The Structural Basis for the Phosphorylation-Induced Activation of Smad Proteins: a Dissertation

The Smad proteins transduce the signal of transforming growth factor-β (TGF-β) and related factors from the cell surface to the nucleus. Following C-terminal phosphorylation by a corresponding receptor kinase, the R-Smad proteins form heteromeric complexes with Smad4. These complexes translocate into the nucleus, bind specific transcriptional activators and DNA, ultimately modulating gene expression. Though studied through a variety of means, the stoichiometry of the R-Smad/Smad4 complex is unclear. We investigated the stoichiometry of the phosphorylation-induced R-Smad/Smad4 complex by using acidic amino acid substitutions to simulate phosphorylation. Size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry analysis revealed that the R-Smad/Smad4 complex is a heterotrimer consisting of two R-Smad subunits and one Smad4 subunit. In addition, a specific mechanism for phosphorylation-induced R-Smad/Smad4 complex formation was studied. Although it had been previously established that part of the mechanism through which phosphorylation induces Smad oligomerization is through relieving MH1-domain mediated autoinhibition of the MH2 (oligomerization) domain, it is also evident that phosphorylation serves to energetically drive Smad complex formation. Through mutational and size exclusion chromatography analysis, we established that phosphorylation induces oligomerization of the Smads by creating an electrostatic interaction between the phosphorylated C-terminal tail of one R-Smad subunit in a Smad trimer with a basic surface on an adjacent R-Smad or Smad4 subunit. The basic surface is defined largely by the L3 loop, a region that had previously been implicated in R-Smad interaction with the receptor kinase. Furthermore, the Smad MH2 domain shares a similar protein fold with the phosphoserine and phosphothreonine-binding FHA domains from proteins like Rad53 and Chk2. Taken together, these results suggest that the Smad MH2 domain may be a distinct phospho serine-binding domain, which utilizes a common basic surface to bind the receptor kinase and other Smads, and takes advantage of phosphorylation-induced allosteric changes dissociate from the receptor kinase and oligomerize with other Smads. Finally, the structural basis for the preferential formation of the R-Smad/Smad4 heterotrimeric complex over the R-Smad homotrimeric complex was explored through X-ray crystallography and isothermal titration calorimetry. Crystal structures of the Smad2/Smad4 and Smad3/Smad4 complexes revealed that specific residue differences in Smad4 compared to R-Smads resulted in highly favorable electrostatic interactions that explain the preference for the interaction with Smad4

Using the VALGENT-3 framework to assess the clinical and analytical performance of the RIATOL qPCR HPV genotyping assay

Background and objective: The VALGENT framework is developed to assess the clinical performance of HPV tests that offer genotyping capability. Samples from the VALGENT-3 panel are used to identify an optimal viral concentration threshold for the RIATOL qPCR HPV genotyping assay (RIATOL qPCR) to assure non-inferior accuracy to detect high-grade cervical intraepithelial neoplasia (CIN), compared to Qiagen Hybrid Capture 2 (HC2), a standard comparator test validated for cervical cancer screening. Study design: The VALGENT-3 panel comprised 1300 samples from women participating in the Slovenian cervical cancer screening programme, enriched with 300 samples from women with abnormal cytology. In follow-up, 126 women were diagnosed with CIN2+ (defined as diseased) and 1167 women had two consecutive negative Pap smears (defined as non-diseased). All 1600 samples were analyzed with the RIATOL qPCR. Viral concentration was expressed as viral log10 of the number of copies/ml. A zone of viral concentration cut-offs was defined by relative ROC analysis where the sensitivity and specificity were not inferior to HC2. Results: The RIATOL qPCR had a sensitivity and specificity for CIN2+ of 97.6% (CI: 93.2-99.5%) and 85.1% (CI: 82.9-87.1%), respectively, when the analytical cut off was used. At a cut off of 6.5, RIATOL qPCR had a sensitivity of 96.0% (CI: 91.0-98.7%) and a specificity of 89.5% (87.6-91.2%). At optimized cut off, accuracy of the qPCR was non-inferior to the HC2 with a relative sensitivity of 1.00 [CI: 0.95-1.05 (p= 0.006)] and relative specificity of 1.00 [CI: 0.98-1.01 (p= 0.0069)]. Conclusions: The RIATOL qPCR has a high sensitivity and specificity for the detection of CIN2+. By using a fixed cut-off based on viral concentration, the test is non-inferior to HC2. HPV tests that provide viral concentration measurements or other quantifiable signals allow flexibility to optimize accuracy required for cervical cancer screening

Phosphorus and Nitrogen Transport in the Binational Great Lakes Basin Estimated Using SPARROW Watershed Models

AbstractEutrophication problems in the Great Lakes are caused by excessive nutrient inputs (primarily phosphorus, P, and nitrogen, N) from various sources throughout its basin. In developing protection and restoration plans, it is important to know where and from what sources the nutrients originate. As part of a binational effort, Midcontinent SPARROW (SPAtially Referenced Regression On Watershed attributes) models were developed and used to estimate P and N loading from throughout the entire basin based on nutrient inputs similar to 2002; previous SPARROW models only estimated U.S. contributions. The new models have a higher resolution (~2‐km2 catchments) enabling improved descriptions of where nutrients originate and the sources at various spatial scales. The models were developed using harmonized geospatial datasets describing the stream network, nutrient sources, and environmental characteristics affecting P and N delivery. The models were calibrated using loads from sites estimated with ratio estimator and regression techniques and additional statistical approaches to reduce spatial correlation in the residuals and have all monitoring sites equally influence model development. SPARROW results, along with interlake transfers and direct atmospheric inputs, were used to quantify the entire P and N input to each lake and describe the importance of each nutrient source. Model results can be used to compare loading and yields from various tributaries and jurisdictions

Elevated levels of the angiogenic cytokines basic fibroblast growth factor and vascular endothelial growth factor in sera of cancer patients.

