Phosphonoacetate Modifications Enhance the Stability and Editing Yields of Guide RNAs for Cas9 Editors.

CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'-O-methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'-O-methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.

Biochemical Characterization of Yeast Xrn1.

Messenger RNA degradation is an important component of overall gene expression. During the final step of eukaryotic mRNA degradation, exoribonuclease 1 (Xrn1) carries out 5' → 3' processive, hydrolytic degradation of RNA molecules using divalent metal ion catalysis. To initiate studies of the 5' → 3' RNA decay machinery in our lab, we expressed a C-terminally truncated version of Saccharomyces cerevisiae Xrn1 and explored its enzymology using a second-generation, time-resolved fluorescence RNA degradation assay. Using this system, we quantitatively explored Xrn1's preference for 5'-monophosphorylated RNA substrates, its pH dependence, and the importance of active site mutations in the molecule's conserved catalytic core. Furthermore, we explore Xrn1's preference for RNAs containing a 5' single-stranded region both in an intermolecular hairpin structure and in an RNA-DNA hybrid duplex system. These results both expand and solidify our understanding of Xrn1, a centrally important enzyme whose biochemical properties have implications in numerous RNA degradation and processing pathways.

MicroRNA-200 family governs ovarian inclusion cyst formation and mode of ovarian cancer spread.

Epidemiologic and histopathologic findings and the laying hen model support the long-standing incessant ovulation hypothesis and cortical inclusion cyst involvement in sporadic ovarian cancer development. MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. Herewith, we show that ovarian surface epithelial (OSE) cells with ectopic miR-200 expression formed stabilized cysts in three-dimensional (3D) organotypic culture with E-cadherin fragment expression and steroid hormone pathway activation, whereas ovarian cancer 3D cultures with miR-200 knockdown showed elevated TGF-ß expression, mitotic spindle disorientation, increased lumenization, disruption of ROCK-mediated myosin II phosphorylation, and SRC signaling, which led to histotype-dependent loss of collective movement in tumor spread. Gene expression profiling revealed that epithelial-mesenchymal transition and hypoxia were the top enriched gene sets regulated by miR-200 in both OSE and ovarian cancer cells. The molecular changes uncovered by the in vitro studies were verified in both human and laying hen ovarian cysts and tumor specimens. As miR-200 is also essential for ovulation, our results of estrogen pathway activation in miR-200-expressing OSE cells add another intriguing link between incessant ovulation and ovarian carcinogenesis.

Douching, Talc Use, and Risk for Ovarian Cancer and Conditions Related to Genital Tract Inflammation.

BACKGROUND: Douching is associated with disorders involving genital tract inflammation and genital talc use with epithelial ovarian cancer (EOC), but their joint effects are infrequently considered. METHODS: From 2,040 cases of EOC and 2,100 controls enrolled in eastern Massachusetts and New Hampshire, we used unconditional logistic regression to estimate risk for EOC associated with douching and/or talc use. In subsets of cases and controls, we also collected information about pelvic inflammatory disease (PID), ectopic pregnancy, and cervical neoplasia to estimate risk for these events from douching and/or talc use. RESULTS: The adjusted OR and 95% confidence interval (CI) for all EOC was 0.94 (0.76-1.16) in women who douched but never used talc and 1.28 (1.09-1.51) in women who used talc but never douched. Compared with women who never regularly douched or used talc, ORs (95% CIs) were 0.83 (0.52-1.33) for women who both used talc and homemade douches and 1.53 (1.11-2.10) for women who both used talc and store-bought douches. Cases who both douched and used talc were more likely to have had PID compared with cases who had used neither [OR = 5.03 (95% CI, 1.61-15.7)]. CONCLUSIONS: Douching is not an independent risk factor for ovarian cancer, but the combination of talc use and store-bought douches may modestly increase the risk for EOC beyond that for talc use alone. IMPACT: The joint effect of talc use and douching, especially with commercial products, should be considered in evaluating risks associated with disorders involving genital tract inflammation or EOC.

