Down-regulation of human topoisomerase IIα expression correlates with relative amounts of specificity factors Sp1 and Sp3 bound at proximal and distal promoter regions

<p>Abstract</p> <p>Background</p> <p>Topoisomerase IIα has been shown to be down-regulated in doxorubicin-resistant cell lines. The specificity proteins Sp1 and Sp3 have been implicated in regulation of topoisomerase IIα transcription, although the mechanism by which they regulate expression is not fully understood. Sp1 has been shown to bind specifically to both proximal and distal GC elements of the human topoisomerase IIα promoter <it>in vitro</it>, while Sp3 binds only to the distal GC element unless additional flanking sequences are included. While Sp1 is thought to be an activator of human topoisomerase IIα, the functional significance of Sp3 binding is not known. Therefore, we sought to determine the functional relationship between Sp1 and Sp3 binding to the topoisomerase IIα promoter <it>in vivo</it>. We investigated endogenous levels of Sp1, Sp3 and topoisomerase IIα as well as binding of both Sp1 and Sp3 to the GC boxes of the topoisomerase IIα promoter in breast cancer cell lines <it>in vivo </it>after short term doxorubicin exposure.</p> <p>Results</p> <p>Functional effects of Sp1 and Sp3 were studied using transient cotransfection assays using a topoisomerase IIα promoter reporter construct. The <it>in vivo </it>interactions of Sp1 and Sp3 with the GC elements of the topoisomerase IIα promoter were studied in doxorubicin-treated breast cancer cell lines using chromatin immunoprecipitation assays. Relative amounts of endogenous proteins were measured using immunoblotting. <it>In vivo </it>DNA looping mediated by proteins bound at the GC1 and GC2 elements was studied using the chromatin conformation capture assay. Both Sp1 and Sp3 bound to the GC1 and GC2 regions. Sp1 and Sp3 were transcriptional activators and repressors respectively, with Sp3 repression being dominant over Sp1-mediated activation. The GC1 and GC2 elements are linked <it>in vivo </it>to form a loop, thus bringing distal regulatory elements and their cognate transcription factors into close proximity with the transcription start site.</p> <p>Conclusion</p> <p>These observations provide a mechanistic explanation for the modulation of topoisomerase IIα and concomitant down-regulation that can be mediated by topoisomerase II poisons. Competition between Sp1 and Sp3 for the same cognate DNA would result in activation or repression depending on absolute amounts of each transcription factor in cells treated with doxorubicin.</p

Paternal low protein diet programs preimplantation embryo gene expression, fetal growth and skeletal development in mice

Defining the mechanisms underlying the programming of early life growth is fundamental for improving adult health and wellbeing. While the association between maternal diet, offspring growth and adult disease risk is well-established, the effect of father's diet on offspring development are largely unknown. Therefore, we fed male mice an imbalanced low protein diet (LPD) to determine the impact on post-fertilisation development and fetal growth. We observed that in preimplantation embryos derived from LPD fed males, expression of multiple genes within the central metabolic AMPK pathway was reduced. In late gestation, paternal LPD programmed increased fetal weight, however, placental weight was reduced, resulting in an elevated fetal:placental weight ratio. Analysis of gene expression patterns revealed increased levels of transporters for calcium, amino acids and glucose within LPD placentas. Furthermore, placental expression of the epigenetic regulators Dnmt1 and Dnmt3L were increased also, coinciding with altered patterns of maternal and paternal imprinted genes. More strikingly, we observed fetal skeletal development was perturbed in response to paternal LPD. Here, while offspring of LPD fed males possessed larger skeletons, their bones comprised lower volumes of high mineral density in combination with reduced maturity of bone apatite. These data offer new insight in the underlying programming mechanisms linking poor paternal diet at the time of conception with the development and growth of his offspring

Single-nucleotide polymorphism-based genetic risk score and patient age at prostate cancer diagnosis

