The Supragingival Biofilm in Early Childhood Caries: Clinical and Laboratory Protocols and Bioinformatics Pipelines Supporting Metagenomics, Metatranscriptomics, and Metabolomics Studies of the Oral Microbiome.

Early childhood caries (ECC) is a biofilm-mediated disease. Social, environmental, and behavioral determinants as well as innate susceptibility are major influences on its incidence; however, from a pathogenetic standpoint, the disease is defined and driven by oral dysbiosis. In other words, the disease occurs when the natural equilibrium between the host and its oral microbiome shifts toward states that promote demineralization at the biofilm-tooth surface interface. Thus, a comprehensive understanding of dental caries as a disease requires the characterization of both the composition and the function or metabolic activity of the supragingival biofilm according to well-defined clinical statuses. However, taxonomic and functional information of the supragingival biofilm is rarely available in clinical cohorts, and its collection presents unique challenges among very young children. This paper presents a protocol and pipelines available for the conduct of supragingival biofilm microbiome studies among children in the primary dentition, that has been designed in the context of a large-scale population-based genetic epidemiologic study of ECC. The protocol is being developed for the collection of two supragingival biofilm samples from the maxillary primary dentition, enabling downstream taxonomic (e.g., metagenomics) and functional (e.g., transcriptomics and metabolomics) analyses. The protocol is being implemented in the assembly of a pediatric precision medicine cohort comprising over 6000 participants to date, contributing social, environmental, behavioral, clinical, and biological data informing ECC and other oral health outcomes.

Intra-species Genomic and Physiological Variability Impact Stress Resistance in Strains of Probiotic Potential.

Large-scale microbiome studies have established that most of the diversity contained in the gastrointestinal tract is represented at the strain level; however, exhaustive genomic and physiological characterization of human isolates is still lacking. With increased use of probiotics as interventions for gastrointestinal disorders, genomic and functional characterization of novel microorganisms becomes essential. In this study, we explored the impact of strain-level genomic variability on bacterial physiology of two novel human Lactobacillus rhamnosus strains (AMC143 and AMC010) of probiotic potential in relation to stress resistance. The strains showed differences with known probiotic strains (L. rhamnosus GG, Lc705, and HN001) at the genomic level, including nucleotide polymorphisms, mutations in non-coding regulatory regions, and rearrangements of genomic architecture. Transcriptomics analysis revealed that gene expression profiles differed between strains when exposed to simulated gastrointestinal stresses, suggesting the presence of unique regulatory systems in each strain. In vitro physiological assays to test resistance to conditions mimicking the gut environment (acid, alkali, and bile stress) showed that growth of L. rhamnosus AMC143 was inhibited upon exposure to alkaline pH, while AMC010 and control strain LGG were unaffected. AMC143 also showed a significant survival advantage compared to the other strains upon bile exposure. Reverse transcription qPCR targeting the bile salt hydrolase gene (bsh) revealed that AMC143 expressed bsh poorly (a consequence of a deletion in the bsh promoter and truncation of bsh gene in AMC143), while AMC010 had significantly higher expression levels than AMC143 or LGG. Insertional inactivation of the bsh gene in AMC010 suggested that bsh could be detrimental to bacterial survival during bile stress. Together, these findings show that coupling of classical microbiology with functional genomics methods for the characterization of bacterial strains is critical for the development of novel probiotics, as variability between strains can dramatically alter bacterial physiology and functionality.

Chemosensitivity of IDH1-Mutated Gliomas Due to an Impairment in PARP1-Mediated DNA Repair.