Migration of Talc From the Perineum to Multiple Pelvic Organ Sites.

OBJECTIVES: Genital talc use is associated with increased risk for ovarian carcinoma in epidemiologic studies. Finding talc in pelvic tissues in women with ovarian carcinoma who have used talc is important in documenting exposure and assessing talc's biologic potential, but tissue-based morphology studies have been rarely reported. METHODS: We report five patient cases with documented perineal talc use, each of whom had talc (by both polarized light and scanning electron microscopy) in multiple pelvic sites distant from the perineum. Six negative-exposure control patients were also analyzed. RESULTS: Talc particles were found in exposed patients, typically within two or more of the following locations: pelvic region lymph nodes, cervix, uterine corpus, fallopian tubes, and ovaries. CONCLUSIONS: Our report adds new insights into the biologic potential of talc and suggests additional anatomic sites that should be closely examined for talc by oncologic surgical pathologists in the setting of perineal talc use.

Correlative polarizing light and scanning electron microscopy for the assessment of talc in pelvic region lymph nodes.

Perineal talc use is associated with ovarian carcinoma in many case-control studies. Such talc may migrate to pelvic organs and regional lymph nodes, with both clinical and legal significance. Our goal was to differentiate talc in pelvic lymph nodes due to exposure, versus contamination with talc in the laboratory. We studied 22 lymph nodes from ovarian tumor patients, some of which had documented talc exposure, to quantify talc using digestion of tissue taken from paraffin blocks and scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX). Talc particles correlated significantly with surface contamination assessments using polarized light microscopy. After adjusting for surface contamination, talc burdens in nodes correlated strongly with perineal talc use. In a separate group of lymph nodes, birefringent particles within the same plane of focus as the tissues in histological sections were highly correlated with talc particles within the tissue by in situ SEM/EDX (r = 0.80; p < 0.0001). We conclude that since talc can be a surface contaminant from tissue collection/preparation, digestion measurements may be influenced by contamination. Instead, because they preserve anatomic landmarks and permit identification of particles in cells and tissues, polarized light microscopy and in situ SEM/EDX are recommended to assess talc in lymph nodes.

Assessment of a Chemotherapy Response Score (CRS) System for Tubo-Ovarian High-Grade Serous Carcinoma (HGSC).

A chemotherapy response score (CRS) system was recently described to assess the histopathologic response and prognosis of patients with tubo-ovarian high-grade serous carcinoma (HGSC) receiving neoadjuvant chemotherapy. The current study was performed as an independent assessment of this CRS system. We retrospectively identified advanced stage HGSC patients who received neoadjuvant chemotherapy and underwent interval debulking. If available, a hemotoxylin and eosin slide from the omentum and the adnexa was selected for the study. Slides were independently scored by 13 pathologists using the 3-tiered CRS system. Reviewers then received web-based training and rescored the slides. Overall survival and progression-free survival were estimated using the Kaplan-Meier method and compared using the log-rank test. A total of 68 patients with omental (n=65) and/or adnexal (n=59) slides were included in the study. Interobserver reproducibility was moderate for omentum (κ, 0.48) and poor for adnexa (κ, 0.40), which improved for omentum (κ, 0.62) but not for adnexa (κ, 0.38) after online training. For omental slides, a consensus CRS of 1/2 was associated with a shorter median progression-free survival (10.9 mo; 95% confidence interval, 9-14) than a CRS of 3 (18.9 mo; 95% CI, 18-24; P=0.020). In summary, a 3-tiered CRS system of hemotoxylin and eosin-stained omental deposits can yield prognostic information for HGSC patients receiving neoadjuvant chemotherapy, and web-based training improved reproducibility but did not alter determination of clinical outcomes. The CRS system may allow oncologists to identify potential nonresponders and triage HGSC patients for heightened observation and/or clinical trials.

