Proteomic analysis of amniotic fluid to identify potential targets predicting preterm delivery

Preterm delivery is a common complication of pregnancy which leads to significant neonatal mortality and morbidity. Identifying predictive markers linked to spontaneous preterm delivery (SPTD) is important for effective treatment and prevention of PTD. To explore potential biomarkers related to SPTD, we performed proteomics analysis in amniotic fluid (AF). In total, we enrolled 30 pregnant women with singleton gestation who underwent clinically indicated amniocentesis at 15–24 weeks of gestation. LC-MS analysis was used to analyze the AF samples of 10 women with SPTD < 34 weeks after cervix cerclage (Preterm group), 10 women with term delivery (TD) ≥ 34 weeks after cervix cerclage (Term group), and 10 women who delivered at term (Normal group). ELISA validation was performed for candidate proteins in a second independent cohort. As a result, we identified 44 differentially expressed proteins (DEPs, P < 0.05) via proteomic analysis, and based on that, 9 primary pathways were also determined in SPTD. Results of the ELISA assay confirmed that the increased concentration of Serpin A1, decreased concentrations of Renin and IGFBP4 were significantly associated with SPTD at ≤34 weeks.     

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.     

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.     

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.     

Proteomic analysis of amniotic fluid for the diagnosis of fetal aneuploidies

Advances in technologies associated with mass spectrometry-based proteomic techniques have added a new dimension to the field of biomedical research. Most of the existing research on human gestation has focused on the application of these high-throughput methodologies in the study of amniotic fluid. In cases of fetal aneuploidies, the use of proteomic platforms has contributed to the identification of relevant protein biomarkers that could potentially change early diagnosis and treatment. The current article focuses on studies of normal amniotic fluid using proteomic technologies and describes alterations noted in the amniotic fluid proteome in the presence of fetal aneuploidies.     

Proteomic analysis of amniotic fluid of pregnant rats with spina bifida aperta

Congenital spina bifida aperta is a common congenital malformation in children and has an incidence of 1‰ to 5‰ in China. However, we currently lack specific biomarkers for screening or prenatal diagnosis and there is no method to entirely cure or prevent such defects. In this study, we used two-dimensional gel electrophoresis (2-DE)/mass spectrometry (MS) to characterize differentially expressed proteins in amniotic-fluid samples (AFSs) of embryonic day (E) 17.5 rat fetuses with spina bifida aperta induced by retinoic acid (RA). We identified five proteins differentially expressed in AFSs of spina bifida aperta, including three upregulated proteins (transferrin, alpha-1 antiproteinase and signal recognition particle receptor, B subunit [SRPRB] 55 kDa), two downregulated proteins (apolipoprotein A IV [APO A4] and Srprb 77 kDa). Specifically, we found 11 alpha-1 fetoprotein (AFP) fragments that were downregulated and 35 AFP fragments that were upregulated in AFSs from embryos with spina bifida aperta. Of the downregulated AFP fragments, 72.7% (8/11) were confined to the AFP N-terminus (amino acids [aas] 25-440) and 77.1% (27/35) of upregulated AFP fragments were confined to the AFP C-terminus (aas 340-596). We also confirmed APO A4 and AFP by immunoblot analysis. This is the first comparative proteomic study of AFSs from rat fetuses with spina bifida aperta. We demonstrate proteomic alterations in the AFS of spina bifida aperta, which may provide new insights in neural tube defects and contribute to the prenatal screening.     

Proteomic analysis of synovial fluid: current and potential uses to improve clinical outcomes

Introduction: Synovial fluid (SF) is in close proximity to tissues which are primarily altered during articular disease and has significant potential to better understand the underlying disease pathogeneses of articular pathologies and biomarker discovery. Although development of mass spectrometry-based methods has allowed faster and higher sensitivity techniques, interrogation of the SF proteome has been hindered by its large protein concentration dynamic range, impeding quantification of lower abundant proteins.

Areas covered: Recent advances have developed methodologies to reduce the large protein concentration dynamic range of SF and subsequently allow deeper exploration of the SF proteome. This review concentrates on methods to overcome biofluid complexity, mass spectrometry proteomics methodologies, extracellular vesicles proteomics and the application of advances within the field in clinical disease, including osteoarthritis, rheumatoid arthritis, spondyloarthritis and juvenile arthritis. A narrative review was conducted with articles searched using PubMed, 1991–2018.

Expert opinion: The SF proteomics field faces various challenges, including the requirement for rigorous and standardised methods of sample collection/storage, the sensitivity and specificity of proteomic assays, techniques to combat the large protein concentration dynamic range and comprehensive data analysis to reduce falsely identified markers. Additionally, there are challenges in developing multi ‘omic’ integration techniques, with computational integration enhancing analysis.     

