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Integrin α6β4 signaling has been shown to be ligand-independent in several model systems where the integrin does not require exogenous ligand to mediate its effects7. In our previous studies, we have found that exogenous laminin is not required to see changes in gene expression associated with enhanced invasion and migration13. Interestingly, the Jones group has demonstrated that integrin α6β4 promotes secretion and deposition of laminin-5, a major laminin isoform implicated in integrin α6β4 signaling, in the extracellular matrix, which in turn promotes motility of keratinocytes28. To test if endogenous secretion of laminin-5 is responsible for ligating integrin α6β4 in the Panc1 model, we plated Panc1-2G6 (low α6β4) and Panc1-3D7 (high α6β4) cells were plated onto collagen-coated coverslips for 4 hours and stained for the laminin γ2 subunit, which is unique to laminin-5. We find that in integrin α6β4 high expressing Panc1-3D7 cells, there is enhanced deposition of laminin, which strongly colocalizes with the integrin β4 subunit (Fig. 1A), which is in line with observations from keratinocyte studies. In contrast, cells with low integrin α6β4 have low detectable levels of laminin (Fig. 1D), which is not deposited extracellularly (Fig. 1B), implicating a deficient integrin α6β4 signaling network. Likewise, our previous studies demonstrate the enhanced migratory abilities of integrin α6β4 high versus low expressing pancreatic cancer cell lines when plated on laminin29. We chose to use these stable subpopulations derived from the Panc1 cell line as our model system for studying the impact of integrin α6β4 on the transcriptome as we have clearly demonstrated both variable levels of the integrin α6β4 and its cognate ligand, laminin-5.Figure 1Integrin α6β4 binds to and promotes secretion of laminin-5 in Panc1 cells. (A–C) Panc1 clones 3D7 (A; high α6β4) and 2G6 (B; low α6β4) were plated on collagen I coated coverslips and allowed to adhere under normal culture conditions for 4 hours. Cells were fixed and immunostained for integrin β4 subunit (red), laminin-5 (γ2 subunit; green), or f-actin (blue) as described in the Methods section. Cells were imaged by TIRF microscopy using the same exposure times and settings. Pearson’s coefficient for colocalization between integrin β4 and laminin-5 were 0.8 for Panc1-3D7 (A) and 0.011 for Panc1-2G6 (B). These values are representative for the 30 cells analyzed for each cell line. (C) Represents a rare β4 expressing cell in the Panc-2G6 cell population. (D) Western blot analysis of whole cell extracts from MDA-MB-231 (positive control), Panc1-2G6 and Panc1-3D7 cells for integrin β4, laminin-5 and actin (loading control).
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Integrin α6β4 binds to and promotes secretion of laminin-5 in Panc1 cells. (A–C) Panc1 clones 3D7 (A; high α6β4) and 2G6 (B; low α6β4) were plated on collagen I coated coverslips and allowed to adhere under normal culture conditions for 4 hours. Cells were fixed and immunostained for integrin β4 subunit (red), laminin-5 (γ2 subunit; green), or f-actin (blue) as described in the Methods section. Cells were imaged by TIRF microscopy using the same exposure times and settings. Pearson’s coefficient for colocalization between integrin β4 and laminin-5 were 0.8 for Panc1-3D7 (A) and 0.011 for Panc1-2G6 (B). These values are representative for the 30 cells analyzed for each cell line. (C) Represents a rare β4 expressing cell in the Panc-2G6 cell population. (D) Western blot analysis of whole cell extracts from MDA-MB-231 (positive control), Panc1-2G6 and Panc1-3D7 cells for integrin β4, laminin-5 and actin (loading control).
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Integrin α6β4 signaling stimulates progression of multiple types of cancer in part by altering the transcriptome. Notably, expression of AREG and EREG positively correlates with expression of and signaling through integrin α6β4 (Fig. 2A), supporting our previous work13. To determine if AREG and EREG expression is regulated by DNA methylation, Panc1-2G6 cells were treated with the DNA methyltransferase inhibitor 5-aza-CdR at indicated concentrations, harvested at indicated time points, and RNA analyzed by QPCR. We found that both AREG and EREG mRNA expression increased in a time and dose dependent manner (Fig. 2B) demonstrating the susceptibility of AREG and EREG to DNA methylation. Furthermore, integrin α6β4 was required for induction of AREG and EREG mediated by 5-aza-CdR, as knocking down the integrin β4 in Panc1-3D7 cells hindered epigenetic induction of AREG and EREG expression (Fig. 2C). Considering that epigenetic changes are reversible, AsPC1 and Suit2 cells, high expressers of integrin α6β4, AREG, and EREG, were treated with the methyl donor S-adenosylmethionine (SAM) and assessed for AREG and EREG expression by QPCR. These data revealed a 50% decrease in expression of AREG and EREG as seen in Fig. 2D. Taken together these data indicate that signaling from integrin α6β4 and DNA demethylation are required to drive AREG and EREG expression.Figure 2AREG and EREG expression is mediated by DNA demethylation in response to signaling from the integrin α6β4. (A) Expression of AREG and EREG was compared in Panc1-2G6 (low α6β4) and cells expressing a dominant negative α6β4 (Panc1-β4ΔCyt), Panc1-3D7, Suit2, and AsPC1 (high α6β4; in order of increasing expression) cell lines. (B) Panc1-2G6 cells (low α6β4) were treated with vehicle only (control) or with 1 μM or 5 μM 5-aza-2′-deoxycitine (5-aza-CdR) in fresh medium daily for 3 or 5 days. (C) Panc1-3D7 stably expressing an shRNA targeting the β4 subunit or a non-targeting (NT) shRNA control vector were treated with 2 μM 5-aza-CdR for 3 days and then assessed for AREG and EREG expression. (D) AsPC1 and Suit2 (high α6β4) were treated with vehicle only (control) or 80 μM S-adenosylmethione (SAM) in fresh medium daily for 5 days (E,F). Panc1-2G6 (E) and Panc1-3D7 (F) cells were treated with 2 μM 5-aza-CdR for 24 or 72 hours, 5-aza-CdR was removed and cells were either collected immediately or maintained in culture for 10 days. (G–I) Cells with high integrin α6β4 were treated with vehicle only (control) or 0.5 μM JQ1 overnight and harvested for analysis by QPCR. For all experiments RT-PCR was used to convert RNA to cDNA and QPCR was used to assess AREG and EREG expression. Data depicted here are representative of at least three different experiments and represent the mean +/− standard deviation. Statistical significance was calculated using a one-tailed t-test in which * denotes P < 0.05 as compared to controls, unless otherwise indicated.
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AREG and EREG expression is mediated by DNA demethylation in response to signaling from the integrin α6β4. (A) Expression of AREG and EREG was compared in Panc1-2G6 (low α6β4) and cells expressing a dominant negative α6β4 (Panc1-β4ΔCyt), Panc1-3D7, Suit2, and AsPC1 (high α6β4; in order of increasing expression) cell lines. (B) Panc1-2G6 cells (low α6β4) were treated with vehicle only (control) or with 1 μM or 5 μM 5-aza-2′-deoxycitine (5-aza-CdR) in fresh medium daily for 3 or 5 days. (C) Panc1-3D7 stably expressing an shRNA targeting the β4 subunit or a non-targeting (NT) shRNA control vector were treated with 2 μM 5-aza-CdR for 3 days and then assessed for AREG and EREG expression. (D) AsPC1 and Suit2 (high α6β4) were treated with vehicle only (control) or 80 μM S-adenosylmethione (SAM) in fresh medium daily for 5 days (E,F). Panc1-2G6 (E) and Panc1-3D7 (F) cells were treated with 2 μM 5-aza-CdR for 24 or 72 hours, 5-aza-CdR was removed and cells were either collected immediately or maintained in culture for 10 days. (G–I) Cells with high integrin α6β4 were treated with vehicle only (control) or 0.5 μM JQ1 overnight and harvested for analysis by QPCR. For all experiments RT-PCR was used to convert RNA to cDNA and QPCR was used to assess AREG and EREG expression. Data depicted here are representative of at least three different experiments and represent the mean +/− standard deviation. Statistical significance was calculated using a one-tailed t-test in which * denotes P < 0.05 as compared to controls, unless otherwise indicated.
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True epigenetic alterations are stable changes maintained across many generations. Since 5-Aza-CdR can modify the epigenetic landscape30, we assessed the impact of short term 5-aza-CdR treatment on AREG and EREG expression by treating cells with the indicated concentrations of 5-Aza-CdR for 24 or 72 hours. 5-Aza-CdR was removed and cells were either harvested immediately or maintained in culture for 10 days. As shown in Fig. 1E, expression of AREG and EREG was not only induced 20–40 fold and maintained in Panc1-2G6 cells following 5-aza-CdR treatment but continued to increase when kept in culture 10 days post 5-aza-CdR removal. Treatment of Panc1-3D7 cells only slightly increased transcription of AREG and EREG (Fig. 2F), suggesting these stable epigenomic modifications have already taken place. These data confirm that the integrin α6β4 contributes to the stable upregulation of pro-tumorigenic molecules AREG and EREG through epigenetic alterations.
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Alterations in DNA methylation strongly impact the activity of enhancers, which activate specific transcriptional profiles through recruitment of transcription factors that interact with the mediator complex31. To determine if enhancer activity is required for AREG and EREG expression in pancreatic cancer cells, we treated cells with JQ1, a BET bromo-domain inhibitor that is specific for BRD432. BRD4 interacts with the elongating factor P-TEFB in Pol II complexes to enhance transcription for both protein-coding and enhancer-derived noncoding RNAs33. We found that AREG and EREG expression markedly decreased with JQ1 treatment, thus indicating their transcriptional dependence on enhancer function (Fig. 2G–I).
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To define DNA demethylation changes that drive expression of AREG and EREG, sodium bisulfite conversion and whole genome sequencing was performed on genomic DNA from pancreatic cancer cells with either high (Panc1-3D7) or low (Panc1-2G6) integrin α6β4 expression. Sequencing reads were aligned to the reference genome, GRCH37, mapped to the AREG and EREG genes, and visualized using the UCSC genome browser. We found that cells with high integrin α6β4 (Fig. 3A and B; bottom panels) have reduced DNA methylation within intronic regions of both EREG (Fig. 3A) and AREG (Fig. 3B), confirming that the integrin α6β4 drives site-specific DNA demethylation, and defining the critical CpG sites of AREG and EREG that become altered downstream of integrin α6β4.Figure 3The integrin α6β4 drives both gene specific and global DNA hypomethylation. (A–C) Genomic DNA from Panc1-2G6 (β4 low; upper panels) and Panc1-3D7 (high β4; lower panels) was processed for high-resolution methyl-seq by the NextGen Sequencing Core at the Norris Comprehensive Cancer Center. Samples were analyzed bioinformatically and percent methylation shown for EREG (A), AREG (B), and AREG pseudogene (C). (D) Percent hypomethylation and hypermethylation per chromosome when comparing Panc1-3D7 vs. Panc1-2G6. (E) Defined regions of interest assessed for changes in DNA methylation (F) Location of DMLs across the genome. (G) Percent of methylation changes located in CpG islands and shores. (H) Location of DMLs associated with H3K27Ac. (H) Distance from TSSs for DMLs (I) Distances from TSSs for DMRs (J) Number of both hypomethylated and hypermethylated regions corresponding to genomic features.