Mutations in isocitrate dehydrogenase (IDH) are the most prevalent genetic abnormalities in lower grade gliomas. The presence of these mutations in glioma is prognostic for better clinical outcomes with longer patient survival. In the present study, we found that defects in oxidative metabolism and 2-HG production confer chemosensitization in IDH1-mutated glioma cells. In addition, temozolomide (TMZ) treatment induced greater DNA damage and apoptotic changes in mutant glioma cells. The PARP1-associated DNA repair pathway was extensively compromised in mutant cells due to decreased NAD+ availability. Targeting the PARP DNA repair pathway extensively sensitized IDH1-mutated glioma cells to TMZ. Our findings demonstrate a novel molecular mechanism that defines chemosensitivity in IDH-mutated gliomas. Targeting PARP-associated DNA repair may represent a novel therapeutic strategy for gliomas. Cancer Res; 77(7); 1709-18. ©2017 AACR.

Metastasis-associated protein 3 (MTA3) regulates G2/M progression in proliferating mouse granulosa cells.

Metastasis-associated protein 3 (MTA3) is a constituent of the Mi-2/nucleosome remodeling and deacetylase (NuRD) protein complex that regulates gene expression by altering chromatin structure and can facilitate cohesin loading onto DNA. The biological function of MTA3 within the NuRD complex is unknown. Herein, we show that MTA3 was expressed highly in granulosa cell nuclei of all ovarian follicle stages and at lower levels in corpora lutea. We tested the hypothesis that MTA3-NuRD complex function is required for granulosa cell proliferation. In the ovary, MTA3 interacted with NuRD proteins CHD4 and HDAC1 and the core cohesin complex protein RAD21. In cultured mouse primary granulosa cells, depletion of endogenous MTA3 using RNA interference slowed cell proliferation; this effect was rescued by coexpression of exogenous MTA3. Slowing of cell proliferation correlated with a significant decrease in cyclin B1 and cyclin B2 expression. Granulosa cell populations lacking MTA3 contained a significantly higher percentage of cells in G2/M phase and a lower percentage in S phase compared with control cells. Furthermore, MTA3 depletion slowed entry into M phase as indicated by reduced phosphorylation of histone H3 at serine 10. These findings provide the first evidence to date that MTA3 interacts with NuRD and cohesin complex proteins in the ovary in vivo and regulates G2/M progression in proliferating granulosa cells.

Lack of functional pregnancy-associated plasma protein-A (PAPPA) compromises mouse ovarian steroidogenesis and female fertility.

The insulin-like growth factor (IGF) system plays an important role in regulating ovarian follicular development and steroidogenesis. IGF binding proteins (IGFBP) mostly inhibit IGF actions, and IGFBP proteolysis is a major mechanism for regulating IGF bioavailability. Pregnancy-associated plasma protein-A (PAPPA) is a secreted metalloprotease responsible for cleavage of IGFBP4 in the ovary. The aim of this study was to investigate whether PAPPA plays a role in regulating ovarian functions and female fertility by comparing the reproductive phenotype of wild-type (WT) mice with mice heterozygous or homozygous for a targeted Pappa gene deletion (heterozygous and PAPP-A knockout [KO] mice, respectively). When mated with WT males, PAPP-A KO females demonstrated an overall reduction in average litter size. PAPP-A KO mice had a reduced number of ovulated oocytes, lower serum estradiol levels following equine chorionic gonadotropin administration, lower serum progesterone levels after human chorionic gonadotropin injection, and reduced expression of ovarian steroidogenic enzyme genes, compared to WT controls. In PAPP-A KO mice, inhibitory IGFBP2, IGFBP3, and IGFBP4 ovarian gene expression was reduced postgonadotropin stimulation, suggesting some compensation within the ovarian IGF system. Expression levels of follicle-stimulating hormone receptor, luteinizing hormone receptor, and genes required for cumulus expansion were not affected. Analysis of preovulatory follicular fluid showed complete loss of IGFBP4 proteolytic activity in PAPP-A KO mice, demonstrating no compensation for loss of PAPPA proteolytic activity by other IGFBP proteases in vivo in the mouse ovary. Taken together, these data demonstrate an important role of PAPPA in modulating ovarian function and female fertility by control of the bioavailability of ovarian IGF.