Changes in culture expanded human amniotic epithelial cells: implications for potential therapeutic applications

Human amniotic epithelial cells (hAEC) isolated from term placenta have stem cell-like properties, differentiate into tissue specific cells and reduce lung and liver inflammation and fibrosis following transplantation into disease models established in mice. These features together with their low immunogenicity and immunosuppressive properties make hAEC an attractive source of cells for potential therapeutic applications. However, generation of large cell numbers required for therapies through serial expansion in xenobiotic-free media may be a limiting factor. We investigated if hAEC could be expanded in xenobiotic-free media and if expansion altered their differentiation capacity, immunophenotype, immunosuppressive properties and production of immunomodulatory factors. Serial expansion in xenobiotic-free media was limited with cumulative cell numbers and population doubling times significantly lower than controls maintained in fetal calf serum. The epithelial morphology of primary hAEC changed into mesenchymal-stromal like cells by passage 4-5 (P4-P5) with down regulation of epithelial markers CK7, CD49f, EpCAM and E-cadherin and elevation of mesenchymal-stromal markers CD44, CD105, CD146 and vimentin. The P5 hAEC expanded in xenobiotic-free medium differentiated into osteocyte and alveolar epithelium-like cells, but not chondrocyte, hepatocyte, α- and β-pancreatic-like cells. Expression of HLA Class IA, Class II and co-stimulatory molecules CD80, CD86 and CD40 remained unaltered. The P5 hAEC suppressed mitogen stimulated T cell proliferation, but were less suppressive compared with primary hAEC at higher splenocyte ratios. Primary and P5 hAEC did not secrete the immunosuppressive factors IL-10 and HGF, whereas TGF-β1 and HLA-G were reduced and IL-6 elevated in P5 hAEC. These findings suggest that primary and expanded hAEC may be suitable for different cellular therapeutic applications.     

The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy

The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140]     

Transport and storage of amniotic fluid samples for prenatal diagnosis of metabolic diseases

Summary 1. A method for freezing uncultured amniotic fluid cells is presented, which allows their use as pregnancy-age matched controls in prenatal diagnosis of metabolic diseases. 2. Amniotic fluid cells were successfully cultured after up to 7 days in transport, which makes prenatal diagnosis available to parents living a long way from specialized centers.

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Zusammenfassung 1. Eine Einfriermethode für nichtkultivierte Fruchtwasserzellen wird beschrieben, wodurch man diese wie Kontrollmuster anwenden kann bei pr?nataler Diagnostik von Stoffwechselkrankheiten. 2. Fruchtwasserzellen wurden erfolgreich kultiviert nach langzeitigem Transport (7 Tage); dieser Befund bringt eine pr?natale Diagnostik im Bereich entfernt von spezialisierten Zentren lebender Eltern.

     

Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential

The main goal of the study was to identify a novel source of human multipotent cells, overcoming ethical issues involved in embryonic stem cell research and the limited availability of most adult stem cells. Amniotic fluid cells （AFCs） are routinely obtained for prenatal diagnosis and can be expanded in vitro; nevertheless current knowledge about their origin and properties is limited. Twenty samples of AFCs were exposed in culture to adipogenic, osteogenic, neurogenic and myogenic media. Differentiation was evaluated using immunocytochemistry, RT-PCR and Western blotting. Before treatments, AFCs showed heterogeneous morphologies. They were negative for MyoD, Myf-5, MRF4, Myogenin and Desmin but positive for osteocalcin, PPARgamma2, GAP43, NSE, Nestin, MAP2, GFAP and beta tubulin III by RT-PCR. The cells expressed Oct-4, Rex-1 and Runx-1, which characterize the undifferentiated stem cell state. By immunocytochemistry they expressed neural-glial proteins, mesenchymal and epithelial markers. After culture, AFCs differentiated into adipocytes and osteoblasts when the predominant cellular component was fibroblastic. Early and late neuronal antigens were still present after 2 week culture in neural specific media even if no neuronal morphologies were detectable. Our results provide evidence that human amniotic fluid contains progenitor cells with multi-lineage potential showing stem and tissue-specific gene/protein presence for several lineages.     

Proteomic applications in ecotoxicology

Within the growing body of proteomics studies, issues addressing problems of ecotoxicology are on the rise. Generally speaking, ecotoxicology uses quantitative expression changes of distinct proteins known to be involved in toxicological responses as biomarkers. Unlike these directed approaches, proteomics examines how multiple expression changes are associated with a contamination that is suspected to be detrimental. Consequently, proteins involved in toxicological responses that have not been described previously may be revealed. Following identification of key proteins indicating exposure or effect, proteomics can potentially be employed in environmental risk assessment. To this end, bioinformatics may unveil protein patterns specific to an environmental stress that would constitute a classifier able to distinguish an exposure from a control state. The combined use of sets of marker proteins associated with a given pollution impact may prove to be more reliable, as they are based not only on a few unique markers which are measured independently, but reflect the complexity of a toxicological response. Such a proteomic pattern might also integrate some of the already established biomarkers of environmental toxicity. Proteomics applications in ecotoxicology may also comprise functional examination of known classes of proteins, such as glutathione transferases or metallothioneins, to elucidate their toxicological responses.     

