Proteomics and Nucleotide Profiling as Tools for Biomarker and Drug Target Discovery.

Proteomics has gone through tremendous development during recent decades [...].

Early pregnancy levels of gingival crevicular fluid matrix metalloproteinases-8 and -9 are associated with the severity of periodontitis and the development of gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) is increasing worldwide and women with a history of GDM are at risk of developing type 2 diabetes which is a risk factor for periodontitis. The aim of this study was to explore the association between the concentrations of matrix metalloproteinase (MMP)-8 and -9 in gingival crevicular fluid (GCF) during early pregnancy with the periodontal diagnosis and the risk of GDM development. METHODS: A prospective cohort study, including 314 women, enrolled at 11 to 14 weeks of pregnancy was conducted. A complete maternal/obstetric and periodontal exam was performed, and GCF samples were obtained for the MMP-8 and -9 determination by Multiplex Elisa Assays. Mann-Whitney test; Spearman's correlation and log-binomial regression model estimated the association between MMPs concentration in GCF and GDM. RESULTS: Fourteen percent of the pregnancies were diagnosed with GDM. An increase in the concentration of MMP-8 and -9 in women with periodontitis stage III and IV compared to periodontitis stage I was observed (99.31 ng/mL [IQR: 85.32] versus 71.95 ng/mL [IQR: 54.04], and 262.4 ng/mL [IQR: 312.55] versus 114.1 ng/mL [IQR: 184.94], respectively). Women who developed GDM showed increased concentrations of MMP-8 and -9 in GCF since the beginning of pregnancy (P = 0.0381; P = 0.0302, respectively). MMP-8 concentration in GCF was associated with GDM (RR: 1.19; P = 0.045; CI 95% 1.00 to 1.40; and RR: 1.20; P = 0.063; CI 95% 0.99 to 1.45 in the adjusted model). CONCLUSION(S): GCF concentrations of MMP-8 and -9 at early of pregnancy are increased in women with severe periodontitis and associated with the GDM development.

Compartmentalized profiling of amniotic fluid cytokines in women with preterm labor.

OBJECTIVE: Amniotic fluid cytokines have been implicated in the mechanisms of preterm labor and birth. Cytokines can be packaged within or on the surface of extracellular vesicles. The main aim of this study was to test whether the protein abundance internal to and on the surface of extracellular vesicles changes in the presence of sterile intra-amniotic inflammation and proven intra-amniotic infection in women with preterm labor as compared to the women with preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection. STUDY DESIGN: Women who had an episode of preterm labor and underwent an amniocentesis for the diagnosis of intra-amniotic infection or intra-amniotic inflammation were classified into three groups: 1) preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection, 2) preterm labor with sterile intra-amniotic inflammation, and 3) preterm labor with intra-amniotic infection. The concentrations of 38 proteins were determined on the extracellular vesicle surface, within the vesicles, and in the soluble fraction of amniotic fluid. RESULTS: 1) Intra-amniotic inflammation, regardless of detected microbes, was associated with an increased abundance of amniotic fluid cytokines on the extracellular vesicle surface, within vesicles, and in the soluble fraction. These changes were most prominent in women with proven intra-amniotic infection. 2) Cytokine changes on the surface of extracellular vesicles were correlated with those determined in the soluble fraction; yet the magnitude of the increase was significantly different between these compartments. 3) The performance of prediction models of early preterm delivery based on measurements on the extracellular vesicle surface was equivalent to those based on the soluble fraction. CONCLUSIONS: Differential packaging of amniotic fluid cytokines in extracellular vesicles during preterm labor with sterile intra-amniotic inflammation or proven intra-amniotic infection is reported herein for the first time. The current study provides insights into the biology of the intra-amniotic fluid ad may aid in the development of biomarkers for obstetrical disease.

Method Development for the Detection of Human Myostatin by High-Resolution and Targeted Mass Spectrometry.