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The integrin α6β4 drives both gene specific and global DNA hypomethylation. (A–C) Genomic DNA from Panc1-2G6 (β4 low; upper panels) and Panc1-3D7 (high β4; lower panels) was processed for high-resolution methyl-seq by the NextGen Sequencing Core at the Norris Comprehensive Cancer Center. Samples were analyzed bioinformatically and percent methylation shown for EREG (A), AREG (B), and AREG pseudogene (C). (D) Percent hypomethylation and hypermethylation per chromosome when comparing Panc1-3D7 vs. Panc1-2G6. (E) Defined regions of interest assessed for changes in DNA methylation (F) Location of DMLs across the genome. (G) Percent of methylation changes located in CpG islands and shores. (H) Location of DMLs associated with H3K27Ac. (H) Distance from TSSs for DMLs (I) Distances from TSSs for DMRs (J) Number of both hypomethylated and hypermethylated regions corresponding to genomic features.
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Importantly, we also found alterations in DNA methylation in an AREG pseudogene, which lies directly downstream of AREG (Fig. 3C). When examining these two regions, both the sequence structure and regulatory similarity were noted as they are 99% homologous when blasted against the reference genome. Since Bismark only reports unique matches, the multi-mapping scenario of AREG and its pseudogene made it difficult to investigate the methylation alternations in these two regions. However, the analysis was possible by masking AREG pseudogene and mapping AREG, and vice-versa for AREG pseudogene. We attempted to investigate this further by using bisulfite conversion with methylation specific PCR to confirm altered CpGs within this region. However, the sequence similarity between these two regions and difficulty designing unique primers for bisulfite converted DNA proved that this analysis was technically unfeasible.
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Regions that had the greatest difference in DNA methylation in both AREG and EREG as a result of integrin α6β4 signaling corresponded to areas enriched in H3K27Ac marks (Fig. 3A,B), as annotated by the ENCODE project, that are reported to mark active enhancer elements34. Additionally, a super-enhancer associated with AREG and EREG expression lies between AREG and the AREG pseudogene35. We found no significant differences in super-enhancer DNA methylation (data not shown), indicating that it is unlikely that DNA methylation of this element is the major driver for enhanced AREG and EREG gene expression. Taken together, these data, along with our observation that BRD4 is required for AREG and EREG expression, indicate that DNA demethylation of enhancer elements localized within the proximal promoters of AREG and EREG drive expression in response to integrin α6β4 signaling.
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Next, we examined the genome wide effects of integrin α6β4 on DNA methylation using our WGBS data. A total of 236,371 differentially methylated loci (DML; 207,168 hypomethylated and 29,203 hypermethylated) were identified comparing Panc1-3D7 vs. Panc1-2G6. Figure 3D illustrates the percentage of hypermethylated and hypomethylated events per chromosome as a percent of the number of DMLs. Of the DMLs identified, 87.6% were hypomethylated and 12.4% were hypermethylated, thus indicating that the integrin α6β4 shifts chromatin to a more hypomethylated state. Further analysis of these data revealed that only 3.1% of these loci were located in promoter regions, 2.1% in exonic regions, 31.1% in intronic regions and 63.1% were in intergenic regions (Fig. 3F). 13,889 differentially methylated regions (DMRs) were identified, of which only about 4% were located in CpG islands, and 5% in CpG shores (Fig. 3G). We found that 40,609 DMLs associated with H3K27Ac marks were hypomethylated as opposed to 13,679 DMLs hypermethylated. These events correspond to 4993 genes that have alterations in methylation within enhancer elements. As seen in Fig. 3H, the majority of these altered DML are localized to intronic and intergenic regions (defined in Fig. 3E) of which the majority are hypomethylated (Fig. 3K). This observation is typical of enhancer elements, as many enhancers are part of non-coding regions of the genome36. Additionally, we found that DMLs and DMRs occur predominantly within the first ten thousand base pairs on either side of the TSS with slightly more occurring after the TSS, as expected (Fig. 2I,J).
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Since our data suggest that AREG and EREG DNA demethylation is an active process, we tested the hypothesis that DNA repair is required to maintain their expression downstream of integrin α6β4 signaling. The NER pathway, including the Xeroderma pigmentosum complementation group proteins XPA, XPG, and XPF, has been implicated in active DNA demethylation by DNA repair37, 38. Accordingly, we targeted molecules critical for and specific to the NER pathway and examined their impact on AREG and EREG expression. When knockdown of XPA (Fig. 4A) was achieved, transcription of AREG and EREG in Panc-3D7 (high α6β4; Fig. 4C) remained unaffected. AREG and EREG transcription in Panc-2G6 (low α6β4; Fig. 4B) showed a statistically significant increase when XPA was knocked down, which implies negative regulation. However, due to very low basal expression of AREG and EREG in these cells (cT value >35) it is unlikely to be biologically significant. Using specific shRNAs we knocked down ERCC4 (XPF; Fig. 4D) and ERCC5 (XPG; Fig. 4G) and demonstrated that effective knockdown of NER genes had relatively little or no effect on AREG and EREG expression (Fig. 4E–F,I–J). Taken together these data indicate that NER is not required to maintain AREG or EREG expression.Figure 4NER is not required for expression of AREG and EREG. Using lentiviral transfection stable knockdown of XPA (A), ERCC4 (XPF) (D), and ERCC5 (XPG) (G) was achieved in Panc1-2G6 (low α6β4) and Panc1-3D7 (high α6β4) cells as confirmed by QPCR. AREG and EREG expression was examined following knockdown in cells with both low α6β4 (B,E,H) and high α6β4 (C,F,I) expression. Data depicted are representative of at least three different experiments and represent the mean +/− standard deviation. Statistical significance was calculated using a one-tailed t-test in which * denotes P < 0.05 as compared to controls.
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NER is not required for expression of AREG and EREG. Using lentiviral transfection stable knockdown of XPA (A), ERCC4 (XPF) (D), and ERCC5 (XPG) (G) was achieved in Panc1-2G6 (low α6β4) and Panc1-3D7 (high α6β4) cells as confirmed by QPCR. AREG and EREG expression was examined following knockdown in cells with both low α6β4 (B,E,H) and high α6β4 (C,F,I) expression. Data depicted are representative of at least three different experiments and represent the mean +/− standard deviation. Statistical significance was calculated using a one-tailed t-test in which * denotes P < 0.05 as compared to controls.
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Gemcitabine is a chemotherapeutic with multiple proposed mechanisms of action, including depletion of deoxynucleotide triphosphates that are necessary for DNA synthesis and completion of DNA repair39. Interestingly, gemcitabine has been shown to specifically inhibit GADD45A mediated gene activation via DNA demethylation and DNA repair40. To investigate the role of DNA repair in expression of AREG and EREG, cells were treated with 10 μM gemcitabine for 72 hours. As demonstrated in Fig. 5A, AREG and EREG expression dramatically decreased in cells with high integrin α6β4 in response to treatment, thus indicating that DNA repair is required to maintain expression. As summarized in Fig. 5B, GADD45A mediated active DNA demethylation is achieved through BER. Therefore, we next investigated the role of TET1, GADD45A, TDG, and PARP1 in the regulation of AREG and EREG as key regulators of DNA repair-mediated DNA demethylation.Figure 5GADD45A is both required for and the rate-limiting step in activation of AREG and EREG expression. (A) Cells were treated with 10 μM Gemcitabine for 72 hours and expression of AREG and EREG measured by QPCR. (B) Summary of current literature for how GADD45A mediated DNA demethylation is achieved. Transient knockdown of GADD45A was achieved using electroporation and specific siRNA (E). Adenovirus was used to overexpress GADD45A in Panc1-2G6 and Panc1-3D7 cells (H). Changes in AREG and EREG expression were measured by QPCR in Panc1-2G6 (C,F) and Panc1-3D7 (D,G). Data depicted here are representative of at least three different experiments and represent the mean +/− standard deviation. Statistical significance was calculated using a one-tailed t-test in which * denotes P < 0.05 as compared to controls.
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GADD45A is both required for and the rate-limiting step in activation of AREG and EREG expression. (A) Cells were treated with 10 μM Gemcitabine for 72 hours and expression of AREG and EREG measured by QPCR. (B) Summary of current literature for how GADD45A mediated DNA demethylation is achieved. Transient knockdown of GADD45A was achieved using electroporation and specific siRNA (E). Adenovirus was used to overexpress GADD45A in Panc1-2G6 and Panc1-3D7 cells (H). Changes in AREG and EREG expression were measured by QPCR in Panc1-2G6 (C,F) and Panc1-3D7 (D,G). Data depicted here are representative of at least three different experiments and represent the mean +/− standard deviation. Statistical significance was calculated using a one-tailed t-test in which * denotes P < 0.05 as compared to controls.
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GADD45A is responsible for identifying residues for DNA demethylation by DNA repair25, 41. We modulated GADD45A in pancreatic cancer cells using either siRNA to knockdown or adenoviral infection to overexpress GADD45A and examined the effects on AREG and EREG expression. As depicted in Fig. 4, knockdown of GADD45A (Fig. 5E) resulted in decreased expression of AREG and EREG regardless of integrin α6β4 expression (Fig. 5C,D). Similarly, overexpression of GADD45A (Fig. 5H) resulted in a further increase in AREG and EREG expression, only in Panc1-3D7 cells (Fig. 5F vs G). These data indicate that GADD45A is a required for and is potentially a rate-limiting step in gene activation of AREG and EREG downstream of integrin α6β4 signaling.
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TET proteins are solely responsible for oxidation of 5-mC to 5-hmC, 5-fC and 5-caC in mammalian DNA42, 43, which provide substrates for further processing to a cytosine by the DNA glycosylases and BER44, 45, with 5-hmC being the most common43. To test the role of the TET proteins, we depleted TET1 using specific shRNAs in Panc1-3D7 cells (Fig. 6A). As demonstrated in Fig. 6B, AREG and EREG expression is robustly decreased following a 70% reduction in TET1.Figure 6BER is necessary for induction of AREG and EREG expression downstream of integrin α6β4 signaling. (A,B) RNA was isolated from Panc1-3D7 cells stably expressing non-targeting or shRNA specific for TET1. QPCR analysis was used to confirm TET1 knockdown (A) and expression of AREG and EREG (B). (C) Nuclei were isolated from Panc1-2G6, Panc1-3D7, and Panc1-3D7 cells expressing specific lentiviral shRNA for TDG. Western blot analysis was performed on nuclear fractions for TDG and Lamin A/C used as a loading control. (D) Cells were collected and AREG and EREG expression measured by QPCR. (E,F) Cells were treated with either 1 μM or 10 μM 3,4-Dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2 H)-isoquinolinone (DPQ) for 72 hours. Expression of AREG and EREG was measured by QPCR in Panc-2G6 (low α6β4; E) and Panc-3D7 (high α6β4; F) cell lines. Data depicted are representative of at least three different experiments and represent the mean +/− standard deviation. Statistical significance was calculated using a one-tailed t-test in which *denotes P < 0.05 as compared to controls.
