Jason S. Lee

Analysis of the CO2 adsorption properties of a well-known series of metal–organic frameworks M2(dobdc) (dobdc4− = 2,5-dioxido-1,4-benzenedicarboxylate; M = Mg, Mn, Fe, Co, Ni, Cu, and Zn) is carried out in tandem with in situ structural studies to identify the host–guest interactions that lead to significant differences in isosteric heats of CO2 adsorption. Neutron and X-ray powder diffraction and single crystal X-ray diffraction experiments are used to unveil the site-specific binding properties of CO2 within many of these materials while systematically varying both the amount of CO2 and the temperature. Unlike previous studies, we show that CO2 adsorbed at the metal cations exhibits intramolecular angles with minimal deviations from 180°, a finding that indicates a strongly electrostatic and physisorptive interaction with the framework surface and sheds more light on the ongoing discussion regarding whether CO2 adsorbs in a linear or nonlinear geometry. This has important implications for proposals that have been made to utilize these materials for the activation and chemical conversion of CO2. For the weaker CO2 adsorbents, significant elongation of the metal–O(CO2) distances are observed and diffraction experiments additionally reveal that secondary CO2 adsorption sites, while likely stabilized by the population of the primary adsorption sites, significantly contribute to adsorption behavior at ambient temperature. Density functional theory calculations including van der Waals dispersion quantitatively corroborate and rationalize observations regarding intramolecular CO2 angles and trends in relative geometric properties and heats of adsorption in the M2(dobdc)–CO2 adducts.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.

Ubiquitination plays a major role in protein degradation. Although phosphorylation-dependent ubiquitination is well known for the regulation of protein stability, methylation-dependent ubiquitination machinery has not been characterized. Here, we provide evidence that methylation-dependent ubiquitination is carried out by damage-specific DNA binding protein 1 (DDB1)/cullin4 (CUL4) E3 ubiquitin ligase complex and a DDB1-CUL4-associated factor 1 (DCAF1) adaptor, which recognizes monomethylated substrates. Molecular modeling and binding affinity studies reveal that the putative chromo domain of DCAF1 directly recognizes monomethylated substrates, whereas critical binding pocket mutations of the DCAF1 chromo domain ablated the binding from the monomethylated substrates. Further, we discovered that enhancer of zeste homolog 2 (EZH2) methyltransferase has distinct substrate specificities for histone H3K27 and nonhistones exemplified by an orphan nuclear receptor, RORα. We propose that EZH2-DCAF1/DDB1/CUL4 represents a previously unrecognized methylation-dependent ubiquitination machinery specifically recognizing "methyl degron"; through this, nonhistone protein stability can be dynamically regulated in a methylation-dependent manner.

Post-translational modifications of DNA and histones are epigenetic mechanisms, which affect the chromatin structure, ultimately leading to gene expression changes. A number of different epigenetic enzymes are actively involved in the addition or the removal of various covalent modifications, which include acetylation, methylation, phosphorylation, ubiquitination, and sumoylation. Deregulation of these processes is a hallmark of cancer. For instance, G9a, a histone methyltransferase responsible for histone H3 lysine 9 (H3K9) mono- and dimethylation, has been observed to be upregulated in different types of cancer and its overexpression has been associated with poor prognosis. Key roles played by these enzymes in various diseases have led to the hypothesis that these molecules represent valuable targets for future therapies. Several small molecule inhibitors have been developed to specifically block the epigenetic activity of these enzymes, representing promising therapeutic tools in the treatment of human malignancies, such as cancer. In this review, the role of one of these epigenetic enzymes, G9a, is discussed, focusing on its functional role in regulating gene expression as well as its implications in cancer initiation and progression. We also discuss important findings from recent studies using epigenetic inhibitors in cell systems in vitro as well as experimental tumor growth and metastasis assays in vivo.

The following is a concise review of the literature that addresses the impact of marine biofilms on two phenomena—ennoblement of corrosion potential and sulfide derivitization due to sulfate-reducing bacteria. A universally defined mechanism of potential ennoblement has not been established. Extent of ennoblement varies among locations and the extent of ennoblement for a particular material cannot be used to predict an increased likelihood of localized corrosion. There is some controversy as to the susceptibility of low- and medium-grade stainless steels. Carbon steel and copper alloys are susceptible to sulfide derivitization but thermodynamic models cannot predict the susceptibility of these materials. Laboratory experiments designed to provide data on susceptibility to sulfide derivitization have produced conflicting results because of the following: (1) laboratory media can contain anions that inhibit localized corrosion, (2) laboratory media can contain yeast extract that interferes with electrochemical measurements, and (3) deaeration procedures can produce environments that are not conducive for the growth of sulfate-reducing bacteria. In general, alloys that undergo ennoblement are not vulnerable to sulfide derivitization and conversely, alloys that are subject to sulfide derivitization do not become ennobled.

Wnt family members play diverse roles in development and disease. Noncanonical Wnt ligands can inhibit canonical Wnt signaling depending on the cellular context; however, the underlying mechanism of this antagonism remains poorly understood. Here we identify a specific mechanism of orphan nuclear receptor RORα-mediated inhibition of canonical Wnt signaling in colon cancer. Wnt5a/PKCα-dependent phosphorylation on serine residue 35 of RORα is crucial to link RORα to Wnt/β-catenin signaling, which exerts inhibitory function of the expression of Wnt/β-catenin target genes. Intriguingly, there is a significant correlation of reduction of RORα phosphorylation in colorectal tumor cases compared to their normal counterpart, providing the clinical relevance of the findings. Our data provide evidence for a role of RORα, functioning at the crossroads between the canonical and the noncanonical Wnt signaling pathways, in mediating transrepression of the Wnt/β-catenin target genes, thereby providing new approaches for the development of therapeutic agents for human cancers.

Lysine methylation within histones is crucial for transcriptional regulation and thus links chromatin states to biological outcomes. Although recent studies have extended lysine methylation to nonhistone proteins, underlying molecular mechanisms such as the upstream signaling cascade that induces lysine methylation and downstream target genes modulated by this modification have not been elucidated. Here, we show that Reptin, a chromatin-remodeling factor, is methylated at lysine 67 in hypoxic conditions by the methyltransferase G9a. Methylated Reptin binds to the promoters of a subset of hypoxia-responsive genes and negatively regulates transcription of these genes to modulate cellular responses to hypoxia.

Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes such as soft tissue reconstruction following mastectomy; however, the ability of tumors to commandeer ASC functions to advance tumor progression is not well understood. Through the integration of physical sciences and oncology approaches we investigated the capability of tumor-derived chemical and mechanical cues to enhance ASC-mediated contributions to tumor stroma formation. Our results indicate that soluble factors from breast cancer cells inhibit adipogenic differentiation while increasing proliferation, proangiogenic factor secretion, and myofibroblastic differentiation of ASCs. This altered ASC phenotype led to varied extracellular matrix (ECM) deposition and contraction thereby enhancing tissue stiffness, a characteristic feature of breast tumors. Increased stiffness, in turn, facilitated changes in ASC behavior similar to those observed with tumor-derived chemical cues. Orthotopic mouse studies further confirmed the pathological relevance of ASCs in tumor progression and stiffness in vivo. In summary, altered ASC behavior can promote tumorigenesis and, thus, their implementation for regenerative therapy should be carefully considered in patients previously treated for cancer.

We analyzed 3,872 common genetic variants across the ESR1 locus (encoding estrogen receptor α) in 118,816 subjects from three international consortia. We found evidence for at least five independent causal variants, each associated with different phenotype sets, including estrogen receptor (ER(+) or ER(-)) and human ERBB2 (HER2(+) or HER2(-)) tumor subtypes, mammographic density and tumor grade. The best candidate causal variants for ER(-) tumors lie in four separate enhancer elements, and their risk alleles reduce expression of ESR1, RMND1 and CCDC170, whereas the risk alleles of the strongest candidates for the remaining independent causal variant disrupt a silencer element and putatively increase ESR1 and RMND1 expression.

Cancer is a disease arising from both genetic and epigenetic modifications of DNA that contribute to changes in gene expression in the cell. Genetic modifications include loss or amplification of DNA, loss of heterozygosity (LOH) as well as gene mutations. Epigenetic changes in cancer are generally thought to be brought about by alterations in DNA and histone modifications that lead to the silencing of tumour suppressor genes and the activation of oncogenic genes. Other consequences that result from epigenetic changes, such as inappropriate expression or repression of some genes in the wrong cellular context, can also result in the alteration of control and physiological systems such that a normal cell becomes tumorigenic. Excessive levels of the enzymes that act as epigenetic modifiers have been reported as markers of aggressive breast cancer and are associated with metastatic progression. It is likely that this is a common contributor to the recurrence and spread of the disease. The emphasis on genetic changes, for example in genome-wide association studies and increasingly in whole genome sequencing analyses of tumours, has resulted in the importance of epigenetic changes having less attention until recently. Epigenetic alterations at both the DNA and histone level are increasingly being recognised as playing a role in tumourigenesis. Recent studies have found that distinct subgroups of poor-prognosis tumours lack genetic alterations but are epigenetically deregulated, pointing to the important role that epigenetic modifications and/or their modifiers may play in cancer. In this review, we highlight the multitude of epigenetic changes that can occur and will discuss how deregulation of epigenetic modifiers contributes to cancer progression. We also discuss the off-target effects that epigenetic modifiers may have, notably the effects that histone modifiers have on non-histone proteins that can modulate protein expression and activity, as well as the role of hypoxia in epigenetic regulation.

Type 1 regulatory T (TR1) cells are Foxp3- interleukin-10 (IL-10)-producing CD4+ T cells with potent immunosuppressive properties, but their requirements for lineage development have remained elusive. We show that TR1 cells constitute the most abundant regulatory population after allogeneic bone marrow transplantation (BMT), express the transcription factor Eomesodermin (Eomes), and are critical for the prevention of graft-versus-host disease. We demonstrate that Eomes is required for TR1 cell differentiation, during which it acts in concert with the transcription factor B lymphocyte-induced maturation protein-1 (Blimp-1) by transcriptionally activating IL-10 expression and repressing differentiation into other T helper cell lineages. We further show that Eomes induction in TR1 cells requires T-bet and donor macrophage-derived IL-27. Thus, we define the cellular and transcriptional control of TR1 cell differentiation during BMT, opening new avenues to therapeutic manipulation.

Identification of any mechanism for microbiologically influenced corrosion (MIC) requires an understanding of the specificity of metal/microbe/electrolyte interactions. Recent advancements in our understanding of MIC are related to recognition of the implications of this specificity. For example, under some circumstances, nutrients can accelerate rates of corrosion. In other cases the oxyanions in nutrients can inhibit localised corrosion. In some environments the absence of oxidisable carbon can force a shift in electron donor and may result in more aggressive corrosion than in the presence of oxidisable carbon. Non-corrosive biofilms can become corrosive with subtle changes in the environment, e.g., addition of electron shuttle compounds. The list of electron donors and acceptors related to MIC has been expanded in recognition of the metabolic flexibility that has been demonstrated for microorganisms. Recent research on microbial fuel cells and microbial batteries has added to our understanding of microbial/metal interactions.

G9a is an epigenetic regulator that methylates H3K9, generally causing repression of gene expression, and participates in diverse cellular functions. G9a is genetically deregulated in a variety of tumor types and can silence tumor suppressor genes and, therefore, is important for carcinogenesis. Although hypoxia is recognized to be an adverse factor in tumor growth and metastasis, the role of G9a in regulating gene expression in hypoxia has not been described extensively. Here, we show that G9a protein stability is increased in hypoxia via reduced proline hydroxylation and, hence, inefficient degradation by the proteasome. This inefficiency leads to an increase in H3K9me2 at its target promoters. Blocking the methyltransferase activity of G9a inhibited cellular proliferation and migration in vitro and tumor growth in vivo. Furthermore, an increased level of G9a is a crucial factor in mediating the hypoxic response by down-regulating the expression of specific genes, including ARNTL, CEACAM7, GATA2, HHEX, KLRG1, and OGN This down-regulation can be rescued by a small molecule inhibitor of G9a. Based on the hypothesis that the changes in gene expression would influence patient outcomes, we have developed a prognostic G9a-suppressed gene signature that can stratify breast cancer patients. Together, our findings provide an insight into the role G9a plays as an epigenetic mediator of hypoxic response, which can be used as a diagnostic marker, and proposes G9a as a therapeutic target for solid cancers.

Highlightsd The activating receptor DNAM-1 identifies two distinct NK

Many pathogens, including Plasmodium spp., exploit the interaction of programmed death-1 (PD-1) with PD-1-ligand-1 (PD-L1) to "deactivate" T cell functions, but the role of PD-L2 remains unclear. We studied malarial infections to understand the contribution of PD-L2 to immunity. Here we have shown that higher PD-L2 expression on blood dendritic cells, from Plasmodium falciparum-infected individuals, correlated with lower parasitemia. Mechanistic studies in mice showed that PD-L2 was indispensable for establishing effective CD4(+) T cell immunity against malaria, because it not only inhibited PD-L1 to PD-1 activity but also increased CD3 and inducible co-stimulator (ICOS) expression on T cells. Importantly, administration of soluble multimeric PD-L2 to mice with lethal malaria was sufficient to dramatically improve immunity and survival. These studies show immuno-regulation by PD-L2, which has the potential to be translated into an effective treatment for malaria and other diseases where T cell immunity is ineffective or short-lived due to PD-1-mediated signaling.

Control of intracellular parasites responsible for malaria requires host IFN-γ+T-bet+CD4+ T cells (Th1 cells) with IL-10 produced by Th1 cells to mitigate the pathology induced by this inflammatory response. However, these IL-10-producing Th1 (induced type I regulatory [Tr1]) cells can also promote parasite persistence or impair immunity to reinfection or vaccination. Here, we identified molecular and phenotypic signatures that distinguished IL-10-Th1 cells from IL-10+Tr1 cells in Plasmodium falciparum-infected people who participated in controlled human malaria infection studies, as well as C57BL/6 mice with experimental malaria caused by P. berghei ANKA. We also identified a conserved Tr1 cell molecular signature shared between patients with malaria, dengue, and graft-versus-host disease. Genetic manipulation of primary human CD4+ T cells showed that the transcription factor cMAF played an important role in the induction of IL-10, while BLIMP-1 promoted the development of human CD4+ T cells expressing multiple coinhibitory receptors. We also describe heterogeneity of Tr1 cell coinhibitory receptor expression that has implications for targeting these molecules for clinical advantage during infection. Overall, this work provides insights into CD4+ T cell development during malaria that offer opportunities for creation of strategies to modulate CD4+ T cell functions and improve antiparasitic immunity.

Pontin is a chromatin remodeling factor that possesses both ATPase and DNA helicase activities. Although Pontin is frequently overexpressed in human cancers of various types and implicated in oncogenic functions, the upstream signaling network leading to the regulation of Pontin that in turn affects transcription of downstream target genes has not been extensively studied. Here, we identify Pontin is methylated by G9a/GLP methyltransferases in hypoxic condition and potentiates HIF-1α-mediated activation by increasing the recruitment of p300 coactivator to a subset of HIF-1α target promoters. Intriguingly, Pontin methylation results in the increased invasive and migratory properties by activating downstream target gene, Ets1. In contrast, inhibition of Pontin methylation results in the suppression of tumorigenic and metastatic properties. Together, our data provide new approaches by targeting Pontin methylation and its downstream targets for the development of therapeutic agents for human cancers.

Abstract Adipose progenitor cells (APCs) are widely investigated for soft tissue reconstruction following tumor resection; however, the long‐term success of current approaches is still limited. In order to develop clinically relevant therapies, a better understanding of the role of cell–microenvironment interactions in adipose tissue regeneration is essential. In particular, the effect of extracellular matrix (ECM) mechanics on the regenerative capability of APCs remains to be clarified. We have used artificial ECMs based on photocrosslinkable RGD‐alginate to investigate the adipogenic and pro‐angiogenic potential of 3T3‐L1 preadipocytes as a function of matrix stiffness. These hydrogels allowed us to decouple matrix stiffness from changes in adhesion peptide density or extracellular Ca 2+ concentration and provided a physiologically relevant 3D culture context. Our findings suggest that increased matrix rigidity promotes APC self‐renewal and angiogenic capacity, whereas, it inhibits adipose differentiation. Collectively, this study advances our understanding of the role of ECM mechanics in adipose tissue formation and vascularization and will aid in the design of efficacious biomaterial scaffolds for adipose tissue engineering applications. Biotechnol. Bioeng. 2011; 108:1683–1692. © 2011 Wiley Periodicals, Inc.

Biodiesels have gained widespread acceptance because they are domestically produced carbon-neutral fuels that ultimately decrease greenhouse gas emissions and reduce dependence on fossil imports. While they are chemically and physically stable fuels, their susceptibility to biological degradation in the absence of oxygen is underexplored. We incubated five anaerobic inocula with biodiesel. The microorganisms originated from fresh and marine environments with differing histories of exposure to hydrocarbons, biodiesel, and oxygen. All inocula were able to biodegrade biodiesel within 1 month. Biodiesel metabolism accelerated the rate of both sulfate reduction and methanogenesis above biodiesel-unamended controls. Metabolite profiling indicated that the methyl esters of biodiesel were readily hydrolyzed to the corresponding suite of fatty acids, and the latter were also metabolized. Electrochemical/corrosion experiments showed that the anaerobic microbial metabolism of biodiesel in coastal seawater samples accelerated the rate of pitting corrosion in carbon steel. The susceptibility of biodiesel to anaerobic biodegradation and its propensity to stimulate biocorrosion suggest caution when integrating this alternate fuel with the existing infrastructure.