Peroxisome proliferator-activated receptor-gamma mediates bisphenol A inhibition of FSH-stimulated IGF-1, aromatase, and estradiol in human granulosa cells.

BACKGROUND: Bisphenol A (BPA), a chemical used as a plasticizer, is a potent endocrine disruptor that, even in low concentrations, disturbs normal development and functions of reproductive organs in different species. OBJECTIVES: We investigated whether BPA affects human ovarian granulosa cell function. METHODS: We treated KGN granulosa cells and granulosa cells from subjects undergoing in vitro fertilization (IVF) with follicle-stimulating hormone (FSH), BPA, or BPA plus FSH in a dose- and time-dependent manner. We then evaluated expression of insulin-like growth factor 1 (IGF-1), aromatase, and transcription factors known to mediate aromatase induction by FSH [including steroidogenic factor-1 (SF-1), GATA4, cAMP response element binding protein-1 (CREB-1), and peroxisome proliferator-activated receptor-gamma (PPARgamma)], as well as 17beta-estradiol (E2) secretion. KGN cells were transfected with a PPARgamma-containing vector, followed by assessment of aromatase and IGF-I expression. RESULTS: BPA reduced FSH-induced IGF-1 and aromatase expression and E2 secretion in a dose-dependent fashion. Similar effects on aromatase were observed in IVF granulosa cells. SF-1 and GATA4, but not CREB-1, were reduced after BPA treatment, although PPARgamma, an inhibitor of aromatase, was significantly up-regulated by BPA in a dose-dependent manner, with simultaneous decrease of aromatase. Overexpression of PPARgamma in KGN cells reduced FSH-stimulated aromatase and IGF-1 mRNAs, with increasing concentrations of the transfected expression vector, mimicking BPA action. Also, BPA reduced granulosa cell DNA synthesis without changing DNA fragmentation, suggesting that BPA does not induce apoptosis. CONCLUSIONS: Overall, the data demonstrate that BPA induces PPARgamma, which mediates down-regulation of FSH-stimulated IGF-1, SF-1, GATA4, aromatase, and E2 in human granulosa cells. These observations support a potential role of altered steroidogenesis and proliferation within the ovarian follicular compartment due to this endocrine disruptor.

The interplay of insulin-like growth factors, gonadotropins, and endocrine disruptors in ovarian follicular development and function.

Over the past 20 years, the expression, signaling mechanisms, and roles of members of the insulin-like growth factor (IGF) family (ligands, receptors, binding proteins, and binding protein proteases and their inhibitors) have been elucidated in ovarian follicle function in humans and other species. In vitro studies with human, nonhuman primate, and farm animal granulosa and thecal cells and genetic approaches using mouse knockout models for IGF family members have revealed that IGFs are key intraovarian regulators of follicle growth, selection, atresia, cellular differentiation, and steroidogenesis, oocyte maturation, and cumulus expansion. Some of these actions are synergistic with gonadotropins, although most are not sustainable with IGFs alone and require gonadotropin actions, thereby designating IGFs as "co-gonadotropins." In the human disorder of polycystic ovarian syndrome, characterized by small antral follicle arrest, the IGF system appears to contribute to the observed resistance to follicle-stimulating hormone action at the level of the granulosa compartment and the persistence of an androgen-dominant milieu in the arrested follicles. Interestingly, recent studies demonstrate that endocrine-disrupting chemicals can compromise IGF activity and signaling in the ovarian follicle, affecting follicle development, steroidogenesis, and oocyte quality. The successful development of a healthy oocyte and appropriate granulosa and theca cell steroidogenesis on a cyclic basis are contingent on multiple factors, including a properly functioning intraovarian IGF system. Disruption of even one component of this system can lead to abnormal follicular development and function and compromised reproductive capacity.

Identification of regulatory elements in the Cyp19 proximal promoter in rat luteal cells.