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BER is necessary for induction of AREG and EREG expression downstream of integrin α6β4 signaling. (A,B) RNA was isolated from Panc1-3D7 cells stably expressing non-targeting or shRNA specific for TET1. QPCR analysis was used to confirm TET1 knockdown (A) and expression of AREG and EREG (B). (C) Nuclei were isolated from Panc1-2G6, Panc1-3D7, and Panc1-3D7 cells expressing specific lentiviral shRNA for TDG. Western blot analysis was performed on nuclear fractions for TDG and Lamin A/C used as a loading control. (D) Cells were collected and AREG and EREG expression measured by QPCR. (E,F) Cells were treated with either 1 μM or 10 μM 3,4-Dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2 H)-isoquinolinone (DPQ) for 72 hours. Expression of AREG and EREG was measured by QPCR in Panc-2G6 (low α6β4; E) and Panc-3D7 (high α6β4; F) cell lines. Data depicted are representative of at least three different experiments and represent the mean +/− standard deviation. Statistical significance was calculated using a one-tailed t-test in which *denotes P < 0.05 as compared to controls.
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TDG has been found in complex with AID and GADD45A in the context of active DNA demethylation and evidence exists that glycosylase activity is necessary for this process45. As shown in Fig. 6C, there was substantially lower nuclear TDG protein expression in Panc1-2G6 compared to Panc1-3D7. As seen in Fig. 6D, this stable knockdown of TDG resulted in marked downregulation of AREG and EREG in Panc1-3D7 cells, indicating that TDG is necessary to maintain expression of AREG and EREG downstream of integrin α6β4, potentially through preferential localization of TDG into the nucleus.
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PARP-1 is required for BER and is implicated in genome-wide and locus specific active DNA demethylation in part through epigenetic regulation of TET146. Using a PARP-1 inhibitor, DPQ, we observed a dramatic decrease in AREG and EREG expression in Panc1-3D7 cells (Fig. 6F). However, in Panc1-2G6 cells, expression of AREG and EREG was relatively unaffected by PARP-1 inhibition (Fig. 6E), indicating that PARP-1 is mediator of AREG and EREG induction regulated by the integrin α6β4.
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We rationalized that if the integrin α6β4 is using the BER pathway to activate specific genes, the integrin may also enhance DNA repair in response to DNA damage. Therefore, we induced oxidative damage, which is repaired by the BER pathway, by exposing cells to 500 μM H2O2 over seven days and measuring cell viability by MTT assay. We observed a modest decrease in cell number in Panc1-3D7 cells; however, this H2O2 treatment nearly abolished Panc1-2G6 cells, indicating a decreased ability to survive insult by oxidative stress (Fig. 7A). To measure DNA repair more directly, we examined NER dependent DNA repair by exposing cells to 30 J/m2 UV light and measuring resolution of 6-4 photoproducts over time. As illustrated in Fig. 7B, Panc1-3D7 cells resolved UV induced lesions more rapidly than Panc1-2G6 cells, with a difference in half-life of about 1 hour. Taken together, these data indicate that the integrin α6β4 can both utilize DNA repair, and enhance the ability of cells to respond to, repair, and survive DNA damage.Figure 7Integrin α6β4 promotes DNA repair and cell survival in response to DNA damage. (A) Cells were treated with 500 μM H2O2 in fresh medium daily for 7 days. Each day cell proliferation was measured by MTT colorimetric assay. (B) Cells were exposed to 30 J/m2 UV light and DNA isolated at indicated time points. Slot blot assay was performed using antibody for 6-4 photoproducts and percent repair compared to damage achieved immediately after exposure (0 hr).
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Integrin α6β4 promotes DNA repair and cell survival in response to DNA damage. (A) Cells were treated with 500 μM H2O2 in fresh medium daily for 7 days. Each day cell proliferation was measured by MTT colorimetric assay. (B) Cells were exposed to 30 J/m2 UV light and DNA isolated at indicated time points. Slot blot assay was performed using antibody for 6-4 photoproducts and percent repair compared to damage achieved immediately after exposure (0 hr).
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While our knowledge of cancer epigenetics has developed rapidly, how dynamic epigenetic regulation is influenced by the tumor microenvironment to foster a metastasis phenotype has yet to be revealed. We find that integrin α6β4 is a critical mediator of DNA demethylation of two pro-invasive molecules, AREG and EREG. These specific changes in DNA demethylation of AREG and EREG occurred at enhancer elements within their proximal promoters that drive their expression downstream of integrin α6β4. Similarly, our data support integrin α6β4 as a modulator of genome-wide DNA methylation patterns, as overexpression of integrin α6β4 resulted in dramatic hypomethylation of the genome, with a significant percentage of these CpGs located in putative enhancer sites. Lastly, our study revealed that integrin α6β4 not only utilizes the BER DNA repair but also facilitates enhanced repair of DNA lesions, as cells with high integrin α6β4 survived better in response to oxidative stress, and directly repaired 6-4 photoproducts more rapidly. Our unique findings provide evidence that places integrin α6β4 as a critical mediator of cancer epigenetics, and thus offer new mechanisms for the integrin’s role in cancer progression.
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Upregulation of invasion promoting molecules and subsequent activation of their downstream signaling targets are critical for the progression of cancer. Here, we demonstrate that AREG and EREG, which are established contributors of tumor progression14, 15, are upregulated downstream of signaling from integrin α6β4 and this upregulation is dependent on active DNA demethylation. This observation builds on our previous data showing that integrin α6β4 stimulates specific DNA demethylation of the S100A4 promoter, ultimately contributing to invasive capabilities of breast cancer cells11. Interestingly, work in squamous cell carcinoma and MDA-MB-231 breast cancer cells demonstrates that ECM content, cell-cell interactions, and 3D environment impact the methylation state of the E-cadherin promoter and this dynamic epigenetic plasticity helps drive EMT47, 48. These observations collectively solidify the role of the tumor microenvironment in regulating specific sites of DNA methylation, thus contributing to invasive growth of cancer cells.
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Our analysis of genome-wide DNA methylation patterns revealed that integrin α6β4 dramatically reshapes the epigenetic landscape, shifting global DNA methylation patterns to a more hypomethylated state. Furthermore, this study shows that changes in specific CpG methylation within the AREG and EREG genes occurred in intronic regions that are not defined by the presence of CpG islands. These sites of altered DNA demethylation within AREG and EREG regulatory region correspond to known sites of H3K27Ac. Coupled with the requirement of BRD4 activity for AREG and EREG expression implicates the necessity for enhancer elements to drive gene expression. Our previous work on S100A4 yielded similar results as specific changes that control gene expression reside in an enhancer element located in a CpG rich region rather than a CpG island11. Similar to our gene specific data, most hypomethylation events induced by integrin α6β4 are not localized to CpG islands or promoter regions, but are instead found in intronic and intergenic elements. In addition, 23% of these regions corresponded to potential sites of H3K27ac, which is indicative of enhancer location34. These changes in DNA methylation are not surprising as hypomethylation of enhancer elements is tightly linked to overexpression of cancer promoting genes and gene profiles, as opposed to promoter methylation49, 50. Therefore, these data suggest that this shift in methylation patterns mediated by integrin α6β4 is indeed a mechanism driving gene expression and progression to a more malignant phenotype in pancreatic cancer cells. While other evidence exists to suggest that the tumor microenvironment can influence epigenetics47, 51, 52, this study is the first to identify a specific mediator of the microenvironment, the integrin α6β4, as a regulator of this process.
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Mounting evidence places the BER pathway as the most common, and context dependent mediator for active DNA demethylation45, 53. Our data support this concept, as we have demonstrated that modulation of multiple components of the BER pathway, including GADD45A, TET1, TDG, and PARP-1, impact transcriptional upregulation of AREG and EREG. Additionally, our confirmation that AREG and EREG enhancers become demethylated downstream of integrin α6β4, supports active DNA demethylation by DNA repair as the mechanism for transcriptional upregulation by the integrin α6β4. More specially, GADD45A acts as an important step in the activation of AREG and EREG and in accordance with the literature, is the coordinating molecule for specific DNA demethylation by BER54. We also show that recruitment of TDG to the nucleus is amplified in cells with high integrin α6β4 expression, suggesting that the integrin coordinates steps in this pathway, potentially through nuclear recruitment or specific targeting of repair factors. These data implicate the integrin α6β4 as a critical amplifier of DNA repair mediated DNA demethylation, identifying a novel mode of transcriptional upregulation in response to this integrin. Finally, we find that not only can the integrin α6β4 utilize BER to promote transcriptional upregulation also enhances the ability of pancreatic cancer cells to respond to and survive in the presence of DNA damage mediated by damaging agents whose damage is repaired by both the BER and NER pathways. This observation supports previous studies demonstrating that tissue architecture mediated by integrin α6β4 promotes resolution of double strand breaks55. Taken together these studies demonstrate that the integrin α6β4 contributes to a multitude of DNA repair pathways, and is a key component for connecting the extracellular environment with enhanced DNA repair.
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| 100.0 |
In conclusion, this study examines a specific sensor of the tumor microenvironment, the integrin α6β4, and provides an exciting new role for this molecule in promoting tumor progression. Our data offer a novel mechanism for the upregulation of tumor promoting genes, alterations in the epigenome, and utilization of DNA repair, and places the integrin α6β4 as a major player in cancer epigenetics. These findings have far reaching impacts on our understanding of pancreatic carcinoma and further analysis of the integrin α6β4’s role in these processes will yield a more comprehensive understanding for how this integrin impacts tumor progression.
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study
| 99.94 |
Panc1 cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (high glucose). Panc1 clones 2G6 (low integrin α6β4) and 3D7 (high integrin α6β4) were characterized and cultured as described previously56. Suit2 (Dr. Takeshi Iwamura, Miyazaki Medical College, Japan) and AsPC1 cells (America Type Culture Collection, ATCC) were maintained in RPMI 1640. Media was supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich, St. Louis, MO), 1% penicillin, 1% streptomycin, and 1% L-glutamine (GIBCO by Life Technologies, Grand Island, NY).
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study
| 99.94 |
Glass coverslips were coated with 10 μg/ml collagen I (BD Biosciences) at 4 °C overnight, then rinsed three times with PBS. Cells were plated on coverslips in normal culture medium and allowed to adhere for 4 hours before fixation. Cells were fixed, permeabilized, and immunostained as described previously57. Briefly, cells were fixed for 15 minutes with 4% paraformaldehyde containing 10 mM PIPES, pH 6.8, 2 mM EGTA, 2 mM MgCl2, 7% sucrose and 100 mM KCl for 15 min at room temperature, and permeabilized with 0.25% Triton X-100. Cells were blocked for 1 hour with 3% BSA + 1% goat serum in PBS. The following primary antibodies were used at indicated concentrations and incubated at 4 °C overnight: rat anti-CD104 (439-9B, BD Pharmingen, 1:100) mouse anti-Laminin-5 (γ2 chain, clone D4B5, Millipore, 1:500 dilution) in 3% BSA in PBST overnight at 4 °C. Cy3-conjugated goat anti-rat and Cy2-conjugated goat anti-mouse (Jackson Immuno Research, 1:500) and Alexa Fluor 647 phalloidin were incubated with 3% + 1% goat serum BSA for 1 hour at room temperature in dark. Coverslips were mounted on glass slides using 50% glycerol solution and sealed with clear polish. Images were acquired by total internal reflection (TIRF) microscopy using a Nikon Eclipse Ti. Images were processed for colocalization analysis and Pearson’s correlation coefficient by NIS Elements AR 3.2 software.