Myostatin, a highly conserved secretory protein, negatively regulates muscle development, affecting both the proliferation and differentiation of muscle cells. Proteolytic processing of the myostatin precursor protein generates a myostatin pro-peptide and mature protein. Dimerization of the mature myostatin protein creates the active form of myostatin. Myostatin dimer activity can be inhibited by noncovalent binding of two monomeric myostatin pro-peptides. This ability for myostatin to self-regulate as well as the altered expression of myostatin in states of abnormal health (e.g., muscle wasting) support the need for specific detection of myostatin forms. Current protein detection methods (e.g., Western blot) rely greatly on antibodies and are semiquantitative at best. Tandem mass spectometry (as in this study) provides a highly specific method of detection, enabling the characterization of myostatin protein forms through the analysis of discrete peptides fragments. Utilizing the scheduled high-resolution multiple reaction monitoring paradigm (sMRMHR; AB SCIEX 5600 TripleTOF) we identified the lower limit of quantitation (LLOQ) of both mature (DFGLDCDEHSTESR) and pro-peptide regions (ELIDQYDVQR) as 0.19 nmol/L. Furthermore, scheduled multiple reaction monitoring (sMRM; AB SCIEX QTRAP 5500) identified a LLOQ for a peptide of the pro-peptide region (LETAPNISK) as 0.16 nmol/L and a peptide of the mature region (EQIIYGK) as 0.25 nmol/L.

Stanford type A aortic dissection with pulmonary arterial intramural hematoma and pulmonary hemorrhage.

A rare complication of a Stanford type A aortic dissection is extension along the pulmonary arteries. We present a case that shows main and right pulmonary artery intramural hematoma and pulmonary hemorrhage in an 80-year-old woman who presented with a type A Stanford aortic dissection. The 11-month follow-up multidetector CT angiogram for this patient showed that the right pulmonary artery had become aneurysmal.

Temporal expression of antioxidants in human cervicovaginal fluid associated with spontaneous labor.

Proteomic analysis of human cervicovaginal fluid (CVF) by 2D electrophoresis revealed significant differential expression of several major antioxidant enzymes during late pregnancy and term labor. Temporal quantitative changes of total antioxidant capacity (TAC), Cu,Zn superoxide dismutase (Cu,Zn SOD) and thioredoxin-1 (Trx-1) with impending term labor were investigated, and the potential of these biomarkers as individual and multiple predictors of labor was determined. The TAC of CVF (n = 193) was 8-fold significantly lower in labor, and approximately 2-fold significantly lower at 0-7, 8-14, 15-21, and 22-28 days, compared with >or=29 days prior to labor onset (p < 0.001). The expression of Cu,Zn SOD (n = 170) was 1.5- to 1.9-fold significantly decreased in labor (p < 0.001). Trx-1 (n = 163) was 2.8- to 5.1-fold significantly lower in labor (p = 0.002). The combination of TAC and Cu,Zn SOD produced the best predictive efficacy with 74% sensitivity and 95% specificity to predict term labor within 3 days of onset. These findings suggest that labor is associated with increased oxidative stress well before its onset and is reflected in the human CVF. The biomarkers identified in this study could serve as predictors of labor and offer potential strategies for novel therapeutics.

The oncogenic and growth-suppressive functions of the integrin-linked kinase are distinguished by JNK1 expression in human cancer cells.

While most reports detail an oncogenic function for the integrin-linked kinase (ILK) in human cancer, few describe a contradictory growth-suppressive function. We previously reported that ILK functions as either a tumor suppressor or an oncogene in rhabdomyosarcoma (RMS), in a manner linked to expression of the c-jun amino terminal kinase-1 (JNK1). However, studies in other tumors are lacking. With the advent of bioavailable small molecule inhibitors of ILK, defining both the function of ILK and biomarkers to predict its behaviour are of critical importance. Here, we studied the role of ILK in a panel of tumor cell lines. We demonstrate that ILK functions as either a growth-promoter or suppressor in numerous tumor cell lines. Further, cell lines in which ILK functioned as a growth suppressor displayed elevated JNK1 expression relative to cells in which ILK functioned as an oncogene. Comparison of endogenous JNK1 and JNK1ß isoform expression levels to the cellular response to ILK overexpression demonstrated that JNK1ß isoforms represent biomarkers differentiating the two functions of ILK. Moreover, RNAi and overexpression-based alteration of JNK1 expression levels was sufficient to switch the function of ILK in both transformed and untransformed cells. These results indicate widespread oncogenic and growth-suppressive functions for ILK in multiple human malignancies and suggest that JNK1 isoforms represent biomarkers for ILK neoplastic activity. These results provide a rationale for stratifying patients to receive ILK kinase inhibitors based on individualized tumor-specific ILK function.