The cytochrome P450 aromatase (Cyp19) gene encodes an enzyme of crucial importance in the synthesis of estradiol. Estradiol is luteotropic in the rat. In this species, luteal Cyp19 expression increases progressively during pregnancy and falls before parturition. The mechanisms that control these changes are unknown. Using gel shift assays, we sought to identify the promoter regions that control Cyp19 expression in the rat corpus luteum (CL). The Cyp19 promoter contains a cAMP response element-like sequence (CLS), two nuclear receptor elements half sites (NREs), a GATA binding site, a Yin Yang-1 (YY1) response element, and an activation protein 3 (AP3) binding site. Nuclear extracts were obtained from CL of rats on days 4, 15, and 23 of pregnancy and from the ovaries of immature rats treated with vehicle or a hormone that induces Cyp19 expression in the follicles. CLS was active in immature ovaries but inactive in the CL of pregnant rats, whereas binding to NREs and GATA was observed in both tissues. YY1 was inactive in all samples tested. In the CL, AP3 binding was higher on day 15 of pregnancy when compared with day 4 and day 23 but it was absent in ovaries of immature rats, whereas luteinization increased AP3 binding activity. Mutation of the AP3 site blunted the stimulation of Cyp19 promoter activity in granulosa cells. Our results indicate that CLS is active only in follicles; whereas in the CL, binding to the GATA, NRE, and AP3 sites associates with changes in Cyp19 expression, suggesting that they control Cyp19 promoter activity in luteal cells.

Follicle-stimulating hormone-induced activation of Gata4 contributes in the up-regulation of Cyp19 expression in rat granulosa cells.

Several studies have suggested that the transcription factor GATA4 plays an important role in ovarian function. This study evaluated the effects of GATA4 on the regulation of the Cyp19 gene in primary rat granulosa cells under basal conditions and in response to stimulation by FSH. A significant increase in GATA4 mRNA, protein, and DNA binding activity was observed in rats treated with pregnant mare serum gonadotropin, a hormone that binds to the FSH receptors, and in granulosa cells incubated with FSH. Enrichment of the Cyp19 promoter was observed in granulosa cells treated with FSH after chromatin precipitation with an anti-GATA4 antibody. Mutation of the GATA binding site on the Cyp19 promoter and inhibition of GATA4 expression with specific small interfering RNA significantly reduced FSH-enhanced Cyp19 expression, whereas overexpression of GATA4 increased Cyp19 promoter activity. A synergistic effect observed between GATA4 overexpression and FSH treatment in Cyp19 expression was abolished by mutating Ser105 in the GATA4 protein or by pretreating granulosa cells with a protein kinase A inhibitor. Inhibition of phosphatidylinositol-dependent kinase (PI3-K)/casein kinase 2 or ERK1/2 attenuated GATA4/FSH synergism, whereas the simultaneous blockade of PI3-K/casein kinase 2 and ERK1/2 activity eliminated Cyp19 stimulation. Finally, we demonstrated that FSH increases GATA4 phosphorylation and that GATA4 activation requires the activation of multiple kinases, including ERK1/2, PI3-K, and protein kinase A. These findings demonstrate that GATA4 contributes in the regulation of Cyp19 expression in the rat ovary and provide the first evidence that FSH regulates GATA4 activity.

Prostaglandin E2 increases cyp19 expression in rat granulosa cells: implication of GATA-4.

The expression of Cyp19, the key gene of estrogen biosynthesis, in granulosa cells (GC) is essential for follicular growth and coordination of the ovulatory process. The goal of this study was to examine the effect of PGE2 and PGF2alpha on Cyp19 expression in undifferentiated and luteinized GC (UGC and LGC). In UGC, PGE2 increased Cyp19 mRNA and Cyp19 protein levels whereas PGF2alpha had no effect. In LGC, PGF2alpha decreased Cyp19 expression whereas PGE2 had no effect. Gene-reporter experiments demonstrated that PGE2 increases Cyp19 transcription in UGC. A protein kinase A inhibitor blocked PGE2-induced increase in Cyp19 promoter activity. PGE2 increased GATA-4 binding to the Cyp19 promoter. Mutation of the GATA binding site resulted in the loss of PGE2 stimulation. This study demonstrates that PGE2 stimulates Cyp19 expression in rat GC and suggests that GATA-4 may mediate (at least in part) the stimulatory effect of PGE2.

