Adherence and cytokine induction in Caco-2 cells by bacterial populations from a three-stage continuous-culture model of the large intestine

Adherence of bacteria to epithelial cells is an important step in colonization and immune modulation in the large bowel. The aims of this study were to use a three-stage continuous-culture system (CCS) to investigate how environmental factors affect bacterial attachment to Caco-2 cells and modulation of cytokine expression by gut microorganisms, including a probiotic Bifidobacterium longum strain, DD2004. The CCS simulated environmental conditions in the proximal large intestine (vessel 1 [V1]) and distal colon (V2 and V3) at two different system retention times (R) within the range of normal colonic transits (20 and 60 h). The model was inoculated with human fecal material, and fluorescence in situ hybridization (FISH) was used to characterize microbial populations and to assess bacterial attachment to Caco-2 cells. Real-time quantitative PCR (qPCR) was employed to measure cytokine gene expression following challenge with bacteria from different components of the CCS in the presence and absence of B. longum. At an R of 60 h, bacterial adherence increased from V1 to V3, but this trend was reversed at an R of 20 h. Atopobia were the predominant adherent organisms detected at both system retention times in each culture vessel. Modulation of transforming growth factor β1 (TGF-β1), interleukin 6 (IL-6), and IL-18 gene expression by CCS bacteria was marked at an R of 60 h, while at an R of 20 h, IL-4, IL-10, TGF-β2, IL-1α, and tumor necrosis factor alpha (TNF-α) were significantly affected. The addition of B. longum affected cytokine expression significantly at both retention times. This study demonstrates that environmental determinants regulate the adherence properties of intestinal bacteria and their abilities to regulate cytokine synthesis.     

A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO(.-) and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO(.-)), it forms methyl radicals ((.-)CH(3)) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO(.-) formation. However, we recently reported that (.-)CH(3) might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated alpha-[4-pyridyl 1]-N-tert-butyl nitrone, d(0)/d(9)-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO(.-) and DMSO. In addition to identifying the two well-known free radicals, (.-)CH(3) and (.-)OCH(3), from this interaction, we also characterized two additional free radicals, (.-)CH(2)OH and (.-)CH(2)S(O)CH(3). Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/(.-)CH(3) was not detected. However, with the addition of the LC/MS technique, we found approximately 0.75 microM POBN/(.-)CH(3) hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.     

Application of the BD ACTOne technology for the high-throughput screening of Gs-coupled receptor antagonists

A novel technology for monitoring the changes of 3,'5'-adenosine cyclic monophosphate (cAMP) in live cells suitable for drug screening relies on the use of cyclic nucleotide-gated channels as biosensors coexpressed with the appropriate target receptor. The technique (termed BD ACTOne) offers measurement of cAMP-dependent calcium influx or membrane depolarization with conventional fluorescent methods both in kinetic and in endpoint modes, optimal for high-throughput and subsequent compound screening. The utility of the technique is reported here based on assay development and high-throughput screening for small-molecule antagonists of the peptide parathyroid hormone 2 receptor (PTH2R). The dual-signaling properties of the receptor were retained in the recombinant system, and the observed pharmacological profile corresponded to data from radiolabeled cAMP determination. The membrane-potential-based high-throughput assay produced reproducible actives and led to the identification of several chemical scaffolds with potential utility as PTH2R ligands.     

Scavenging of reactive oxygen species by some nonsteroidal anti-inflammatory drugs and fenofibrate

Ketoprofen and tolmetin are widely used nonsteroidal anti-inflammatory drugs, whereas fenofibrate belongs to a family of hypolipidemic drugs used in the prevention of cardiovascular diseases. The aim of this study was to assess effect of these drugs on reactions generating reactive oxygen species (ROS). The following generators of ROS were used: 18-crown-6/KO(2) dissolved in DMSO as a source of superoxide radical (O(.-)(2), the Fenton-like reaction (Cu/H(2)O(2)) for hydroxyl radical (HO(.)), 2,2'-azobis (2-amidino-propane) dichloride (AAPH) as peroxyl radical (ROO(.)) generator, and a mixture of alkaline aqueous H(2)O(2) and acetonitrile for singlet oxygen ((1)O(2)). Measurements were done using chemiluminescence, fluorescence, and spin-trapping with 2,2,6,6-tetramethylpiperidine combined with electron spin resonance spectroscopy (ESR), and a deoxyribose assay based on the spectrophotometry. The results obtained demonstrated that all tested drugs were active against O(.-)(2). There was a clear ranking of drug inhibition effects on chemiluminescence from the O(.-)(2) system: ketoprofen > tolmetin > fenofibrate. The examined compounds inhibited the HO(.)-dependent deoxyribose degradation and scavenged the ROO(.) concentration dependently with an order of potencies similar to that of the superoxide radical system. Hence, these results indicate that the studied drugs show broad ROS scavenging property and, as a consequence, might decrease tissue damage due to the ROS and thus to contribute to anti-inflammatory therapy.     