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study
| 99.9 |
5-Aza-2′deoxycytidine (5-aza-CdR; Sigma-Aldrich) and S-adenosylmethionine (SAM; NEB, Ipswich, MA) was added to cells in fresh medium daily at indicated concentrations for 3 or 5 days or equal volume DMSO or 0.005 M H2SO4 plus 10% ETOH respectively. JQ1 (250–500 nM; Bradner Lab; Dana-Farber Cancer Institute) or DMSO was added to cells for 16 hours. Gemcitabine (Sigma-Aldrich) or 3,4-Dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2 H)-isoquinolinone (DPQ; Sigma-Aldrich) treatment or equal volume vehicle control was added once for 72 hours.
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study
| 99.94 |
For H2O2 treatment (Sigma-Aldrich), cells were plated in a 96-well plate at 2000 cells/well. Medium was changed each day to normal growth 500 μM H2O2 containing medium. Cell density was measured using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Fisher Scientific) according to manufacturer’s protocol.
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study
| 99.94 |
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) per manufacturer’s protocol. cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and target expression was assessed using available probes, reagents, and the StepOnePlus Real-Time PCR System from Applied Biosystems, as performed previously13. Target expression (2−ΔΔCT) was normalized to endogenous reference (18S or β-actin) and reported relative to control samples. Each QPCR experiment was performed a minimum of three times and consistent trends across biologically replicated experiments were observed. The representative experiments shown in most figures are from experiments that generally best reflected the average QPCR data of these three experiments. We chose representative data over averaging the individual experiments together since significant variability often existed between experiments. This variability occurred because of the amplification of small differences present in the mRNA when values of one of the conditions are exceptionally low (as we see in the Panc-2G6 cells with AREG and EREG expression). The exceptions are data from Figs 4B,H and 5F, which showed less inter-experimental variability and were averaged in order to demonstrate significance of the findings.
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study
| 100.0 |
Whole genomic DNA was isolated from cell lines using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). DNA was processed for high-resolution methyl-seq by the NextGen Sequencing Core at the Norris Comprehensive Cancer Center. Whole genome sequencing was done on an Illumina NextSeq and each library sequenced with paired-end runs for 150-bp read length analysis.
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study
| 99.9 |
DNA reads were aligned against GRCH37 using Bismark58 software version 0.14.3, permitting at most one mismatch, considering both sequence and bisulfite conversion mismatches. Methylation calls for each CpG were extracted using Bismark methylation extractor tool. Read alignment revealed that many reads could be mapped to both AREG and the AREG pseudogene due to high degree of homology. To allow mapping of AREG, the AREG pseudogene was masked during analysis, and vice-versa. Differential methylation analysis comparing Panc1-3D7 and Panc1-2G6 was performed using Bioconductor DSS software version 2.10.059. Differentially methylated loci (DML) were determined by >0.99 posterior probability of the difference in mean methylation levels being >0.3. Differentially methylated regions (DMR) were also detected by joining DMLs with p-value less than 0.01. DMRs have a minimum length >50 bps, minimum number of DML >3 and >50% of CpG sites with p-value < 0.01. DMRs with distance less than 100 bps were merged. DMLs and DMRs were annotated using methylKit60 version 0.9.5, where we defined the promoters as +/− 1000 bp from TSS and CpG shores +/− 2000 bp flanking each side of the CpG island.
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study
| 100.0 |
For shRNA, lentivirus was produced by combining MISSION constructs for packaging (psPAX2), envelope (pDM2G) and targeting shRNA or a non-targeting vector (pLKO.1), at a 4:2:1 ratio (Sigma Aldrich, St. Louis, MO). Polyethylenimine (PEI; Polysciences) was combined with DNA at a 3:1 ratio, and added to 70% confluent HEK 293LTV cells. Conditioned media was collected 24 and 48 hrs post transfection by centrifugation, and viral supernatant added to cells with 8 μg/ml hexadimethrine bromide (polybrene, Sigma-Aldrich). Gene expression was measured by QPCR 24 hrs following puromycin selection (2 μg/ml).
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study
| 99.94 |
Cells were washed 2x with cold PBS and collected with 400 μl cold Buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). Nuclei were lysed using 10% NP-40 and nuclear pellet resuspended in cold Buffer C (20 mM Hepes pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF). Nuclear extracts were collected by centrifugation, separated using 10% SDS-PAGE, transferred and immunoblotted for TDG (Genetex, GT622), and Lamin A/C (EMD Millipore). Alternatively, whole cell lysates were collected using RIPA buffer, separated using 10% or 12.5% SDS-PAGE and then immunoblotted for integrin β4 (BD Transduction Labs, #611232), laminin γ2 chain for laminin-5 (Millipore, clone D4B5) or β-actin (Sigma-Aldrich). Uncropped western blot images can be found in the supplemental information.
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| 99.94 |
Immuno-slot-blot analysis was performed as described previously61. Briefly, cells (70% confluent) were exposed to 30 J/m2 UV and harvested immediately or indicated times post-treatment. Cells were lysed with 10 mM Tris pH 8.0, 1 mM EDTA, 0.05% SDS, 100 μg/ml fresh proteinase K) and DNA isolated. DNA was bound to a nitrocellulose membrane using a slot blot apparatus and probed using antibody for 6-4 photoproducts (6-4 PP; Cosmobio). Data are reported as percent repair compared to the amount of initial damage (0 hr time point).
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study
| 100.0 |
Toxic chemicals have currently been released into the environment by accidental spills and the improper management of chemical industries. These toxic chemicals include inorganic products such as heavy metals and organic products such as benzene, toluene, ethylbenzene, biphenyl, and styrene, accidental release of which into environment are a significant threat to the environment. Heavy metals and oil products are difficult to remove from the environment and cannot be easily degraded. Thus, they are ultimately indestructible and constitute a global environmental hazard. As a result, soil and groundwater contamination has become a major problem at these polluted sites and requires urgent remediation technology to protect the environment.
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other
| 99.94 |
Over the past few decades, several technologies based on novel analytical methods have been developed to remove certain metals and organic pollutants from the environment . Unfortunately, many conventional techniques have been found to be ineffective and/or expensive due to low permeability, different subsurface conditions, and contaminant mixtures. Owing to the limitations of traditional methods, researchers have focused on in situ bioremediation, which uses microorganisms to degrade petroleum products or immobilize heavy metal contaminants. Bioremediation strategies have been proposed as potential alternatives for the removal of organic and inorganic pollutants due to their safety, speed, low cost, and high efficiency in removing pollutants from the environment.
|
review
| 99.9 |
The central principle of bioremediation is that microorganisms are able to produce energy they need to grow and reproduce by degrading hazardous contaminants. In some cases, bioremediation occurs spontaneously because the essential materials required for bacterial growth are naturally present at the contaminated sites. More often, bioremediation requires an engineered bacterial system to accelerate the tailor-made biodegradation of organic compounds or bio-adsorption of inorganic elements as we desired [2, 3]. It is also needed to further optimize the environmental conditions, in which the microorganisms carry out the detoxification reactions by employing several engineered microorganism systems such as cell surface display- and secretion-based strategies to remediate the contaminated environment. Cell surface display technologies have widely been used in both pharmaceutical and bioremediation applications such as live vaccine development, antibody production, peptide library screening, biosensors, bio-adsorption of organic and inorganic pollutants, and whole-cell biocatalysis (Fig. 1) [4–7].Fig. 1Application of different cell surface display technologies in A antibody production, B peptide library screening, C biosensors, D biocatalysts, E bio-adsorption, and F vaccine development
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review
| 99.9 |
Heavy metals are common pollutants that are byproducts of various industrial activities. Microorganisms usually mobilize metals from one location and scavenge metals from another. Recently, recombinant bacterial systems displaying chimeric proteins on the cell surface have been developed for use in the bio-adsorption of specific heavy metals. To address organic products, microorganisms have been engineered to produce extracellular enzymes or display enzymes as outer membrane proteins, and they act as a whole-cell catalyst to break down petroleum hydrocarbons and their derivatives. However, all of these constructs require expensive inducers, or the constitutive expression of a membrane protein on the cell surface may affect the growth of the host system. Additionally, none of these engineered bacteria can sense the particular bio-component to be degraded. Therefore, engineered bacteria should be designed to monitor the environmental pollutants, and the design should also include a well-defined removal system. The engineered bacterial system should behave normally until it senses the target in the environment. Once the target is detected, the system should modulate bacterial genes in response. In this way, the genes needed to remove the target are only transcribed and expressed when required. Therefore, it is essential to construct an inexpensive system that can efficiently examine and remove hazardous materials present in the environment.
|
review
| 99.7 |
Nature has provided an excellent solution to this problem. Interestingly, cells have evolved many intricate sensory apparatuses to control cellular growth and behavior. Thus, some cells not only sense light, temperature, oxygen, and pH, but also detect the toxic status of the external environment. An essential requirement for a biosensor or bioremediation process is promoting contact between the contaminants and microbes. As a result of this contact, the microbes adapt their cellular functions in response to the surrounding environmental conditions and then express the relevant genes when needed. If the aim is to monitor and remove an individual toxic compound from the environment, then a synthetic biological strategy will be more feasible because the necessary genetic circuits can be assembled to sense and reduce the level of the exogenous toxin. These synthetic genetic circuits can be assembled using a two-component regulatory system (TCRS) in bacteria .
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review
| 99.06 |
Two-component regulatory systems are widely found in prokaryotes, but only a few have been identified in eukaryotic organisms that can be coupled to environmental stimuli for an appropriate cellular response. This system senses environmental changes and regulates cellular metabolism in response to these changes thereby allowing bacteria to grow, thrive and adapt in different environments. A prototypical TCRS has two components: a histidine kinase (HK) and a response regulator (RR). The HK sensor is a homodimeric integral membrane protein that contains a sensor domain as an extracellular loop located between two membrane-spanning segments (TM1 and TM2) and a transmitter domain located in the last transmembrane segment confined to the cytoplasm. All HK domains contain two highly conserved domains: dimerization and histidine phosphotransfer domain (DHp) and catalytic ATP-binding domain (CA). The periplasmic or extracellular region serves mostly as the signal recognition domain. The DHp and CA domains are responsible for the molecular recognition of the cognate RR as well as the hydrolysis of ATP. The transmitter domain, which serves as a signal transmitter linking the periplasmic and cytoplasmic regions, contains three domains that are named after the proteins where they were first discovered: PAS (Periodic circadian proteins, Aryl hydrocarbon nuclear translocator proteins and Single-minded proteins), HAMP (HKs, Adenylate cyclases, Methyltransferases, and Phosphodiesterases), and GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and formate hydrogenases). These domains can either transmit signals from the periplasmic region or directly recognize the cytoplasmic signals. Therefore, the HK senses stimuli from the external environment and autophosphorylates conserved histidine residues in the kinase itself. The RR is regulated by the HK, which phosphorylates aspartate residues on the RR. The phosphorylated RR generates output by binding to promoters and thus activates or represses gene expression .