Genome-wide association studies (GWASs) have revealed increased breast cancer risk associated with multiple genetic variants at 5p12. Here, we report the fine mapping of this locus using data from 104,660 subjects from 50 case-control studies in the Breast Cancer Association Consortium (BCAC). With data for 3,365 genotyped and imputed SNPs across a 1 Mb region (positions 44,394,495–45,364,167; NCBI build 37), we found evidence for at least three independent signals: the strongest signal, consisting of a single SNP rs10941679, was associated with risk of estrogen-receptor-positive (ER+) breast cancer (per-g allele OR ER+ = 1.15; 95% CI 1.13–1.18; p = 8.35 × 10−30). After adjustment for rs10941679, we detected signal 2, consisting of 38 SNPs more strongly associated with ER-negative (ER−) breast cancer (lead SNP rs6864776: per-a allele OR ER− = 1.10; 95% CI 1.05–1.14; p conditional = 1.44 × 10−12), and a single signal 3 SNP (rs200229088: per-t allele OR ER+ = 1.12; 95% CI 1.09–1.15; p conditional = 1.12 × 10−05). Expression quantitative trait locus analysis in normal breast tissues and breast tumors showed that the g (risk) allele of rs10941679 was associated with increased expression of FGF10 and MRPS30. Functional assays demonstrated that SNP rs10941679 maps to an enhancer element that physically interacts with the FGF10 and MRPS30 promoter regions in breast cancer cell lines. FGF10 is an oncogene that binds to FGFR2 and is overexpressed in ∼10% of human breast cancers, whereas MRPS30 plays a key role in apoptosis. These data suggest that the strongest signal of association at 5p12 is mediated through coordinated activation of FGF10 and MRPS30, two candidate genes for breast cancer pathogenesis. Genome-wide association studies (GWASs) have revealed increased breast cancer risk associated with multiple genetic variants at 5p12. Here, we report the fine mapping of this locus using data from 104,660 subjects from 50 case-control studies in the Breast Cancer Association Consortium (BCAC). With data for 3,365 genotyped and imputed SNPs across a 1 Mb region (positions 44,394,495–45,364,167; NCBI build 37), we found evidence for at least three independent signals: the strongest signal, consisting of a single SNP rs10941679, was associated with risk of estrogen-receptor-positive (ER+) breast cancer (per-g allele OR ER+ = 1.15; 95% CI 1.13–1.18; p = 8.35 × 10−30). After adjustment for rs10941679, we detected signal 2, consisting of 38 SNPs more strongly associated with ER-negative (ER−) breast cancer (lead SNP rs6864776: per-a allele OR ER− = 1.10; 95% CI 1.05–1.14; p conditional = 1.44 × 10−12), and a single signal 3 SNP (rs200229088: per-t allele OR ER+ = 1.12; 95% CI 1.09–1.15; p conditional = 1.12 × 10−05). Expression quantitative trait locus analysis in normal breast tissues and breast tumors showed that the g (risk) allele of rs10941679 was associated with increased expression of FGF10 and MRPS30. Functional assays demonstrated that SNP rs10941679 maps to an enhancer element that physically interacts with the FGF10 and MRPS30 promoter regions in breast cancer cell lines. FGF10 is an oncogene that binds to FGFR2 and is overexpressed in ∼10% of human breast cancers, whereas MRPS30 plays a key role in apoptosis. These data suggest that the strongest signal of association at 5p12 is mediated through coordinated activation of FGF10 and MRPS30, two candidate genes for breast cancer pathogenesis. Strong evidence for the existence of a breast cancer (MIM: 114480) susceptibility locus at 5p12 has been observed through a GWAS in Iceland (SNP rs7703618),1Stacey S.N. Manolescu A. Sulem P. Rafnar T. Gudmundsson J. Gudjonsson S.A. Masson G. Jakobsdottir M. Thorlacius S. Helgason A. et al.Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer.Nat. Genet. 2007; 39: 865-869Crossref PubMed Scopus (633) Google Scholar in the Breast Cancer Association Consortium (BCAC; SNP rs981782, 371 Kb centromeric),2Easton D.F. Pooley K.A. Dunning A.M. Pharoah P.D. Thompson D. Ballinger D.G. Struewing J.P. Morrison J. Field H. Luben R. et al.SEARCH collaboratorskConFabAOCS Management GroupGenome-wide association study identifies novel breast cancer susceptibility loci.Nature. 2007; 447: 1087-1093Crossref PubMed Scopus (1933) Google Scholar and in the Cancer GEnetic Markers of Susceptibility study (CGEMS; SNP rs4866929; 352 Kb centromeric; r2 = 0.18).3Hunter D.J. Kraft P. Jacobs K.B. Cox D.G. Yeager M. Hankinson S.E. Wacholder S. Wang Z. Welch R. Hutchinson A. et al.A genome-wide association study identifies alleles in FGFR2 associated with risk of sporadic postmenopausal breast cancer.Nat. Genet. 2007; 39: 870-874Crossref PubMed Scopus (1249) Google Scholar A subsequent study, using 22 SNPs in ∼5,000 case subjects and ∼33,000 control subjects of European ancestry, reported that risk at this locus could be explained by two SNPs: rs4415084 and rs10941679.4Stacey S.N. Manolescu A. Sulem P. Thorlacius S. Gudjonsson S.A. Jonsson G.F. Jakobsdottir M. Bergthorsson J.T. Gudmundsson J. Aben K.K. et al.Common variants on chromosome 5p12 confer susceptibility to estrogen receptor-positive breast cancer.Nat. Genet. 2008; 40: 703-706Crossref PubMed Scopus (391) Google Scholar More recently, a BCAC study confirmed that rs10941679 was associated with risk of lower-grade, progesterone receptor (PGR [MIM: 607311])-positive breast cancer tumors.5Milne R.L. Goode E.L. García-Closas M. Couch F.J. Severi G. Hein R. Fredericksen Z. Malats N. Zamora M.P. Arias Pérez J.I. et al.GENICA NetworkkConFab InvestigatorsAOCS GroupConfirmation of 5p12 as a susceptibility locus for progesterone-receptor-positive, lower grade breast cancer.Cancer Epidemiol. Biomarkers Prev. 2011; 20: 2222-2231Crossref PubMed Scopus (25) Google Scholar Here, we report the comprehensive fine-scale mapping of this locus in 104,660 subjects from 50 case-control studies participating in BCAC, including 41 studies from populations of European ancestry and nine of East Asian ancestry, and we explore the functional mechanisms underlying the associations in this region. Genotyping was conducted with the COGS array, a custom array comprising approximately 200,000 SNPs.6Michailidou K. Hall P. Gonzalez-Neira A. Ghoussaini M. Dennis J. Milne R.L. Schmidt M.K. Chang-Claude J. Bojesen S.E. Bolla M.K. et al.Breast and Ovarian Cancer Susceptibility CollaborationHereditary Breast and Ovarian Cancer Research Group Netherlands (HEBON)kConFab InvestigatorsAustralian Ovarian Cancer Study GroupGENICA (Gene Environment Interaction and Breast Cancer in Germany) NetworkLarge-scale genotyping identifies 41 new loci associated with breast cancer risk.Nat. Genet. 2013; 45: 353-361, e1–e2Crossref PubMed Scopus (836) Google Scholar After quality-control exclusions, we analyzed data from 48,155 case subjects and 43,612 control subjects of European ancestry and 6,269 case subjects and 6,624 control subjects of Asian ancestry. Estrogen receptor (ESR1 [MIM: 133430]) status of the primary tumor was available for 27,748 European and 4,997 Asian case subjects; of these, 7,646 (22%) European and 1,623 (32%) Asian case subjects were ER−. We examined a 1 Mb region (positions 44,394,495–45,364,167; NCBI build 37 assembly) in which the 1000 Genomes Project cataloged 1,811 variants (March 2010 Pilot version 60 CEU project data). We aimed to genotype all 628 SNPs with minor allele frequency (MAF) > 2% and correlated with rs981782 and rs10941679 at r2 > 0.1 (n = 424), plus a set of SNPs designed to tag all remaining SNPs with r2 > 0.9 (n = 184), but we managed to include 563 SNPs with a designability score (DS) > 0.9 and which passed QC.6Michailidou K. Hall P. Gonzalez-Neira A. Ghoussaini M. Dennis J. Milne R.L. Schmidt M.K. Chang-Claude J. Bojesen S.E. Bolla M.K. et al.Breast and Ovarian Cancer Susceptibility CollaborationHereditary Breast and Ovarian Cancer Research Group Netherlands (HEBON)kConFab InvestigatorsAustralian Ovarian Cancer Study GroupGENICA (Gene Environment Interaction and Breast Cancer in Germany) NetworkLarge-scale genotyping identifies 41 new loci associated with breast cancer risk.Nat. Genet. 2013; 45: 353-361, e1–e2Crossref PubMed Scopus (836) Google Scholar IMPUTE v.2.0 was used to impute genotypes of all known SNPs in the region using the 1000 Genome Project data (March 2012 version) as a reference panel. Case-control analyses were conducted on 3,365 SNPs (563 genotyped and 2,776 imputed at r2 > 0.3). In European-ancestry women, 461 of these SNPs were associated with overall breast cancer risk, 489 with ER+ and 38 with ER− breast cancer risk (p < 10−4; Table S1). SNP rs10941679 showed the strongest overall association (MAF = 0.27, per-minor (g) allele: OR = 1.12; 95% CI 1.10–1.14; p = 2.55 × 10−26; Figure 1, Tables 1 and S1). To identify additional association signals at this region, we conducted a forward stepwise logistic regression examining SNPs with univariate p < 0.1 (n = 1,040).6Michailidou K. Hall P. Gonzalez-Neira A. Ghoussaini M. Dennis J. Milne R.L. Schmidt M.K. Chang-Claude J. Bojesen S.E. Bolla M.K. et al.Breast and Ovarian Cancer Susceptibility CollaborationHereditary Breast and Ovarian Cancer Research Group Netherlands (HEBON)kConFab InvestigatorsAustralian Ovarian Cancer Study GroupGENICA (Gene Environment Interaction and Breast Cancer in Germany) NetworkLarge-scale genotyping identifies 41 new loci associated with breast cancer risk.Nat. Genet. 2013; 45: 353-361, e1–e2Crossref PubMed Scopus (836) Google Scholar The most parsimonious model included three variants: SNP1 rs10941679 (signal 1), SNP2 rs6864776 (signal 2; conditional p = 6.22 × 10−11), and SNP3 rs200229088 (signal 3; conditional p = 1.12 × 10−5, borderline significance; Table S2). SNP1 and SNP3 are weakly correlated (r2 = 0.15) but SNP2 was uncorrelated with the other two (r2 = 0.07 and 0.05).Table 1Associations of the Top SNPs from Each Signal with Overall Breast Cancer Risk and Breast Cancer Stratified by ER StatusSigSNPComMinMAF∗OR Overall 95% CIp OverallConditional p ValueOR ER−p ER−OR ER+p ER+Europeans1rs10941679AG0.271.12 (1.10–1.14)2.55 × 10−266.55 × 10−241.04 (1–1.08)0.0591.15 (1.13–1.18)8.35 × 10−302rs6864776GA0.231.04 (1.02–1.06)7.84 × 10−41.44 × 10−121.10 (1.05–1.14)2.5 × 10−51.02 (0.99–1.05)0.083rs200229088TTGT0.311.09 (1.07–1.12)2.28 × 10−121.12 × 10−51.03 (0.99–1.09)0.111.12 (1.09–1.15)7.51 × 10−14Asians1rs10941679AG0.501.09 (1.04–1.15)9.12 × 10−40.08591.03 (0.95–1.11)0.531.11 (1.04–1.18)1.32 × 10−32rs6864776GA0.320.94 (0.89–1.00)3.47 × 10−20.89010.95 (0.87–1.04)0.280.94 (0.89–1.00)6.24 × 10−23rs200229088TTGT0.371.09 (1.02–1.15)6.52 × 10−30.91491.04 (0.95–1.14)0.431.08 (1.00–1.16)3.65 × 10−2Abbreviations are as follows: Com, common alleles; Min, minor alleles; MAF, minor allele frequency; OR, per-allele odds ratios (OR); 95% CI, 95% confidence intervals and 1 degree of freedom; p, significance levels for overall breast cancer are indicated in European and Asian case-control studies, and separately for ER+ and ER− disease. Open table in a new tab Abbreviations are as follows: Com, common alleles; Min, minor alleles; MAF, minor allele frequency; OR, per-allele odds ratios (OR); 95% CI, 95% confidence intervals and 1 degree of freedom; p, significance levels for overall breast cancer are indicated in European and Asian case-control studies, and separately for ER+ and ER− disease. The top signal, SNP1 rs10941679, is markedly more significant than any other SNP in the locus (likelihood ratio > 10,000:1). Hence, the most parsimonious explanation is that this SNP is causally related to risk. The next most strongly associated SNP, after adjustment for signal 1 SNP rs10941679, was rs6864776, representing signal 2 (OR per minor allele = 1.04; 95% CI 1.02–1.06; p = 7.84 × 10−4; conditional p = 1.44 × 10−12). Within signal 2, a further 37 SNPs correlated with rs6864776 at r2 > 0.6, had likelihood ratios of <100:1 relative to rs6864776, and hence could not be excluded from being causative statistically (Table S2). After adjustment for both signal 1 SNP rs10941679 and signal 2 top SNP rs6864776, a single SNP remained: rs200229088 (OR overall = 1.09, 95%; CI 1.07–1.12; p = 2.28 × 10−12; conditional p = 1.12 × 10−5). There are no other SNPs correlated with rs200229088 that could explain this association. All other SNPs were excluded from causality (likelihood ratio > 10,000:1; Table S2). Two of the excluded variants had been previously postulated as likely causative variants4Stacey S.N. Manolescu A. Sulem P. Thorlacius S. Gudjonsson S.A. Jonsson G.F. Jakobsdottir M. Bergthorsson J.T. Gudmundsson J. Aben K.K. et al.Common variants on chromosome 5p12 confer susceptibility to estrogen receptor-positive breast cancer.Nat. Genet. 2008; 40: 703-706Crossref PubMed Scopus (391) Google Scholar, 7Quigley D.A. Fiorito E. Nord S. Van Loo P. Alnæs G.G. Fleischer T. Tost J. Moen Vollan H.K. Tramm T. Overgaard J. et al.The 5p12 breast cancer susceptibility locus affects MRPS30 expression in estrogen-receptor positive tumors.Mol. Oncol. 2014; 8: 273-284Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar and so we investigated these in more depth. We found both SNPs to be partially correlated with all three signals and consequently display initially inflated effects, which are adjusted by the conditional analyses. Thus, SNP rs44150844Stacey S.N. Manolescu A. Sulem P. Thorlacius S. Gudjonsson S.A. Jonsson G.F. Jakobsdottir M. Bergthorsson J.T. Gudmundsson J. Aben K.K. et al.Common variants on chromosome 5p12 confer susceptibility to estrogen receptor-positive breast cancer.Nat. Genet. 2008; 40: 703-706Crossref PubMed Scopus (391) Google Scholar (r2 with signal 1 SNP rs10941679 = 0.51, with signal 2 SNP rs6864776 = 0.11, and with signal 3 SNP rs200229088 = 0.37) has odds against causality > 10 million:1 versus signal 1 candidate rs10941679. Similarly, SNP rs7716600, which is an eQTL for MRPS30 expression7Quigley D.A. Fiorito E. Nord S. Van Loo P. Alnæs G.G. Fleischer T. Tost J. Moen Vollan H.K. Tramm T. Overgaard J. et al.The 5p12 breast cancer susceptibility locus affects MRPS30 expression in estrogen-receptor positive tumors.Mol. Oncol. 2014; 8: 273-284Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar (r2 with SNP rs10941679 = 0.77, with SNP rs6864776 = 0.05, and with SNP rs200229088 = 0.12) has odds against causality >160,000:1 versus signal 1 candidate rs10941679. These exclusions of former causal candidates highlight the need for fine-mapping studies before conducting functional analyses. Haplotype analyses were conducted using the above three signal-representative variants, which generated eight haplotypes (Table 2). Haplotypes carrying the rare allele of signal 3 SNP rs200229088 conferred higher risks than corresponding haplotypes carrying the common allele, consistent with this allele having an independent effect. Haplotype G, carrying the minor alleles of both the signal 1 and 2 representative SNPs, is very rare and reveals that their risk alleles are negatively correlated, which is also consistent with our finding that signal 2 top SNP rs6864776 increases in significance after conditioning on signal 1 SNP rs10941679 (Table 1).Table 2Haplotype Analysis across the BCAC StudiesHaplotypesrs10941679 Signal 1rs6864776 Signal 2rs200229088 Signal 3Haplotype FrequencyORp ValueA1110.395440––B1120.1200991.06 (1.02–1.10)1.49 × 10−3C1210.1995991.10 (1.06–1.13)7.76 × 10−11D1220.0186651.15 (1.04–1.27)5.03 × 10−3E2110.0981691.14 (1.09–1.19)1.45 × 10−11F2120.1545251.20 (1.16–1.24)2.72 × 10−30G2210.0042480.91 (0.72–1.15)4.15 × 10−1H2220.0092531.28 (1.10–1.48)1.14 × 10−3Each haplotype was compared to the ancestral haplotype carrying the common alleles of signal 1 SNP rs10941679, signal 2 SNP rs6864776, and signal 3 SNP rs200229088 (haplotype A). Open table in a new tab Each haplotype was compared to the ancestral haplotype carrying the common alleles of signal 1 SNP rs10941679, signal 2 SNP rs6864776, and signal 3 SNP rs200229088 (haplotype A). We examined the associations of these three SNPs in the Asian case-control studies within BCAC. SNP1 and SNP3 both replicated in the Asian studies and the relative risk estimates with overall breast cancer were consistent with those seen in the European population: per g-allele OR (rs10941679) = 1.09; 95% CI 1.04–1.15; p = 0.0009, conditional p = 0.0859 and per t-allele OR (rs200229088) = 1.09; 95% CI 1.02–1.15; p = 0.0065, conditional p = 0.9149 (Table 1). SNP2 was not replicated in Asians (per a-allele OR = 0.94; 95% CI 0.89–1.00; p = 0.034, conditional p = 0.8901) (Table 1). We investigated the associations of these three signals with tumor subtypes based on ER status. SNP1 rs10941679 was largely associated with ER+ breast cancer (OR ER+ = 1.15; 95% CI 1.13–1.18; p = 8.35 × 10−30 versus OR ER− disease = 1.04; 95% CI 1.00–1.08; p = 0.059; p heterogeneity = 1.5 × 10−5; Table 1) as was SNP3 rs200229088 (OR ER+ = 1.12; 95% CI 1.09–1.15; p = 7.51 × 10−14 versus OR ER− = 1.03; 95% CI 0.99–1.09; p = 0.11, p heterogeneity = 0.02). By contrast, SNP2 rs6864776 was moderately associated with ER− but not ER+ tumors (OR ER− = 1.10; 95% CI 1.05–1.14; p = 2.55 × 10−5 versus OR ER+ = 1.02; 95% CI 0.99–1.05; p = 0.08; p heterogeneity = 0.01; Table 1). Candidate SNPs 1–3 span a 1.7 Mb region on 5p12 that includes three annotated genes—FGF10 (MIM: 602115), MRPS30 (MIM: 611991), and HCN1 (MIM: 602780)—and several putative long noncoding RNAs (lncRNAs; Figure 1). To identify potential target gene(s), we examined the associations of the three lead SNPs with expression levels of genes located within 1 Mb in three different studies: (1) 116 normal breast samples and 241 breast tumors from the Norwegian Breast Cancer Study (NBCS),8Ghoussaini M. Edwards S.L. Michailidou K. Nord S. Cowper-Sal Lari R. Desai K. Kar S. Hillman K.M. Kaufmann S. Glubb D.M. et al.Australian Ovarian Cancer Management GroupAustralian Ovarian Cancer Management GroupEvidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation.Nat. Commun. 