Different value of coronary calcium score to predict obstructive coronary artery disease in patients with and without moderate chronic kidney disease

Purpose

The coronary calcium score (CCS) predicts significant coronary artery disease (CAD) in the general population. While moderate chronic kidney disease (CKD) is associated with high CCS, the use of CCS to predict significant CAD in these patients is unknown.

Methods

A total of 704 patients underwent computed tomography coronary angiography for the assessment of CCS and CAD. Sixty-nine (10 %) patients had moderate CKD, defined by an estimated glomerular filtration rate (eGFR) between 30 and 59 mL/min/1.73m², and the remaining patients were considered to be without significant CKD (eGFR?≥?60 mL/min/1.73m²).

Results

Patients with moderate CKD were older, had a higher CCS, and a higher prevalence of obstructive CAD than patients without significant CKD. Receiver-operator curve analysis showed that CCS predicted the presence of obstructive CAD in both patients with moderate CKD and those without significant CKD. In patients with moderate CKD, the optimal cut-off value of CCS to diagnose obstructive CAD was 140 (sensitivity 73 % and specificity of 70 %), and is 2.8 fold higher than in patients without significant CKD (cut-off value?=?50; sensitivity 75 % and specificity 75 %).

Conclusion

The present results demonstrate that CCS can predict obstructive CAD in patients with moderate CKD, although the optimal cut-off value is higher than in patients without significant CKD.     

The effect of intermittent hypobaric hypoxic exposure and sea level training on submaximal economy in well-trained swimmers and runners.

To evaluate the effect of intermittent hypobaric hypoxia combined with sea level training on exercise economy, 23 well-trained athletes (13 swimmers, 10 runners) were assigned to either hypobaric hypoxia (simulated altitude of 4,000-5,500 m) or normobaric normoxia (0-500 m) in a randomized, double-blind design. Both groups rested in a hypobaric chamber 3 h/day, 5 days/wk for 4 wk. Submaximal economy was measured twice before (Pre) and after (Post) the treatment period using sport-specific protocols. Economy was estimated both from the relationship between oxygen uptake (V(.-)o2) and speed, and from the absolute V(.-)o2 at each speed using sport-specific protocols. V(.-)o2 was measured during the last 60 s of each (3-4 min) stage using Douglas bags. Ventilation (V(.-)E), heart rate (HR), and capillary lactate concentration ([La(-)]) were measured during each stage. Velocity at maximal V(.-)o2 (velocity at V(.-)o2max) was used as a functional indicator of changes in economy. The average V(.-)o2 for a given speed of the Pre values was used for Post test comparison using a two-way, repeated-measures ANOVA. Typical error of measurement of V(.-)o2 was 4.7% (95% confidence limits 3.6-7.1), 3.6% (2.8-5.4), and 4.2% (3.2-6.9) for speeds 1, 2, and 3, respectively. There was no change in economy within or between groups (ANOVA interaction P = 0.28, P = 0.23, and P = 0.93 for speeds 1, 2, and 3). No differences in submaximal HR, [La-], Ve, or velocity at V(.-)o2(max) were found between groups. It is concluded that 4 wk of intermittent hypobaric hypoxia did not improve submaximal economy in this group of well-trained athletes.     

Prevention of damage in acute spinal cord injury by peptides and pharmacologic agents