Mevastatin inhibits proliferation of rat ovarian theca-interstitial cells by blocking the mitogen-activated protein kinase pathway.

OBJECTIVE: To evaluate mechanisms involved in mevastatin-induced inhibition of proliferation of ovarian theca-interstitial cells. DESIGN: In vitro study. SETTING: Academic laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): Ovarian theca-interstitial cells were cultured without and with mevastatin in the presence and absence of serum, mevalonic acid, and/or insulin. MAIN OUTCOME MEASURE(S): Proliferation was assessed by determination of DNA synthesis by thymidine incorporation assay. Activation of extracellular signal-regulated kinase (Erk1/2) and of Akt/protein kinase B (PKB) was determined by ELISA. RESULT(S): Mevastatin induced a concentration-dependent inhibition of theca-interstitial cell proliferation in the absence and in the presence of serum. Inhibitory effects of mevastatin were partly abrogated by mevalonic acid and by insulin. Mevastatin blocked basal and insulin-induced phosphorylation of ERK1/2. In contrast, mevastatin had no significant effect on either basal or insulin-induced phosphorylation of Akt/PKB. CONCLUSION(S): Mevastatin inhibits proliferation of theca-interstitial cells by a mechanism that involves depletion of mevalonic acid and selective inhibition of basal and insulin-induced activity of Erk1/2 pathway, but not Akt/PKB pathway. These effects of mevastatin may be a result of decreased isoprenylation of small GTPases.

Statins inhibit growth of human endometrial stromal cells independently of cholesterol availability.

Endometriosis is characterized by ectopic growth of endometrial tissues. Statins, inhibitors of 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMGCR), have been shown to decrease proliferation of several mesenchymal tissues. Actions of statins may be related to decreased availability of cholesterol as well as intermediate metabolites of the mevalonate pathway downstream of HMGCR. This study was designed to evaluate effects of statins on growth of endometrial stromal cells and to investigate mechanisms of these effects. Human endometrial stromal cells were cultured in the absence and in the presence of serum and with or without mevastatin and simvastatin. DNA synthesis and viable cell numbers were determined. Effects of statins were also evaluated in the presence of mevalonate and squalene. Furthermore, effects on phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) (also known as extracellular signal-regulated kinase [ERK1/2]) were determined. Mevastatin and simvastatin induced a concentration-dependent inhibition of DNA synthesis and viable cell count in chemically defined media and in the presence of serum. Mevalonate, but not squalene, abrogated inhibitory effects of statins on cell proliferation. Statins inhibited MAPK3/1 phosphorylation. This is the first study demonstrating that statins inhibit growth of endometrial stromal cells. This effect is also demonstrable in the presence of a supply of cholesterol and may be related to decreased activation of MAPK3/1. The present observations may be relevant to potential therapeutic use of statins in conditions such as endometriosis.

Insulin and oxidative stress modulate proliferation of rat ovarian theca-interstitial cells through diverse signal transduction pathways.

Insulin and moderate oxidative stress stimulate proliferation of ovarian theca-interstitial cells. The effects of these agents on selected signal transduction pathways were examined. PD98059 (inhibitor of MAP2K1, also known as MEK-1, upstream of extracellular signal-regulated protein kinases MAPK3/1, also known as ERK1/2), wortmannin (inhibitor of PIK3C2A, also known as PI3K), and rapamycin (inhibitor of FRAP1, also known as mTOR, upstream of RPS6KB1) each significantly decreased insulin and oxidative stress-induced proliferation of theca-interstitial cells. The greatest inhibition was observed in the presence of rapamycin; this effect occurred without a significant change in cell viability. Phosphorylation of AKT was stimulated by insulin only, while phosphorylation of MAPK3/1 and RPS6KB1 was increased by insulin and oxidative stress. Insulin-induced and oxidative stress-induced phosphorylation of RPS6KB1 was partly inhibited by wortmannin and partly by PD98059; the greatest inhibition was observed in the presence of a combination of wortmannin plus PD98059. Effects of insulin and oxidative stress on phosphorylation of RPS6KB1 were confirmed by kinase activity assays. These findings indicate that actions of insulin and oxidative stress converge on MAPK3/1 and RPS6KB1. Furthermore, we speculate that activation of RPS6KB1 may be in part induced via the MAPK3/1 pathway.