Importance: Few studies have evaluated the association between a single-nucleotide polymorphism-based genetic risk score (GRS) and patient age at prostate cancer (PCa) diagnosis. Objectives: To test the association between a GRS and patient age at PCa diagnosis and to compare the performance of a GRS with that of family history (FH) in PCa risk stratification. Design, Setting, and Participants: A cohort study of 3225 white men was conducted as a secondary analysis of the Reduction by Dutasteride of Prostate Cancer Events (REDUCE) chemoprevention trial, a 4-year, randomized, double-blind, placebo-controlled multicenter study conducted from March 2003 to April 2009 to evaluate the safety and efficacy of dutasteride in reducing PCa events. Participants were confirmed to be cancer free by prostate biopsy (6-12 cores) within 6 months prior to the study and underwent 10 core biopsies every 2 years per protocol. The dates for performing data analysis were from July 2016 to October 2019. Interventions: A well-established, population-standardized GRS was calculated for each participant based on 110 known PCa risk-associated single-nucleotide polymorphisms, which is a relative risk compared with the general population. Men were classified into 3 GRS risk groups based on predetermined cutoff values: low (\u3c0.50), average (0.50-1.49), and high (≥1.50). Main Outcomes and Measures: Prostate cancer diagnosis-free survival among men of different risk groups. Results: Among 3225 men (median age, 63 years [interquartile range, 58-67 years]) in the study, 683 (21%) were classified as low risk, 1937 (60%) as average risk, and 605 (19%) as high risk based on GRS alone. In comparison, 2789 (86%) were classified as low or average risk and 436 (14%) as high risk based on FH alone. Men in higher GRS risk groups had a PCa diagnosis-free survival rate that was worse than that of those in the lower GRS risk group (χ2 = 53.3; P \u3c .001 for trend) and in participants with a negative FH of PCa (χ2 = 45.5; P \u3c .001 for trend). Combining GRS and FH further stratified overall genetic risk, indicating that 957 men (30%) were at high genetic risk (either high GRS or positive FH), 1667 men (52%) were at average genetic risk (average GRS and negative FH), and 601 men (19%) were at low genetic risk (low GRS and negative FH). The median PCa diagnosis-free survival was 74 years (95% CI, 73-75 years) for men at high genetic risk, 77 years (95% CI, 75 to \u3e80 years) for men at average genetic risk, and more than 80 years (95% CI, \u3e80 to \u3e80 years) for men at low genetic risk. In contrast, the median PCa diagnosis-free survival was 73 years (95% CI, 71-76 years) for men with a positive FH and 77 years (95% CI, 76-79 years) for men with a negative FH. Conclusions and Relevance: This study suggests that a GRS is significantly associated with patient age at PCa diagnosis. Combining FH and GRS may better stratify inherited risk than FH alone for developing personalized PCa screening strategies

Opinion: more mouse models and more translation needed for ALS

Amyotrophic lateral sclerosis is a complex disorder most of which is 'sporadic' of unknown origin but approximately 10% is familial, arising from single mutations in any of more than 30 genes. Thus, there are more than 30 familial ALS subtypes, with different, often unknown, molecular pathologies leading to a complex constellation of clinical phenotypes. We have mouse models for many genetic forms of the disorder, but these do not, on their own, necessarily show us the key pathological pathways at work in human patients. To date, we have no models for the 90% of ALS that is 'sporadic'. Potential therapies have been developed mainly using a limited set of mouse models, and through lack of alternatives, in the past these have been tested on patients regardless of aetiology. Cancer researchers have undertaken therapy development with similar challenges; they have responded by producing complex mouse models that have transformed understanding of pathological processes, and they have implemented patient stratification in multi-centre trials, leading to the effective translation of basic research findings to the clinic. ALS researchers have successfully adopted this combined approach, and now to increase our understanding of key disease pathologies, and our rate of progress for moving from mouse models to mechanism to ALS therapies we need more, innovative, complex mouse models to address specific questions

Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis.

Most BRCA1-associated breast tumours are basal-like yet originate from luminal progenitors. BRCA1 is best known for its functions in double-strand break repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions fully explains the cell lineage-specific tumorigenesis. In vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Here we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 5' end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1-knockout mouse mammary epithelium. Our studies show that Pol II pausing is an important contributor to BRCA1-associated R-loop accumulation and breast cancer development

Nanophase-photocatalysis: loading, storing, and release of H2O2 using graphitic carbon nitride

A blue light mediated photochemical process using solid graphitic carbon nitride (g-C3N4) in ambient air/isopropanol vapour is suggested to be linked to "nanophase" water inclusions and is shown to produce approx. 50 μmol H2O2 per gram of g-C3N4, which can be stored in the solid g-C3N4 for later release for applications, for example, in disinfection or anti-bacterial surfaces

