment and proteomic analysis of brain proteins with signaling function by heparin chromatography.

Detection of low-abundance proteins with signaling function is essential for the identification of biomarkers and novel drug targets. We present a protocol for specific enrichment of secreted proteins with signaling function by combining subcellular fractionation with heparin chromatography. The subcellular fractionation includes the preparation of a fraction enriched in cytosolic proteins. A further enrichment was achieved by heparin chromatography. The proteins eluted from the heparin column were analyzed by MudPIT tandem mass spectrometry and identified with the use of an in silico algorithm. Forty-eight percent of the identified proteins (188 out of 391) bound to the heparin matrix. Fifty-four percent of them (101) are secreted proteins with signaling function and 23% (44) of the enriched signaling proteins had not been detected by 2D PAGE without application of the heparin enrichment step. The heparin chromatography method can be combined with other proteomics enrichment approaches, such as ion exchange or reversed phase chromatography.

Potential involvement of MYC- and p53-related pathways in tumorigenesis in human oral squamous cell carcinoma revealed by proteomic analysis.

Oral squamous cellular carcinoma is a malignant tumor with poor prognosis. Discovery of early markers to discriminate between malignant and normal cells is of high importance in clinical diagnosis. Subcellular fractions from 10 oral squamous cell carcinoma and corresponding control samples, enriched in mitochondrial and cytosolic proteins, as well as blood from the tumor were analyzed by proteomics, two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Three-hundred and fifty different gene products were identified. Twenty proteins showed deranged levels in oral squamous cell carcinoma in comparison with the control samples and are potentially involved in tumor growth and metastasis. Of these, 16 proteins were upregulated. By applying pathway analysis, we found 8 of the upregulated gene products to be linked to three main locus genes, p53, MYC, and MYCN, and could be candidate biomarkers for OSCC. The findings of this pilot study show that OSCC gene ontology combined with proteomic analysis is a powerful tool in systems biology for the elucidation of the complexity of expression profiles in cellular processes. Application of such pathway analysis has the potential to generate new insights into complex molecular mechanisms underlying disease related processes and could therefore significantly contribute to the efficient performance of the entire discovery process.

Protein fractionation by preparative electrophoresis.

Preparative electrophoresis is a protein fractionation approach useful for the enrichment of low-abundance gene products. Preparative electrophoresis is usually performed in the PrepCell apparatus. Proteins are separated according to their size in a cylindrical gel in the presence of an ionic detergent. The method is particularly efficient for the enrichment of low-molecular-mass gene products. Preparative electrophoresis can be followed by proteomic analysis, and the proteins eluted from the preparative gel can be separated by two-dimensional gel electrophoresis and identified by mass spectrometry.

Proteomic analysis of amniotic fluid in pregnancies with Turner syndrome fetuses.

Turner syndrome, occurring in 1:2500 female births, is caused by the complete or partial absence of one X chromosome. Amniotic fluid supernatant proteins from five second trimester pregnancies with Turner syndrome fetuses and five normal ones were analyzed by 2DE, MALDI-TOF-MS, and Western blot. Serotransferin, lumican, plasma retinol-binding protein, and apolipoprotein A-I were increased in Turner syndrome, while kininogen, prothrombin, and apolipoprotein A-IV were decreased. Since differentially expressed proteins are likely to cross the placenta barrier and be detected in maternal plasma, proteomic analysis may enhance research for noninvasive prenatal diagnosis of Turner syndrome.

Marked defects in the expression and glycosylation of alpha2-HS glycoprotein/fetuin-A in plasma from neonates with intrauterine growth restriction: proteomics screening and potential clinical implications.