Cats were used as models of traumatic spinal cord injury. Each experimental animal received a 500 g-cm force to the exposed dura at the level of thoracic fourth vertebra. Somatosensory evoked potentials (SEPs), carotid arterial blood pressure (BP), and abdominal aorta blood flow in the treated groups were compared with those of the control group. The three treated groups received naloxone (5 mg/kg), TRH (5 mg/kg), and a combination of methyl-prednisolone sodium succinate (MP, 35 mg/kg) and epsilon-aminocaproic acid (EACA, 350 mg/kg). The SEPs which were done only in the naloxone treated group approached "normalcy" 24-26 hours after trauma as compared with the absence of SEPs in traumatized untreated group. In all three groups, the treatment increased the blood flow in abdominal aorta significantly. Morphine sulfate increased substance P (SP) immunoreactivity in the dorsal and ventral gray matter. Naloxone not only reversed this effect, it depleted SP below the saline control level. In order to establish that lipid free radicals are responsible for damage to biological membranes, their effects were also investigated in vitro: 14C-GABA uptake by mouse cortical slices which had decreased by 33% in the presence of superoxide (. O-2) generating system, horseradish peroxidase (HRP), was reduced only by 9% when superoxide dismutase was added to the medium. The latter also protected the nerve endings from damage by (. O-2) as examined by electron microscopy. It is concluded that the agents used in this study produce their ameliorating effects by virtue of their anti-inflammatory, anti-oxidant, and membrane stabilizing properties in addition to their effect on enhancing the regional microcirculation. The release of SP by naloxone may be responsible for the increase in blood flow. The consequences of traumatic injury as depicted in Fig. 1 are discussed at length.     

Properties of the radical intermediate obtained on oxidation of 2',7'-dichlorodihydrofluorescein, a probe for oxidative stress

Reduced "leuco" dyes such as dichlorodihydrofluorescein (DCFH(2)) are widely used as profluorescent probes for oxidative stress, although they require a catalyst to be oxidized by hydrogen peroxide and react indiscriminately with oxidizing radicals and the fluorescent product (DCF) is a potential photosensitizer of superoxide generation. In this study, key properties of the radical intermediate in oxidation ("semiquinone," DCFH(.-)/DCF(.)(-)) were measured, to help understand the reactions that can occur in biological systems. The intermediate was generated by oxidizing DCFH(2) or reducing DCF by radiolytically generated radicals and monitoring the reactions using kinetic spectrophotometry. The semiquinone showed pH-sensitive absorption spectral changes, decay kinetics (both in the absence and in the presence of oxygen), and reduction potential, all corresponding to prototropic dissociations with pK(a)'s of approximately 7.1 and 9.0. DCFH(2) has pK(a)'s in a similar region (8-9) and hence pH variations are potentially important in the use of this probe. The rate constant for reaction of the semiquinone with oxygen at pH 7.4 is 5.3 x 10(8) M(-1) s(-1): this reaction, rather than disproportionation of DCFH(.-)/DCF(.)(-), generates DCF in biological systems, concomitantly forming superoxide and hence H(2)O(2) to cycle the catalyst. The midpoint reduction potential of the couple DCF,H(+)/DCFH() is approximately -0.75 V vs. NHE at pH 7.4; DCF is unlikely to be reduced rapidly by common flavoprotein reductases.     

Are Immigrants and Nationals Born to Immigrants at Higher Risk for Delayed or No Lifetime Breast and Cervical Cancer Screening? The Results from a Population-Based Survey in Paris Metropolitan Area in 2010

Objectives

This study aims to compare breast cancer screening (BCS) and cervical cancer screening (CCS) practices of French women born to French parents with those of immigrants and nationals born to immigrants, taking their socioeconomic status into account.

Methods

The study is based on data collected in 2010 in the Paris metropolitan area among a representative sample of 3000 French-speaking adults. For women with no history of breast or cervical cancer, multivariate logistic regressions and structural equation models were used to investigate the factors associated with never having undergone BCS or CCS.

Results

We confirmed the existence of a strong gradient, with respect to migration origin, for delaying or never having undergone BCS or CCS. Thus, being a foreign immigrant or being French of immigrant parentage were risk factors for delayed and no lifetime screening. Interestingly, we found that this gradient persisted (at least partially) after adjusting for the women’s socioeconomic characteristics. Only the level of income seemed to play a mediating role, but only partially. We observed differences between BCS and CCS which suggest that organized CCS could be effective in reducing socioeconomic and/or ethnic inequities.

Conclusion

Socioeconomic status partially explained the screening nonparticipation on the part of French women of immigrant origin and foreign immigrants. This was more so the case with CCS than with BCS, which suggests that organized prevention programs might reduce social inequalities.     

