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Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants and recognized developmental toxicants that are detectable in placental tissues. Higher levels of in utero PBDE exposure have been associated with an increased risk of adverse birth outcomes. During pregnancy, cytotrophoblasts (CTBs) from the placenta play critical roles in the formation of the maternal-fetal interface via uterine invasion and vascular remodeling. The differentiation of these cells towards an invasive phenotype is crucial for proper placental development. We previously have shown that BDE-47 can impact CTB viability and hinder the ability of these cells to migrate and invade. To expand on potential toxicological mechanisms, we utilized quantitative proteomic approaches to identify changes in the global proteome of mid-gestation primary human CTBs after exposure to BDE-47. Using sequential window acquisition of all theoretical fragment-ion spectra (SWATH), we identified 3024 proteins in our CTB model of differentiation/invasion. Over 200 proteins were impacted as a function of BDE-47 exposure (1 μM and 5 μM) across the treatment period (15, 24, and 39 h). The differentially expressed molecules displayed time- and concentration-dependent changes in expression and were enriched in pathways associated with aggregatory and adhesive processes. Network analysis identified CYFIP1, a molecule previously unexplored in a placental context, to be dysregulated at BDE-47 concentrations previously seen to impact CTB migration/invasion. Our SWATH-MS dataset thus demonstrates BDE-47 impacts the global proteome of differentiating CTBs and serves as a valuable resource for further understanding of the relationship between environmental chemical exposures and placental development and function. AVAILABILITY OF DATA AND MATERIAL: Raw chromatograms are deposited on the MassIVE proteomic database (https://massive.ucsd.edu) under accession number MSV000087870. Normalized relative abundances are also available as Table S1.

Wicked problems are large, complex problems involving multiple perspectives that present substantial future challenges. These challenges can be overwhelming for learners and pose difficulties in teaching for instructors. Herein a solutions-oriented teaching strategy that amalgamates proven active learning strategies is presented along with a step-by-step guide and materials list. Evidence of student learning is provided. This strategy provides students the opportunity to view complex, wicked problems from multiple perspectives and to visualize their role in future solutions.

Reversing brain aging may be possible through systemic interventions such as exercise. We found that administration of circulating blood factors in plasma from exercised aged mice transferred the effects of exercise on adult neurogenesis and cognition to sedentary aged mice. Plasma concentrations of glycosylphosphatidylinositol (GPI)-specific phospholipase D1 (Gpld1), a GPI-degrading enzyme derived from liver, were found to increase after exercise and to correlate with improved cognitive function in aged mice, and concentrations of Gpld1 in blood were increased in active, healthy elderly humans. Increasing systemic concentrations of Gpld1 in aged mice ameliorated age-related regenerative and cognitive impairments by altering signaling cascades downstream of GPI-anchored substrate cleavage. We thus identify a liver-to-brain axis by which blood factors can transfer the benefits of exercise in old age.

Interdisciplinary (i.e., university-wide programming) and disciplinary (i.e., programming open to participants from one college or department) teaching development programs for graduate students have been used for many years in higher education. Currently, research on the benefits of these teaching models remains scant in terms of a contextualized understanding, and empirical studies are needed. The purpose of this study was to determine graduate students' perspectives related to interdisciplinary and disciplinary teaching and learning experiences. Two online surveys were used: a quantitative survey and a qualitative follow-up survey. Three participatory focus groups were also conducted to allow for further in-depth exploration in both an interdisciplinary and disciplinary group setting that represented seven distinct colleges. Statistical and thematic analyses were conducted with survey responses, and thematic analyses were conducted on focus group data. Similar themes emerged from the survey and focus group data identifying perceived benefits of participation in either interdisciplinary or disciplinary teaching development. Respondents' perceived benefits were related to: (a) becoming a better teacher; (b) social learning; and (c) that while the perceived benefits of the models vary, the outcomes of both experiences are shared. The lived experiences of these graduate students expand the characterization of interdisciplinary and disciplinary programming. This study points to the need for graduate student programs--specifically teaching development offered by educational development units--to provide both interdisciplinary and disciplinary teaching development opportunities that achieve a blend of benefits for learners.