Characterization of MicroRNA-200 pathway in ovarian cancer and serous intraepithelial carcinoma of fallopian tube.

BACKGROUND: Ovarian cancer is the leading cause of death among gynecologic diseases in Western countries. We have previously identified a miR-200-E-cadherin axis that plays an important role in ovarian inclusion cyst formation and tumor invasion. The purpose of this study was to determine if the miR-200 pathway is involved in the early stages of ovarian cancer pathogenesis by studying the expression levels of the pathway components in a panel of clinical ovarian tissues, and fallopian tube tissues harboring serous tubal intraepithelial carcinomas (STICs), a suggested precursor lesion for high-grade serous tumors. METHODS: RNA prepared from ovarian and fallopian tube epithelial and stromal fibroblasts was subjected to quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) to determine the expression of miR-200 families, target and effector genes and analyzed for clinical association. The effects of exogenous miR-200 on marker expression in normal cells were determined by qRT-PCR and fluorescence imaging after transfection of miR-200 precursors. RESULTS: Ovarian epithelial tumor cells showed concurrent up-regulation of miR-200, down-regulation of the four target genes (ZEB1, ZEB2, TGFß1 and TGFß2), and up-regulation of effector genes that were negatively regulated by the target genes. STIC tumor cells showed a similar trend of expression patterns, although the effects did not reach significance because of small sample sizes. Transfection of synthetic miR-200 precursors into normal ovarian surface epithelial (OSE) and fallopian tube epithelial (FTE) cells confirmed reduced expression of the target genes and elevated levels of the effector genes CDH1, CRB3 and EpCAM in both normal OSE and FTE cells. However, only FTE cells had a specific induction of CA125 after miR-200 precursor transfection. CONCLUSIONS: The activation of the miR-200 pathway may be an early event that renders the OSE and FTE cells more susceptible to oncogenic mutations and histologic differentiation. As high-grade serous ovarian carcinomas (HGSOC) usually express high levels of CA125, the induction of CA125 expression in FTE cells by miR-200 precursor transfection is consistent with the notion that HGSOC has an origin in the distal fallopian tube.

Cyclin A1 expression and paclitaxel resistance in human ovarian cancer cells.

BACKGROUND: The development of intrinsic and acquired resistance to antineoplastic agents is a major obstacle to successful chemotherapy in ovarian cancers. Identification and characterisation of chemoresponse-associated biomarkers are of paramount importance for novel therapeutic development. METHODS: Global RNA expression profiles were obtained by high-throughput microarray analysis. Cell cycle, proliferation rate, and paclitaxel sensitivity of ovarian cancer cells harbouring cyclin A1-inducible expression construct were compared with and without tetracycline induction, as well as when the cyclin A1 expression was suppressed by short inhibiting RNA (siRNA). Cellular senescence was evaluated by ß-galactosidase activity staining. RESULTS: Global RNA expression profiling and subsequent correlation studies of gene expression level and drug response has identified that elevated expression of cyclin A1 (CCNA1) was significantly associated with cellular resistance to paclitaxel, doxorubicin and 5-fluorouracil. The role of cyclin A1 in paclitaxel resistance was confirmed in ovarian cancer cells that harbour an inducible cyclin A1 expression construct, which showed reduced paclitaxel-mediated growth inhibition and apoptosis when cyclin A1 expression was induced, whereas downregulation of cyclin A1 expression in the same cell lines using cyclin A1-specific siRNAs sensitised the cells to paclitaxel toxicity. However, ovarian cancer cells with ectopic expression of cyclin A1 demonstrated slowdown of proliferation and senescence-associated ß-galactosidase activity. CONCLUSIONS: Our profiling and correlation studies have identified cyclin A1 as one chemoresistance-associated biomarker in ovarian cancer. The results of the characterisation studies suggest that cyclin A1 functions as an oncogene that controls proliferative and survival activities in tumourigenesis and chemoresistance of ovarian cancer.