Effects of oxidants and antioxidants on proliferation of endometrial stromal cells.

OBJECTIVE: To evaluate the effects of oxidative stress and antioxidants on proliferation of endometrial stromal cells. DESIGN: In vitro study. SETTING: Academic laboratory. PATIENT(S): Women, with and without endometriosis, of reproductive age. INTERVENTION(S): Culture of endometrial stromal cells with antioxidants or with agents inducing oxidative stress. MAIN OUTCOME MEASURE(S): Proliferation of endometrial stromal cells as determined by thymidine incorporation assay and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay. RESULT(S): Antioxidants induced a dose-dependent inhibition of thymidine incorporation: vitamin E succinate was inhibitory at 10-100 microM (by 43%-95%), ebselen at 10-30 microM (by 29%-77%), and N-acetylcysteine at 10-30 mM (by 52%-85%). In contrast, modest oxidative stress induced by hypoxanthine/xanthine oxidase (1 mM/3-30 microU/mL) stimulated proliferation by 40%-62%. H2O2 (1 microM) increased DNA synthesis by 56%. Comparable findings were obtained using MTT proliferation assay. Antioxidants inhibited proliferation: vitamin E succinate (100 microM) by 91%, ebselen (30 microM) by 81%, and N-acetylcysteine (30 mM) by 95%. Hypoxanthine/xanthine oxidase (1 mM/30 microU/mL) and H2O2 (1 microM) stimulated growth by 122% and 58%, respectively. CONCLUSION(S): Reactive oxygen species may modulate growth of endometrial stroma. Under pathologic conditions such as endometriosis, increased oxidative stress and depletion of antioxidants may contribute to excessive growth of endometrial stromal cells.

Mevastatin inhibits ovarian theca-interstitial cell proliferation and steroidogenesis.

OBJECTIVE: Statins reduce cardiovascular risks by improving hypercholesterolemia, reducing vascular smooth muscle proliferation, and ameliorating inflammation. Polycystic ovary syndrome (PCOS) is associated with increased cardiovascular risks and is characterized by ovarian theca-interstitial hyperplasia and hyperandrogenism. This study tested the hypothesis that mevastatin limits theca-interstitial proliferation and decreases steroidogenesis. DESIGN: In vitro study. SETTING: Academic laboratory. PATIENT(S): None. INTERVENTION(S): Effects of mevastatin on cultured theca-interstitial cells. MAIN OUTCOME MEASURE(S): Proliferation was evaluated by determination of DNA synthesis using thymidine incorporation assay and by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay. Production of P and T was determined by specific radioimmunoassays. RESULT(S): Mevastatin induced a profound concentration-dependent inhibition of DNA synthesis. At the highest concentration (30 microM), mevastatin inhibited DNA synthesis by 92%. Similarly, in the MTT proliferation assay, mevastatin induced a concentration-dependent decrease in cell number. Mevastatin decreased production of P (by up to 49%) and T (by up to 52%); these effects remained significant when the effect on cell culture protein content was accounted for. CONCLUSION(S): Mevastatin inhibits proliferation of theca-interstitial cells; it also inhibits P and T production independently of the effects on cell growth. These findings provide a foundation for studies evaluating statins as potential therapeutic agents in the treatment of ovarian mesenchymal hyperplasia and hyperandrogenism characteristic of PCOS.