The concentration of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) was determined in the serum of 90 untreated and 42 treated metastatic cancer patients, including patients with colorectal, breast, ovarian and renal carcinomas, with an enzyme-linked immunosorbent assay (ELISA). Levels higher than the 95th percentile of the concentrations of a control group, i.e. 7.5 pg ml(-1) for bFGF and 500 pg ml(-1) for VEGF, were identified as 'elevated'. One measurement during follow-up was included into the analysis per patient. For 19 treated patients, consecutive serum samples were analysed. Fifty-seven per cent of all untreated patients had elevated serum levels of one or both angiogenic factors. The fraction of patients with elevated serum levels of bFGF and/or VEGF was similar in the different tumour types. Agreement of bFGF levels and VEGF levels, classified in relation to their respective cut-off values, was present in 67% of all patients. Fifty-eight per cent of the patients with progressive disease during treatment compared with 15% of the patients showing response to treatment (chi-squared test P < 0.05) had elevated bFGF and/or VEGF serum levels. When consecutive serum samples were analysed, two-thirds of the patients showing progressive disease had increasing serum levels of the angiogenic factors compared with less than one-tenth of the patients showing response (chi-squared test P < 0.05). The lack of association between the serum bFGF and VEGF levels and the tumour type may suggest an aspecific host reaction responsible for solid tumour-related angiogenesis. The main determinants of the serum bFGF and VEGF concentration are the progression kinetics of the metastatic carcinomas

Impact of the admitting ward on care quality and outcomes in non-ST-segment elevation myocardial infarction (NSTEMI) : insights from a national registry

Peer reviewedPostprin

Steam consumption minimization using genetic algorithm optimization method: an industrial case study

yesCondensate stabilization is a process where hydrocarbon condensate recovered from natural gas reservoirs is processed to meet the required storage, transportation, and export specifications. The process involves stabilizing of hydrocarbon liquid by separation of light hydrocarbon such as methane from the heavier hydrocarbon constituents such as propane. An industrial scale back-up condensate stabilization unit was simulated using Aspen HYSYS software and validated with the plant data. The separation process consumes significant amount of energy in form of steam. The objectives of the paper are to find the minimum steam consumption of the process and conduct sensitivity and exergy analyses on the process. The minimum steam consumption was found using genetic algorithm optimization method for both winter and summer conditions. The optimization was carried out using MATLAB software coupled with Aspen HYSYS software. The optimization involves six design variables and four constraints, such that realistic results are achieved. The results of the optimization show that savings in steam consumption is 34% as compared to the baseline process while maintaining the desired specifications. The effect of natural gas feed temperature has been investigated. The results show that steam consumption is reduced by 46% when the natural gas feed temperature changes from 17.7 to 32.7°C. Exergy analysis shows that exergy destruction of the optimized process is 37% less than the baseline process

Detection of mammaglobin mRNA in peripheral blood is associated with high grade breast cancer: Interim results of a prospective cohort study

<p>Abstract</p> <p>Background</p> <p>We sought to examine the detection rate of cancer cells in peripheral blood (PBL) and in bone marrow (BM) using an established 7-gene marker panel and evaluated whether there were any definable associations of any individual gene with traditional predictors of prognosis.</p> <p>Methods</p> <p>Patients with T1-T3 primary breast cancer were enrolled into a prospective, multi-institutional cohort study. In this interim analysis 215 PBL and 177 BM samples were analyzed by multimarker, real-time RT-PCR analysis designed to detect circulating and disseminated breast cancer cells.</p> <p>Results</p> <p>At a threshold of three standard deviations from the mean expression level of normal controls, 63% (136/215) of PBL and 11% (19/177) of BM samples were positive for at least one cancer-associated marker. Marker positivity in PBL demonstrated a statistically significant association with grade II-III (vs. grade I; p = 0.0083). Overexpression of the mammaglobin (<it>mam</it>) gene alone had a statistically significant association with high tumor grade (p = 0.0315), and showed a trend towards ER-negative tumors and a high risk category. There was no association between marker positivity in PBL and the pathologic (H&E) and/or molecular (RT-PCR) status of the axillary lymph nodes (ALN).</p> <p>Conclusion</p> <p>This study suggests that molecular detection of circulating cancer cells in PBL detected by RT-PCR is associated with high tumor grade and specifically that overexpression of the <it>mam </it>gene in PBL may be a poor prognostic indicator. There was no statistically significant association between overexpression of cancer-associated genes in PBL and ALN status, supporting the concept of two potentially separate metastatic pathways.</p