We used isobaric mass tagging (iTRAQ) and lectin affinity capture mass spectrometry (MS)-based workflows for global analyses of parotid saliva (PS) and whole saliva (WS) samples obtained from patients diagnosed with primary Sjögren's Syndrome (pSS) who were enrolled in the Sjögren's International Collaborative Clinical Alliance (SICCA) as compared with two control groups. The iTRAQ analyses revealed up- and down-regulation of numerous proteins that could be involved in the disease process (e.g., histones) or attempts to mitigate the ensuing damage (e.g., bactericidal/permeability increasing fold containing family (BPIF) members). An immunoblot approach applied to independent sample sets confirmed the pSS associated up-regulation of β2-microglobulin (in PS) and down-regulation of carbonic anhydrase VI (in WS) and BPIFB2 (in PS). Beyond the proteome, we profiled the N-glycosites of pSS and control samples. They were enriched for glycopeptides using lectins Aleuria aurantia and wheat germ agglutinin, which recognize fucose and sialic acid/N-acetyl glucosamine, respectively. MS analyses showed that pSS is associated with increased N-glycosylation of numerous salivary glycoproteins in PS and WS. The observed alterations of the salivary proteome and N-glycome could be used as pSS biomarkers enabling easier and earlier detection of this syndrome while lending potential new insights into the disease process.

Common environmental contaminants such as bisphenols and phthalates and persistent contaminants such as polychlorinated biphenyls are thought to influence tissue homeostasis and carcinogenesis by acting as disrupters of endocrine function. In this study we investigated the direct effects of exposure to bisphenol A (BPA), mono-n-butyl phthalate (Pht), and polychlorinated biphenyl 153 (PCB153) on the proteome of primary organotypic cultures of the mouse mammary gland. At low-nanomolar doses each of these agents induced distinct effects on the proteomes of these cultures. Although BPA treatment produced effects that were similar to those induced by estradiol, there were some notable differences, including a reduction in the abundance of retinoblastoma-associated protein and increases in the Rho GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle protein CDC42. Both Pht and PCB153 induced changes that were distinct from those induced by estrogen, including decreased levels of the transcriptional corepressor C-terminal binding protein 1. Interestingly, the three chemicals appeared to alter the abundance of distinct splice forms of many proteins as well as the abundance of several proteins that regulate RNA splicing. Our combined results indicate that the three classes of chemical have distinct effects on the proteome of normal mouse mammary cultures, some estrogen-like but most estrogen independent, that influence diverse biological processes including apoptosis, cell adhesion, and proliferation.

Endometriosis, ectopic growth of the uterine lining (endometrium), which affects 6-11% of reproductive age women, is associated with pelvic pain and infertility. We investigated the peritoneal fluid (PF), urine and omental fat (OF) proteomes of women with endometriosis vs. individuals with no surgically visualized endometriosis. All participants were enrolled in the NICHD-funded ENDO Study. A two-step proteomic study was performed. The first, a broad survey, employed a semi-quantitative gel LC-mass spectrometry (MS) workflow: SDS PAGE fractionation, trypsin digestion and LC-MS/MS. The results showed sample integrity but failed to detect any differences between women with and without endometriosis. The second step was a quantitative analysis of OF samples. We employed another sample set (n=30) from women ± disease and isobaric mass-tag (iTRAQ) chemistry to label peptides and 2D LC-MS/MS for protein identification and quantification. Three proteins-matrix metalloproteinase-9, neutrophil elastase, and FAM49B-were significantly lower in abundance in samples from women with endometriosis. Interestingly, neutrophil elastase and FAM49B levels were associated with higher levels of a subset of endocrine disrupting chemicals (EDCs) that were previously measured in the same samples. The results of these experiments showed the feasibility of associating endometriosis with changes in the OF protein repertoire and EDC levels.Biological significanceEndometriosis, pathological growth of the uterine lining, is associated with significant morbidities, including pain and infertility. However, the causes of this common condition are poorly understood. This study determined whether endometriosis was associated with changes in the protein composition of peritoneal fluid, urine and/or omental fat. A protein of unknown function (FAM49B) and two proteinases (metalloproteinase-9, neutrophil elastase) were down regulated in OF samples from women with versus without endometriosis. These findings suggested proteinase imbalances at sites that were distant from the endometriotic lesions. Additionally, FAM49B and neutrophil elastase levels were associated with higher levels of a subset of environmental chemicals that were quantified in the same samples, suggesting other possible associations. Thus, this work generated hypotheses that will be tested in further studies.