Intrauterine growth restriction (IUGR) has been associated with increased perinatal morbidity and mortality and increased morbidity and metabolic abnormalities later in life. IUGR is characterized as the failure of a fetus to achieve his or her genetic growth potential in utero. Altered protein expression profiles associated with IUGR may be informative on the pathologic mechanisms of this condition and might reveal potential markers for postnatal complications. The aim of this study was to compare protein profiles of umbilical cord plasma from IUGR and appropriate for gestational age full-term neonates. Blood samples from doubly clamped umbilical cord at delivery from 10 IUGR and 10 appropriate for gestational age full-term neonates were analyzed by two-dimensional electrophoresis and MS. Prominent changes of the alpha2-HS glycoprotein/fetuin-A were observed in IUGR cases. Specifically we showed that these changes occur primarily at the level of post-translational modifications of the protein. Using a combination of mass spectrometry and classical biochemical assays, single and heavy chain forms of fetuin-A were found to lack the normally present O-linked sialic acids in IUGR neonates. Fetuin A is a glycoprotein that has been associated with promotion of in vitro cell replication, fetal growth and osteogenesis, and protection from Gram-negative bacterial endotoxins. Prominent defects in glycosylation/sialylation of fetuin-A revealed by our study might be responsible for impaired function of fetuin-A, leading to deficient fetal growth, especially osteogenesis, and/or to the development of complications frequently seen later in the lives of IUGR neonates.

The normal human amniotic fluid supernatant proteome.

Proteomic analysis combining two-dimentional electrophoresis (2DE) and mass spectrometry (MS) has the potential for a wide range of applications in biological and medical sciences, as protein screening in tissues obtained from healthy and diseased conditions can determine drug targets and diagnostic markers. Conventionally, amniotic fluid (AF) samples are routinely used for prenatal diagnosis of a wide range of fetal abnormalities. Proteomics have already been applied in the analysis of tissues from fetuses with Down's syndrome, in order to detect differences in their protein profile as compared to the normal profiles and to determine possible diagnostic tools. A detailed protein 2DE for the normal human AF has not been reported. In the present study, the 2D protein database of the normal human AF supernatant (AFS) was constructed. Ten AFS samples from women carrying normal fetuses were analysed by 2DE. A mean of 412 spots per gel were analyzed and protein identification was carried out by MALDI-MS and MALDI-MS-MS. A 2D protein map comprising of 136 different gene products was constructed. The majority of the identified proteins are regulatory proteins, enzymes, secreted proteins, carriers and immunoglobulins. Twelve hypothetical proteins were also included. The normal AFS proteome map is a valuable tool for the study of aberrant protein expression and the search for proteins as possible markers for the prediction of abnormal fetuses.

HUPO Brain Proteome Project: summary of the pilot phase and introduction of a comprehensive data reprocessing strategy.

The Human Proteome Organisation (HUPO) initiated several projects focusing on the proteome analysis of distinct human organs. The Brain Proteome Project (BPP) is the initiative dedicated to the brain, its development and correlated diseases. Two pilot studies have been performed aiming at the comparison of techniques, laboratories and approaches. With the help of the results gained, objective data submission, storage and reprocessing workflow have been established. The biological relevance of the data will be drawn from the inter-laboratory comparisons as well as from the re-calculation of all data sets submitted by the different groups. In the following, results of the single groups as well as the centralised reprocessing effort will be summarised and compared, showing the added value of this concerted work.

Proteomic analysis of amniotic fluid in pregnancies with Down syndrome.

Proteomic analysis is widely used for the detection of diagnostic markers. In the present study amniotic fluid supernatants (AFS) from pregnancies with Down syndrome (DS) fetuses and from chromosomally normal fetuses in the 17th week of gestation were analyzed by 2-DE. Gel comparison revealed significant differences in the two groups. Spots with different expression levels were excised and proteins were identified by MALDI-MS and nano-ESI-MS/MS. Splicing factor arginine/serine-rich 4 (SFRS4; Q08170) was present only in AFS from DS fetuses and completely absent in the control group. Quantitative differences were detected for alpha-1-microglobulin (AMBP; P02760), collagen alpha 1 (I) chain (CO1A1; P02452), collagen alpha 1 (III) chain (CO3A1; P02461), collagen alpha 1 (V) chain d (CO5A1; P20908), and basement membrane-specific heparin sulfate proteoglycan core protein (PGBM; P98160). These proteins were increased in cases with DS, whereas protein IBP-1 (P08833) was decreased by 40% compared with chromosomally normal fetuses. Four proteins, CO1A1, CO3A1, CO5A1, and PGBM, appeared as fragments. As differentially expressed proteins were present in all pregnancies with DS tested, they may represent useful potential markers for prenatal diagnosis. However, for protein biomarkers to be of any clinical utility, systematic analysis of the maternal serum should be conducted.