Oncogenic ILK, tumor suppression and all that JNK.

In neoplastic cells, proteins exert either pro or anti-tumorigenic functions. However, some proteins exhibit both properties, commonly dependent on specific aberrations occurring in a tumor-specific context. Recently, we demonstrated that the integrin-linked kinase (ILK), generally characterized as an oncogenic protein kinase, functions as a tumor suppressor protein in vitro and in vivo in the aggressive pediatric tumor, rhabdomyosarcoma (RMS). Other studies have similarly demonstrated both growth and tumor suppressive functions for ILK in normal and transformed tissues. The mechanism of ILK tumor suppression in RMS relies on expression levels of another kinase, the c-jun amino terminal kinase-1 (JNK1). These findings support a model in which ILK tumor suppression is mediated in part by elevated JNK1 expression, and indicate both a rationale for stratification of patients to receive anti-ILK therapies, and a need to better understand the context in which ILK displays its seemingly contradictory functions. This review discusses the complex roles of ILK in tumorigenesis, and offers arguments to harness ILK and JNK signaling as novel targets for anti-cancer therapy.

Protein profiling the effects of in vitro hyperoxic exposure on fetal rabbit lung.

The aim of this study was to test the hypothesis that acute in vitro exposure of prematurely delivered fetal rabbit lungs to hyperoxic conditions will induce the expression of an adaptive cassette of proteins that mediates antioxidant and inflammatory processes. To test this hypothesis, ex situ fetal rabbit lung explants were prepared from New Zealand white rabbits delivered by cesarean section on day 29 of gestation and incubated under air (21% O2; 5% CO2) or hyperoxic (95% O2; 5% CO2) atmospheres. Total tissue protein was extracted following incubation and subjected to 2-DE. Using this technique, 1500-2000 protein spots were resolved per gel. Treatment-dependent, differentially expressed proteins were identified by image analysis (Melanie II) and MALDI-TOF MS and MALDI-MS/MS. The analysis identified 12 protein spots that were differentially expressed by 1.5-fold or more (p<0.05) by exposure to hyperoxic conditions. Six of these differentially expressed proteins were identified as vimentin, annexin I, inorganic pyrophosphatase, prohibitin, an N-terminal fragment of ATP synthase and heat shock protein 27. The data obtained are consistent with the roles of these proteins in mediating cellular response to oxidative stress and in regulating cell proliferation.

Haplotype structure, LD blocks, and uneven recombination within the LRP5 gene.

Patterns of linkage disequilibrium (LD) in the human genome are beginning to be characterized, with a paucity of haplotype diversity in "LD blocks," interspersed by apparent "hot spots" of recombination. Previously, we cloned and physically characterized the low-density lipoprotein-receptor-related protein 5 (LRP5) gene. Here, we have extensively analysed both LRP5 and its flanking three genes, spanning 269 kb, for single nucleotide polymorphisms (SNPs), and we present a comprehensive SNP map comprising 95 polymorphisms. Analysis revealed high levels of recombination across LRP5, including a hot-spot region from intron 1 to intron 7 of LRP5, where there are 109 recombinants/Mb (4882 meioses), in contrast to flanking regions of 14.6 recombinants/Mb. This region of high recombination could be delineated into three to four hot spots, one within a 601-bp interval. For LRP5, three haplotype blocks were identified, flanked by the hot spots. Each LD block comprised over 80% common haplotypes, concurring with a previous study of 14 genes that showed that common haplotypes account for at least 80% of all haplotypes. The identification of hot spots in between these LD blocks provides additional evidence that LD blocks are separated by areas of higher recombination.