2014; 4: 4999Crossref PubMed Scopus (87) Google Scholar (2) 93 normal and 765 breast cancer tissues from the TCGA study (germline genotype data from Affymetrix SNP 6 array were obtained from TCGA dbGAP data portal9Li Q. Seo J.H. Stranger B. McKenna A. Pe’er I. Laframboise T. Brown M. Tyekucheva S. Freedman M.L. Integrative eQTL-based analyses reveal the biology of breast cancer risk loci.Cell. 2013; 152: 633-641Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), and (3) 183 normal breast samples from the Genotype-Tissue Expression (GTEx) project.10Consortium G.T. GTEx ConsortiumThe Genotype-Tissue Expression (GTEx) project.Nat. Genet. 2013; 45: 580-585Crossref PubMed Scopus (4308) Google Scholar The SNP1 rs10941679 risk-associated g-allele was moderately associated with increased FGF10 mRNA expression in NBCS normal breast (p = 0.013, p corrected = 0.39) and breast tumors (p = 0.005, p corrected = 0.38) as well as in GTEx normal breast (p corrected = 0.02; Figures 2A and S1A). The effect in TCGA was in the same direction, though not significant (normal breast p = 0.353, p corrected = 0.95 and breast tumors p = 0.057, p corrected = 0.41; Figure S1B). The g-allele was also associated with increased expression of MRPS30 in the NBCS normal (p = 0.002, p corrected = 0.36) and breast tumors (p = 0.049, p corrected = 0.43), in GTEx normal breast (p corrected = 0.002), and in TCGA (normal breast p = 6.86 × 10−5, p corrected = 5.31 × 10−3 and breast tumors p = 7.21 × 10−6, p corrected = 9.35 × 10−4; Figures 2B, S1A, and S1C). No associations were observed with SNP2 rs6864776 or SNP3 variant rs200229088. We also measured endogenous levels of FGF10, MRPS30, and nearby lncRNAs FGF10-AS1, BRCAT54, RP11-503D12.1, and RP11-473L15.3 mRNA in breast cell lines homozygous (A/A or G/G) or heterozygous (A/G) for the common allele of SNP1 (Table S3, Figures 2C, 2D, S2, and S3). Total RNA from cell lines was extracted using Trizol and complementary DNA synthesized using random primers as per manufacturers’ instructions. Quantitative PCR (qPCR) were performed using TaqMan assays for FGF10 and MRPS30 normalized against beta-glucuronidase (GUSB [MIM: 611499]) or with SYTO9 for lncRNAs normalized against TATA box-binding protein (TBP [MIM: 600075]; primers are listed in Table S4). Although the number of ER+ breast cell lines carrying the risk allele was limited, FGF10 and MRPS30 mRNA levels were significantly higher in the BT474 heterozygous cell line (Figures 2C and 2D). BRCAT54 was detected in the majority of cell lines but its expression appears to be genotype independent (Figure S3A). FGF10-AS1, RP11-503D12.1, and RP11-473L15.3 transcripts were either expressed at very low levels or not detected in the cell lines analyzed (Figures S3B–S3D). Therefore, although we cannot rule out the possibility that the risk SNPs may influence local lncRNA expression, the low or absent transcript levels precluded any further evaluation. Candidate causal SNPs were then explored using publicly available datasets from ENCODE,11Birney E. Stamatoyannopoulos J.A. Dutta A. Guigó R. Gingeras T.R. Margulies E.H. Weng Z. Snyder M. Dermitzakis E.T. Thurman R.E. et al.ENCODE Project ConsortiumNISC Comparative Sequencing ProgramBaylor College of Medicine Human Genome Sequencing CenterWashington University Genome Sequencing CenterBroad InstituteChildren’s Hospital Oakland Research InstituteIdentification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.Nature. 2007; 447: 799-816Crossref PubMed Scopus (4118) Google Scholar which includes information such as the location of promoter and enhancer histone marks, open chromatin, bound proteins, and altered motifs for the MCF7 breast cancer cell line, and from Hnisz et al.12Hnisz D. Abraham B.J. Lee T.I. Lau A. Saint-André V. Sigova A.A. Hoke H.A. Young R.A. Super-enhancers in the control of cell identity and disease.Cell. 2013; 155: 934-947Abstract Full Text Full Text PDF PubMed Scopus (2102) Google Scholar and Corradin et al.13Corradin O. Saiakhova A. Akhtar-Zaidi B. Myeroff L. Willis J. Cowper-Sal lari R. Lupien M. Markowitz S. Scacheri P.C. Combinatorial effects of multiple enhancer variants in linkage disequilibrium dictate levels of gene expression to confer susceptibility to common traits.Genome Res. 2014; 24: 1-13Crossref PubMed Scopus (247) Google Scholar to identify the location of likely enhancers and their gene targets in a cell-specific context. Analysis of cis enhancer-gene interactions via PreSTIGE13Corradin O. Saiakhova A. Akhtar-Zaidi B. Myeroff L. Willis J. Cowper-Sal lari R. Lupien M. Markowitz S. Scacheri P.C. Combinatorial effects of multiple enhancer variants in linkage disequilibrium dictate levels of gene expression to confer susceptibility to common traits.Genome Res. 2014; 24: 1-13Crossref PubMed Scopus (247) Google Scholar showed evidence of putative regulatory elements (PREs) surrounding the top risk-associated SNPs in MCF7 breast cancer cells, but no histone-marked elements harboring a risk SNP in this cell line or in a range of cell lines and tissues analyzed in Roadmap (Figures 1 and S4). However, it is possible that certain epigenetic marks may be detected only in a specific cell subtype such as breast stem cells or in response to an external stimulus. To identify target gene(s), we performed chromatin conformation capture (3C) assays in ER+ MCF7, BT474, and MDA-MB-361 and ER− MDA-MB-231 breast cancer cell lines and Bre80 normal breast cells (Table S5).8Ghoussaini M. Edwards S.L. Michailidou K. Nord S. Cowper-Sal Lari R. Desai K. Kar S. Hillman K.M. Kaufmann S. Glubb D.M. et al.Australian Ovarian Cancer Management GroupAustralian Ovarian Cancer Management GroupEvidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation.Nat. Commun. 2014; 4: 4999Crossref PubMed Scopus (87) Google Scholar 3C libraries were created by cross-linking the chromatin from cell lines; DNA was then digested with EcoRI, which flanks 12 contiguous fragments that cover the PRE, and the FGF10, MRPS30, and HCN1 promoters (Table S6); DNA was religated and decrosslinked; and qPCR with primers for the bait (gene promoters) and interactors (12 PRE fragments) was performed to detect the presence of ligation products, representing gene loops. BAC clones covering the regions of interest were used to normalize for PCR efficiency. These assays showed that the PRE containing SNP1 frequently interacted with the FGF10 and MRPS30 promoter regions in MCF7 and BT474 breast cancer cell lines, but only with MRPS30 in the MDA-MB-361, MDA-MB-231, and Bre80 cell lines. This latter result was expected because FGF10 is not expressed or expressed at very low levels in these cell lines (Figures 2C, 3A , S5, and S6). Notably, both genes share a bidirectional promoter with the lncRNAs FGF10-AS1 and BRCAT54, raising the possibility that these transcripts are also targets of the PRE (Figure 3A). No additional interactions were detected between the PRE and other annotated genes within 1 Mb of the PRE, including HCN1 (Figure S5). To assess the potential impact of SNP1 on the identified chromatin interactions, allele-specific 3C was performed in heterozygous BT474 cell lines.8Ghoussaini M. Edwards S.L. Michailidou K. Nord S. Cowper-Sal Lari R. Desai K. Kar S. Hillman K.M. Kaufmann S. Glubb D.M. et al.Australian Ovarian Cancer Management GroupAustralian Ovarian Cancer Management GroupEvidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation.Nat. Commun. 2014; 4: 4999Crossref PubMed Scopus (87) Google Scholar However, the sequence profiles revealed that SNP1 had no significant effect on chromatin looping (Figure S7). The regulatory capability of the PRE, combined with the effect of SNP1, was further examined in reporter assays. Promoter-driven luciferase reporter constructs were generated by the insertion of PCR-amplified fragments containing FGF10, FGF10-AS1, MRPS30, or BRCAT54 promoters into pGL3-Basic.14Glubb D.M. Maranian M.J. Michailidou K. Pooley K.A. Meyer K.B. Kar S. Carlebur S. O’Reilly M. Betts J.A. Hillman K.M. et al.GENICA NetworkkConFab InvestigatorsNorwegian Breast Cancer StudyFine-scale mapping of the 5q11.2 breast cancer locus reveals at least three independent risk variants regulating MAP3K1.Am. J. Hum. Genet. 2015; 96: 5-20Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar A 1,736-bp PRE fragment (containing either the common or minor allele of rs10941679) was then generated by PCR and cloned downstream of the modified pGL3-promoter constructs (Table S7). MCF7 and BT474 breast cancer cell lines plus Bre80 normal breast cells were transfected with the reporter plasmids and luciferase activity was measured 24 hr after transfection. To correct for any differences in transfection efficiency or cell lysate preparation, Firefly luciferase activity was normalized to Renilla. Notably, the “Ref PRE” acted as a transcriptional enhancer, leading to a 2- to 3-fold increase in FGF10, MRPS30, and BRCAT54 promoter activity, but had no effect on the FGF10-AS1 promoter in MCF7 and BT474 cells (Figures 3B and S8). The enhancer activity was also observed for the MRPS30 and BRCAT54 promoters in Bre80 cells (Figure S8). In all cell lines, inclusion of the SNP1 risk (g) allele had no significant effect on the PRE enhancer activity. Although this appears to rule out an effect of this SNP on transactivation, it is possible that SNP1 affects the recruitment of key proteins required for the epigenetic modification of the enhancer, which would not be observed in a reporter assay. Another possibility is that the SNP effect may be observed only under certain biological conditions such as growth factor stimulation. To seek further evidence that SNP1 lies within an enhancer element, we performed electrophoretic mobility shift assays (EMSAs) for both the protective (a) and risk (g) alleles.15Dunning A.M. Michailidou K. Kuchenbaecker K.B. Thompson D. French J.D. Beesley J. Healey C.S. Kar S. Pooley K.A. Lopez-Knowles E. et al.EMBRACEGEMO Study CollaboratorsHEBONkConFab InvestigatorsBreast cancer risk variants at 6q25 display different phenotype associations and regulate ESR1, RMND1 and CCDC170.Nat. Genet. 2016; 48: 374-386Crossref PubMed Scopus (98) Google Scholar Nuclear lysates were prepared from ER+ BT474, MCF7, and MDA-MB-361 or ER− MDA-MB-231 and Hs578T cells using the NE-PER nuclear and cytoplasmic extraction reagents. Biotinylated oligonucleotide duplexes were prepared by combining sense and antisense oligonucleotides, heat annealing, and slow cooling. Duplex-bound complexes were transferred onto Zeta-Probe positively charged nylon membranes by semi-dry transfer then cross-linked onto the membranes. Membranes were processed with the LightShift Chemiluminescent EMSA kit as per the manufacturer’s instructions, and signals were visualized with the C-DiGit blot scanner. For SNP1, we observed allele-specific binding by nuclear proteins only in the ER+ BT474, MCF7, and MDA-MB-361 extracts (Figures 3C and S9). The protein-DNA complexes were shown to be specific, as demonstrated by increasing amounts of cold self-competitor (Figures 3C and S9 and Table S8). Further EMSAs using competitor DNA or antibody supershifts against predicted transcription factors (TFs) suggested four proteins bound to the SNP site including FOXA1, FOXA2, CEBPB, and OCT1 (Figure S10 and Table S9). To confirm TF binding in vivo, we performed chromatin immunoprecipitation (ChIP) in heterozygous BT474 cells as previously described (Table S10).15Dunning A.M. Michailidou K. Kuchenbaecker K.B. Thompson D. French J.D. Beesley J. Healey C.S. Kar S. Pooley K.A. Lopez-Knowles E. et al.EMBRACEGEMO Study CollaboratorsHEBONkConFab InvestigatorsBreast cancer risk variants at 6q25 display different phenotype associations and regulate ESR1, RMND1 and CCDC170.Nat. Genet. 2016; 48: 374-386Crossref PubMed Scopus (98) Google Scholar When compared to an IgG control antibody, we observed a moderate enrichment in FOXA1 and OCT1 binding to DNA overlapping SNP rs10941679, but no difference between alleles in this cell line (Figure S11). In addition, western blot analysis indicated that FOXA1 protein expression was restricted to the ER+ breast cancer cell lines analyzed, whereas OCT1 was more widely expressed (Figure S12). FOXA1 is a pioneer factor and master regulator of ER activity due to its ability to open local chromatin and recruit ER to target gene promoters.16Hurtado A. Holmes K.A. Ross-Innes C.S. Schmidt D. Carroll J.S. FOXA1 is a key determinant of estrogen receptor function and endocrine response.Nat. Genet. 2011; 43: 27-33Crossref PubMed Scopus (616) Google Scholar Notably, breast cancer-associated SNPs are enriched for FOXA1 binding17Cowper-Sal lari R. Zhang X. Wright J.B. Bailey S.D. Cole M.D. Eeckhoute J. Moore J.H. Lupien M. Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression.Nat. Genet. 2012; 44: 1191-1198Crossref PubMed Scopus (286) Google Scholar and several studies have linked cooperative binding of FOXA1, ER, and OCT1 to increased gene transcription.18Meyer K.B. Maia A.T. O’Reilly M. Teschendorff A.E. Chin S.F. Caldas C. Ponder B.A. Allele-specific up-regulation of FGFR2 increases susceptibility to breast cancer.PLoS Biol. 2008; 6: e108Crossref PubMed Scopus (230) Google Scholar, 19Belikov S. Astrand C. Wrange O. FoxA1 binding directs chromatin structure and the functional response of a glucocorticoid receptor-regulated promoter.Mol. Cell. Biol. 2009; 29: 5413-5425Crossref PubMed Scopus (42) Google Scholar Consistent with our eQTL data, it is tempting to speculate that in specific ER+ cell subtypes and/or conditions, rs10941679 alters FOXA1 affinity and OCT1 recruitment leading to target gene activation. In conclusion, we have provided evidence for at least three independent causal SNPs with effects on the risk of breast cancer at this locus. The minor g-allele of signal 1 SNP rs10941679 conferred a 15% increased risk of ER+ breast cancer and higher expression levels of the MRPS30 and FGF10 genes and was the most strongly associated SNP with MRPS30 expression in this 1 Mb region. MRPS30—also called PDCD9 (Programmed Cell Death protein 9)—encodes a mitochondrial ribosomal protein involved in apoptosis.20Cavdar Koc E. Ranasinghe A. Burkhart W. Blackburn K. Koc H. Moseley A. Spremulli L.L. A new face on apoptosis: death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins.FEBS Lett. 2001; 492: 166-170Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Although the role of mitochondria in apoptosis remains unclear, it is well established that cytochrome c and other pro-apoptotic proteins are released during cell death initiation.20Cavdar Koc E. Ranasinghe A. Burkhart W. Blackburn K. Koc H. Moseley A. Spremulli L.L. A new face on apoptosis: death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins.FEBS Lett. 2001; 492: 166-170Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Clearly, further investigation of the function of this protein is now merited. By contrast, FGF10 is an extensively studied gene with compelling data suggesting its involvement in breast tumorigenesis. FGF10 is a member of the fibroblast growth factor (FGF) family and encodes a glycoprotein that specifically binds to FGFR2 (splice FGFR2IIIb) to control signaling pathways including cell differentiation, proliferation, and apoptosis.21Turner N. Grose R. Fibroblast growth factor signalling: from development to cancer.Nat. Rev. Cancer. 2010; 10: 116-129Crossref PubMed Scopus (1849) Google Scholar Variants regulating FGFR2 (MIM: 176943) have the strongest association with ER+ breast cancer susceptibility identified to date.22Meyer K.B. O’Reilly M. Michailidou K. Carlebur S. Edwards S.L. 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FGF10: type III epithelial mesenchymal transition and invasion in breast cancer cell lines.J. Cancer. 2014; 5: 537-547Crossref PubMed Scopus (28) Google Scholar, 25Chioni A.M. Grose R. Negative regulation of fibroblast growth factor 10 (FGF-10) by polyoma enhancer activator 3 (PEA3).Eur. J. Cell Biol. 2009; 88: 371-384Crossref PubMed Scopus (7) Google Scholar It signals through multiple downstream pathways including MAPK and WNT and genes such as FGFR2, CCND1 (MIM: 168461), and TGFB1 (MIM: 190180),21Turner N. Grose R. Fibroblast growth factor signalling: from development to cancer.Nat. Rev. Cancer. 2010; 10: 116-129Crossref PubMed Scopus (1849) Google Scholar, 24Abolhassani A. Riazi G.H. Azizi E. Amanpour S. Muhammadnejad S. Haddadi M. Zekri A. Shirkoohi R. FGF10: type III epithelial mesenchymal transition and invasion in breast cancer cell lines.J. Cancer. 2014; 5: 537-547Crossref PubMed Scopus (28) Google Scholar all known to play key roles in breast cancer. Therapeutic targeting of FGFs and their receptors (FGFRs) is currently a major area of drug development research, and the identification of a subgroup of individuals diagnosed with breast cancer with alterations in these pathways may open new avenues for personalized medicine and pathway-targeted treatments. Download .pdf (3.27 MB) Help with pdf files Document S1. Supplemental Acknowledgments, Figures S1–S12, Tables S1–S10, and Consortia Information 1000 Genomes, http://www.1000genomes.orgCancer Cell Line Encyclopedia (CCLE), https://portals.broadinstitute.org/ccle/homeENCODE, https://www.encodeproject.org/GEO, http://www.ncbi.nlm.nih.gov/geo/GTEx Portal, http://www.gtexportal.org/home/OMIM, http://www.omim.org/PreSTIGE, http://genetics.case.edu/prestige/The Cancer Genome Atlas, http://cancergenome.nih.gov/