Endosalpingiosis: More than just an incidental finding at the time of gynecologic surgery?

OBJECTIVE: To describe the clinical characteristics of patients with endosalpingiosis (ES) and examine its association with endometriosis and gynecologic malignancies. METHODS: We queried the medical record for patients who underwent gynecologic surgery (Gynecologic Surgery Cohort (GSC), n=58,161) from 1998 to 2013 at a single institution for the presence of "endosalpingiosis" (ES). Demographic and clinical characteristics were collected for patients with pathologically confirmed ES (n=838). Within GSC, we compared the frequency of endometriosis and gynecologic malignancies with and without ES. We estimated the expected distribution of ovarian cancer subtypes using cases from the New England Case Control Study (NECC). We used chi-square tests to test for significant differences in frequency distributions and unconditional logistic regression to calculate multivariate odds ratios for the association between ES and ovarian cancer subtypes. RESULTS: We observed concurrent endometriosis (p<0.0001), uterine cancer (p<0.0001), and ovarian cancer (p<0.0001) more frequently in women with ES. Women from the GSC with ES and ovarian cancer were more likely to have serous borderline (OR=10.2, 95% CI=5.1-20.7), clear cell (OR=3.0, 95% CI=1.1-8.0), and invasive mucinous tumors (OR=5.0, 95% CI=1.5-16.6) as compared to ovarian cancer cases from the NECC without ES, after accounting for age, race, menopausal status, parity, tubal ligation, and endometriosis. CONCLUSION: Women with ES are more likely to also be diagnosed with endometriosis, uterine, and ovarian cancers. Further study is needed to understand these associations so we may appropriately counsel patients with ES diagnosed at time of gynecologic surgery.

Pinin interacts with C-terminal binding proteins for RNA alternative splicing and epithelial cell identity of human ovarian cancer cells.

Unlike many other human solid tumors, ovarian tumors express many epithelial markers at a high level for cell growth and local invasion. The phosphoprotein Pinin plays a key role in epithelial cell identity. We showed that clinical ovarian tumors and ovarian cancer cell lines express a high level of Pinin when compared with normal ovarian tissues and immortalized normal ovarian surface epithelial cell lines. Pinin co-localized and physically interacted with transcriptional corepressor C-terminal binding proteins, CtBP1 and CtBP2, in the nuclei of cancer cells. Knockdown of Pinin in ovarian cancer cells resulted in specific reduction of CtBP1 protein expression, cell adhesion, anchorage-independent growth, and increased drug sensitivity. Whole transcriptomic comparison of next-generation RNA sequencing data between control ovarian cancer cell lines and cancer cell lines with respective knockdown of Pinin, CtBP1, and CtBP2 expression also showed reduced expression of CtBP1 mRNA in the Pinin knockdown cell lines. The Pinin knockdown cell lines shared significant overlap of differentially expressed genes and RNA splicing aberrations with CtBP1 knockdown and in a lesser degree with CtBP2 knockdown cancer cells. Hence, Pinin and CtBP are oncotargets that closely interact with each other to regulate transcription and pre-mRNA alternative splicing and promote cell adhesion and other epithelial characteristics of ovarian cancer cells.

Pore diameter effects on phase behavior of a gas condensate in graphitic one-and two-dimensional nanopores.