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study
| 99.44 |
Aside from the application of TCRSs in the development of engineered microorganisms for coupled detection and degradation of environmental pollutants, recently, the potential application of TCRSs to metabolically engineered microorganisms has also been extensively examined for different biotechnological purposes. Thus, the recent advances in TCRS-based biosensors designed for cell-mediated bioremediation in response to different environmental pollutants are discussed along with the potential application of TCRSs for the development of engineered host microorganisms in biorefinery process to produce bio-based chemicals.
|
review
| 99.9 |
Two-component regulatory systems can detect a broad range of environmental signals, such us light, oxygen, pH, temperature, and even some heavy metals and organic contaminants . Many types of TCRS-based environmental biosensors have been reported, but only a few heavy metal- and organic pollutant-based sensors have been developed to date (Fig. 2). Bacteria use several TCRSs to sense specific heavy metals. Because heavy metals are cations that are both toxic and essential, bacterial cells use TCRSs to regulate the homeostasis of these metal cations. A HydHG TCRS (also known as ZraSR) was identified in Escherichia coli that senses and controls the expression of zraP gene encoding zinc efflux protein under high concentrations of Zn2+ and Pb2+ in aerobic condition . HydH protein is tightly bound to the cell membrane and is assumed to be responsible for sensing high periplasmic Zn2+ and Pb2+ concentration. Then, in the presence of a phosphoryl donor, HydG binds to the intergenic region within zraP-hydHG resulting in the upregulated expression of ZraP . Likewise, the CusRS (ylcA, ybcZ) TCRS found in E. coli K-12 is responsive to Cu2+ ions and is required for the inducible expression of pcoE, belonging to the plasmid-borne pco operon, the induction of the genes in this operon activates the copper efflux system thereby allowing the excess Cu2+ to exit the cell . Some TCRS can regulate the expression of several specific genes in an operon or a whole operon. The SilRS TCRS increases the resistance of Salmonella enterica to silver cations through the coupled sensing and activation expression of the periplasmic silver-specific binding protein, SilE encoded by silE gene and two parallel efflux pumps, SilP and SilCBA . This is also in the case of NrsSR TCRS identified in Synechocystis sp. PCC6803. NrsSR senses Ni2+ and Co2+ ions and regulates the expression of the nrsBACD operon that encodes proteins involved in Ni2+ resistance . In another study, a PfeS/R TCRS senses ferric enterobactin and induces the production of the enterobactin receptor PfeA in Pseudomonas aeruginosa .Fig. 2 a Domain structure of bacterial two-component regulatory systems (TCRS). Typical two-component phosphotransfer systems contain a sensor domain and a cytoplasmic response regulator (RRs). b A multi-component phosphorelay system containing the HAMP, PAS, and phosphotransfer domains. The periplasmic metal-sensing receptors sense heavy metals and phosphorylate the HK domain and activate the corresponding RR. The RR activates the synthetic genetic circuit of the TCRS resulting in the expression of the reporter protein. The genetic circuit shown in gray can be developed as a biosensor
|
review
| 99.7 |
a Domain structure of bacterial two-component regulatory systems (TCRS). Typical two-component phosphotransfer systems contain a sensor domain and a cytoplasmic response regulator (RRs). b A multi-component phosphorelay system containing the HAMP, PAS, and phosphotransfer domains. The periplasmic metal-sensing receptors sense heavy metals and phosphorylate the HK domain and activate the corresponding RR. The RR activates the synthetic genetic circuit of the TCRS resulting in the expression of the reporter protein. The genetic circuit shown in gray can be developed as a biosensor
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study
| 53.47 |
Aromatic compounds are the most abundant organic contaminants. However, utilizing these compounds is disruptive to most bacteria. Due to the genetic and metabolic flexibility of bacteria, some microorganisms can use organic contaminants as their sole carbon source. Several TCRSs have been identified to be involved in catabolizing aromatic compounds by inducing and activating the aromatic metabolism pathways. The TodST TCRS of Pseudomonas putida can be induced by different aromatic substrates such as toluene, xylene, benzene, and ethylbenzene. This TCRS modulates the expression of the tod genes, which encode enzymes for the catabolism of these aromatic compounds . The StySR TCRS identified in Pseudomonas sp. strain Y2 activates the expression of the styABCD genes in response to changes in styrene concentration in the environment . Another TCRS, BpdST, potentially controls biphenyl or polychlorobiphenyl degradation in Rhodococcus sp. .
|
study
| 100.0 |
One of the best approaches to a biosensor-based method is to use a genetically modified microorganism that emits a clear signal when the microbes encounter a target molecule [17, 18]. To date, many metal-specific and a few petroleum product-based bacterial sensors have been developed [19–23]. Based on the nature of the cells used, a variety of TCRS-based environmental contaminant sensors has been constructed by several research groups. However, to remediate environmental pollutants, new synthetic genetic circuits are needed so that the bacterial system can have both sensor and remediation activities. Future research on the application of biosensors in bioremediation should focus on the development of such TCRSs. Some of the TCRS-based heavy metal biosensors for use in bioremediation applications have been developed and are reviewed below.
|
review
| 99.9 |
A zinc adsorption system was developed by using the ZraSR TCRS and chimera Zinc binding OmpC. In normal microbial system, ZraSR detects and induces the membrane protein ZraP, which is responsible for the efflux of Zn2+ ions. Engineered zinc adsorption system was based on normal ZraSR TCRS, in which ZraS is used for detecting Zn2+ ions, but the ZraR activates the ompC-Zinc binding peptide chimeric gene under the ZraP promoter instead of native ZraP. The zinc binding peptides displayed in the cell surface can adsorb exogenous Zinc. This system is sensitive to zinc even at low concentrations (0.001 mM) .
|
study
| 100.0 |
In the same manner, simultaneous detection and removal of copper ions in the bacterial surface was achieved through the combined application of CuSR TCRS and cell surface displayed copper binding peptides (CBP) fused to the membrane protein OmpC. In this system, CuSR induces the expression of the chimera OmpC-CBP upon sensing Cu2+ ions. Then, the chimera proteins expressed in bacterial cell surface can adsorb the copper ions .
|
study
| 100.0 |
An interesting feature of these adsorption systems is that the expression of the chimeric OmpC with the metal binding site is induced by heavy metals (Table 1). Hence, the construction of a heavy metal biosensor in combination with a bio-adsorption system would complement analytical heavy metal detection methodologies and enable the rapid monitoring and removal of toxic levels of bioavailable metal contaminants in industrial settings. The above biosensor combined with bio-adsorption was able to absorb heavy metals efficiently without any induction system. Following this scheme, this synthetic bacterial system is an excellent paradigm for developing multifunctional synthetic systems that can be applied both in the efficient removal and recovery of the target compound.Table 1Two-component regulatory systems based on microbial biosensors coupled with bio-adsorptionField of applicationTCRSFunctionHost chassisPromoter-reporterChemical targetDetection range (mM)ReferencesBioremediationZraSR (also known as HydHG) Biosensor E. coli XL1-blue zraP-gfp-HydGZinc0.01–1CuSRBiosensor E. coli XL1-blue cusC-gfp-CusRCopper0.004–1ZraSR and CusSRBiosensor coupled with bio-adsorption E. coli XL1-blue zraP-gfp, cusC-gfp Zinc and Copper0.05–1ZraSRBiosensor coupled with bio-adsorption E. coli TOP10 zraP-gfp-ompC Lead0.3–1ZraSR Biosensor coupled with bio-adsorption E. coli XL1-blue zraP-gfp Zinc0.1–1BiorefineryDcuSZ (Chimeric)Biosensor E. coli BL21(DE3) ompC-gfp Fumarate0.1–10MalKZ (Chimeric)Biosensor E. coli BL21(DE3) ompC-gfp Malate0.1–10AauSZ (Chimeric)Biosensor E. coli BL21(DE3) ompC-gfp Acidic amino acid0.05–10Tazl (Chimeric)Biosensor E. coli RU1012 ompC-lacZ Aspartate0.2–1
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study
| 99.94 |
The successful design and construction of TCRS provide a better understanding of the system to obtain a chimeric TCRS customized for achieving a desired input/output. The HK domain, which has a variety of signal recognition capabilities, may be used to couple or shuffle a broad range of input signals to the appropriate output responses through a conserved phosphotransfer process. This shuffling can be achieved by cross-linking the domains of evolutionarily distinct TCRSs, and a chimeric TCRS with the desired sensing ability can be obtained. Most of the domain shuffling required for rational design of chimeric proteins is between HKs and rarely between RRs. At present, several research groups have successfully constructed a chimeric two-component sensor protein by fusing the HK domain to the sensory domain of another kinase or a completely unrelated protein. These studies improve our understanding of the molecular events that occur during signal transduction across membranes in these organisms.
|
review
| 68.56 |
Engineering receptor kinases mainly involve a domain swapping or shuffling strategy in which a receptor protein or another HK contributes their functional module. The domain swapping in HKs implies that these proteins are flexible, allowing the construction of new kinases using a rational design strategy. The domain swapping strategy has been used to produce chimeric TCRSs that include chemotaxis proteins. There are several periplasmic chemotactic receptors, such as Tsr, Tar, Trg, and Aer, that recognize specific chemicals, and they can be coupled with the cytoplasmic domain of EnvZ to allow signal transduction . EnvZ is the most studied HK protein that regulates the phosphorylation state of OmpR in response to osmolarity changes. OmpR is an RR protein responsible for the controlled expression of ompF and ompC genes encoding for the membrane porin proteins OmpF and OmpC, respectively. Aside from OmpR, EnvZ can also regulate the phosphotransfer of 11 different RRs found in E. coli . Because the EnvZ–ompR complex in E. coli is a well-studied TCRS that is widely-distributed in bacteria, the DHp and CA domains of EnvZ are commonly used for the domain swapping strategy. A good example of this is the hybridization of Tar, a chemoreceptor transmembrane protein that can detect aspartate and EnvZ. By replacing the cytoplasmic signaling domain of Tar protein with the cytoplasmic kinase/phosphatase domain of EnvZ, the hybridized proteins were able to carry out both the sensing capability of Tar for aspartate and the regulation capability of EnvZ towards OmpR thereby consequently activating ompC . This strategy also worked in the hybridization of Trg protein and EnvZ, allowing the recognition of ribose-binding peptides and activation expression of ompC . In addition to functioning as chemotactic receptors, HK domains are also involved in light sensing, and kinases that sense C4-dicarboxylate, sugar, aspartate, and acidic amino acids have been engineered with the EnvZ cytoplasmic domain to provide a better sensing ability for the desired substance (Table 1). This approach to engineering novel two-component sensor proteins not only acts as a high throughput screening system but also provides knowledge of the newly identified two-component signaling pathways.
|
review
| 99.7 |
In line with the depletion of fossil fuels, renewable biomass is being exploited as a sustainable substitute for petroleum. Among the renewable biomass resources, lignocellulosic biomass is one of the most promising due to its abundance. Lignocellulosic biomass undergoes different pretreatment methods that result in a hydrolysate containing mixed sugars and inhibitors that can be detrimental to the growth of microbial cells during fermentation .