Proteomic analysis using protein chips to detect biomarkers in cervical and amniotic fluid in women with intra-amniotic inflammation

Intra-amniotic inflammation (IAI) may cause preterm birth with poor neonatal out-come. To identify novel biomarkers for IAI, we analyzed amniotic and cervical fluid samples from 27 patients with signs of threatening preterm birth with or without IAI by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Seventeen proteins were significantly overexpressed in amniotic fluid from IAI cases and more often in women with preterm labor than those with rupture of membranes. Five of these were identified as human neutrophil protein 1-3, calgranulin A and B.     

Biomedical applications and potential health risks of nanomaterials: molecular mechanisms

Nanotechnologies, defined as techniques aimed to conceive, characterize and produce material at the nanometer scale, represent a fully expanding domain, and one can predict without risk that production and utilization of nanomaterials will increase exponentially in the coming years. Applications of nanotechnologies are numerous, in constant development, and their potential use in the medical field as diagnosis and therapeutics tools is very attractive. The size particularity of these nanomaterials gives them novel properties, allowing them to adopt new comportments because of the laws of quantum physics that exist at this scale. However, worries are expressed regarding the exact properties that make these nanomaterials attractive, and questions are raised regarding their potential toxicity, their long-term secondary effects or their biodegradability, particularly when thinking of their use in the (nano)medical field. These questions are justified by the knowledge of the toxic effects of atmospheric pollution micrometric particles on health, and the fear to get an amplification of these effects because of the size of the materials blamed. In this paper, we first expose the sensed medical applications of nanomaterials, and the physicochemical and molecular determinants potentially responsible for nanomaterials biological effects. Finally, we present a synthesis of the actual knowledge regarding toxicological effects of nanomaterials. It is clear that, in regard to the almost empty field of what is known on the subject, there's an urge to better understand biological effects of nanomaterials, which will allow their safe use, in particular in the nanomedicine field.     

Gut microbes in cardiovascular diseases and their potential therapeutic applications

Microbial ecosystem comprises a complex community in which bacteria interact with each other.The potential roles of the intestinal microbiome play in human health have gained considerable attention.The imbalance of gut microbial community has been looked to multiple chronic diseases.Cardiovascular diseases(CVDs)are leading causes of morbidity worldwide and are influ-enced by genetic and environmental factors.Recent advances have provided scientific evidence that CVD may also be attributed to gut microbiome.in this review,we highlight the complex interplay between microbes,their metabolites,and the potential influence on the generation and development of CVDs.The therapeutic potentiai of using intestinal microbiomes to treat CVD is also discussed.it is quite possible that gut microbes may be used for clinical treatments of CVD in the near future.     

Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.     

Direct tissue proteomics in human diseases: potential applications to melanoma research

Although the rates of cancer are stabilizing, the number of new invasive melanoma continues to rise. Melanoma represents only 4% of all skin cancers, but nearly 80% of skin cancer deaths. In loss of potential productive life-years, it is second only to adult leukemia. Once melanoma spreads to regional and distant sites, the chance of cure decreases significantly. Unfortunately, current diagnostic and prognostic methods are often inadequate. More precise staging and disease characterization will lead to new and more rational approaches to treatment. Proteomics is a fast-growing discipline in biomedicine that can be defined as the global characterization and differential expression of the entire protein complement of a cell, tissue or organism. Despite major advances in molecular approaches to the diagnosis and prognostication of human diseases such as melanoma, there remain significant obstacles in applying the proteomic technologies to clinical samples to extract important biological information. The application of a shotgun-based technique termed direct tissue proteomics with improved extraction protocol of proteins from formalin-fixed paraffin-embedded tissue would enable retrospective biomarker investigations of the vast archive of pathologically characterized clinical samples that exist worldwide. Combination of this direct tissue proteomics method with laser-capture microdissection may assist in the discovery of new biomarkers and may lead to new diagnostic tests, risk assessment and staging tools as well as improvement in therapeutics. In addition, these tools can provide a molecular characterization of melanoma, which may enable individualized molecular therapy.     

Direct tissue proteomics in human diseases: potential applications to melanoma research

Although the rates of cancer are stabilizing, the number of new invasive melanoma continues to rise. Melanoma represents only 4% of all skin cancers, but nearly 80% of skin cancer deaths. In loss of potential productive life-years, it is second only to adult leukemia. Once melanoma spreads to regional and distant sites, the chance of cure decreases significantly. Unfortunately, current diagnostic and prognostic methods are often inadequate. More precise staging and disease characterization will lead to new and more rational approaches to treatment. Proteomics is a fast-growing discipline in biomedicine that can be defined as the global characterization and differential expression of the entire protein complement of a cell, tissue or organism. Despite major advances in molecular approaches to the diagnosis and prognostication of human diseases such as melanoma, there remain significant obstacles in applying the proteomic technologies to clinical samples to extract important biological information. The application of a shotgun-based technique termed direct tissue proteomics with improved extraction protocol of proteins from formalin-fixed paraffin-embedded tissue would enable retrospective biomarker investigations of the vast archive of pathologically characterized clinical samples that exist worldwide. Combination of this direct tissue proteomics method with laser-capture microdissection may assist in the discovery of new biomarkers and may lead to new diagnostic tests, risk assessment and staging tools as well as improvement in therapeutics. In addition, these tools can provide a molecular characterization of melanoma, which may enable individualized molecular therapy.