Molecular dynamics (MD) simulations were performed on a hydrocarbon mixture representing a typical gas condensate composed mostly of methane and other small molecules with small fractions of heavier hydrocarbons, representative of mixtures found in tight shale reservoirs. The fluid was examined both in bulk and confined to graphitic nano-scale slits and pores. Numerous widths and diameters of slits and pores respectively were examined under variable pressures at 300 K in order to find conditions in which the fluid at the center of the apertures would not be affected by capillary condensation due to the oil-wet walls. For the bulk fluid, retrograde phase behavior was verified by liquid volumes obtained from Voronoi tessellations. In cases of both one and two-dimensional confinement, for the smallest apertures, heavy molecules aggregated inside the pore space and compression of the gas outside the solid structure lead to decreases in density of the confined fluid. Normal density/pressure relationships were observed for slits having gaps of above 3 nm and pores having diameters above 6 nm. At 70 bar, the minimum gap width at which the fluid could pass through the center of slits without condensation effects was predicted to be 6 nm and the corresponding diameter in pores was predicted to be 8 nm. The models suggest that in nanoscale networks involving pores smaller than these limiting dimensions, capillary condensation should significantly impede transmission of natural gases with similar composition.

Loss of E-cadherin disrupts ovarian epithelial inclusion cyst formation and collective cell movement in ovarian cancer cells.

Increased inclusion cyst formation in the ovary is associated with ovarian cancer development. We employed in vitro three-dimensional (3D) organotypic models formed by normal human ovarian surface epithelial (OSE) cells and ovarian cancer cells to study the morphologies of normal and cancerous ovarian cortical inclusion cysts and the molecular changes during their transitions into stromal microenvironment. When compared with normal cysts that expressed tenascin, the cancerous cysts expressed high levels of laminin V and demonstrated polarized structures in Matrigel; and the cancer cells migrated collectively when the cyst structures were positioned in a stromal-like collagen I matrix. The molecular markers identified in the in vitro 3D models were verified in clinical samples. Network analysis of gene expression of the 3D structures indicates concurrent downregulation of transforming growth factor beta pathway genes and high levels of E-cadherin and microRNA200 (miR200) expression in the cancerous cysts and the migrating cancer cells. Transient silencing of E-cadherin expression in ovarian cancer cells disrupted cyst structures and inhibited collective cell migration. Taken together, our studies employing 3D models have shown that E-cadherin is crucial for ovarian inclusion cyst formation and collective cancer cell migration.

The Association Between Talc Use and Ovarian Cancer: A Retrospective Case-Control Study in Two US States.

BACKGROUND: Multiple studies of ovarian cancer and genital talc use have led only to consensus about possible carcinogenicity. Seeking greater clarity, we examined this association in 2,041 cases with epithelial ovarian cancer and 2,100 age- and-residence-matched controls. METHODS: We defined genital talc use as regular application to the genital/rectal area directly, on sanitary napkins, tampons, or underwear. To estimate "talc-years," we multiplied applications per year by years used. Unconditional logistic regression, Wald statistics, likelihood-ratio tests, and polytomous logistic regression were used to calculate adjusted odds ratios (OR) and 95% confidence intervals (CI), trends, effect-modification, and heterogeneity by ovarian cancer histologic subtype. RESULTS: Overall, genital talc use was associated with an OR (95% CI) of 1.33 (1.16, 1.52), with a trend for increasing risk by talc-years. Women who used talc were more likely to be older, heavier, asthma sufferers, and regular analgesic users--none of which was a confounder. Dose-responses were more apparent for premenopausal women, especially nonsmokers and those heavier or postmenopausal users of menopausal hormones (hormone therapy [HT]). Subtypes of ovarian cancer more likely to be associated with talc included invasive serous and endometrioid tumors and borderline serous and mucinous tumors. Premenopausal women and postmenopausal HT users with these subtypes who had accumulated >24 talc-years had ORs (95% CI) of 2.33 (1.32, 4.12) and 2.57 (1.51, 4.36), respectively. CONCLUSION: Risks for epithelial ovarian cancer from genital talc use vary by histologic subtype, menopausal status at diagnosis, HT use, weight, and smoking. These observations suggest that estrogen and/or prolactin may play a role via macrophage activity and inflammatory response to talc.

Prognostic significance and predictors of the neutrophil-to-lymphocyte ratio in ovarian cancer.