|
review
| 74.44 |
Metabolic engineering strategies have been developed in systems level for the development of metabolically engineered microorganisms as host strains in biorefinery processes to produce bio-based fuels [31–35], chemicals [36–41] and polymers [42–47] from renewable resources. Also, engineered strains that have high levels of growth and tolerance in the presence of high concentrations of sugars and inhibitors are extensively being developed to utilize biomass-derived renewable resources [48–53]. Therefore, it is important to develop a high-throughput screening method to identify the high-producing strains. High-producing strains can be screened using a riboselector, which is composed of a riboswitch that can detect the target compound and a selection module such as tetA, which will enable favorable growth of a lysine-accumulating cell in the presence of selection pressure (NiCl2) . Likewise, chimeric TCRS can be potentially used in screening for high-producing strains (Fig. 3). DcuSZ is an EnvZ/OmpR-based chimeric TCRS that was constructed by fusing the DcuS HK sensory domain with the cytoplasmic domain of EnvZ. The chimeric DcuSZ is highly specific to fumarate in such a way that the expression of the gfp gene under the control of the ompR-regulated ompC promoter is proportional to different fumarate concentrations in the medium . Other chimeric TCRSs based on EnvZ/OmpR were constructed by fusing the HK sensory domain of MalK and AauS to the EnvZ catalytic domain to detect high malate- and aspartate-producing strains, respectively [56, 57].Fig. 3Application of TCRSs in bioremediation and microbial biorefinery. TCRSs serve as a regulatory system for the expression of genes encoding enzymes for the degradation of the detected target pollutant compound or for genes encoding enzymes for the production of the target chemical product
|
study
| 99.7 |
Application of TCRSs in bioremediation and microbial biorefinery. TCRSs serve as a regulatory system for the expression of genes encoding enzymes for the degradation of the detected target pollutant compound or for genes encoding enzymes for the production of the target chemical product
|
other
| 99.9 |
Two-component regulatory systems may also be used to develop tightly regulated gene expression systems. Tightly regulated gene expression is important in engineering metabolic pathways to avoid leaky expression that may cause a metabolic burden to the microbial cell. Typical induction strategies include the use of isopropyl-β-d-thiogalactopyranoside (IPTG). However, IPTG is expensive and can be toxic to cells at high concentrations. An example of tightly regulated gene expression induced by an inexpensive substrate is the invertible promoter system. In this system, the promoter is active or ‘ON’ when the target substrate that serves as an inducer is present and ‘OFF’ (inverted orientation) when absent . Based on this invertible promoter system’s mechanism, the coupled sensing and regulating activities of TCRSs can be modified to achieve tightly regulated gene expression.
|
study
| 99.94 |
To date, some TCRSs have been identified that sense organic compounds (benzene, toluene, ethylbenzene, biphenyl, styrene, fumarate, and malate) and regulate the gene expression of proteins involved in catabolic pathways. These compounds can be metabolized and used as a carbon source for most groups of microorganisms . In TCRSs, the signal recognized by the sensor kinase domain catalyzes the ATP-dependent phosphorylation of a conserved histidine residue in the protein. The phosphoryl group is then transferred from the histidine to an aspartate residue located in the RR. The phosphorylated RR binds to specific promoter sequences to either activate or repress transcription. At present, a wide range of synthetic genetic circuits has been developed that can couple a sensor output to a desired biological activity . In addition, numerous genetic switches are also available to turn on gene expression once a target molecule has reached its activation threshold. A switch can be assembled using transcriptional repressors or activators, which allows the connection between the sensor output and regulation of the biological response . Several switch types have been developed to control the cellular response: inverter switches that produce a reciprocal response ; biphasic switches that use both negative and positive regulation and respond to small amounts of input ; toggle switches that use two repressors that cross-regulate each other’s promoters ; and riboswitches that regulate gene expression by inhibiting protein synthesis . Likewise, many logic gate types have been developed for biological circuits, including ‘NAND’, ‘NOT IF’ and ‘NOR’ .
|
review
| 99.9 |
Integrated approaches provide a better perspective for developing a specific biosensor designed to catalyze the production and/or degradation of the desired compound. To achieve this, it is necessary to rewire the genetic circuits of bacteria using the above synthetic devices. Design of the engineered system should be based on strategies for building sensory regulation components that incorporate a target substrate-responsive TCRS in any desired host (Fig. 4). Introducing a sensory regulation device in a host cell enables it to sense the target compound and trigger the genetic circuit, achieving real-time monitoring of the compound present and upregulation of the effector protein’s gene expression. Use of engineered TCRSs in bacteria would prevent the production of redundant proteins at the initial growth phase and avoid the use of toxic and costly chemical inducers.Fig. 4Synthetic TCRS with integrated biosensing and bioremediating functions for the detection of the target compound and upregulation of the effector protein that allow real-time detection of controlled gene expression
|
review
| 62.6 |
Although a large number of accessible sensor parts are available for TCRSs, employing these sensors in a domain shuffling strategy can be challenging. To attach the sensor domain to the HK domain of the protein, structural and functional information on both proteins is needed . When designing chimeric TCRS-based biosensors, great care is required in domain swapping to maximize the kinase activity of the chimeric protein. In the majority of the chimeric TCRS-based biosensors, monitoring of the extracellular targets and the response to these targets is achieved by producing a reporter protein [55–57]. Moreover, biosensors have also been modified with other synthetic biology tools such as the bio-absorption of heavy metals with a cell surface display system and expression of an extracellular enzyme to degrade aromatic compounds. Therefore, such a synthetic genetic circuit can be switched on when a signal is detected to remove certain pollutants, and after the input signal disappears, the microbes behave like normal bacteria.
|
review
| 99.6 |
In this review, we have discussed numerous TCRSs engineered in different prokaryotic species that can sense inorganic and organic pollutants, and examined the recent developments in cellular biosensors coupled with bioremediation. The TCRS-based biosensor coupled with bioremediation approach has the potential to advance even further using the recent developments in bioengineering in strain development. However, only a few studies on TCRS-based biosensors have been reported, and much effort is needed to obtain a complete picture of the TCRS-based control of downstream catabolic pathways. To achieve these goals, a thorough understanding of TCRS mechanisms is essential to engineer strains for use in efficient biosensor systems coupled with bio-degradation or bio-adsorption functionality. Moreover, more studies are required to extend its use in food, pharmaceutical and industrial biotechnology applications.
|
review
| 99.9 |
The effect of menopausal hormone therapy (MHT), previously known as hormone replacement therapy, on cardiovascular health in post-menopausal women remains controversial and unclear. Extensive observational data had suggested MHT to be cardioprotective [1–3], leading to MHT being routinely prescribed for both primary and secondary prevention of coronary heart disease (CHD). However, subsequent data from the Women’s Health Initiative (WHI) and Heart and Estrogen/Progestin Replacement Study (HERS) studies cast doubt on the beneficial cardiovascular effects of MHT [4–6]; this was reflected in learned societies’ clinical guidance concerning MHT’s role in CHD prevention . The most recent randomised trial data on the subject arose from the Danish Osteoporosis Prevention Study , which indicated that women taking MHT had a reduced risk of the composite endpoint of mortality, heart failure and myocardial infarction but the study has been subject to criticism . In more recent work, again from the WHI, there was no difference in cardiovascular mortality in MHT users compared to placebo, although the authors themselves state that cause-specific mortality data should be interpreted “cautiously” . it has been suggested that commencement of MHT in the perimenopausal transition or early menopause is not associated with increased risk of CHD compared to when treatment is administered at a later stage. This is known as the “timing hypothesis” .
|
review
| 99.9 |
The UK Biobank is an ongoing, large-scale, population-based study designed to examine determinants of health in middle and old age . Besides extensive collection of health questionnaire data, biological samples and physical measurements, it has incorporated cardiovascular magnetic resonance (CMR) imaging–the gold standard for analysis of cardiac structure and function–to provide detailed imaging phenotypes . At present, there is a paucity of data on the effects of MHT on left ventricular (LV) and left atrial (LA) volumes and function, alterations in which are markers of subclinical cardiovascular disease and have prognostic implications.
|
study
| 96.8 |
The UK Biobank is a versatile scientific resource, in which questionnaire data, physical measurements and biological samples were collected from over 500,000 individuals aged 40–69 between 2006 and 2010 registered with the UK National Health Service; the study protocol has been described in detail previously . Additionally, the UK Biobank imaging enhancement study is ongoing with the aim of performing, in a single visit, brain, heart, whole body, carotid artery, bone and joint imaging in 100,000 of the original 500,000 participants. Cardiovascular magnetic resonance imaging (CMR) was selected as the modality of choice for heart imaging. The study population presented here consists of 1604 individuals, a subset of the 5,065 individuals who underwent CMR examination as part of the pilot phase (April 2014 –August 2015) of the UK Biobank imaging enhancement. All participants provided written consent; UK Biobank’s scientific protocol and operational procedures were reviewed and approved by the North West Multi-centre Research Ethics Committee in the UK . The research presented here was conducted under access application 2964 and was approved by the UK Biobank access committee.
|
study
| 99.94 |
Male participants (n = 2356), female participants not reporting having undergone menopause (n = 693), participants reporting myocardial infarction, angina, heart failure, arrhythmias (including atrial fibrillation), cardiomyopathy, stroke or peripheral vascular disease (n = 76), and participants using MHT for < 3 years or with missing duration data (n = 246) were excluded from the analysis leaving a study population of 1604.
|
study
| 99.94 |
The UK Biobank CMR protocol has been described in detail elsewhere . Briefly, all examinations were performed on a wide-bore 1.5 Tesla scanner (MAGNETOM Aera, Syngo Platform VD13A, Siemens Healthcare, Erlangen, Germany). For cardiac function, long axis cines and a complete short axis stack of balanced steady-state free precession (bSSFP) cines, covering the left and right ventricle were acquired.
|
study
| 99.9 |
Analysis of the cardiac chambers for all CMR examinations was performed manually across two core laboratories according to pre-approved standard operating procedures using cvi42 post-processing software (Version 5.1.1, Circle Cardiovascular Imaging Inc., Calgary, Canada) by observers blinded to all exposures. LV papillary muscles were included in blood pool volumes and excluded from LV mass. Detailed descriptions of analysis methodology, including reference ranges, exemplar contours and intra- and inter-observer variability, have been previously described . The CMR parameters examined in this study were left ventricular end-diastolic volume, end-systolic volume, stroke volume, ejection fraction and mass and left atrial maximal volume.
|
study
| 100.0 |
To reliably assess the impact of MHT on cardiac structure and function, only women using MHT for ≥ 3 years were included in the analysis. Data concerning MHT use was derived from UK Biobank fields 3546 (age last used MHT) and 3536 (age started MHT). Duration of MHT use was calculated by subtracting values in these two fields. Where women indicated that they were still currently using MHT, age at the time of imaging visit was used to determine duration. Duration of menopause was calculated by subtracting age at menopause (data field 3581) from age at time of imaging visit. To assess the impact of the “timing hypothesis”, a timing variable was created defined as age of menopause subtracted from age started MHT expressed in years.
|
study
| 100.0 |
Descriptive statistics for continuous variables were presented as mean ± standard deviation or median and interquartile range (IQR) whilst categorical variables were presented as number (percentage). Differences in means were tested using unpaired t-test or Mann-Whitney-U test and differences in percentages using chi-squared test. CMR parameters used as dependent variables were LV end-diastolic volume, LV end-systolic volume, LV stroke volume, LV ejection fraction, LV mass, and LA maximal volume. All dependent variables were assessed for normality using histograms and quantile-quantile plots; natural logarithmic transformation was performed for all dependent variables barring LV ejection fraction. For each dependent variable, outliers were defined as measurements more than three interquartile ranges below the first quartile or above the third quartile and removed from analysis. With respect to missing values, the data presented is a complete case analysis.