Carbon Nitride as a Ligand: Selective Hydrogenation of Terminal Alkenes using [(η5-C5Me5)IrCl(g-C3N4-κ2N, N')]Cl : Selective Hydrogenation of Terminal Alkenes using [(η5-C5Me5)IrCl(g-C3N4-κ2N, N')]Cl

Anchoring a homogeneous catalyst onto a heterogeneous support facilitates separation of the product from the catalyst, and catalyst-substrate interactions can also modify reactivity. Herein we describe the synthesis of composite materials comprising carbon nitride (g-C 3 N 4 ) as the heterogeneous support and the well established homogeneous catalyst moiety [Cp*IrCl] + (where Cp* = η 5 -C 5 Me 5 ), commonly used for catalytic hydrogenation. Coordination of [Cp*IrCl] + to g-C 3 N 4 occurs directly at exposed edge sites with a κ 2 N, N' binding motif, leading to a primary inner coordination sphere analogous to known homogeneous complexes of the general class [Cp*IrCl(NN-κ 2 N, N' )] + (where N, N' = a bidentate nitrogen ligand). Hydrogenation of unsaturated substrates using the composite catalyst is selective for terminal alkenes, which is attributed to the restricted steric environment of the outer coordination sphere at the edge-sites of g-C 3 N 4

Highly selective and solvent-dependent reduction of Nitrobenzene to N-phenylhydroxylamine, azoxybenzene, and aniline catalyzed by phosphino-modified polymer immobilized ionic liquid-stabilized AuNPs

Gold nanoparticles stabilized by phosphine-decorated polymer immobilized ionic liquids (AuNP@PPh2-PIILP) is an extremely efficient multiproduct selective catalyst for the sodium borohydride-mediated reduction of nitrobenzene giving N-phenylhydroxylamine, azoxybenzene, or aniline as the sole product under mild conditions and a very low catalyst loading. The use of a single nanoparticle-based catalyst for the partial and complete reduction of nitroarenes to afford three different products with exceptionally high selectivities is unprecedented. Under optimum conditions, thermodynamically unfavorable N-phenylhydroxylamine can be obtained as the sole product in near quantitative yield in water, whereas a change in reaction solvent to ethanol results in a dramatic switch in selectivity to afford azoxybenzene. The key to obtaining such a high selectivity for N-phenylhydroxylamine is the use of a nitrogen atmosphere at room temperature as reactions conducted under an inert atmosphere occur via the direct pathway and are essentially irreversible, while reactions in air afford significant amounts of azoxy-based products by virtue of competing condensation due to reversible formation of N-phenylhydroxylamine. Ultimately, aniline can also be obtained quantitatively and selectively by adjusting the reaction temperature and time accordingly. Introduction of PEG onto the polyionic liquid resulted in a dramatic improvement in catalyst efficiency such that N-phenylhydroxylamine could be obtained with a turnover number (TON) of 100 000 (turnover frequency (TOF) of 73 000 h–1, with >99% selectivity), azoxybenzene with a TON of 55 000 (TOF of 37 000 h–1 with 100% selectivity), and aniline with a TON of 500 000 (TOF of 62 500 h–1, with 100% selectivity). As the combination of ionic liquid and phosphine is required to achieve high activity and selectivity, further studies are currently underway to explore whether interfacial electronic effects influence adsorption and thereby selectivity and whether channeling of the substrate by the electrostatic potential around the AuNPs is responsible for the high activity. This is the first report of a AuNP-based system that can selectively reduce nitroarenes to either of two synthetically important intermediates as well as aniline and, in this regard, is an exciting discovery that will form the basis to develop a continuous flow process enabling facile scale-up

Guiding Ethical Principles in Engineering Biology Research

Engineering biology is being applied toward solving or mitigating some of the greatest challenges facing society. As with many other rapidly advancing technologies, the development of these powerful tools must be considered in the context of ethical uses for personal, societal, and/or environmental advancement. Researchers have a responsibility to consider the diverse outcomes that may result from the knowledge and innovation they contribute to the field. Together, we developed a Statement of Ethics in Engineering Biology Research to guide researchers as they incorporate the consideration of long-term ethical implications of their work into every phase of the research lifecycle. Herein, we present and contextualize this Statement of Ethics and its six guiding principles. Our goal is to facilitate ongoing reflection and collaboration among technical researchers, social scientists, policy makers, and other stakeholders to support best outcomes in engineering biology innovation and development