OBJECTIVE: To investigate the neutrophil-to-lymphocyte ratio (NLR) from peripheral blood, a general measure of inflammation, in ovarian cancer. METHODS: White cell counts and CA125 levels before treatment, tumor features, and questionnaire data on 519 women with ovarian cancer at two Boston hospitals were recorded. Counts were log-transformed and effects on these by tumor features and epidemiologic variables assessed by analysis of variance and generalized linear models. Cox proportional hazards models were used to assess effects on overall survival. RESULTS: Greater NLR was associated with higher tumor stage and grade, presence of ascites, and bilateral disease and correlated with risk factors including Jewish ethnicity, taller height, more ovulatory cycles, and family history of cancer in premenopausal women and talc use in all women. CA125 was positively correlated with neutrophil count, monocyte count, and NLR and inversely correlated with lymphocyte count. In a multivariate adjusted analysis, high NLR predicted poorer survival and high lymphocyte count better survival. CONCLUSION: An elevated NLR before treatment signals more aggressive disease and correlates with risk factors for ovarian cancer. CA125 directly correlates with neutrophils which may reflect secretion of both CA125 and neutrophilic growth factors by the tumor. CA125 inversely correlates with lymphocytes which may reflect the ability of some neutrophilic factors to induce lymphopenia and/or binding of CA125 to lymphocytes removing CA125 from the serum pool. Links between NLR, CA125, and epidemiologic factors may provide new clues about the pathogenesis and progression of ovarian cancer.

Endosalpingiosis as it relates to tubal, ovarian and serous neoplastic tissues: an immunohistochemical study of tubal and Müllerian antigens.

OBJECTIVE: The origins and clinical significance of endosalpingiosis (ES), ectopic tubal epithelium, are not well understood. These investigations aim to characterize ES as it relates to normal fallopian tube, ovarian surface and serous neoplasms. METHODS: A retrospective review of pathology reports from all prophylactic gynecologic surgeries from 2000 to 2010 was performed to assess the frequency of ES. Twenty-one archival specimens of ES, 6 normal fallopian tubes, 9 normal ovaries, 21 serous neoplasms and a commercially available ovarian tissue microarray were subjected to immunohistochemistry (IHC) with 11 tubal and Müllerian antigens. IHC staining was evaluated with a quantitative scoring system and scores were analyzed using MINITAB statistical software. RESULTS: ES was noted in 3.5% of pathologic specimens from 464 prophylactic surgeries. The majority of antigens showed no significant differences (p > 0.05) in median IHC scores between ES and normal fallopian tube epithelium (nFTE), while they were significantly different (p < 0.05) from the ovarian surface epithelium (OSE). Median IHC scores were unchanged in ES tissues regardless of the location of ES or the presence of a concurrent serous neoplasm. Three antigens emerged as contemporary tubal and ES biomarkers: phospho-Smad2, BCL2 and FOXJ1. All 3 biomarkers were expressed in ES, nFTE and serous neoplasms, but not in OSE or other tumor types. CONCLUSION: This study provides immunophenotypic evidence that ES is more similar to the nFTE than OSE. Further, ES biomarker expression closely resembles serous neoplasms strengthening the growing body of evidence that all Müllerian serous carcinomas arise from tubal-like epithelium.

Insight into the packing pattern of ß2 fibrils: a model study of glutamic acid rich oligomers with 13C isotopic edited vibrational spectroscopy.