A critical component of the DNA damage response is the p53 tumor suppressor, and aberrant p53 function leads to uncontrolled cell proliferation and malignancy. Several molecules have been shown to regulate p53 stability; however, genome-wide systemic approaches for determining the affected, specific downstream target genes have not been extensively studied. Here, we first identified an orphan nuclear receptor, RORα, as a direct target gene of p53, which contains functional p53 response elements. The functional consequences of DNA damage-induced RORα are to stabilize p53 and activate p53 transcription in a HAUSP/Usp7-dependent manner. Interestingly, microarray analysis revealed that RORα-mediated p53 stabilization leads to the activation of a subset of p53 target genes that are specifically involved in apoptosis. We further confirmed that RORα enhances p53-dependent, in vivo apoptotic function in the Drosophila model system. Together, we determined that RORα is a p53 regulator that exerts its role in increased apoptosis via p53.

Experiments were designed to evaluate the nature and extent of microbial contamination and the potential for microbiologically influenced corrosion of alloys exposed in a conventional high sulfur diesel (L100) and alternative fuels, including 100% biodiesel (B100), ultra-low sulfur diesel (ULSD) and blends of ULSD and B100 (B5 and B20). In experiments with additions of distilled water, all fuels supported biofilm formation. Changes in the water pH did not correlate with observations related to corrosion. In all exposures, aluminum 5052 was susceptible to pitting while stainless steel 304L exhibited passive behavior. Carbon steel exhibited uniform corrosion in ULSD and L100, and passive behavior in B5, B20, and B100.

Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.

Post-translational modifications play an important role in regulating protein activity by altering their functions. Sumoylation is a highly dynamic process which is tightly regulated by a fine balance between conjugating and deconjugating enzyme activities. It affects intracellular localization and their interaction with their binding partners, thereby changing gene expression. Consequently, these changes in turn affect signaling mechanisms that regulate many cellular functions, such as cell growth, proliferation, apoptosisapoptosis , DNA repairDNA repair , and cell cancer survival. It is becoming apparent that deregulation in the SUMO pathway contributes to oncogenic transformation by affecting sumoylation/desumoylation of many oncoproteins and tumor suppressors. Loss of balance between sumoylation and desumoylation has been reported in a number of studies in a variety of disease types including cancer. This chapter summarizes the mechanisms and functions of the deregulated SUMO pathway affecting oncogenes and tumor suppressor genes.

G9a histone methyltransferase exerts oncogenic effects in several tumor types and its inhibition promotes anticancer effects. However, the impact on checkpoint inhibitor blockade response and the utility of G9a or its target genes as a biomarker is poorly studied. We aimed to examine whether G9a inhibition can augment the efficacy of checkpoint inhibitor blockade and whether LC3B, a G9a target gene, can predict treatment response.Clinical potential of LC3B as a biomarker of checkpoint inhibitor blockade was assessed using patient samples including tumor biopsies and circulating tumor cells from liquid biopsies. Efficacy of G9a inhibition to enhance checkpoint inhibitor blockade was examined using a mouse model.Patients with melanoma who responded to checkpoint inhibitor blockade were associated with not only a higher level of tumor LC3B but also a higher proportion of cells expressing LC3B. A higher expression of MAP1LC3B or LC3B protein was associated with longer survival and lower incidence of acquired resistance to checkpoint inhibitor blockade, suggesting LC3B as a potential predictive biomarker. We demonstrate that G9a histone methyltransferase inhibition is able to not only robustly induce LC3B level to augment the efficacy of checkpoint inhibitor blockade, but also induces melanoma cell death.Checkpoint inhibitor blockade response is limited to a subset of the patient population. These results have implications for the development of LC3B as a predictive biomarker of checkpoint inhibitor blockade to guide patient selection, as well as G9a inhibition as a strategy to extend the proportion of patients responding to immunotherapy.

PURPOSE Establishing accurate age-related penetrance figures for the broad range of cancer types that occur in individuals harboring a pathogenic germline variant in the TP53 gene is essential to determine the most effective clinical management strategies. These figures also permit optimal use of cosegregation data for classification of TP53 variants of unknown significance. Penetrance estimation can easily be affected by bias from ascertainment criteria, an issue not commonly addressed by previous studies. MATERIALS AND METHODS We performed a maximum likelihood penetrance estimation using full pedigree data from a multicenter study of 146 TP53-positive families, incorporating adjustment for the effect of ascertainment and population-specific background cancer risks. The analysis included pedigrees from Australia, Spain, and United States, with phenotypic information for 4,028 individuals. RESULTS Core Li-Fraumeni syndrome (LFS) cancers (breast cancer, adrenocortical carcinoma, brain cancer, osteosarcoma, and soft tissue sarcoma) had the highest hazard ratios of all cancers analyzed in this study. The analysis also detected a significantly increased lifetime risk for a range of cancers not previously formally associated with TP53 pathogenic variant status, including colorectal, gastric, lung, pancreatic, and ovarian cancers. The cumulative risk of any cancer type by age 50 years was 92.4% (95% CI, 82.2 to 98.3) for females and 59.7% (95% CI, 39.9 to 81.3) for males. Females had a 63.3% (95% CI, 35.6 to 90.1) cumulative risk of developing breast cancer by age 50 years. CONCLUSION The results from maximum likelihood analysis confirm the known high lifetime risk for the core LFS-associated cancer types providing new risk estimates and indicate significantly increased lifetime risks for several additional cancer types. Accurate cancer risk estimates will help refine clinical recommendations for TP53 pathogenic variant carriers and improve TP53 variant classification.

Posttranslational modification by small ubiquitin-like modifier (SUMO) controls diverse cellular functions of transcription factors and coregulators and participates in various cellular processes including signal transduction and transcriptional regulation. Here, we report that pontin, a component of chromatin-remodeling complexes, is SUMO-modified, and that SUMOylation of pontin is an active control mechanism for the transcriptional regulation of pontin on androgen-receptor target genes in prostate cancer cells. Biochemical purification of pontin-containing complexes revealed the presence of the Ubc9 SUMO-conjugating enzyme that underlies its function as an activator. Intriguingly, 5alpha-dihydroxytestosterone treatments significantly increased the SUMOylation of pontin, and SUMOylated pontin showed further activation of a subset of nuclear receptor-dependent transcription and led to an increase in proliferation and growth of prostate cancer cells. These data clearly define a functional model and provide a link between SUMO modification and prostate cancer progression.

Abstract The term microbiologically influenced corrosion (MIC) is used to designate corrosion due to the presence and activities of microorganisms, ie, those organisms that cannot be seen individually with the unaided human eye, including microalgae, archaea, bacteria, and fungi. Microorganisms can accelerate rates of partial reactions in corrosion processes or shift the mechanism for corrosion. Microorganisms can influence pitting, dealloying, enhanced erosion corrosion, enhanced galvanic corrosion, stress corrosion cracking, and hydrogen embrittlement. Microbiologically influenced corrosion has been reported for all engineering metals and alloys with the exception of predominantly titanium and high chromium–nickel alloys. It has been documented for metals and nonmetals exposed to seawater, freshwater, distilled/demineralized water, crude and distillate hydrocarbon fuels, process chemicals, foodstuffs, soils, human plasma, saliva, and sewage. The following sections describe the estimated costs of MIC, mechanisms, and causative microorganisms contributing to MIC; techniques for diagnosing, measuring, and monitoring; engineering practices that influence MIC and strategies to prevent or mitigate MIC.

Surfaces of carbon steel (CS) exposed to mixed cultures of iron-oxidizing bacteria (FeOB) and dissimilatory iron-reducing bacteria (FeRB) in seawater media under aerobic conditions were rougher than surfaces of CS exposed to pure cultures of either type of microorganism. The roughened surface, demonstrated by profilometry, is an indication of loss of metal from the surface. In the presence of CS, aerobically grown FeOB produced tight, twisted helical stalks encrusted with iron oxides. When CS was exposed anaerobically in the presence of FeRB, some surface oxides were removed. However, when the same FeOB and FeRB were grown together in an aerobic medium, FeOB stalks were less encrusted with iron oxides and appeared less tightly coiled. These observations suggest that iron oxides on the stalks were reduced and solubilized by the FeRB. Roughened surfaces of CS and denuded stalks were replicated with culture combinations of different species of FeOB and FeRB under three experimental conditions. Measurements of electrochemical polarization resistance established different rates of corrosion of CS in aerobic and anaerobic media, but could not differentiate rate differences between sterile controls and inoculated exposures for a given bulk concentration of dissolved oxygen. Similarly, total iron in the electrolyte could not be used to differentiate treatments. The experiments demonstrate the potential for iron cycling (oxidation and reduction) on corroding CS in aerobic seawater media.

Metal-organic frameworks (MOFs) have gained much attention as next-generation porous media for various applications, especially gas separation/storage, and catalysis. New MOFs are regularly reported; however, to develop better materials in a timely manner for specific applications, the interactions between guest molecules and the internal surface of the framework must first be understood. A combined experimental and theoretical approach is presented, which proves essential for the elucidation of small-molecule interactions in a model MOF system known as M2 (dobdc) (dobdc(4-) = 2,5-dioxido-1,4-benzenedicarboxylate; M = Mg, Mn, Fe, Co, Ni, Cu, or Zn), a material whose adsorption properties can be readily tuned via chemical substitution. It is additionally shown that the study of extensive families like this one can provide a platform to test the efficacy and accuracy of developing computational methodologies in slightly varying chemical environments, a task that is necessary for their evolution into viable, robust tools for screening large numbers of materials.

Genome-wide association studies have identified 196 high confidence independent signals associated with breast cancer susceptibility. Variants within these signals frequently fall in distal regulatory DNA elements that control gene expression.We designed a Capture Hi-C array to enrich for chromatin interactions between the credible causal variants and target genes in six human mammary epithelial and breast cancer cell lines. We show that interacting regions are enriched for open chromatin, histone marks for active enhancers, and transcription factors relevant to breast biology. We exploit this comprehensive resource to identify candidate target genes at 139 independent breast cancer risk signals and explore the functional mechanism underlying altered risk at the 12q24 risk region.Our results demonstrate the power of combining genetics, computational genomics, and molecular studies to rationalize the identification of key variants and candidate target genes at breast cancer GWAS signals.

Rationale: Epigenetic mechanisms are fundamental processes that can modulate gene expression, allowing cellular adaptation to environmental conditions.Hypoxia is an important factor known to initiate the metastatic cascade in cancer, activating cell motility and invasion by silencing cell adhesion genes.G9a is a histone methyltransferase previously shown to accumulate in hypoxic conditions.While its oncogenic activity has been previously reported, not much is known about the role G9a plays in the hypoxia-mediated metastatic cascade.Methods: The role of G9a in cell motility in hypoxic condition was determined by inhibiting G9a either by short-hairpin mediated knock down or pharmacologically using a small molecule inhibitor.Through gene expression profiling, we identified CDH10 to be an important G9a target that regulates breast cancer cell motility.Lung metastasis assay in mice was used to determine the physiological significance of G9a. Results:We demonstrate that, while hypoxia enhances breast cancer migratory capacity, blocking G9a severely reduces cellular motility under both normoxic and hypoxic conditions and prevents the hypoxia-mediated induction of cellular movement.Moreover, inhibition of G9a histone methyltransferase activity in mice using a specific small molecule inhibitor significantly reduced growth and colonisation of breast cancer cells in the lung.We identify the type-II cadherin CDH10 as being a novel hypoxia-dependent gene, directly repressed by G9a through histone methylation.CDH10 overexpression significantly reduces cellular movements in breast cancer cell lines and prevents the hypoxia-mediated increase in cell motility.In addition, we show that CDH10 expression is prognostic in breast cancer and that it is inversely correlated to EHMT2 (G9a) transcript levels in many tumor-types, including breast cancer. Conclusion:We propose that G9a promotes cellular motility during hypoxic stress through the silencing of the cell adhesion molecule CDH10 and we describe CDH10 as a novel prognostic biomarker for breast cancer.

Silibinin is a polyphenolic flavonoid isolated from the milk thistle (Silybum marianum) and is reported to exhibit anticancer properties. Recently, it has been reported that silibinin inhibits hypoxia-inducible factor-1alpha (HIF-1alpha) expression in cancer cells. However, the precise mechanism by which silibinin decreases HIF-1 expression is not fully understood. In this study, silibinin inhibited basal and hypoxia induced expression levels of HIF-1alpha protein in LNCaP and PC-3 prostate cancer cells, while the rate of HIF-1alpha protein degradation and mRNA levels were not affected. We found that the decrease in HIF-1 protein by silibinin correlated with suppression of de novo synthesis of HIF-1alpha protein. Silibinin inhibited global protein synthesis coincided with reduction of eIF4F complex formation and induction of phosphorylation of the translation initiation factor 2alpha (eIF-2alpha) which can cause inhibition of general protein synthesis. These results suggest that silibinin's activity to inhibit HIF-1alpha protein expression is associated with the suppression of global protein translation.

The relationship between corrosion and biodegradation of bio- and petroleum-based fuels was evaluated using aerobic seawater, fuel and unprotected carbon steel coupons under stagnant conditions to simulate a potential fuel storage condition. Aerobic respiration and corrosion reactions consumed oxygen in the incubations in a short time. The transient oxygen influenced the microbial biodegradation of all fuels and resulted in a suite of characteristic metabolites, including catechols. The corrosion was believed to be the result of biogenic sulfide production and in all cases, the black corrosion products contained chlorine and sulfur (presumed chloride and sulfide) in addition to iron. There were few differences in electrochemically measured corrosion rates in incubations amended with any of the fuels or their blends. Clone library analysis demonstrated higher proportions of Firmicutes, Deltaproteobacteria (primarily sulfate-reducing bacteria), Chloroflexi, and Lentisphaerae in incubations exposed to fuels than the original seawater. Relative proportions of sequences affiliated with these bacterial groups varied with fuel. Methanogen sequences similar to those of Methanolobus were also found in multiple incubations. Despite the dominance of characteristically anaerobic taxa, sequences coding for an alkane monooxygenase from marine hydrocarbon-degrading genera and aerobically produced intermediates were observed, indicative that organisms with this metabolic potential were active at some point during the incubation. Aerobic oxidation of fuel components resulted in the formation of a series of intermediates that could be used by anaerobic seawater microbial communities to support metabolism, sulfide production, and carbon steel corrosion.

Women with epithelial ovarian cancer (EOC) are usually treated with platinum/ taxane therapy after cytoreductive surgery but there is considerable inter-individual variation in response.To identify germline single-nucleotide polymorphisms (SNPs) that contribute to variations in individual responses to chemotherapy, we carried out a multi-phase genome-wide association study (GWAS) in 1,244 women diagnosed with serous EOC who were treated with the same first-line chemotherapy, carboplatin and paclitaxel.We identified two SNPs (rs7874043 and rs72700653) in TTC39B (best P=7x10 -5 , HR=1.90, for rs7874043) associated with progression-free survival (PFS).Functional analyses show that both SNPs lie in a putative regulatory element (PRE) that physically interacts with the promoters of PSIP1, CCDC171 and an alternative promoter of TTC39B.The C allele of rs7874043 is associated with poor PFS and showed increased binding of the Sp1 transcription factor, which is critical for chromatin interactions with PSIP1.Silencing of PSIP1 significantly impaired DNA damage-induced Rad51 nuclear foci and reduced cell viability in ovarian cancer Oncotarget

Exposure to oil from the Deepwater Horizon spill may have lasting impacts on preservation of historic shipwrecks in the Gulf of Mexico. Submerged steel structures, including shipwrecks, serve as artificial reefs and become hotspots of biodiversity in the deep-sea. Marine biofilms on submerged structures support settlement of micro- and macrobiota and may enhance and protect against corrosion. Disruptions in the local environment, including oil spills, may impact the role that biofilms play in reef preservation. To determine how the Deepwater Horizon spill potentially impacted shipwreck biofilms and the functional roles of the biofilm microbiome, experiments containing carbon steels disks (CSDs) were placed at five historic shipwreck sites located within, and external to the benthic footprint of the Deepwater Horizon spill. The CSDs were incubated for 16 weeks to enable colonization by biofilm-forming microorganisms and to provide time for in situ corrosion to occur. Biofilms from the CSDs, as well as sediment and water microbiomes, were collected and analyzed by 16S rRNA amplicon gene sequencing to describe community composition and determine the source of taxa colonizing biofilms. Biofilm metagenomes were sequenced to compare differential gene abundances at spill-impacted and reference sites. Biofilms were dominated by Zeta-, Alpha-, Epsilon and Gammaproteobacteria. Sequences affiliated with the Mariprofundus and Sulfurimonas genera were prolific, and Roseobacter, and Colwellia genera were also abundant. Analysis of 16S rRNA sequences from sediment, water, and biofilms revealed sediment to be the main known source of taxa to biofilms at impacted sites. Differential gene abundance analysis revealed the two-component response regulator CreC, a gene involved in environmental stress response, to be elevated at reference sites compared to impacted sites within the spill plume fallout area on the seafloor. Genes for chemotaxis, motility, and alcohol dehydrogenases were differentially abundant at reference vs. impacted sites. Metal loss on CSDs was elevated at sites within the spill fallout plume. Time series images reveal that metal loss at a heavily impacted site, the German Submarine U-166, has accelerated since the spill in 2010. This study provides evidence that spill residues on the seafloor may impact biofilm communities and the preservation of historic steel shipwrecks.

Although d-glucosamine has been reported as an inhibitor of tumor growth both in vivo and in vitro, the mechanism for the anticancer effect of d-glucosamine is still unclear. Since there are several reports suggesting d-glucosamine inhibits protein synthesis, we examined whether d-glucosamine affects p70S6 K activity, an important signaling molecule involved in protein translation. In the present study, we found d-glucosamine inhibited the activity of p70S6K and the proliferation of DU145 prostate cancer cells and MDA-MB-231 breast cancer cells. d-Glucosamine decreased phosphorylation of p70S6K, and its downstream substrates RPS6, and eIF-4B, but not mTOR and 4EBP1 in DU145 cells, suggesting that d-glucosamine induced inhibition of p70S6K is not through the inhibition of mTOR. In addition, d-glucosamine enhanced the growth inhibitory effects of rapamycin, a specific inhibitor of mTOR. These findings suggest that d-glucosamine can inhibit growth of cancer cells through dephosphorylation of p70S6K.

Abstract IL-6 mediates broad physiological and pathological effects through its receptor signal transducing unit gp130. Due to the reportedly wide cellular expression of gp130, IL-6 is thought to signal ubiquitously via gp130 complex formation with membrane-bound IL-6Rα or soluble IL-6Rα. gp130 signaling primarily induces p-STAT3 and p-STAT1. In contrast to the previous dogma, we show in this article that circulating mouse and human granulocytes are unable to induce p-STAT3 or p-STAT1 after stimulation with IL-6 or an IL-6/soluble IL-6R complex. Furthermore, we demonstrate that this is due to a lack of gp130 expression on mouse and human granulocytes, despite their expression of membrane-bound IL-6R. Importantly, the absence of gp130 is not only a feature of mature granulocytes in healthy individuals, it is also observed after allogeneic stem cell transplantation. Moreover, granulocyte gp130 expression is lost during maturation, because granulocyte-monocyte progenitor cells express gp130 and respond to IL-6. Given that granulocytes constitute 50–70% of circulating leukocytes, this indicates a significantly smaller scope of IL-6 signaling than previously anticipated and has important implications for therapeutic IL-6 inhibition and the mechanisms of action thereof.