|
study
| 99.94 |
To examine the impact of MHT use on cardiac structure and function, multivariable linear regression models were fitted for each cardiac (dependent) variable. With our sample size, the study has 80% power at the 5% significance level to detect a 0.15 standard deviation difference in any of the continuous variables; this would be considered a small effect size . Co-variates included in the model (Model 1) were age, age at menopause, ethnicity, height, weight, systolic blood pressure, diastolic blood pressure, smoking status, regular alcohol use, presence of raised cholesterol, presence of diabetes, Townsend deprivation index and income. Height and weight were included as covariates in the model rather than indexing the dependent variables, as the use of ratios in regression analysis can lead to spurious results and misinterpretation . The adjustment made ensured all variables in the model were appropriately adjusted for body composition. The variance inflation factor was calculated to test for multicollinearity. Where cardiac variables had been log-transformed, the beta coefficients were anti-logged and expressed as a percentage difference.
|
study
| 100.0 |
To determine whether the effect of MHT use on each of the cardiac outcomes differed by age, multivariable regression models were constructed with a cross-product of age and MHT fitted as an interaction term. Co-variates included: duration of MHT use fitted as thirds, ethnicity, height, weight, systolic blood pressure, diastolic blood pressure, smoking status, regular alcohol use, presence of raised cholesterol, presence of diabetes, Townsend deprivation index and income (Model 2). Interactions were tested using age as a continuous variable. For ease of interpretation we also fitted the interaction using tertile of age and presented effect sizes for MHT use by tertile.
|
study
| 100.0 |
In sensitivity analyses, to examine differences between the MHT use ≥ 3 years and no MHT use groups on CMR parameters, propensity-score matching was used. Matching was performed using all co-variates used in Model 1 at a one-to-one ratio. Differences between MHT use ≥ 3 years and propensity-matched controls was assessed using paired t-test.
|
study
| 100.0 |
To examine the effect of missing data, multiple imputation by chained equations was used to impute 20 complete datasets on which the analysis was repeated and the results pooled. Predictive mean matching with five nearest neighbours was used for continuous variables and logistic regression for binary variables. Plots were examined to assess convergence and plausibility of estimates.
|
study
| 99.94 |
A total of 1,604 participants were included in this study, 513 post-menopausal women who had used MHT for ≥ 3 years and 1,091 post-menopausal women who had never used MHT; case selection is depicted in Fig 1. The mean number of outliers for CMR variables was 2 (range = 0–7).
|
study
| 100.0 |
Baseline characteristics for the study population, divided by never used MHT vs MHT use ≥ 3 years, are presented in Table 1. The mean age (65.4±5.7 vs 61.3±6.4; P<0.0001) was higher and the median age at menopause (50 [IQR = 45–52] vs 51 [IQR = 48–53]; p<0.0001) was lower in the MHT cohort compared to the never used MHT cohort. For those using MHT, the median number of years of MHT use was 8 (IQR = 5–11) and the mean age of commencement was 47.6±5.3. At the time of CMR examination, 15.2% (n = 78) were still on treatment. There was no significant difference in socioeconomic status measures between MHT users and non-users including Townsend deprivation index, household income or educational attainment.
|
study
| 100.0 |
Mean CMR parameters for each cohort are presented in Table 2. Before adjustment, women who had used MHT ≥ 3 years had significantly smaller LV end-diastolic volume (117±22 ml vs 124±22 ml; p<0.0001), LV end-systolic volume (46±12 ml vs 48±12 ml; p <0.005), LV stroke volume (71±14 ml vs 75±14 ml; p<0.0001) and LA maximal volume (57±17 ml vs 61±17 ml; p<0.0001) compared to the cohort who had never used MHT. There was no significant difference in LV mass between the two groups.
|
study
| 100.0 |
The effect of MHT use on LV and LA CMR parameters in fully adjusted models is detailed in Table 3. Use of MHT for ≥ 3 years was associated with a significant reduction in LV end-diastolic volume (123 ml vs 120 ml, effect size = -2.4%, 95% confidence interval [CI]: -4.2% to -0.5%; p = 0.013), LV stroke volume (74 ml vs 72 ml, effect size = -3.1%, 95% CI: -5.1% to -1.0%; p = 0.004) and LA maximal volume (60 ml vs 58 ml, effect size = -4.5%, 95% CI: -7.8% to -1.0%; p = 0.012). These associations remained significant at a false discovery rate of 10%. To further examine these associations produced by complete case analysis, multiple imputation of missing values was performed and the analysis repeated. The same CMR variables demonstrated significant associations with MHT use; these results are detailed in S1 Table.
|
study
| 100.0 |
Results of interaction analyses using age*MHT as an interaction term in our regression models are presented in Table 4 and Fig 2. Age significantly modified the association between MHT use and CMR parameters with smaller chambers observed with advancing tertiles of age: LV end-diastolic volume (47–60 years: β = 3.2%, 95% CI: -0.6% to 7.2%; 61–66 years: β = -2.9%, 95% CI: -5.8% to 0.2%; 67–77 years: β = -6.2%, 95% CI: -9.0% to -3.3%; p for interaction = 0.0005), LV end-systolic volume (47–60 years: β = 7.7%, 95% CI: 1.6% to 14.1%; 61–66 years: β = -3.1%, 95% CI: —7.6% to 1.6%; 67–77 years: β = -5.9%, 95% CI: -10.1% to -1.4%; p for interaction = 0.001), LV stroke volume (47–60 years: β = 0.3%, 95% CI: -3.8% to 4.5%; 61–66 years: β = -2.6%, 95% CI: -5.9% to 0.8%; 67–77 years: β = -6.4%, 95% CI: -9.4% to -3.2%; p for interaction = 0.033), and LA maximal volume (47–60 years: β = 0.1%, 95% CI: -6.6% to 7.3%; 61–66 years: β = -1.2%, 95% CI: -6.6% to 4.6%; 67–77 years: β = -10.1%, 95% CI: -15.0% to -5.0%; p for interaction = 0.006).
|
study
| 100.0 |
For every ten-year increment in age, there is a reduction in LV end-diastolic volume, LV end-systolic volume, LV stroke volume and LA maximal volume. The relationship between age and CMR outcomes is of greater magnitude amongst MHT users than that amongst non-users.
|
study
| 99.9 |
Effect sizes presented following adjustment for: duration of MHT use fitted as tertiles, age at menopause, ethnicity, height, weight, systolic blood pressure, diastolic blood pressure, smoking status, regular alcohol use, presence of raised cholesterol, presence of diabetes, Townsend score and income.
|
study
| 99.94 |
Results from sensitivity analyses examining the difference between CMR parameters in MHT users ≥ 3 years and propensity-matched controls are presented in Table 5. There were 429 matched participants in each group. As in multivariable regression models (Table 3), LV end-diastolic volume (MHT users = 117.5 ml vs never users = 121.7 ml, mean difference = -3.2%, 95% CI: -5.5% to -0.9%; p = 0.007), LV stroke volume (MHT users = 70.9 ml vs never users = 73.8 ml, mean difference = -3.7%, 95% CI: -6.1% to -1.3%; p = 0.003) and LA maximal volume (MHT users = 56.8 ml vs never users = 59.4 ml, mean difference = -4.9%, 95% CI: -8.9% to -0.8%; p = 0.019) were significantly lower in MHT users ≥3 years. S2 Table details the balance between the co-variates both before and after propensity-matching.
|
study
| 100.0 |
Propensity-matched controls are selected using the following variables: age, age at menopause, ethnicity, height, body mass index, systolic blood pressure, diastolic blood pressure, smoking status, regular alcohol use, presence of raised cholesterol and presence of diabetes, Townsend deprivation index and income.
|
study
| 98.9 |
In a population-based cohort of 1604 post-menopausal women free of known cardiovascular disease, the present study identified the following: firstly, LV end-diastolic volumes and LA maximal volumes were lower in women using MHT ≥ 3 years compared to those who had never used MHT after accounting for potential confounders. Secondly, MHT use significantly modified the relationship between advancing age and LV end-diastolic volume, LV end-systolic volume and LA maximal volume. Thirdly, timing of commencement of MHT in relation to the onset of menopause had no discernible impact of LV or LA volumes.
|
study
| 99.94 |
This study describes the relationship between MHT use and prognostically important cardiac phenotypes and indicates lower LV end-diastolic and LA maximal volumes in women who used MHT ≥ 3 years compared to those who never received treatment. Increases in LV volumes–LV dilatation–is associated with cardiac decompensation and poor prognosis in a range of cardiovascular diseases. Increases in end-diastolic volume and end-systolic volume have been demonstrated to be associated with an increased risk of heart failure in asymptomatic individuals whilst reduction in LV volumes–indicative of reverse cardiac remodelling–is associated with favourable outcomes . Equivalently, LA enlargement, as determined by LA maximal volume, is a robust and independent predictor of incident cardiovascular events whilst reduction in LA volumes (reverse remodelling) is associated with lower mortality and risk of heart failure . There was no significant difference in LV mass between MHT users ≥ 3 years and those who had never used MHT. LV mass is one of the most important cardiovascular imaging-derived phenotypes with increases in LV mass predicting a higher incidence of cardiovascular events and mortality .
|
study
| 99.94 |
This study demonstrates that use of MHT significantly impacts upon age-related reduction in LV and LA volumes. Recently published data detailing reference ranges for CMR imaging derived from a strictly healthy UK Biobank cohort has demonstrated that LV end-diastolic volume, end-systolic volume and LA maximal volume all decrease with advancing age , in keeping with findings from the never used MHT cohort. It is noteworthy, however, that use of MHT results in a much more marked rate of diminishment in LV and LA volumes, even after accounting for duration of MHT in addition to other confounders. Given that only 15% of women were using MHT at the time of CMR examination, it appears that this is an effect that persists, rather than being a contemporaneous result of being on treatment.
|
study
| 99.94 |
CMR is the most accurate and reproducible cardiac imaging modality and, when coupled with a large cohort size, permits detection of subclinical changes in cardiac structure and function. Whilst these subtle alterations may not result in any discernible impact to an individual at a single point in time, if they persist it is possible that they will eventually lead to prognostically relevant changes in cardiovascular outcomes. To our knowledge, this study is the first to explore the relationship between MHT use and subclinical changes in cardiac structure and function. Previous studies examining the impact of MHT on the cardiovascular system at the subclinical stage have focused on atherosclerotic burden as detected by cardiac computed tomography (CT). The most influential of these was a substudy of the Women’s Health Initiative which reported the coronary artery calcified-plaque burden was lower in women assigned to MHT than in those assigned to placebo . The more recent Early versus Late Intervention Trial with Estradiol (ELITE) study , designed specifically to investigate the “timing hypothesis” in relation to atherosclerosis progression in post-menopausal women, did not show any difference in plaque burden as assessed by cardiac CT in either early (<6 years) or late (≥10 years) menopause when compared to placebo although the authors did state that their sample size may be insufficient to detect any difference.