Polyglutamic acid at low pH forms aggregates and self-assembles into a spiral, fibril-like superstructure formed as a ß2-type sheet conformation that has a more compact intersheet packing than commonly found. This is stabilized by three-centered bifurcated hydrogen bonding of the amide carbonyl involving the protonated glutamic acid side chain. We report vibrational spectroscopic results and analyses for oligopeptides rich in glutamic acid enhanced with (13)C isotope labeling in a study modeling low pH poly-Glu self-assembly. Our results indicate bifurcated H-bonding and ß2 aggregation can be attained in these model decamers, confirming they have the same conformations as poly-Glu. We also prepared conventional ß1-sheet aggregates by rapid precipitation from the residual peptides in the higher pH supernatant. By comparing the isotope-enhanced IR and VCD spectra with theoretical predictions, we deduced that the oligo-Glu ß2 structure is based on stacked, twisted, antiparallel ß-sheets. The best fit to theoretical predictions was obtained for the strands being out of register, sequentially stepped by one residue, in a ladder-like fashion. The alternate ß1 conformer for this oligopeptide was similarly shown to be antiparallel but was less ordered and apparently had a different registry in its aggregate structure.

Puerperal mastitis: a reproductive event of importance affecting anti-mucin antibody levels and ovarian cancer risk.

PURPOSE: Test the hypothesis that puerperal mastitis may alter immunity related to the mucin (MUC) family of glycoproteins and lower risk of ovarian cancer. METHODS: In two case-control studies conducted in New England between 1998 and 2008, we examined the association between self-reported mastitis and ovarian cancer in 1,483 women with epithelial ovarian cancer and 1,578 controls. IgG1 antibodies against (MUC1) CA15.3 and (MUC16) CA125 were measured using electrochemiluminescence assays in a subset of controls (n = 200). Preoperative CA125 was recorded in 649 cases. The association between ovarian cancer and mastitis was assessed using unconditional logistic regression to calculate adjusted odds ratios, OR, and 95 % confidence intervals (CI). Associations between mastitis and anti-CA15.3 and anti-CA125 antibodies and preoperative CA125 levels were evaluated using adjusted linear regression models. RESULTS: Prior mastitis was associated with a significantly lower risk of ovarian cancer: OR (and 95 % CI) of 0.67 (0.48, 0.94) adjusted for parity, breastfeeding, and other potential confounders. The association was strongest with 2 or more episodes of mastitis, and risk declined progressively with increasing number of children and episodes of mastitis. Among controls, prior mastitis was associated with significantly higher anti-CA15.3 and anti-CA125 antibody levels and, among cases, with significantly lower preoperative CA125 levels. CONCLUSION: Puerperal mastitis may produce long-lasting anti-mucin antibodies that may lower the risk of ovarian cancer, plausibly through enhanced immune surveillance. Studying immune reactions related to MUC1 and MUC16 in the 10-20 % of breastfeeding women who develop mastitis may suggest ways to duplicate its effects through vaccines based on both antigens.

Structural analyses of experimental 13C edited amide I' IR and VCD for peptide ß-sheet aggregates and fibrils using DFT-based spectral simulations.

In the preceding paper, computational models based on density functional theory (DFT) were presented to characterize the sensitivity of vibrational spectroscopic methods (IR, VCD, and Raman) to structural features of ß-sheets. Isotopically edited amide I' IR for peptides labeled with (13)C in multiple different sites provides the most structurally distinct signatures of strand alignment, while VCD is sensitive to the sheet twist and intersheet stacking. In this report, we simulate the IR and VCD spectra for models approximating structures of four ß-sheet forming peptides previously experimentally studied using these methods with (13)C isotopic editing. Various register alignments are tested. Agreement with experiment is evaluated based on frequency shifts of both the (12)C and (13)C IR amide I' signals, relative intensity patterns, and VCD spectra where available. While for the simulation of IR spectra canonical planar sheets provide a sufficient model system, for VCD simulation twisted, stacked sheets are required in order to reproduce strong couplet-like amide I' VCD. Effects of the solvent (water) and amino acid side chains are also tested by using a simplified, electrostatic solvent model and atomic partial charges for the side chains. Very good agreement with experimental spectra is obtained, particularly for the relative (12)C and (13)C band frequencies. All four peptide models are shown to be antiparallel as had previously been assumed. However, in some cases our simulations are consistent with different register alignment of strands than originally proposed.