Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1

Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.     

Breast Tissue Composition and Immunophenotype and Its Relationship with Mammographic Density in Women at High Risk of Breast Cancer

Aim

To investigate the cellular and immunophenotypic basis of mammographic density in women at high risk of breast cancer.

Methods

Mammograms and targeted breast biopsies were accrued from 24 women at high risk of breast cancer. Mammographic density was classified into Wolfe categories and ranked by increasing density. The histological composition and immunophenotypic profile were quantified from digitized haematoxylin and eosin-stained and immunohistochemically-stained (ERα, ERβ, PgR, HER2, Ki-67, and CD31) slides and correlated to mammographic density.

Results

Increasing mammographic density was significantly correlated with increased fibrous stroma proportion (rs (22) = 0.5226, p = 0.0088) and significantly inversely associated with adipose tissue proportion (rs (22) = -0.5409, p = 0.0064). Contrary to previous reports, stromal expression of ERα was common (19/20 cases, 95%). There was significantly higher stromal PgR expression in mammographically-dense breasts (p=0.026).

Conclusions

The proportion of stroma and fat underlies mammographic density in women at high risk of breast cancer. Increased expression of PgR in the stroma of mammographically dense breasts and frequent and unexpected presence of stromal ERα expression raises the possibility that hormone receptor expression in breast stroma may have a role in mediating the effects of exogenous hormonal therapy on mammographic density.     

Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major

Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2’-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and k_cat values for dThd of 1.1 μM and 2.62 s^-1 and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5’-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP₅dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs.     

Selective Allosteric Inhibition of MMP9 Is Efficacious in Preclinical Models of Ulcerative Colitis and Colorectal Cancer

Expression of matrix metalloproteinase 9 (MMP9) is elevated in a variety of inflammatory and oncology indications, including ulcerative colitis and colorectal cancer. MMP9 is a downstream effector and an upstream mediator of pathways involved in growth and inflammation, and has long been viewed as a promising therapeutic target. However, previous efforts to target matrix metalloproteinases (MMPs), including MMP9, have utilized broad-spectrum or semi-selective inhibitors. While some of these drugs showed signs of efficacy in patients, all MMP-targeted inhibitors have been hampered by dose-limiting toxicity or insufficient clinical benefit, likely due to their lack of specificity. Here, we show that selective inhibition of MMP9 did not induce musculoskeletal syndrome (a characteristic toxicity of pan-MMP inhibitors) in a rat model, but did reduce disease severity in a dextran sodium sulfate-induced mouse model of ulcerative colitis. We also found that MMP9 inhibition decreased tumor growth and metastases incidence in a surgical orthotopic xenograft model of colorectal carcinoma, and that inhibition of either tumor- or stroma-derived MMP9 was sufficient to reduce primary tumor growth. Collectively, these data suggest that selective MMP9 inhibition is a promising therapeutic strategy for treatment of inflammatory and oncology indications in which MMP9 is upregulated and is associated with disease pathology, such as ulcerative colitis and colorectal cancer. In addition, we report the development of a potent and highly selective allosteric MMP9 inhibitor, the humanized monoclonal antibody GS-5745, which can be used to evaluate the therapeutic potential of MMP9 inhibition in patients.     

The Impact of Different Levels of Adaptive Iterative Dose Reduction 3D on Image Quality of 320-Row Coronary CT Angiography: A Clinical Trial

Purpose

The aim of this study was the systematic image quality evaluation of coronary CT angiography (CTA), reconstructed with the 3 different levels of adaptive iterative dose reduction (AIDR 3D) and compared to filtered back projection (FBP) with quantum denoising software (QDS).

Methods

Standard-dose CTA raw data of 30 patients with mean radiation dose of 3.2 ± 2.6 mSv were reconstructed using AIDR 3D mild, standard, strong and compared to FBP/QDS. Objective image quality comparison (signal, noise, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), contour sharpness) was performed using 21 measurement points per patient, including measurements in each coronary artery from proximal to distal.

Results

Objective image quality parameters improved with increasing levels of AIDR 3D. Noise was lowest in AIDR 3D strong (p≤0.001 at 20/21 measurement points; compared with FBP/QDS). Signal and contour sharpness analysis showed no significant difference between the reconstruction algorithms for most measurement points. Best coronary SNR and CNR were achieved with AIDR 3D strong. No loss of SNR or CNR in distal segments was seen with AIDR 3D as compared to FBP.

Conclusions

On standard-dose coronary CTA images, AIDR 3D strong showed higher objective image quality than FBP/QDS without reducing contour sharpness.

Trial Registration

Clinicaltrials.gov NCT00967876     

DNA methylation age of blood predicts all-cause mortality in later life

BackgroundDNA methylation levels change with age. Recent studies have identified biomarkers of chronological age based on DNA methylation levels. It is not yet known whether DNA methylation age captures aspects of biological age.ResultsHere we test whether differences between people’s chronological ages and estimated ages, DNA methylation age, predict all-cause mortality in later life. The difference between DNA methylation age and chronological age (Δ_age) was calculated in four longitudinal cohorts of older people. Meta-analysis of proportional hazards models from the four cohorts was used to determine the association between Δ_age and mortality. A 5-year higher Δ_age is associated with a 21% higher mortality risk, adjusting for age and sex. After further adjustments for childhood IQ, education, social class, hypertension, diabetes, cardiovascular disease, and APOE e4 status, there is a 16% increased mortality risk for those with a 5-year higher Δ_age. A pedigree-based heritability analysis of Δ_age was conducted in a separate cohort. The heritability of Δ_age was 0.43.ConclusionsDNA methylation-derived measures of accelerated aging are heritable traits that predict mortality independently of health status, lifestyle factors, and known genetic factors.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0584-6) contains supplementary material, which is available to authorized users.     

A comparison of the predictive accuracy of three screening models for pulmonary arterial hypertension in systemic sclerosis

IntroductionThere is evidence that early screening for pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc) improves outcomes. We compared the predictive accuracy of two recently published screening algorithms (DETECT 2013 and Australian Scleroderma Interest Group (ASIG) 2012) for SSc-associated PAH (SSc-PAH) with the commonly used European Society of Cardiology/European Respiratory Society (ESC/ERS 2009) guidelines.MethodsWe included 73 consecutive SSc patients with suspected PAH undergoing right heart catheterization (RHC). The three screening models were applied to each patient. For each model, contingency table analysis was used to determine sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values for PAH. These properties were also evaluated in an ‘alternate scenario analysis’ in which the prevalence of PAH was set at 10%.ResultsRHC revealed PAH in 27 (36.9%) patients. DETECT and ASIG algorithms performed equally in predicting PAH with sensitivity and NPV of 100%. The ESC/ERS guidelines had sensitivity of 96.3% and NPV of only 91%, missing one case of PAH; these guidelines could not be applied to three patients who had absent tricuspid regurgitant (TR) jet. The ASIG algorithm had the highest specificity (54.5%). With PAH prevalence set at 10%, the NPV of the models was unchanged, but the PPV dropped to less than 20%.ConclusionsIn this cohort, the DETECT and ASIG algorithms out-perform the ESC/ERS guidelines, detecting all patients with PAH. The ESC/ERS guidelines have limitations in the absence of a TR jet. Ultimately, the choice of SSc-PAH screening algorithm will also depend on cost and ease of application.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0517-5) contains supplementary material, which is available to authorized users.