Iron (Fe)- and manganese (Mn)-oxidizing bacteria are often cited individually and collectively as putative microorganisms for microbiologically influenced corrosion (MIC). The two groups of microorganisms have in common the ability to attach to surfaces and produce macroscopic accumulations (deposits) of metal oxides/hydroxides/oxyhydroxides that can influence corrosion of some metals and alloys in some environments. In all cases, once initiated, the corrosion is independent of the activities of the colonizing species. Despite the phylogenetic diversity of Fe-oxidizing bacteria (FeOB), the following sections will deal with corrosion mechanisms attributed to neutrophilic, lithotrophic, microaerophilic FeOB. The mineralogy of biologically oxidized Fe is consistent over a wide range of environments. All FeOB produce dense deposits that can cause corrosion of low alloy stainless steels (SS) directly, i.e., under-deposit corrosion. Association of Mn-oxidizing bacteria (MnOB) and other microorganisms may stabil...

G9a and EZH2 are two histone methyltransferases commonly upregulated in several cancer types, yet the precise roles that these enzymes play cooperatively in cancer is unclear. We demonstrate here that frequent concurrent upregulation of both G9a and EZH2 occurs in several human tumors. These methyltransferases cooperatively repressed molecular pathways responsible for tumor cell death. In genetically distinct tumor subtypes, concomitant inhibition of G9a and EZH2 potently induced tumor cell death, highlighting the existence of tumor cell survival dependency at the epigenetic level. G9a and EZH2 synergistically repressed expression of genes involved in the induction of endoplasmic reticulum (ER) stress and the production of reactive oxygen species. IL24 was essential for the induction of tumor cell death and was identified as a common target of G9a and EZH2. Loss of function of G9a and EZH2 activated the IL24-ER stress axis and increased apoptosis in cancer cells while not affecting normal cells. These results indicate that G9a and EZH2 promotes the evasion of ER stress-mediated apoptosis by repressing IL24 transcription, therefore suggesting that their inhibition may represent a potential therapeutic strategy for solid cancers.These findings demonstrate a novel role for G9a and EZH2 histone methyltransferases in suppressing apoptosis, which can be targeted with small molecule inhibitors as a potential approach to improve solid cancer treatment.

Despite overall improvement in breast cancer patient outcomes from earlier diagnosis and personalised treatment approaches, some patients continue to experience recurrence and incurable metastases. It is therefore imperative to understand the molecular changes that allow transition from a non-aggressive state to a more aggressive phenotype. This transition is governed by a number of factors.As crosstalk with extracellular matrix (ECM) is critical for tumour cell growth and survival, we applied high throughput shRNA screening on a validated '3D on-top cellular assay' to identify novel growth suppressive mechanisms.A number of novel candidate genes were identified. We focused on COMMD3, a previously poorly characterised gene that suppressed invasive growth of ER + breast cancer cells in the cellular assay. Analysis of published expression data suggested that COMMD3 is normally expressed in the mammary ducts and lobules, that expression is lost in some tumours and that loss is associated with lower survival probability. We performed immunohistochemical analysis of an independent tumour cohort to investigate relationships between COMMD3 protein expression, phenotypic markers and disease-specific survival. This revealed an association between COMMD3 loss and shorter survival in hormone-dependent breast cancers and in particularly luminal-A-like tumours (ER+/Ki67-low; 10-year survival probability 0.83 vs. 0.73 for COMMD3-positive and -negative cases, respectively). Expression of COMMD3 in luminal-A-like tumours was directly associated with markers of luminal differentiation: c-KIT, ELF5, androgen receptor and tubule formation (the extent of normal glandular architecture; p < 0.05). Consistent with this, depletion of COMMD3 induced invasive spheroid growth in ER + breast cancer cell lines in vitro, while Commd3 depletion in the relatively indolent 4T07 TNBC mouse cell line promoted tumour expansion in syngeneic Balb/c hosts. Notably, RNA sequencing revealed a role for COMMD3 in copper signalling, via regulation of the Na+/K+-ATPase subunit, ATP1B1. Treatment of COMMD3-depleted cells with the copper chelator, tetrathiomolybdate, significantly reduced invasive spheroid growth via induction of apoptosis.Overall, we found that COMMD3 loss promoted aggressive behaviour in breast cancer cells.

The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR) is thought to act as a suppressor of tumor growth by binding the mitogenic peptide IGF-II and modulating its extracellular levels via degradation. This receptor has been found to be absent or nonfunctional in a high proportion of breast tumors as a result of LOH and mutation of the gene. In our study, we have examined the effect of increasing expression of M6P/IGF-IIR on breast cancer cell tumorigenicity. MDA-MB-231 breast cancer cells stably transfected with M6P/IGF-IIR cDNA exhibited not only a greatly reduced ability to form tumors but also a markedly reduced growth rate in nude mice. In vitro, increased M6P/IGF-IIR expression resulted in 2-fold reduced uptake of IGF-II and was associated with reduced cellular invasiness and motility. Cells with increased M6P/IGF-IIR expression exhibited reduced phosphorylation of IGF-I receptor and p44/42 MAPK compared to vector transfectants, or wild-type MDA-MB-231 cells. These results therefore suggest that M6P/IGF-IIR levels can modulate breast cancer cell tumorigenicity by a mechanism that may involve altered IGF-I receptor signaling.

Phaleria macrocarpa, also known as Mahkota dewa, is an Indonesian native plant that has been used as a remedy for many diseases. However, the molecular mechanism of Phaleria macrocarpa is still limited. In this study, we evaluate its molecular mechanism using a bioactivity-guided DLBS1425, an extract of Phaleria macrocarpa on MDA-MB-231 breast cancer cell line. DLBS1425 exhibited inhibition of proliferative, migratory and invasive potential of MDA-MB-231 in a dose-dependent manner, and significantly reduced phosphoinositide-3 (PI3)-kinase/protein kinase B (AKT) signalling by reducing PI3K transcript level and subsequent reduction in AKT phosphorylation. Further, it induced pro-apoptotic genes including BAX, BAD and PUMA and consequently induces cellular death signal by caspase-9 activation, promoting PARP cleavage and DNA fragmentation. Our results suggest that DLBS1425 is a potential anticancer agent which targets genes involved in both cell survival and apoptosis in MDA-MB-231 breast cancer cells.

The cytotoxic enhancement of cisplatin by magnetic fluid hyperthermia (MFH) was investigated in human colon adenocarcinoma cells (Caco-2). A nanoparticle platform based on iron oxide functionalized with carboxymethyl dextran was employed to produce heat at the nanoscale. To assess the synergistic effect of hyperthermia and the anticancer drug cis-Diamminedichloroplatinum, commonly known as cisplatin (CIS), cell viability was measured 24, 48, and 72 hours after three different combined hyperthermia and CIS exposure sequences. These included CIS incubation prior to hyperthermia or magnetic fluid hyperthermia, CIS exposure only during hyperthermia or MFH, and additional CIS incubation following hyperthermia or MFH. Additional incubation of CIS after hyperthermia treatment appears to be more effective than prior CIS incubation for both hyperthermia treatments. Viability data also indicated that MFH combined with CIS is significantly more effective than hot water hyperthermia at the same temperature. A CIS concentration an order of magnitude lower than the calculated IC50 was found to be very effective in reducing cell viability. Such dramatic differences suggest that MFH may enhance the passive transport of CIS.

Despite obvious differences in morphology, substratum chemistry and the electrolyte in which they form, accumulations of iron corrosion products have the following characteristics in common: stratification of iron oxides/hydroxides with a preponderance of α-FeOOH (goethite) and accumulation of metals. Bacteria, particularly iron-oxidizing and sulfate-reducing bacteria have been identified in some accumulations. Both biotic and abiotic mechanisms have been used to rationalize observations for particular sets of environmental data. This review is the first to compare observations and interpretations.

Addition of 1 g/L yeast extract (YE) to sterile, aerobic (approximately 21% dissolved oxygen) and deoxygenated (&amp;lt;0.0001% dissolved oxygen) natural seawater fixed the corrosion potential (Ecorr) of 316L (UNS S31603) stainless steel. YE contains riboflavin and other B vitamins that can act as redox mediators, sorb to surfaces, and chelate metal ions. As demonstrated, YE alters the pH of buffered media, including natural seawater. These same activities are typically attributed to microorganisms and are related to microbiologically influenced corrosion (MIC) mechanisms. Despite the prevalent use of YE to stimulate microbial growth in MIC experiments, the potential impact of YE on the outcome of those experiments has not been examined.

Biodeterioration of plasticized fabrics is a serious problem leading to degradation of materials used in military and civilian applications. This study aimed to characterize the composition of the microbial communities present on six plasticized fabrics exposed to a harsh tropical environment and explore their role in biodeterioration. Metagenomics, bioinformatics, light and scanning electron microscopy (SEM), and solid-phase microextraction GC-MS were used to characterize the fabric-associated microbial communities and plasticizer degradation. SEM analysis showed multi-layered biofilms containing bacteria and a high abundance of fungal structures and yeast cells. Shotgun metagenomics with a multifaceted bioinformatic pipeline generated 3,314,688 contigs and 120 microbial genomes. The microbial genomes were classified into three domains with the majority belonging to fungi followed by bacteria, and archaea. Functional gene annotation revealed that the fabric-associated microbiomes harbor important pathways for energy generation, stress tolerance, and compounds degradation including esterases, lipases and monooxygenases, suggesting a role of these microorganisms in degradation. The results showed that black yeasts were the keystone species of microbiomes affecting the fabrics. Information from this study will help to understand the effect of different microbial communities and species on the biodeterioration of fabrics and may help support the development of new antimicrobial materials.

Experiments were designed to evaluate the corrosion-related consequences of storing/transporting fatty acid methyl ester (FAME) alternative diesel fuel in contact with natural seawater. Coastal Key West, FL (KW), and Persian Gulf (PG) seawaters, representing an oligotrophic and a more organic- and inorganic mineral-rich environment, respectively, were used in 60 day incubations with unprotected carbon steel. The original microflora of the two seawaters were similar with respect to major taxonomic groups but with markedly different species. After exposure to FAME diesel, the microflora of the waters changed substantially, with Clostridiales (Firmicutes) becoming dominant in both. Despite low numbers of sulphate-reducing bacteria in the original waters and after FAME diesel exposure, sulphide levels and corrosion increased markedly due to microbial sulphide production. Corrosion morphology was in the form of isolated pits surrounded by an intact, passive surface with the deepest pits associated with the fuel/seawater interface in the KW exposure. In the presence of FAME diesel, the highest corrosion rates measured by linear polarization occurred in the KW exposure correlating with significantly higher concentrations of sulphur and chlorine (presumed sulphide and chloride, respectively) in the corrosion products.