|
study
| 99.9 |
Despite landmark randomised trials and systematic reviews declaring that MHT does not provide any protective effects from cardiac events or mortality, this has been challenged by more recent studies examining both clinical end-points and surrogate markers of atherosclerosis progression. What is clear is that there remains significant confusion regarding MHT’s benefit, or lack thereof, in relation to cardiovascular health. Discouragingly, it has been noted that it is “unlikely” that additional large, prospective trials will be performed investigating MHT’s impact on cardiovascular disease due to existing controversy, fear of potential harm and the expense associated with longitudinal follow-up . We hope that our utilisation of biomarkers provided by CMR in the context of a large-scale, population-based study such as UK Biobank has provided a useful and novel method of examining MHT’s influence on the cardiovascular system.
|
review
| 99.9 |
This study used a large population-based cohort, with uniform assessment of exposures, covariates and outcomes. Importantly, there was no difference in socioeconomic status between the two groups; previous studies have highlighted that women prescribed MHT are of higher social class and educational attainment–and therefore, in general, healthier–than women who do not receive MHT, thereby confounding results . This is something that we have attempted to control for in this data. However, there are several limitations that should be considered in the interpretation of the findings. Firstly, the analysis was cross-sectional, thus causality cannot be inferred from the associations demonstrated. Secondly, it was not possible to explore longitudinal change in cardiac structure in relation to MHT use. Thirdly, all menopause and MHT data was self-reported. Finally, we were not able to provide data on type of MHT due to significant amount of missing data.
|
study
| 99.94 |
In a large, population-based cohort of post-menopausal women free of cardiovascular disease, use of MHT is not associated with adverse, subclinical changes in cardiac structure and function. Indeed, we demonstrate significantly smaller LV and LA chamber volumes which have been linked to favourable cardiovascular outcomes in other settings. Our findings provide a novel way to examine the impact of MHT on the cardiovascular system; future work will focus upon linkage of MHT use and CMR parameters to cardiovascular outcome data.
|
study
| 99.94 |
Data was partially missing for 275/1604 (17%) of participants. Between those with missing and no missing data, the groups were similar for all co-variates other than BMI (25.8 kg/m2 vs. 26.8 kg/m2, p = 0.003) and systolic blood pressure (133 mmHg vs. 137 mmHg, p = 0.009) which were higher in those with missing data. After multiple imputation of missing values, the effect sizes are similar to those from complete case analysis with the same CMR parameters realising significance. The complete case analysis detailed in the main manuscript provides more conservative results.
|
study
| 100.0 |
Mutations in the sodium phosphate co-transporters NPT2a [1–3] and NPT2c [4, 5] have been associated with intraluminal stones (nephrolithiasis) and mineral deposits in the renal parenchyma (nephrocalcinosis) in patients with familial forms of hypophosphatemia. In genome-wide association studies, NPT2a has also been associated with nephrolithiasis and altered renal function [7, 8]. With both genetic abnormalities affected individuals show renal phosphate-wasting, high circulating levels of 1,25(OH)2D, and absorptive hypercalciuria as a result of increased intestinal uptake of calcium [4, 5, 9, 10], and oral phosphate supplements are currently thought to reduce the risk for renal mineralization by lowering circulating levels of 1,25(OH)2D and absorptive hypercalciuria . However, the relative contribution of genotype, dietary calcium and phosphate, and modifiers of mineralization to the formation of renal mineral deposits is unclear. Our recent work suggests that reduced levels of osteopontin (Opn), an extracellular matrix factor affecting binding of phosphate to hydroxyapatite crystals, contribute to the development of nephrocalcinosis in Npt2a-/- mice . This may be due to the fact that Npt2a-/- mice respond differently to dietary phosphate when compared to WT mice . Further evaluation in the Npt2a-/- cohort on different diets suggests that urinary calcium excretion, plasma phosphate, and FGF23 levels appear to be positively correlated to renal mineral deposit formation, while urine phosphate levels and the urine anion gap, an indirect measure of ammonia excretion, appear to be inversely correlated . In addition, local tissue levels of Pi generated by tissue nonspecific alkaline phosphatase (Tnsalp) and ectonucleoside triphosphate diphosphohydrolase 5 (Entpd5) may be important as suggested by decreased skeletal mineralization in the absence of these enzymes [14, 15].
|
study
| 99.94 |
PPi is present in plasma at a concentration of 1–6 μM and in urine levels are around 10 μM . Calcium phosphate stone formers appear to have reduced urinary PPi excretion when compared with control subjects [20–23]. Intravenous 32PPi is rapidly hydrolyzed in plasma by tissue nonspecific alkaline phosphatase (Tnsalp) that is expressed in the proximal tubules of the kidneys and less than 5% of intravenous 32PPi appears in urine. These data indicate that urine PPi is generated locally in the kidneys [25, 26].
|
study
| 100.0 |
Extracellular nucleotide pyrophosphatase phosphodiesterase 1 (Enpp1) hydrolyzes extracellular ATP into AMP and PPi and may be an important source of extracellular PPi in the body [27, 28]. Enpp1 is the founding member of the ENPP or NPP family of enzymes . It has phosphodiesterase activity and is a type II extracellular membrane bound glycoprotein located on the mineral-depositing matrix vesicles of osteoblasts and chondrocytes and the vascular surface of cerebral capillaries . Enpp1 is also expressed in the kidney collecting duct and possibly other segments . The second source of PPi generation in the kidney is the mevalonate pathway inside mitochondria . Intracellular PPi is released into the interstitium and the urine by the transporter progressive ankylosis gene product (Ank) . Ank is located at the apical membrane of collecting ducts suggesting that it may function to inhibit mineralization within the tubule lumen. Additionally, ecto-5-prime nucleotidase (Nt5E/CD73), which inhibits Tnsalp by further hydrolyzing AMP to adenosine, and adenosine triphosphate-binding cassette , and subfamily C, member 6 (Abcc 6), recently shown to secrete ATP from hepatocytes , may both be involved in PPi generation.
|
study
| 99.94 |
In the present study, we used Npt2a-/- mice to model these disorders. Renal mineral deposits in Npt2a-/- mice are found at intraluminal and interstitial sites, they contain calcium, phosphorus and osteopontin, and it has been suggested that they ultrastructurally resemble the composition of Randall’s plaques [33, 34]. The extent of renal mineralization is highest between newborn and weaning age Npt2a-/- mice . Mineralization resolves subsequently on 0.3–0.6% dietary phosphate, but persists beyond weaning age when diets are supplemented with 1.65% phosphate or 1.2% phosphate [12, 36]. Ablation of 25(OH)-vitamin D-1-alpha hydroxylase (Cyp27a1) prevents renal mineralization, as shown in Cyp27a1-/-/Npt2a-/- double-knockout mice .
|
study
| 100.0 |
We here report that urine PPi levels are increased in Npt2a-/- mice when compared to WT mice, possibly to protect from renal mineralization in the setting of hyperphosphaturia. Presence of two hypomorphic Enpp1asj/asj alleles decreases urine PPi and worsens renal calcium phosphate deposit formation in Npt2a-/- mice. Conversely, development of mineral deposits in these mice can be reduced by intraperitoneal administration of sodium pyrophosphate. These studies suggest that PPi may be a thus far unrecognized factor modulating the development of renal calcifications in Npt2a-/- mice which may be, if confirmed in humans, of diagnostic and therapeutic relevance for phosphaturic disorders.
|
study
| 100.0 |
Male and female C57BL/6 mice were obtained from Charles River Laboratory, MA. Male and female Npt2a-/- mice (B6.129S2-Slc34a1tm1Hten/J, Stock No: 004802), and Enpp1asj/asj mice (C57BL/6J-Enpp1asj/GrsrJ, Stock No: 012810) were purchased from The Jackson Laboratory, ME. The Enpp1asj allele is partially active and shows approximately 15% level of Enpp1 activity compared to wild-type controls . Mice were genotyped by PCR amplification of genomic DNA extracted from tail clippings as described [29, 38–40]. Mice were weaned at 3 weeks of age and allowed free access to water and regular chow (1.0% calcium, 0.7% phosphorus, of which 0.3% phosphorus is readily available for absorption, Harlan Teklad TD.2018S). Mice received daily intraperitoneal (i.p.) injection of Hanks Buffered Saline (Gibco, Life Sciences) or sodium pyrophosphate in HBSS for two weeks until age four weeks as previously described (160 micromole/Kg/day) according to . To determine whether renal mineral deposits persist beyond weaning age mice were followed for an additional 10 weeks of age after weaning on regular chow. The background of all mouse lines is C57Bl6, use of littermates for controls further reduced bias based on genetic background. No difference in renal mineral deposits was observed between sexes as previously reported by us [12, 36] and thus genders were combined here.
|
study
| 100.0 |
Mice were euthanized following orbital exsanguination in deep anesthesia with isoflurane and vital organs were removed as described [12, 36]. The research under IACUC protocol 2014–11635 was first approved Oct. 22 2014 by the Yale Institutional Animal Care and Use Committee (IACUC), was renewed Sept. 7 2016, and is valid through Sept. 30 2017. Yale University has an approved Animal Welfare Assurance (#A3230-01) on file with the NIH Office of Laboratory Animal Welfare. The Assurance was approved May 5, 2015.
|
other
| 99.9 |
Biochemical analyses were done on blood samples (taken by orbital exsanguination) and spot urines collected following an overnight fast at the same time of day between 10 AM and 2 PM. Following deproteinization of heparinized plasma by filtration (NanoSep 300 K, Pall Corp., Ann Arbor, MI), plasma and urinary total pyrophosphate (PPi) concentrations were determined using a fluorometric probe (AB112155, ABCAM, Cambridge, MA). Urine PPi was corrected for urine creatinine, which was measured by LC-MS/MS or by ELISA using appropriate controls to adjust for inter-assay variability.
|
study
| 100.0 |
Left kidneys were fixed in 4% formalin/PBS at 4°C for 12 h and then dehydrated with increasing concentration of ethanol and xylene, followed by paraffin embedding. Mineral deposits were determined on 10 um von Kossa stained sections counterstained with 1% methyl green. Hematoxyline/eosin was used as counterstain for morphological evaluation. Histomorphometric evaluation of sagittal kidney sections that includes cortex, medulla and pelvis was performed blinded by two independent observers using an Osteomeasure System (Osteometrics, Atlanta, GA). Percent calcified area was determined using the formula: % calc. area = 100*calcified area/total area (including cortex, medulla and pelvic lumen), and is dependent on number of observed areas per section. Mineralization size was determined using the formula: calc. size = calcified area/number of observed calcified areas per section.
|
study
| 100.0 |
For transmission electron microscopy, a 1 mm3 block of the left kidney was fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in phosphate buffered saline for 2 hrs., followed by post-fixation in 1% osmium liquid for 2 hours. Dehydration was carried out using a series of ethanol concentrations (50% to 100%). Renal tissue was embedded in epoxy resin, and polymerization was carried out overnight at 60°C. After preparing a thin section (50 nm), the tissues were double stained with uranium and lead and observed using a Tecnai Biotwin (LaB6, 80 kV) (FEI, Thermo Fisher, Hillsboro, OR) at the Yale Center for Cellular and Molecular Imaging (YCCMI).
|
study
| 99.75 |
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