Six protein biomarkers from two strains of Escherichia coli O157:H7 and one non-O157:H7, nonpathogenic strain of E. coli have been identified by matrix-assisted laser desorption ionization time-of-flight time-of-flight tandem mass spectrometry (MALD1-TOF--TOF-MS/MS) and top-down proteomics. Proteins were extracted from bacterial cell lysates, ionized by MALD1, and analyzed by MS/MS. Protein biomarker ions were identified from their sequence-specific fragment ions by comparison to a database of in silico fragment ions derived from bacterial protein sequences. Web-based software, developed in-house, was used to rapidly compare the mass-to-charge (m/z) of MS/MS fragment ions to the m/z of in silico fragment ions derived from hundreds of bacterial protein sequences. A peak matching algorithm and a p-value algorithm were used to independently score and rank identifications on the basis of the number of MS/MS-in silico matches. The six proteins identified were the acid stress chaperone-like proteins, HdeA and HdeB; the cold shock protein, CspC; the YbgS (or homeobox protein); the putative stress-response protein YjbJ (or CsbD family protein); and a protein of unknown function, YahO. HdeA, HdeB, YbgS, and YahO proteins were found to be modified post-translationally with removal of an N-terminal signal peptide. Gene sequencing of hdeA, hdeB, cspC, ybgS, yahO, and yjbJ for 11 strains of E. coli Ol57:H7 and 7 strains of the 'near-neighbor' serotype O55:H7 revealed a high degree sequence homology between these two serotypes. Although it was not possible to distinguish O157:H7 from O55:H7 from these six biomarkers, it was possible to distinguish E. coli O157:H7 from a nonpathogenic E. coli by top-down proteomics of the YahO and YbgS. In the case of the YahO protein, a single amino acid residue substitution in its sequence (resulting in a molecular weight difference of only 1 Da) was sufficient to distinguish E. coli O157:H7 from a non-O157:H7, non-pathogenic E. coli by MALDI-TOF--TOF-MS/MS, whereas this would be difficult to distinguish by MALDI-TOF-MS. Finally, a protein biomarker ion at m/z ~9060 observed in the MS spectra of non-O157:H7 E. coli strains but absent from MS spectra of E. coli O157:H7 strains was identified by top-down analysis to be the HdeB acid stress chaperone-like protein consistent with previous identifications by gene sequencing and bottom-up proteomics. 10.1021/ac902455d

During human pregnancy, cytotrophoblasts (CTBs) from the placenta differentiate into specialized subpopulations that play crucial roles in proper fetal growth and development. A subset of these CTBs differentiate along an invasive pathway, penetrating the decidua and anchoring the placenta to the uterus. A crucial hurdle in pregnancy is the ability of these cells to migrate, invade and remodel spiral arteries, ensuring adequate blood flow to nourish the developing fetus. Although advances continue in describing the molecular features regulating the differentiation of these cells, assessment of their global proteomic changes at mid-gestation remain undefined. Here, using sequential window acquisition of all theoretical fragment-ion spectra (SWATH), which is a data-independent acquisition strategy, we characterized the protein repertoire of second trimester human CTBs during their differentiation towards an invasive phenotype. This mass spectrometry-based approach allowed identification of 3026 proteins across four culture time points corresponding to sequential stages of differentiation, confirming the expression dynamics of established molecules and offering new information into other pathways involved. The availability of a SWATH CTB global spectral library serves as a beneficial resource for hypothesis generation and as a foundation for further understanding CTB differentiation dynamics. [ABSTRACT FROM AUTHOR]

Web-based software and databases developed in house in the U.S. department of agriculture (USDA) were used to identify 10 protein biomarkers from three pure strains of Campylobacter by sequence-specific fragmentation. The findings from fragment ion error analysis might be successfully applied to confirm algorithm identification by distinguishing systematic fragment ion error caused by drift in the time of flight (TOF) calibration from random error caused by random matches between tandem mass spectrometry (MS-MS) fragmentation and in silico fragment ions.