The gene(s) whose expression is regulated by allergy risk variants is unknown for many loci identified through genome-wide association studies. Addressing this knowledge gap might point to new therapeutic targets for allergic disease. The aim of this study was to identify the target gene(s) and the functional variant(s) underlying the association between rs7009110 on chromosome 8q21 and allergies. Eight genes are located within 1 Mb of rs7009110. Multivariate association analysis of publicly available exon expression levels from lymphoblastoid cell lines (LCLs) identified a significant association between rs7009110 and the expression of a single gene, PAG1 (p = 0.0017), 732 kb away. Analysis of histone modifications and DNase I hypersensitive sites in LCLs identified four putative regulatory elements (PREs) in the region. Chromosome conformation capture confirmed that two PREs interacted with the PAG1 promoter, one in allele-specific fashion. To determine whether these PREs were functional, LCLs were transfected with PAG1 promoter-driven luciferase reporter constructs. PRE3 acted as a transcriptional enhancer for PAG1 exclusively when it carried the rs2370615:C allergy predisposing allele, a variant in complete linkage disequilibrium with rs7009110. As such, rs2370615, which overlaps RelA transcription factor (TF) binding in LCLs and was found to disrupt Foxo3a binding to PRE3, represents the putative functional variant in this locus. Our studies suggest that the risk-associated allele of rs2370615 predisposes to allergic disease by increasing PAG1 expression, which might promote B cell activation and have a pro-inflammatory effect. Inhibition of PAG1 expression or function might have therapeutic potential for allergic diseases. The gene(s) whose expression is regulated by allergy risk variants is unknown for many loci identified through genome-wide association studies. Addressing this knowledge gap might point to new therapeutic targets for allergic disease. The aim of this study was to identify the target gene(s) and the functional variant(s) underlying the association between rs7009110 on chromosome 8q21 and allergies. Eight genes are located within 1 Mb of rs7009110. Multivariate association analysis of publicly available exon expression levels from lymphoblastoid cell lines (LCLs) identified a significant association between rs7009110 and the expression of a single gene, PAG1 (p = 0.0017), 732 kb away. Analysis of histone modifications and DNase I hypersensitive sites in LCLs identified four putative regulatory elements (PREs) in the region. Chromosome conformation capture confirmed that two PREs interacted with the PAG1 promoter, one in allele-specific fashion. To determine whether these PREs were functional, LCLs were transfected with PAG1 promoter-driven luciferase reporter constructs. PRE3 acted as a transcriptional enhancer for PAG1 exclusively when it carried the rs2370615:C allergy predisposing allele, a variant in complete linkage disequilibrium with rs7009110. As such, rs2370615, which overlaps RelA transcription factor (TF) binding in LCLs and was found to disrupt Foxo3a binding to PRE3, represents the putative functional variant in this locus. Our studies suggest that the risk-associated allele of rs2370615 predisposes to allergic disease by increasing PAG1 expression, which might promote B cell activation and have a pro-inflammatory effect. Inhibition of PAG1 expression or function might have therapeutic potential for allergic diseases. To date, genome-wide association studies (GWASs) have identified 41 genetic associations with allergic diseases (Table S1), including asthma (MIM: 600807), hay fever or allergic rhinitis (MIM: 607154), and atopic dermatitis or eczema (MIM: 603165). The identification of allergy risk variants is expected to provide new insights into the molecular pathways involved in disease pathophysiology and, in this way, facilitate the development of new disease treatments. However, these expectations have been hard to meet and this represents a major bottleneck in the field. There are two main reasons for this. First, for many loci, there is no functional evidence linking the risk variant with changes in the expression or protein sequence of any nearby genes; the likely target gene(s) is therefore unknown. This is the case for 27 of the 41 allergy risk loci discovered to date; of note, for 10 of these 27 loci, experimental mouse models of allergic disease implicate nearby genes in disease pathophysiology. Second, often there is little or no information available to determine whether and how disruption of the expression or sequence of the target gene impacts cellular function and, ultimately, disease pathophysiology. Addressing these knowledge gaps is critical to help translate GWAS findings into clinically useful information. In a recent GWAS, we compared 6,685 individuals with both asthma and hay fever against 14,091 control subjects free of asthma and hay fever and identified two new risk loci for allergic disease: 8q21 and 16p13.1Ferreira M.A. Matheson M.C. Tang C.S. Granell R. Ang W. Hui J. Kiefer A.K. Duffy D.L. Baltic S. Danoy P. et al.Australian Asthma Genetics Consortium CollaboratorsGenome-wide association analysis identifies 11 risk variants associated with the asthma with hay fever phenotype.J. Allergy Clin. Immunol. 2014; 133: 1564-1571Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Variants in both were associated with the individual risks of asthma and hay fever, but the association was stronger with the combined asthma and hay fever phenotype. In the 16p13 locus, evidence from gene expression studies suggests that DEXI is the most likely target gene of this association;2Zeller T. Wild P. Szymczak S. Rotival M. Schillert A. Castagne R. Maouche S. Germain M. Lackner K. Rossmann H. et al.Genetics and beyond--the transcriptome of human monocytes and disease susceptibility.PLoS ONE. 2010; 5: e10693Crossref PubMed Scopus (483) Google Scholar, 3Davison L.J. Wallace C. Cooper J.D. Cope N.F. Wilson N.K. Smyth D.J. Howson J.M. Saleh N. Al-Jeffery A. Angus K.L. et al.Cardiogenics ConsortiumLong-range DNA looping and gene expression analyses identify DEXI as an autoimmune disease candidate gene.Hum. Mol. Genet. 2012; 21: 322-333Crossref PubMed Scopus (88) Google Scholar functional studies of this gene are now warranted to understand how variation in its expression might affect disease risk. Currently, the gene whose expression is regulated by allergy risk variants in the 8q21 locus is unknown. Therefore, the aim of this study was to use both population genetics and functional approaches to identify the target gene(s) and likely causal variant(s) underlying the 8q21 association with allergy risk. The study procedures used were approved by the Human Research Ethics Committee from the QIMR Berghofer Medical Research Institute. Eight genes and one miRNA are located within 1 Mb of the sentinel SNP rs7009110, which has a 36% risk allele frequency in Europeans and was associated with a 1.14 per-allele odds of disease,1Ferreira M.A. Matheson M.C. Tang C.S. Granell R. Ang W. Hui J. Kiefer A.K. Duffy D.L. Baltic S. Danoy P. et al.Australian Asthma Genetics Consortium CollaboratorsGenome-wide association analysis identifies 11 risk variants associated with the asthma with hay fever phenotype.J. Allergy Clin. Immunol. 2014; 133: 1564-1571Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar the nearest gene being ZBTB10 (Figure 1A). rs7009110 was the variant with the strongest association with disease risk after imputation of unmeasured variants from the 1000 Genomes Project.1Ferreira M.A. Matheson M.C. Tang C.S. Granell R. Ang W. Hui J. Kiefer A.K. Duffy D.L. Baltic S. Danoy P. et al.Australian Asthma Genetics Consortium CollaboratorsGenome-wide association analysis identifies 11 risk variants associated with the asthma with hay fever phenotype.J. Allergy Clin. Immunol. 2014; 133: 1564-1571Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Forty-three gene expression quantitative trait loci (eQTLs) have been reported in this 2 Mb region across six relevant tissue or cell types, but rs7009110 is not in linkage disequilibrium (LD) with any of these (Table S2). However, rs7009110 is in modest LD with two nearby risk variants associated with eczema (rs7000782, r2 = 0.51)4Paternoster L. Standl M. Chen C.M. Ramasamy A. Bonnelykke K. Duijts L. Ferreira M.A. Alves A.C. Thyssen J.P. Albrecht E. et al.Meta-analysis of genome-wide association studies identifies three new risk loci for atopic dermatitis.Nat. Genet. 2011; 44: 187-192Crossref PubMed Scopus (261) Google Scholar and rheumatoid arthritis (MIM: 180300; rs998731, r2 = 0.20);5Okada Y. Wu D. Trynka G. Raj T. Terao C. Ikari K. Kochi Y. Ohmura K. Suzuki A. Yoshida S. et al.RACI consortiumGARNET consortiumGenetics of rheumatoid arthritis contributes to biology and drug discovery.Nature. 2014; 506: 376-381Crossref PubMed Scopus (1418) Google Scholar in Europeans, the risk alleles for these three variants (rs7009110:T, rs7000782:A, and rs998731:T) are on the same haplotype. The associations at this locus with asthma, hay fever, eczema, and rheumatoid arthritis suggest that the underlying disrupted molecular mechanism affects a component of the immune system that is shared between allergic and auto-immune diseases. The most plausible candidate target genes are TPD52 (MIM: 604068), which is involved in B cell maturation;6Tiacci E. Orvietani P.L. Bigerna B. Pucciarini A. Corthals G.L. Pettirossi V. Martelli M.P. Liso A. Benedetti R. Pacini R. et al.Tumor protein D52 (TPD52): a novel B-cell/plasma-cell molecule with unique expression pattern and Ca(2+)-dependent association with annexin VI.Blood. 2005; 105: 2812-2820Crossref PubMed Scopus (39) Google Scholar PAG1 (MIM: 605767), involved in T and B cell activation;7Smida M. Cammann C. Gurbiel S. Kerstin N. Lingel H. Lindquist S. Simeoni L. Brunner-Weinzierl M.C. Suchanek M. Schraven B. Lindquist J.A. PAG/Cbp suppression reveals a contribution of CTLA-4 to setting the activation threshold in T cells.Cell Commun. Signal. 2013; 11: 28Crossref PubMed Scopus (9) Google Scholar, 8Itoh K. Sakakibara M. Yamasaki S. Takeuchi A. Arase H. Miyazaki M. Nakajima N. Okada M. Saito T. Cutting edge: negative regulation of immune synapse formation by anchoring lipid raft to cytoskeleton through Cbp-EBP50-ERM assembly.J. Immunol. 2002; 168: 541-544Crossref PubMed Scopus (151) Google Scholar, 9Davidson D. Bakinowski M. Thomas M.L. Horejsi V. Veillette A. Phosphorylation-dependent regulation of T-cell activation by PAG/Cbp, a lipid raft-associated transmembrane adaptor.Mol. Cell. Biol. 2003; 23: 2017-2028Crossref PubMed Scopus (165) Google Scholar, 10Kalland M.E. Solheim S.A. Skånland S.S. Taskén K. Berge T. Modulation of proximal signaling in normal and transformed B cells by transmembrane adapter Cbp/PAG.Exp. Cell Res. 2012; 318: 1611-1619Crossref PubMed Scopus (8) Google Scholar and ZBTB10, a putative repressor of the Sp1 transcription factor (TF),11Tillotson L.G. RIN ZF, a novel zinc finger gene, encodes proteins that bind to the CACC element of the gastrin promoter.J. Biol. Chem. 1999; 274: 8123-8128Crossref PubMed Scopus (44) Google Scholar which regulates multiple immune-related genes.12Tone M. Powell M.J. Tone Y. Thompson S.A. Waldmann H. IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3.J. Immunol. 2000; 165: 286-291Crossref PubMed Scopus (218) Google Scholar, 13Takahashi K. Hayashi N. Shimokawa T. Umehara N. Kaminogawa S. Ra C. Cooperative regulation of Fc receptor gamma-chain gene expression by multiple transcription factors, including Sp1, GABP, and Elf-1.J. Biol. Chem. 2008; 283: 15134-15141Crossref PubMed Scopus (28) Google Scholar We first considered the possibility that the lack of a published association between rs7009110 and the expression of nearby genes could represent a false-negative finding, arising because (1) a true association did not exceed the false discovery rate threshold in the original eQTL studies and so was not reported; and/or (2) most published eQTL studies surveyed used expression microarrays, which have incomplete coverage of gene expression patterns. To test this possibility, we analyzed RNA-seq data obtained by the Geuvadis Project14Lappalainen T. Sammeth M. Friedländer M.R. ’t Hoen P.A. Monlong J. Rivas M.A. Gonzàlez-Porta M. Kurbatova N. Griebel T. Ferreira P.G. et al.Geuvadis ConsortiumTranscriptome and genome sequencing uncovers functional variation in humans.Nature. 2013; 501: 506-511Crossref PubMed Scopus (1212) Google Scholar for lymphoblastoid cell line (LCL) samples of 373 individuals of European descent from the 1000 Genomes Project.15Abecasis G.R. Auton A. Brooks L.D. DePristo M.A. Durbin R.M. Handsaker R.E. Kang H.M. Marth G.T. McVean G.A. 1000 Genomes Project ConsortiumAn integrated map of genetic variation from 1,092 human genomes.Nature. 2012; 491: 56-65Crossref PubMed Scopus (5677) Google Scholar LCLs are derived from peripheral blood B cells and therefore represent a practical and effective in vitro model to study gene expression patterns relevant to immune-related conditions. Genotype and RNA-seq data were downloaded from EBI ArrayExpress: E-GEUV-1 and E-GEUV-2. As in Lappalainen,14Lappalainen T. Sammeth M. Friedländer M.R. ’t Hoen P.A. Monlong J. Rivas M.A. Gonzàlez-Porta M. Kurbatova N. Griebel T. Ferreira P.G. et al.Geuvadis ConsortiumTranscriptome and genome sequencing uncovers functional variation in humans.Nature. 2013; 501: 506-511Crossref PubMed Scopus (1212) Google Scholar we selected the exon (not the gene) as the quantification unit and restricted the analysis to exons expressed in >90% of all the individuals. Read counts normalized by library depth and with technical variation removed by PEER normalization14Lappalainen T. Sammeth M. Friedländer M.R. ’t Hoen P.A. Monlong J. Rivas M.A. Gonzàlez-Porta M. Kurbatova N. Griebel T. Ferreira P.G. et al.Geuvadis ConsortiumTranscriptome and genome sequencing uncovers functional variation in humans.Nature. 2013; 501: 506-511Crossref PubMed Scopus (1212) Google Scholar were available for all exons of six of the eight genes located within a 1 Mb region around rs7009110. Read counts were quantile normalized and adjusted for ancestry informative covariates and genotype imputation status, as in Lappalainen.14Lappalainen T. Sammeth M. Friedländer M.R. ’t Hoen P.A. Monlong J. Rivas M.A. Gonzàlez-Porta M. Kurbatova N. Griebel T. Ferreira P.G. et al.Geuvadis ConsortiumTranscriptome and genome sequencing uncovers functional variation in humans.Nature. 2013; 501: 506-511Crossref PubMed Scopus (1212) Google Scholar For each of these six genes, the association between rs7009110 allelic dosage and variation in the expression of all exons was tested simultaneously via a multivariate test of association that improves power over the alternative strategy of testing each exon individually.16Ferreira M.A. Purcell S.M. A multivariate test of association.Bioinformatics. 2009; 25: 132-133Crossref PubMed Scopus (169) Google Scholar The remaining two genes, ZNF704 and STMN2 (MIM: 600621), were expressed in only 56% and 12% of samples, respectively. Given their low relative abundance and because there was no association between dichotomized read counts (expressed versus not expressed) and rs7009110 genotype (not shown), both genes were excluded from further analysis. Likewise, the expression of miR5708 was not associated with rs7009110 (not shown) and so this miRNA was not considered further. Multivariate association was tested between rs7009110 and the expression levels of five exons in each of HEY1 (MIM: 602953), MRPS28 (MIM: 611990), and FABP5 (MIM: 605168); seven in ZBTB10; nine in PAG1; and ten in TPD52. In this analysis, rs7009110 was significantly associated with the expression of PAG1 (p = 0.0017) but with no other gene (Table S3). The weights attributed to individual PAG1 exons in the multivariate analysis were consistent with an effect of rs7009110 on the expression of exons 1, 2, and 3; this observation was confirmed with individual univariate analyses of these three exons (Table S4). The rs7009110:T allele that increases the risk of allergic disease was associated with an increased expression of PAG1 (Figure S1), explaining 2.6% of the observed variation. To help fine-map the eQTL results for PAG1, we extended the multivariate association analysis to an additional 167 common variants (MAF ≥ 5%, call rate > 95%, Hardy-Weinberg equilibrium test p value > 10−6) located in the 69.4 kb core region of association with allergic disease, delimited by the left-most (rs7822328, chr8: 81,246,659) and right-most (rs4739746, chr8: 81,316,034) variants in LD (r2 ≥ 0.6) with rs7009110. This is the region most likely to include the underlying causal variant(s); most variants were in LD with rs7009110 (70% with r2 ≥ 0.6). Multiple SNPs in high LD (r2 ≥ 0.8) with rs7009110 were associated with the expression of PAG1 (Figure 1B). In general, the strength of the association increased with the physical proximity to rs7009110 but did not exceed that seen with this SNP. However, no SNPs in LD (r2 ≥ 0.6) with rs7009110 showed an association with the expression of the other five genes tested (p > 0.05, Figure S2). Although the association between 8q21 allergy risk variants and PAG1 expression in LCLs was relatively modest, it pointed to the possibility that a regulatory element in this region might control the expression of PAG1. Thus, we next queried genome-wide maps of epigenetic profiles to search for putative regulatory elements (PREs) in the 8q21 core region of association. Analysis of histone modifications associated with regulatory activity (e.g., H3K4me1/2) and DNase I hypersensitive sites assayed by the ENCODE Project17ENCODE Project ConsortiumAn integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (11031) Google Scholar in an LCL (GM12878) identified four PREs in this region, named PRE1 to PRE4 (Figure 1C). Consistent with these results, Seumois et al.18Seumois G. Chavez L. Gerasimova A. Lienhard M. Omran N. Kalinke L. Vedanayagam M. Ganesan A.P. Chawla A. Djukanović R. et al.Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility.Nat. Immunol. 2014; 15: 777-788Crossref PubMed Scopus (124) Google Scholar detected a significant gain in H3K4me2, which marks both active and poised enhancers, in PRE1 and PRE2 when comparing primary human CD4+ memory T cells against naive CD4+ T cells. Similarly, Hnisz et al.19Hnisz D. Abraham B.J. Lee T.I. Lau A. Saint-André V. Sigova A.A. Hoke H.A. Young R.A. Super-enhancers in the control of cell identity and disease.Cell. 2013; 155: 934-947Abstract Full Text Full Text PDF PubMed Scopus (2102) Google Scholar predicted PRE3 to be an enhancer in multiple human immune cell types. Of the 118 SNPs that are in LD (r2 ≥ 0.6) with rs7009110, 35 overlap one of the four PREs identified (Table S5); rs7009110 overlaps PRE3. We therefore hypothesized that (1) one (or more) of these four PREs in this region regulates the expression of PAG1 and (2) rs7009110 or a correlated variant disrupts the function of the PRE. To test the first hypothesis, we used Chromosome Conformation Capture (3C) as described previously20Ghoussaini M. Edwards S.L. Michailidou K. Nord S. Cowper-Sal Lari R. Desai K. Kar S. Hillman K.M. Kaufmann S. Glubb D.M. et al.Australian Ovarian Cancer Management GroupAustralian Ovarian Cancer Management GroupEvidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation.Nat. Commun. 2014; 4: 4999Crossref PubMed Scopus (87) Google Scholar to quantify the frequency of long-range chromatin interactions that take place between the core region of association and the promoter of PAG1, 710 kb apart. In brief, 3C libraries were created by cross-linking the chromatin from LCLs of individuals homozygous for the rs7009110:T risk allele (n = 2) or the C protective allele (n = 2); DNA was then digested with EcoRI, which flanks 17 contiguous fragments that cover the core region of association, as well as the PAG1 promoter (Table S6); DNA was religated and decrosslinked; and quantitative PCR (qPCR) with primers for the bait (PAG1 promoter) and interactors (17 fragments) was then performed to detect the presence of ligation products, which represent gene loops. BAC clones covering the regions of interest were used to normalize for PCR efficiency. The frequency of chromatin interactions between the PAG1 promoter and 15 of the 17 fragments that covered the core region of association was low and consistent with being due to chance (Figure 2A). However, for two fragments—numbered F9 and F11—the interaction frequency was significantly above the background level. The size of the interaction products amplified was confirmed by gel electrophoresis and their sequence verified by Sanger sequencing; similar results were also obtained in two additional independent replicates of each sample (Figure S3). Fragment F9 is 1.1 kb long and overlaps the centromeric end of PRE2, including the peak region of H3K4me2 gain observed by Seumois et al.18Seumois G. Chavez L. Gerasimova A. Lienhard M. Omran N. Kalinke L. Vedanayagam M. Ganesan A.P. Chawla A. Djukanović R. et al.Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility.Nat. Immunol. 2014; 15: 777-788Crossref PubMed Scopus (124) Google Scholar in memory CD4+ T cells. Interactions with fragment F9 were observed in LCLs homozygote for the rs7009110:C protective allele but not (or less frequently) for the T predisposing allele. Consistent with this, allele-specific 3C from two LCLs heterozygous for rs11783496, a SNP near fragment F9 and in LD with rs7009110 (r2 = 0.75), indicate that the protective C allele of rs11783496 is more strongly associated with looping of PRE2 to the PAG1 promoter (Figures 2B and S4). These results suggest that an allergy protective allele (rs11783496:C or another on the same haplotype) is associated with the establishment of long-range chromatin interactions between PRE2 and the PAG1 promoter. The interaction observed with fragment F11—which is 10.6 kb long and overlaps both the telomeric end of PRE2 and the centromeric end of PRE3—was detected in all four samples, irrespective of rs7009110 genotype. This suggests that allergy-associated variants do not influence the establishment of chromatin looping between the PAG1 promoter and PRE3. Having established that two independent DNA fragments overlapping PRE2 and PRE3 physically interact with the promoter of PAG1, next we tested the hypothesis that the regulatory ability of these two PREs is modulated by allergy risk variants. To assess this, a PAG1 promoter-driven luciferase reporter construct was generated by inserting a 1,660 bp fragment containing the PAG1 promoter into pGL3-basic, as described previously.21French J.D. Ghoussaini M. Edwards S.L. Meyer K.B. Michailidou K. Ahmed S. Khan S. Maranian M.J. O’Reilly M. Hillman K.M. et al.GENICA NetworkkConFab InvestigatorsFunctional variants at the 11q13 risk locus for breast cancer regulate cyclin D1 expression through long-range enhancers.Am. J. Hum. Genet. 2013; 92: 489-503Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar A fragment overlapping PRE2 (1,893 bp) or PRE3 (1,560 bp) and containing the major (i.e., allergy protective) allele for SNPs in LD (r2 > 0.6) with rs7009110 was inserted downstream of luciferase. The coordinates of these fragments were selected such that they coincided with the two interaction peaks observed in the 3C experiments (fragments F9 and F11) and, within these regions, with the highest density of histone modifications and DNase I hypersensitive sites from ENCODE (Figures 1D and S5). To assess the effect of individual SNPs on PRE activity, the minor (i.e., allergy predisposing) allele for SNPs in LD with rs7009110 was individually incorporated into PRE2 (rs11783496, rs11786685, rs11786704, or rs13275449) and PRE3 (rs4739737, rs10957979, or rs2370615) (Figure 1D) via standard DNA cloning. All constructs were sequenced to confirm variant incorporation (AGRF). Primers used to generate all constructs are listed in Table S7. LCLs were electroporated with the reporter plasmids and luciferase activity was measured 24 hr post-transfection. To correct for any differences in transfection efficiency or cell lysate preparation, Firefly luciferase activity was normalized to Renilla luciferase. The activity of each test construct was calculated relative to PAG1 promoter construct. Our 3C studies provided evidence for allele-specific chromatin looping between PRE2 and PAG1. Despite this, PRE2 did not enhance or silence the PAG1 promoter in reporter assays (Figure 3A), making it unclear of the functional consequence of allele-specific chromatin looping. For PRE3, which interacted with the PAG1 promoter irrespective of genotype, we found that the construct containing the major (allergy protective) allele at the three SNPs (rs4739737, rs10957979, and rs2370615) in LD with rs7009110 also did not increase PAG1 promoter activity (Figure 3B). A similar lack of regulatory activity was observed for PRE3 when the fragments cloned contained the minor (allergy predisposing) allele for rs4739737 or rs10957979. However, the PRE3 construct containing the minor allele for rs2370615 increased PAG1 promoter activity by 1.8-fold when compared to the promoter-only construct (p = 0.0051) and by 2.3-fold when compared to the construct with the promoter plus the enhancer containing the major alleles (p = 0.0005). These results demonstrate that PRE3 acts as a transcriptional enhancer in the presence of the rs2370615:C allergy predisposing allele. Consistent with this effect, rs2370615:C was associated with increased expression of PAG1 in the eQTL analyses described above (p = 0.0018); this variant is in complete LD (r2 = 1) with rs7009110. Collectively, these results confirm that PAG1 is a target gene of 8q21 allergy risk variants and suggest that rs2370615 represents the underlying putative functional variant. Based on ENCODE ChIP-seq data for LCLs,17ENCODE Project ConsortiumAn integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (11031) Google Scholar two transcription factors (TFs) bind to PRE3 and overlap the putative functional variant rs2370615: the RelA (or p65 [MIM: 164014]) subunit of the nuclear factor κB (NF-κB) TF, which is critical for innate and adaptive immune responses,22Bonizzi G. Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity.Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2073) Google Scholar and the POU domain class 2 transcription factor 2 (POU2F2 or Oct-2 [MIM: 164176]), which regulates B-cell-specific genes.23Matthias P. Lymphoid-specific transcription mediated by the conserved octamer site: who is doing what?.Semin. Immunol. 1998; 10: 155-163Crossref PubMed Scopus (66) Google Scholar These TFs have been shown to co-occur in LCLs24Zhao B. Barrera L.A. Ersing I. Willox B. Schmidt S.C. Greenfeld H. Zhou H. Mollo S.B. Shi T.T. Takasaki K. et al.The NF-κB genomic landscape in lymphoblastoid B cells.Cell Rep. 2014; 8: 1595-1606Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar and to synergistically regulate enhancer activity in B cells.25Sepulveda M.A. Emelyanov A.V. Birshtein B.K. NF-kappa B and Oct-2 synergize to activate the human 3′ Igh hs4 enhancer in B cells.J. Immunol. 2004; 172: 1054-1064Crossref PubMed Scopus (34) Google Scholar Furthermore, TNF-α (MIM: 191160)-induced recruitment of RelA to enhancers involved in long-range looping interactions is associated with transcriptional induction of target genes.26Jin F. Li Y. Dixon J.R. Selvaraj S. Ye Z. Lee A.Y. Yen C.A. Schmitt A.D. Espinoza C.A. Ren B. A high-resolution map of the three-dimensional chromatin interactome in human cells.Nature. 2013; 503: 290-294PubMed Google Scholar These observations suggest that binding of RelA and Oct-2 to PRE3 might be required for long-range activation of PAG1 transcription. However, despite RelA binding to PRE3 being highest precisely over rs2370615 (Figure S5), this T/C polymorphism is not predicted to disrupt the binding motifs reported for either RelA or Oct-2.24Zhao B. Barrera L.A. Ersing I. Willox B. Schmidt S.C. Greenfeld H. Zhou H. Mollo S.B. Shi T.T. Takasaki K. et al.The NF-κB genomic landscape in lymphoblastoid B cells.Cell Rep. 2014; 8: 1595-1606Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 27Staudt L.M. Clerc R.G. Singh H. LeBowitz J.H. Sharp P.A. Baltimore D. Cloning of a lymphoid-specific cDNA encoding a protein binding the regulatory octamer DNA motif.Science. 1988; 241: 577-580Crossref PubMed Scopus (194) Google Scholar Based on these observations, it is possible that the rs2370615:C allele alters the binding affinity of another transcription factor, which is involved in RelA recruitment to PRE3 and so promotes long-range activation of PAG1 transcription. Notably, the rs2370615:C allele is predicted to disrupt the binding motif (TTGTTTAC) for five Forkhead box (Fox) TFs,28Ward L.D. Kellis M. HaploReg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants.Nucleic Acids Res. 2012; 40: D930-D934Crossref PubMed Scopus (1655) Google Scholar namely Foxo3a (MIM: 602681), an NF-κB antagonist that inhibits lymphocyte activation and proliferation.29Lin L. Hron J.D. Peng S.L. Regulation of NF-kappaB, Th activation, and autoinflammation by the forkhead transcription factor Foxo

Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.