Characterization of the human urine proteome by preparative electrophoresis in combination with 2-DE.

The protein components of urine are useful indicators of renal function and human health in general. Urine samples are easily attainable making them ideal substrates for biomarker research. Analysis of the urine proteome however, has been hindered by the great variability of the urine specimens, and the presence of various proteins in low abundance or modified forms. To alleviate some of these problems urine samples from five different individuals were pooled, concentrated and the proteome characterized by a combination of preparative electrophoresis and 2-DE, followed by PMF. A total of 778 protein spots corresponding to 141 different gene products were identified. In comparison, 171 spots corresponding to 44 unique proteins were identified in the unfractionated starting material. Among the proteins identified from the preparative electrophoresis were many of low abundance such as proteins involved in signal transduction. Furthermore, the median molecular mass of the identified proteins from the preparative electrophoresis was significantly lower in comparison to the proteins identified from the unfractionated starting material (39 886 Da versus 71 317 Da, respectively). Concluding, application of this methodology provides a coherent analysis of the urine proteome and contributes to the generation of the urine protein map in health and disease.

Pro-invasive gene regulating effect of irradiation and combined temozolomide-radiation treatment on surviving human malignant glioma cells.

The current chemotherapeutic treatment of glioblastoma patients has minor success. Little is known about the molecular and cellular mechanisms of the resistance of gliomas towards current therapies. This study investigated both suppressive cellular effects and regulation of extracellular matrix remodeling proteins with pro-invasive activity in surviving human glioblastoma cells under clinically relevant treatments. All cellular and molecular biological investigations were performed on the genetically well-defined and clinically relevant p53-wild type U87Mg glioma cells. Malignant glioma cells underwent either radiation or temozolomide treatments alone, or combined chemo/radio treatment. Protein expression patterns were investigated by two-dimensional polyacrylamide gel electrophoresis followed by protein spot identification using tandem mass spectrometry analysis. Specific expression levels were quantified by Western-blotting. Extracellular gelatinase activities for both metalloproteinases MMP-2 and MMP-9 were determined by zymogramms. Survival curves indicated no effective suppression of glioma cells under all treatment conditions tested. Morphological changes demonstrated sub-lethal effect of both temozolomide and combined treatment. Expression of MMP-2, MMP-9, and membrane type 1 matrix metalloproteinases (MT1-MMP) was differentially up-regulated by increasing cellular density and treatment conditions. A significantly enhanced extracellular degrading activity under all treatment conditions tested was demonstrated for MMP-2 only. Being a marker for brain tumour progression and angiogenesis, lysozyme c was highly up-regulated under the combined chemo/radio treatment. The activation of proteins with pro-invasive activity indicates an increasing malignancy grade of surviving glioma cells under treatment conditions tested correlating well with more aggressive tumour phenotypes observed clinically in recurrences of treated glioblastomas.

Genetics, transcriptomics, and proteomics of Alzheimer's disease.