Abstract: Underground storage tanks (UST(s)) are a critical infrastructure for the storage of petroleum and other hazardous substances. As with much of the nationwide infrastructure, USTs are aging beyond their intended lifetime. In 1985, the average age of a UST removed for replacement or closeout was 17 years old; USTs removed today are on average 33 years old. Corrosion in UST systems can lead to tank failure resulting in leaks which can contaminate soil and ground water and also result in vapor intrusion in nearby buildings. Presently, there are approximately 65,000 leaking USTs in the U.S. With increased flooding, both inland and coastal, there is greater potential for accelerated corrosion where there are approximately 33,000 USTs within FEMA's 100 year floodplain. With changes in the fuel supply through the introduction of alternative fuels, unintended consequences to fuel storage and delivery infrastructure have been observed. Biofouling and corrosion can be exacerbated by additions of relatively small volumes of alternative fuels. The current standards for monitoring the potential leakage events of USTs are wholly inadequate in terms of predictive capabilities. In this present work, the past, current and future of UST infrastructure are detailed. The materials used in the construction of the USTs including plastics and steels are reviewed as well as their compatibilities to the current and future fuels. A geospatial database application developed by the Environmental Protection Agency is highlighted for insights into correlations between UST data (e.g. age, type, location, fuel) and natural disasters (e.g. flooding, fires) in order to identify potentially vulnerable systems.

Steel marine structures provide foci of biodiversity when they develop into artificial reefs. Development begins with deposition of a biofilm. The effects of contaminants from oil spills on biofilm microbiomes, microbially-induced corrosion (MIC) and metal loss may impact preservation of marine metal structures. A microcosm experiment exposed biofilms on carbon steel disks (CSDs) to crude oil, dispersant, and dispersed oil to address their impacts on bacterial composition and metal loss and pitting. Biofilm diversity increased over time in all exposures. Community composition in dispersant and dispersed oil treatments deviated from the controls for the duration of a 12-week experiment. As biofilms matured, Pseudomonadaceae increased while Rhodobacteraceae decreased in abundance in dispersed oil treatments compared to the controls and dispersant treatments. Greatest mass loss and deepest pitting on CSDs were observed in dispersed oil treatments, suggesting impacts manifest as a consequence of increased MIC potential on carbon steel.

Genetic and epigenetic factors contribute to the development of cancer. Epigenetic dysregulation is common in gynaecological cancers and includes altered methylation at CpG islands in gene promoter regions, global demethylation that leads to genome instability and histone modifications. Histones are a major determinant of chromosomal conformation and stability, and unlike DNA methylation, which is generally associated with gene silencing, are amenable to post-translational modifications that induce facultative chromatin regions, or condensed transcriptionally silent regions that decondense resulting in global alteration of gene expression. In comparison, other components, crucial to the manipulation of chromatin dynamics, such as histone modifying enzymes, are not as well-studied. Inhibitors targeting DNA modifying enzymes, particularly histone modifying enzymes represent a potential cancer treatment. Due to the ability of epigenetic therapies to target multiple pathways simultaneously, tumours with complex mutational landscapes affected by multiple driver mutations may be most amenable to this type of inhibitor. Interrogation of the actionable landscape of different gynaecological cancer types has revealed that some patients have biomarkers which indicate potential sensitivity to epigenetic inhibitors. In this review we describe the role of epigenetics in gynaecological cancers and highlight how it may exploited for treatment.

KAI1 is a metastasis suppressor gene known to inhibit cancer metastasis without affecting primary tumorigenicity. Although KAI1 expression has been reported to undergo transcriptional regulation, how its expression is up- or down-regulated by specific upstream signaling pathways has not been studied in detail. In this study, we characterized the regulatory elements within the 500 bp upstream region of mouse KAI1 gene and identified a functional hypoxia-response element (HRE) within the promoter region. Hypoxia-dependent induction of KAI1 was directly mediated by hypoxia-inducible factor (HIF)-1α binding on the promoter, which subsequently caused increased recruitment of RNA polymerase II for transcriptional activation. The failure of HIF-1α recruitment to the KAI1 promoter was observed in Hif-1α knockout mouse embryonic fibroblasts. Furthermore, KAI1 protein synthesis was markedly increased in ischemic tissues, suggesting that KAI1 is a hypoxia target gene in vivo.

Two coastal seawaters (Key West, FL, USA and the Persian Gulf, Bahrain, representing oligotrophic and eutrophic environments, respectively) were used to evaluate potential biodegradation and corrosion problems during exposure to alternative and conventional fuels. Uncoated carbon steel was exposed at the fuel/seawater interface and polarization resistance was monitored. Under typical marine storage conditions, dioxygen in natural seawater exposed to fuel and carbon steel was reduced to <0.1parts-per-million within 2d due to consumption by corrosion reactions and aerobic microbial respiration. Sulfides, produced by anaerobic sulfate-reducing bacteria, and chlorides were co-located in corrosion products. Transient dioxygen influenced both metabolic degradation pathways and resulting metabolites. Catechols, indicative of aerobic biodegradation, persisted after 90d exposures. Detection of catechols suggested that initial exposure to dioxygen resulted in the formation of aerobic metabolites that exacerbated subsequent corrosion processes.

Understanding the effect of conventional and alternative fuels on the marine bacterial community is crucial, as it pertains to the impact, biodegradation, and final fate of these fuels in the environment. Metagenomics analysis demonstrated that conventional and alternative fuels promoted the growth of Proteobacteria. Marinobacter and Desulfovibrio were predominant in seawater exposed to conventional jet propellant-5 (JP-5), while Hyphomonas and Rhodovulum were most abundant in seawater with hydroprocessed renewable jet fuel (HRJ) and conventional F-76 diesel, respectively. The phyla Bacteroidetes, Firmicutes, and Lentisphaerae were underrepresented in samples with fuel, and these phyla were largely comprised of unclassified bacteria. Culture-dependent tests isolated several of the same genera detected in high abundance by metagenomics DNA sequencing, including Marinobacter, Rhodovulum, and Halobacillus. Growth studies in fuel and gas chromatography analysis demonstrated that isolates grew in fuel and metabolized hydrocarbons efficiently. The hydrocarbon degradation profile of each bacterium was conserved from conventional to alternative fuels. The study indicated that bacteria must out-compete others to get established and proliferate. Competition between hydrocarbon degraders was an important factor affecting the bioremediation process. This study provides insights into the growth characteristics of hydrocarbon-degrading bacteria and the effects of fuel on marine bacterial communities.

Student-run free clinics (SRFCs) have the capacity to decrease health care inequity in underserved populations. These facilities can benefit from improved patient experience and outcomes. We implemented a series of quality improvement interventions with the objectives to decrease patient wait times and to increase the variety of services provided.A needs assessment was performed. Problems related to time management, communication between staff and providers, clinic resources, and methods for assessing clinic performance were identified as targets to reduce wait times and improve the variety of services provided. Seventeen interventions were designed and implemented over a 2-month period.The interventions resulted in improved efficiency for clinic operations and reduced patient wait times. The number of specialty providers, patient visits for specialty care, lifestyle education visits for disease prevention and treatment, free medications, and free laboratory investigations increased to achieve the goal of improving the availability and the variety of services provided.We demonstrated that it is feasible to implement successful quality improvement interventions in SRFCs to decrease patient wait times and to increase the variety of services provided. We believe that the changes we implemented can serve as a model for other SRFCs to improve their performance.

Up to 80% of endometrial and breast cancers express oestrogen receptor alpha (ERα). Unlike breast cancer, anti-oestrogen therapy has had limited success in endometrial cancer, raising the possibility that oestrogen has different effects in both cancers. We investigated the role of oestrogen in endometrial and breast cancers using data from The Cancer Genome Atlas (TCGA) in conjunction with cell line studies. Using phosphorylation of ERα (ERα-pSer118) as a marker of transcriptional activation of ERα in TCGA datasets, we found that genes associated with ERα-pSer118 were predominantly unique between tumour types and have distinct regulators. We present data on the alternative and novel roles played by SMAD3, CREB-pSer133 and particularly XBP1 in oestrogen signalling in endometrial and breast cancer.

Functional role of nuclear receptors and numerous coregulators have been studied in terms of regulating transcriptional control of genes that play critical roles in various pathways. There is growing evidence that nuclear receptors and their coregulators control inflammatory programs of gene expression and progression of hormone-dependent cancer. This review provides a general overview of the interrelationship between nuclear receptor signalling, inflammation and cancer. These insights provide inflammatory genes as attractive targets for the development of cancer therapeutics.

Knee function deficits may persist after anterior cruciate ligament reconstruction (ACLR). Return to sport (RTS) testing batteries assess recovery after ACLR and can guide RTS progression, but the ideal test components are debatable. The single leg vertical hop for height (SLVH) test using a commercially available jump mat may provide a valuable assessment of knee function.The purpose of this study was to compare the limb symmetry index (LSI) of SLVH to horizontal hop testing in a cohort of National Collegiate Athletic Association (NCAA) Division 1 collegiate athletes after ACLR. The hypothesis was the SLVH would elicit significantly lower LSI than horizontal hop tests.Cross-Sectional Study.Eighteen National Collegiate Athletic Association (NCAA) Division 1 collegiate athletes (7 males, 11 females) at 7.33 ± 2.05 months after ACLR were included in this retrospective study. LSI was calculated for single hop for distance (SHD), triple hop for distance (THD), cross-over hop for distance (CHD), timed 6-meter hop (T6H), and SLVH. A repeated measures ANOVA was performed to identify differences in LSI for each test. Spearman's Rho correlation coefficient was calculated to examine the relationship between LSIs for each test.The LSI for SLVH (84.48% ± 11.41%) was significantly lower than LSI for SHD (95.48 ± 8.02%, p = 0.003), THD (94.40 ± 3.70%, p = 0.002), CHD (95.85 ± 7.00, p = 0.007), and T6H (97.69 ± 6.60%, p = 0.001). The correlation of LSI between SLVH and the horizontal hop tests was weak and non-significant for SHD (rs = 0.166, p = 0.509), CHD (rs = 0.199, p = 0.428), and T6H (rs = 0.211, p = 0.401) and moderate and non-significant for THD (rs = 0.405, p = 0.096).Individuals after ACLR had lower LSI on the SLVH than on horizontal hop tests and weak to moderate correlations between the tests suggest SLVH detects performance deficits not identified by the horizontal hop tests.3.

The COVID-19 pandemic has led to a proliferation of intubation barriers designed to protect healthcare workers from infection. We developed the Suction-Assisted Local Aerosol Containment Chamber (SLACC) and tested it in the operating room. The primary objectives were to determine the ease and safety of airway management with SLACC, and to measure its efficacy of aerosol containment to determine if it significantly reduces exposure to health care workers. In this randomized clinical trial, adult patients scheduled to undergo elective surgery with general endotracheal anesthesia were screened and informed consent obtained from those willing to participate. Patients were randomized to airway management either with or without the SLACC device. Patients inhaled nebulized saline before and during anesthesia induction to simulate the size and concentration of particles seen with severe symptomatic SARS-CoV-2 infection. 79 patients were enrolled and randomized. Particle number concentration (PNC) at the patients' and healthcare workers’ locations were measured and compared between the SLACC vs. control groups during airway management. Ease and success of tracheal intubation were recorded for each patient. All intubations were successful and time to intubation was similar between the two groups. Healthcare workers were exposed to significantly lower particle number concentrations (#/cm3) during airway management when SLACC was utilized vs. control. The particle count outside SLACC was reduced by 97% compared to that inside the device. The SLACC device does not interfere with airway management and significantly reduces healthcare worker exposure to aerosolized particles during airway management.

We examine cost shifting within the context of Medicare payment policy. We briefly review economic theory and available data and discuss the importance of cost shifting for policy. Then we present four central findings on cost shifting based on the views of former high-level policymakers. First, Medicare's early (pre-prospective) payment policy was a boon to hospitals. Second, Medicare payment policy is a "top-down" affair, driven by budgetary and special-interest politics. Third, federal policymakers may not consciously consider cost shifting, but state policymakers do. Fourth, Medicare payment policy requires constant adjustment, but we are "getting it right" most of the time.

Iron (Fe) oxides/oxyhydroxides in drinking water distribution systems (DWDS), produced by electrochemical, chemical, and biological reactions, can adsorb toxic metal ions, including strontium, lead, arsenic, and vanadium that, if desorbed, generate pulses of drinking water with elevated toxic metal ion concentrations. To illustrate that potential, sorption data for strontium (cation) and vanadium (oxyanion) in functioning DWDS are reviewed. In addition, the influence of flow/no flow on adsorption and desorption of strontium in a model DWDS is included. The reactions that influence adsorption and desorption within a DWDS are extremely complicated and poorly understood. The sorption capacity of Fe oxhydroxides varies with surface area, which in turn varies with source water and disinfectant. Desorption and release can be triggered by changes in source water, disinfection chemicals, or flow. Because of the interrelatedness of adsorption/desorption and Fe corrosion products, subtle changes in DWDS operating p...

The mechanism by which acid zeolites catalyze the formation of aromatic species is not fully understood and is important in an array of industrial processes such as the methanol to gasoline reaction. The so-called "carbon pool" mechanism is generally agreed to be the main channel for the formation of hydrocarbons from methanol. There is, however, no agreed sequence of elementary steps that explains how linear intermediates transform to cyclic intermediates, let alone aromatic rings. Recent work suggests the formation of conjugated trienes during zeolite-catalyzed aromatization, but mechanisms involving triene-derived carbocations have never been investigated using modern computational tools. In this work, we propose a new mechanism for cyclization of hexatriene over the Brønsted acid site of faujasite zeolite. Microkinetic models (MKM) using the results of Density Functional Theory (DFT) calculations predict selectivity for neutral 5-membered-ring intermediates over 6-membered-ring intermediates, as suggested by infrared and UV–vis spectroscopic results reported by others. Given that the products of aromatization are 6-membered rings, this result suggests that triene cyclization can only explain how linear hydrocarbons become cyclic intermediates but not the mechanisms that ultimately lead to the aromatic rings seen in industrial zeolite-catalyzed hydrocarbon processes.

Ennoblement, a positive shift in corrosion potential, due to biofilm formation is the basis of patents for biofilm monitoring and power generating devices. Ennoblement is a global phenomenon that is routinely cited as a mechanism for microbiologically influenced corrosion of some passive alloys. Increased corrosion is attributed to acceleration of the oxygen reduction reaction via several potential mechanisms that have been debated for decades. Because the phenomenon is predictable and reproducible at specific locations, ennoblement is the basis for patented methods and devices for monitoring biofilm formation and relating ennobled potentials to increased likelihood of corrosion and for evaluating cleaning and biocide treatments. Furthermore, when anodes and cathodes can be separated, as in a microbial fuel cell, biofilm formation on the cathode increases the potential difference between the two and the resulting power output. Most patented fuel cells using metal cathodes do not refer specifically to ennoblement in the disclosures. Keywords: Biofilms, ennoblement, microbial fuel cell, microbiologically induced corrosion, microbiologically influenced corrosion, oxygen reduction reaction, redox-active compounds, seawater battery, microbial film, MICROBE-BASED POWER SOURCES

Abstract The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1β and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8+ T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11–deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8+ T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation.

Chapter 27 Microbiologically Influenced Corrosion Brenda J. Little, Brenda J. Little Naval Research Laboratory, Stennis Space Center, Mississippi, USASearch for more papers by this authorJason S. Lee, Jason S. Lee Naval Research Laboratory, Stennis Space Center, Mississippi, USASearch for more papers by this author Brenda J. Little, Brenda J. Little Naval Research Laboratory, Stennis Space Center, Mississippi, USASearch for more papers by this authorJason S. Lee, Jason S. Lee Naval Research Laboratory, Stennis Space Center, Mississippi, USASearch for more papers by this author Book Editor(s):R. Winston Revie, R. Winston RevieSearch for more papers by this author First published: 07 April 2015 https://doi.org/10.1002/9781119019213.ch27 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary The term microbiologically influenced corrosion (MIC) is used to designate corrosion due to the presence and activities of microorganisms, that is, those organisms that cannot be seen individually with the unaided human eye. Information in this chapters related to internal MIC is limited to petroleum-based hydrocarbon fuels in low-alloy steel piping. The focus of most testing, monitoring, and research related to MIC in the oil and gas industry for internal and external pipeline surfaces is on sulfate-reducing bacteria (SRB). Consequently, any discussion of causative microorganisms, in the chapter, is dominated by references to SRB. Microorganisms have developed several strategies for survival in natural environments: (1) spore formation (2) biofilm formation (3) dwarf cells, and (4) a viable, but non-culturable state. Pitting is the typical type of internal corrosion in pipelines. All line pipe is externally coated and can be further protected with cathodic protection (CP). Oil and Gas Pipelines RelatedInformation

Chemotherapy is used as a standard-of-care against cancers that display high levels of inherent genome instability. Chemotherapy induces DNA damage and intensifies pressure on the DNA repair pathways that can lead to deregulation. There is an urgent clinical need to be able to track the emergence of DNA repair driven chemotherapy resistance and tailor patient staging appropriately. There have been numerous studies into chemoresistance but to date no study has elucidated in detail the roles of the key DNA repair components in resistance associated with the frontline clinical combination of anthracyclines and taxanes together. In this study, we hypothesized that the emergence of chemotherapy resistance in triple negative breast cancer was driven by changes in functional signaling in the DNA repair pathways. We identified that consistent pressure on the non-homologous end joining pathway in the presence of genome instability causes failure of the key kinase DNA-PK, loss of p53 and compensation by p73. In-turn a switch to reliance on the homologous recombination pathway and RAD51 recombinase occurred to repair residual double strand DNA breaks. Further we demonstrate that RAD51 is an actionable target for resensitization to chemotherapy in resistant cells with a matched gene expression profile of resistance highlighted by homologous recombination in clinical samples.

Phenoxybenzamine (nonselective, noncompetitive alpha-blocker) is the preferred drug for preoperative treatment of pheochromocytoma, but doxazosin (selective, competitive alpha-blocker) may be equally effective. We compared the efficacy of doxazosin vs phenoxybenzamine.We conducted a prospective study of patients undergoing pheochromocytoma or paraganglioma resection by randomizing pretreatment with phenoxybenzamine or doxazosin at a single tertiary referral center. The high cost of phenoxybenzamine led to high crossover to doxazosin. Randomization was halted, and a consecutive historical cohort of phenoxybenzamine patients was included for a case-control study design. The efficacy of alpha-blockade was assessed with preinduction infusion of incremental doses of phenylephrine. The primary outcomes were mortality, cardiovascular complications, and intensive care unit admission. The secondary outcomes were hemodynamic instability index (proportion of operation outside of hemodynamic goals), adequacy of blockade by the phenylephrine titration test, and drug costs.Twenty-four patients were prospectively enrolled (doxazosin, n = 20; phenoxybenzamine, n = 4), and 15 historical patients treated with phenoxybenzamine were added (total phenoxybenzamine, n = 19). No major cardiovascular complications occurred in either group. The phenylephrine dose-response curves showed less blood pressure rise in the phenoxybenzamine than in the doxazosin group (linear regression coefficient = 0.008 vs 0.018, P = .01), suggesting better alpha-blockade in the phenoxybenzamine group. The median hemodynamic instability index was 14% vs 13% in the phenoxybenzamine and doxazosin groups, respectively (P = .56). The median highest daily cost of phenoxybenzamine was $442.20 compared to $5.06 for doxazosin.Phenoxybenzamine may blunt intraoperative hypertension better than doxazosin, but this difference did not translate to fewer cardiovascular complications and is offset by a considerably increased cost.

This study employed exploratory and confirmatory factor analysis techniques to probe the dimensionality of measures of home environment. Thirty-five (35) demographic, attitudinal, and behavioral indicators were derived from telephone interviews with a probability sample of 2000 parents of seventh-graders participating in a national longitudinal study. The measures included such frequently used indicators as socioeconomic status, home resources, parent expectations, and parent-child communication. LISREL analysis with cross-validation supported a two-factor structure over alternative specifications. The factors were interpreted as family resources and communication-interaction. Results indicate that comprehensive measures of home environment are needed in studies of familial influence on educational and psychological outcomes.

Amid escalating health care costs and a managed care backlash, employers are considering traditional cost control methods from the pre-managed care era. We use an actuarial model to estimate the premium-reducing effects of two such methods: increasing employee cost sharing and reducing benefits. Starting from a baseline plan with rich benefits and low cost sharing, estimated premium savings as a result of eliminating five specific benefits were about 22 percent. The same level of savings was also achieved by increasing cost sharing from a 15 dollars copayment with no deductible to 20 percent coinsurance and a 250 dollars deductible. Further increases in cost sharing produced estimated savings of up to 50 percent. We discuss possible market- and individual-level effects of the proliferation of plans with high cost sharing and low benefits.

Free Access Wiley Series in Corrosion Brenda J. Little, Naval Research Laboratory, Stennis Space Center, MS, USASearch for more papers by this authorJason S. Lee, Naval Research Laboratory, Stennis Space Center, MS, USASearch for more papers by this author Book Author(s):Brenda J. Little, Naval Research Laboratory, Stennis Space Center, MS, USASearch for more papers by this authorJason S. Lee, Naval Research Laboratory, Stennis Space Center, MS, USASearch for more papers by this author First published: 01 August 2006 https://doi.org/10.1002/9780470112458.scard AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat No abstract is available for this article. Microbiologically Influenced Corrosion RelatedInformation

Abstract : Models for corrosion influenced by iron-oxidizing bacteria (IOB) in fresh water are specific for material/environment combinations, i.e., 300 series stainless steel exposed to oxygenated chloridecontaining potable water and carbon steel exposed in oxygenated fresh water ([Cl~] < 20 ppb) containing dissolved copper. Reports of IOB influenced corrosion in marine environments have been limited to rusticle formation on shipwrecks. IOB involved in corrosion in fresh water include Gallionella, Leptothrix, and Siderocapsa. Historically these organisms have also been thought to be active in marine environments. New isolation and molecular identification techniques are demonstrating the presence of novel IOB in both freshwater and marine environments, and expanding our understanding of their potential role in microbiologically influenced corrosion.

Women with epithelial ovarian cancer (EOC) are usually treated with platinum/ taxane therapy after cytoreductive surgery but there is considerable inter-individual variation in response.To identify germline single-nucleotide polymorphisms (SNPs) that contribute to variations in individual responses to chemotherapy, we carried out a multi-phase genome-wide association study (GWAS) in 1,244 women diagnosed with serous EOC who were treated with the same first-line chemotherapy, carboplatin and paclitaxel.We identified two SNPs (rs7874043 and rs72700653) in TTC39B (best P=7x10 -5 , HR=1.90, for rs7874043) associated with progression-free survival (PFS).Functional analyses show that both SNPs lie in a putative regulatory element (PRE) that physically interacts with the promoters of PSIP1, CCDC171 and an alternative promoter of TTC39B.The C allele of rs7874043 is associated with poor PFS and showed increased binding of the Sp1 transcription factor, which is critical for chromatin interactions with PSIP1.Silencing of PSIP1 significantly impaired DNA damage-induced Rad51 nuclear foci and reduced cell viability in ovarian cancer Oncotarget

Abstract : The experiments described in this paper demonstrate changes in the chemistries and microflora of two natural coastal seawaters collected from Key West, Florida, and the Persian Gulf as a result of storage and environmental conditions. Exposure to an anaerobic atmosphere containing a mixed gas of nitrogen, carbon dioxide, and hydrogen generated the highest microflora concentration, especially sulfate-reducing bacteria (SRB). Biotic dissolved sulfide levels were also highest in the mixed gas atmosphere. In contrast, sulfides were not detected in seawater maintained anaerobic with bubbled nitrogen. Separate introductions of carbon steel and agitation also affected chemistries and microflora. Key West seawater consistently had equal or greater bacterial numbers in all conditions when compared with Persian Gulf seawater. Bubbling nitrogen into natural seawater to achieve an anaerobic condition is not conducive to the growth of SRB and the resulting biotic sulfide. Laboratory experiments that mimic anaerobic conditions by bubbling nitrogen may not produce conditions found in the field due to pH changes. Therefore, removal of oxygen is not the only consideration when attempting to reproduce anaerobic conditions. A followup paper is planned to address the influences of chemistry and microflora on corrosivity.

ABSTRACT Genome-wide association studies have identified 196 high confidence independent signals associated with breast cancer susceptibility. Variants within these signals frequently fall in distal regulatory DNA elements that control gene expression. We designed a Capture Hi-C array to enrich for chromatin interactions between the credible causal variants and target genes in six human mammary epithelial and breast cancer cell lines. We show that interacting regions are enriched for open chromatin, histone marks for active enhancers and transcription factors relevant to breast biology. We exploit this comprehensive resource to identify candidate target genes at 139 independent breast cancer risk signals, and explore the functional mechanism underlying altered risk at the 12q24 risk region. Our results demonstrate the power of combining genetics, computational genomics and molecular studies to rationalize the identification of key variants and candidate target genes at breast cancer GWAS signals.

In this study, we described Millennial anesthesiology residents' learning preferences and study habits and how they correlate with performance on the In-Training Exam (ITE).A confidential questionnaire including personal characteristics, previous examination performance, study habits, study material preferences, and perceived residency program support was emailed to 1047 anesthesiology residents from 30 ACGME-accredited residency programs across the United States.Four hundred and twelve residents (39.4%) responded to the survey, and 240 of those respondents (58.3%) self-reported their 2017 ITE scores. The majority (95.9%) were Millennials. Respondents preferentially used online multiple-choice questions (92.3%) to prepare for the ITE, but many also used traditional anesthesiology textbooks (35.5%) and review books (46.7%). Respondents preferred independent study (94.6%) to group study (5.4%), and handwritten notes (69.4%) to taking notes on a laptop (26.8%) or tablet (3.8%). Less than half (47.5%) of respondents felt supported by their residency program in exam preparation, and 30.7% felt lack of support. Factors correlated with ITE scores on univariate analysis included prior USMLE 1 scores (p < .0000) and USMLE 2 scores (p < .0000), clinical anesthesia year (p < .0000), test anxiety score (p = .0004), prior failure of the basic exam (p = .0026), and prior failure of any board exam (p = .0124).Millennial learners have consistent performance on ITE exams regardless of personal characteristics, preferred study methods, or materials used. Prior exam performance is the most important predictor of future performance. Currently available study materials are meeting residents' needs and preferences, and while residency program offerings do not affect ITE performance, residents would like to feel more supported.

Microbiologically influenced corrosion (MIC) is the result of specific metal/microbe/electrolyte interactions, making it impossible to generalize. The relationship between microorganisms and minerals can be used to determine mechanisms, causative microorganisms, and the environment in which the corrosion occurred, providing definitive evidence for MIC. In this chapter, the vulnerability of metals and alloys to MIC in the presence of specific microorganisms is supported by mineralogical studies.

This chapter contains sections titled: Introduction Ennoblement Concentration Cells Reactions within Biofilms Inactivation of Corrosion Inhibitor Alteration of Anion Ratios Summary References

Abstract : Experiments were designed to evaluate the nature and extent of microbial contamination and the potential for microbiologically influenced corrosion in biodiesel (B100), ultra-low sulfur diesel (ULSD) and mixtures of the two (B5 and B20). In experiments with additions of distilled water, B100 had the highest propensity for biofouling while the highest corrosion rates were measured in ultra-low-sulfur diesel.

Abstract : Experiments were designed to evaluate corrosion-related consequences of storing/transporting biodiesel in contact with natural seawater under anaerobic conditions. Coastal Key West, FL (KW) and Persian Gulf (PG) natural seawaters were used in these 60-day studies. The highest corrosion rates measured by electrochemical techniques were for unprotected carbon steel exposed to natural KW seawater with biodiesel addition. However, the deepest pits were measured in biodiesel with PG seawater. Microbial sulfide production was stimulated in both seawaters by the presence of biodiesel either as a separate phase or as a fuel-in-water emulsion. The presence of seawater influenced the chemistry of the biodiesel, contributing both sulfur and chloride.

The authors examined the physiochemical and microbiological properties of archived rusticles from World War II shipwrecks in the Gulf of Mexico. Rusticles, iron (Fe)-rich accumulations on shipwrecks in marine environments, have long been assumed to be the result of low alloy steel corrosion. In many cases the assumed corrosion has been attributed to biodeterioration because of the presence of specific types of bacteria associated with the rusticles. However, archived rusticles from WWII shipwrecks in the Gulf of Mexico (GOM) do not have the mineralogical layering typical of iron corrosion products. Moreover, spatial relationships between bacteria and rusticles cannot be interpreted as biodeterioration. The authors concluded that environmental Fe plays a role in rusticle formation and differences in Fe concentrations can be used to explain differences in rusticle size and distribution with depth in the GOM. Both biotic and abiotic mechanisms for Fe accumulation are provided.

Abstract : Microbiologically influenced corrosion (MIC) is the terminology applied when biological parameters play a role in the corrosion process. In literature, terms such as microbial corrosion, biocorrosion, microbially influenced/induced corrosion, and biodegradation are often applied. All descriptions express that microorganisms (bacteria, archaea, and fungi) influence the corrosion process of a given material. In this chapter, an overview of the common MIC mechanisms encountered in the oil and gas industry is presented.

Abstract : Invasions by non-native species have resulted in significant ecological changes and enormous economic cost. Although there are several vectors that can transport aquatic invaders, by far the most important is through ship ballast water. Currently, however, there is no environmentally friendly ballast water treatment that is truly effective at reducing introductions and yet also acceptable to the shipping industry in terms of safety, time, and cost. Deoxygenation may however be such a treatment with benefit for ship owners through corrosion prevention, while simultaneously limiting the number of aquatic organisms surviving transport in ballast tanks. Our current investigations are providing the critical information required to evaluate the efficacy and feasibility of deoxygenation as a ballast water treatment to prevent aquatic invasions and tank corrosion. Specifically, we are: (1) evaluating the Venturi Oxygen Stripping (trademark) system developed by NEI Treatment Systems, Inc. to optimize the deoxygenation process, (2) examining the impact of this oxygen stripping technique on the immediate and long-term survival of natural Chesapeake Bay planktonic organisms, and (3) quantifying corrosion rates and establishing the corrosion mechanism under deoxygenated conditions (with particular emphasis on microbiologically influenced corrosion and the production of hydrogen sulfide). These results will ultimately lead to a full-scale shipboard evaluation of deoxygenation as a cost-saving ballast water treatment.

Pneumonia and influenza (P&I) are the sixth leading cause of death in the United States. Despite universal coverage under Medicare, one-half to three-quarters of elderly adults fail to get vaccinated against P&I disease. Hepatitis B vaccine is also widely underutilized by adults. Although more than 100 times as many adults as children die from vaccine-preventable disease, the Centers for Disease Control and Prevention (CDC) currently allocates the vast majority of federal immunization funds to childhood programs. Top CDC officials say this is in accordance with the will of the Congress and the President. However, analysis of legislative documents shows that there is no legal bar or restriction to the use of federal funds to support adult immunization. CDC has the authority to use federal immunization funds to enhance adult immunization services, but the agency has yet to make adult immunization a priority. A commentary follows.

Glycol/seawater mixtures containing > 50% glycol inhibit corrosion of 316L stainless steel and do not support bacterial growth. The results indicate bacteria are able to use low concentrations of glycol (10%) as a growth medium, but bacterial growth decreased with increasing glycol concentration. Pitting potential, determined by anodic polarization, was used to evaluate susceptibility of 316L SS to corrosion in seawater-contaminated glycol. Mixture containing a minimum concentration of 50% propylene glycol-based coolant inhibited pitting corrosion. A slightly higher minimum concentration (55%) was needed for corrosion protection in ethylene glycol mixtures.

Abstract : Methods for handling and storing natural coastal seawater, particularly methods for deaeration over time, influenced the chemistry and microflora. Bubbling nitrogen into natural seawater to achieve an anaerobic condition was not conducive to the growth of sulfate-reducing bacteria (SRB). Despite a higher initial dissolved sulfate concentration and higher total organic carbon in Persian Gulf seawater, higher dissolved sulfide concentrations and SRB populations were measured in Key West, Florida, seawater under all exposure conditions.

&lt;p&gt;Supplementary Tables&lt;/p&gt;

&lt;p&gt;Supplementary Figures&lt;/p&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;G9a histone methyltransferase exerts oncogenic effects in several tumor types and its inhibition promotes anticancer effects. However, the impact on checkpoint inhibitor blockade response and the utility of G9a or its target genes as a biomarker is poorly studied. We aimed to examine whether G9a inhibition can augment the efficacy of checkpoint inhibitor blockade and whether &lt;i&gt;LC3B&lt;/i&gt;, a G9a target gene, can predict treatment response.&lt;/p&gt;Experimental Design:&lt;p&gt;Clinical potential of LC3B as a biomarker of checkpoint inhibitor blockade was assessed using patient samples including tumor biopsies and circulating tumor cells from liquid biopsies. Efficacy of G9a inhibition to enhance checkpoint inhibitor blockade was examined using a mouse model.&lt;/p&gt;Results:&lt;p&gt;Patients with melanoma who responded to checkpoint inhibitor blockade were associated with not only a higher level of tumor LC3B but also a higher proportion of cells expressing LC3B. A higher expression of &lt;i&gt;MAP1LC3B&lt;/i&gt; or LC3B protein was associated with longer survival and lower incidence of acquired resistance to checkpoint inhibitor blockade, suggesting LC3B as a potential predictive biomarker. We demonstrate that G9a histone methyltransferase inhibition is able to not only robustly induce LC3B level to augment the efficacy of checkpoint inhibitor blockade, but also induces melanoma cell death.&lt;/p&gt;Conclusions:&lt;p&gt;Checkpoint inhibitor blockade response is limited to a subset of the patient population. These results have implications for the development of LC3B as a predictive biomarker of checkpoint inhibitor blockade to guide patient selection, as well as G9a inhibition as a strategy to extend the proportion of patients responding to immunotherapy.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;G9a histone methyltransferase exerts oncogenic effects in several tumor types and its inhibition promotes anticancer effects. However, the impact on checkpoint inhibitor blockade response and the utility of G9a or its target genes as a biomarker is poorly studied. We aimed to examine whether G9a inhibition can augment the efficacy of checkpoint inhibitor blockade and whether &lt;i&gt;LC3B&lt;/i&gt;, a G9a target gene, can predict treatment response.&lt;/p&gt;Experimental Design:&lt;p&gt;Clinical potential of LC3B as a biomarker of checkpoint inhibitor blockade was assessed using patient samples including tumor biopsies and circulating tumor cells from liquid biopsies. Efficacy of G9a inhibition to enhance checkpoint inhibitor blockade was examined using a mouse model.&lt;/p&gt;Results:&lt;p&gt;Patients with melanoma who responded to checkpoint inhibitor blockade were associated with not only a higher level of tumor LC3B but also a higher proportion of cells expressing LC3B. A higher expression of &lt;i&gt;MAP1LC3B&lt;/i&gt; or LC3B protein was associated with longer survival and lower incidence of acquired resistance to checkpoint inhibitor blockade, suggesting LC3B as a potential predictive biomarker. We demonstrate that G9a histone methyltransferase inhibition is able to not only robustly induce LC3B level to augment the efficacy of checkpoint inhibitor blockade, but also induces melanoma cell death.&lt;/p&gt;Conclusions:&lt;p&gt;Checkpoint inhibitor blockade response is limited to a subset of the patient population. These results have implications for the development of LC3B as a predictive biomarker of checkpoint inhibitor blockade to guide patient selection, as well as G9a inhibition as a strategy to extend the proportion of patients responding to immunotherapy.&lt;/p&gt;&lt;/div&gt;