OBJECTIVE: To provide an updated overview of the methods used in genetic, transcriptomic, and proteomic studies in Alzheimer's disease and to demonstrate the importance of those methods for the improvement of the current diagnostic and therapeutic possibilities. DATA SOURCES: MEDLINE-based search of 233 peer-reviewed articles published between 1975 and 2006. DATA SYNTHESIS: Alzheimer's disease is a genetically heterogeneous disorder. Rare mutations in the amyloid precursor protein, presenilin 1, and presenilin 2 genes have shown the importance of the amyloid metabolism for its development. In addition, converging evidence from population-based genetic studies, gene expression studies, and protein profile studies in the brain and in the cerebrospinal fluid suggest the existence of several pathogenetic pathways such as amyloid precursor protein processing, beta-amyloid degradation, tau phosphorylation, proteolysis, protein misfolding, neuroinflammation, oxidative stress, and lipid metabolism. CONCLUSIONS: The development of high-throughput genotyping methods and of elaborated statistical analyses will contribute to the identification of genetic risk profiles related to the development and course of this devastating disease. The integration of knowledge derived from genetic, transcriptomic, and proteomic studies will greatly advance our understanding of the causes of Alzheimer's disease, improve our capability of establishing an early diagnosis, help define disease subgroups, and ultimately help to pave the road toward improved and tailored treatments.

Is current therapy of malignant gliomas beneficial for patients? Proteomics evidence of shifts in glioma cells expression patterns under clinically relevant treatment conditions.

The most common human brain tumours - gliomas - have poor prognosis with and without treatment. The current therapy conditions act sub-lethally and cannot effectively suppress the proliferation of glioma cells. Here we show differential protein expression patterns in surviving human malignant U87-MG glioma cells under clinically relevant chemo/radiotherapy. In parallel experiments, the cells underwent either irradiation (2 Gy, 200 KV X-ray) or chemotreatment with 30 microg/mL of temozolomide in the cultivation medium or combined chemo/radiation treatment. The cell cultures were treated during 5 days from day 4 until day 9 of growth. Modulated expression patterns of vimentin and RhoA GTPase indicate a potentially increasing grade of malignancy in treated cell fractions correlating well with extremely aggressive tumour phenotypes observed clinically at recidivation of treated malignant gliomas.

Proteomic analysis of the mouse brain following protein enrichment by preparative electrophoresis.

Proteomics is a powerful technology to study the identity and levels of brain proteins. Changes of protein levels as well as modifications that occur in neurological disorders may be informative for the pathogenesis of these disorders and could result in the identification of potential drug targets and disease markers. To increase the capability of characterizing complex protein profiles, protein mixtures should be separated into simpler fractions, thus increasing the likelihood of detecting low-abundance proteins. Considering that low-abundance proteins are thought to be involved in important biological processes, identification of those low-copy-number gene products appears to be a scientific challenge. In the present study, proteomic analysis of adult mouse brain tissue was performed following enrichment by preparative electrophoresis. This was performed using the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Samples were electrophoresed in a cylindrical polyacrylamide gel and the proteins of the fractions collected were first analyzed by 1-D and then by 2-DE. Protein identification was performed by MALDI-TOF-MS. The present analysis resulted in the identification of 360 different gene products. Among those were transport proteins, transcription activators, signal transduction molecules as well as proteins with a number of other functions. Preparative electrophoresis is an efficient method for the enrichment of proteins of low molecular mass and may be useful in the investigation of disorders of the central nervous system.

Proteomics-driven progress in neurodegeneration research.

Proteomics technologies have been widely used in the investigation of neurodegenerative and psychiatric disorders, and in particular in the detection of differences between healthy individuals and patients suffering from such diseases. Thus, brain and cerebrospinal fluid (CSF) samples from patients with Alzheimer's disease, Down syndrome, Pick's disease, Parkinson's disease, schizophrenia, and other disorders as well as brain and CSF from animals serving as models of neurological disorders have been analyzed by proteomics. 2-DE followed by MALDI-TOF-MS has been mainly applied as this proteomics approach provides the possibility of convenient quantification of protein levels and detection of post-translational modifications. About 330 unique proteins with deranged levels and modifications have been detected by proteomics approaches to be related to neurodegeneration and psychiatric disorders. They are mainly involved in metabolism pathways, cytoskeleton formation, signal transduction, guidance, detoxification, transport, and conformational changes. In this article, we provide a summary of the major contributions of proteomics technologies in the study of neurodegenerative and psychiatric diseases, in particular, in the detection of changes in protein levels and modifications related to these disorders.