Capillary array reversed-phase liquid chromatography-based multidimensional separation system coupled with MALDI-TOF-TOF-MS detection for high-throughput proteome analysis

A high-throughput on-line capillary array-based two-dimensional liquid chromatography (2D-LC) system coupled with MALDI-TOF-TOF-MS proteomics analyzer for comprehensive proteomic analyses has been developed, in which one capillary strong-cation exchange (SCX) chromatographic column was used as the first separation dimension and 18 parallel capillary reversed-phase liquid chromatographic (RPLC) columns were integrated as the second separation dimension. Peptides bound to the SCX phase were "stepped" off using multiple salt pulses followed by sequentially loading of each subset of peptides onto the corresponding precolumns. After salt fractionation, by directing identically split solvent-gradient flows into 18 channels, peptide fractions were concurrently back-flushed from the precolumns and separated simultaneously with 18 capillary RP columns. LC effluents were directly deposited onto the MALDI target plates through an array of capillary tips at a 15-s interval, and then alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution was added to each sample spot for subsequent MALDI experiments. This new system allows an 18-fold increase in throughput compared with serial-based 2D-LC system. The high efficiency of the overall system was demonstrated by the analysis of a tryptic digest of proteins extracted from normal human liver tissue. A total of 462 proteins was identified, which proved the system's promising potential for high-throughput analysis and application in proteomics.     

Measuring genetic stability in bacteria of potential use in genetic engineering

Four commonly used conjugation techniques, colony cross streak (CCS), broth mating (BM), combined spread plate (CSP), and membrane filtration (MF), were compared with each other regarding reliability, sensitivity, and complexity in evaluating the transfer of conjugative plasmids. Five plasmids representing several incompatibility groups plus a variety of laboratory and environmental isolates were used as mating pairs. The suitability of each method was evaluated for use in a routine assessment of the genetic stability of genetically engineered microorganisms. By the CSP and MF techniques with laboratory strains such as Escherichia coli and Pseudomonas species as recipients, transconjugants were usually produced in 100% of the mating trials. However, when environmental strains isolated from plants and soil were used as recipients, transconjugants were detected in 100% of some crosses and in as little as 30% in other crosses depending on the plasmid and recipient used. In general, differences in the percentage of successful matings between the CSP and MF techniques compared with the BM and CCS techniques were not statistically significant at the P less than or equal to 0.05 level. Occasionally, certain mating pairs consistently produced transconjugants by CCS or BM but not by CSP or MF. Since any single conjugation mating technique is not completely reliable in detecting transconjugants, we have developed a combined mating technique which integrates the CCS, CSP, BM, and MF methods as a single procedure to assess the mobility of plasmid DNA of genetically engineered microorganisms.     

Measuring genetic stability in bacteria of potential use in genetic engineering.

Four commonly used conjugation techniques, colony cross streak (CCS), broth mating (BM), combined spread plate (CSP), and membrane filtration (MF), were compared with each other regarding reliability, sensitivity, and complexity in evaluating the transfer of conjugative plasmids. Five plasmids representing several incompatibility groups plus a variety of laboratory and environmental isolates were used as mating pairs. The suitability of each method was evaluated for use in a routine assessment of the genetic stability of genetically engineered microorganisms. By the CSP and MF techniques with laboratory strains such as Escherichia coli and Pseudomonas species as recipients, transconjugants were usually produced in 100% of the mating trials. However, when environmental strains isolated from plants and soil were used as recipients, transconjugants were detected in 100% of some crosses and in as little as 30% in other crosses depending on the plasmid and recipient used. In general, differences in the percentage of successful matings between the CSP and MF techniques compared with the BM and CCS techniques were not statistically significant at the P less than or equal to 0.05 level. Occasionally, certain mating pairs consistently produced transconjugants by CCS or BM but not by CSP or MF. Since any single conjugation mating technique is not completely reliable in detecting transconjugants, we have developed a combined mating technique which integrates the CCS, CSP, BM, and MF methods as a single procedure to assess the mobility of plasmid DNA of genetically engineered microorganisms.     

Development of a new EPR spin trap, DOD-8C (N-[4-dodecyloxy-2-(7'-carboxyhept-1'-yloxy)benzylidene]-N-tert-butylamine N-oxide), for the trapping of lipid radicals at a predetermined depth within biological membranes

We report on the development of the first member of a new family of EPR spin-trapping agents designed to trap radicals at a predetermined depth within biological membranes. By analogy to the use of nitroxide spin labels to 'report' on the environment at specific depths within biological membranes, we set out to prepare similar reporter molecules, but with a nitrone in place of the nitroxide function. The prototype compounds were tested in a model system consisting of large unilamellar vesicles exposed to a copper-dependent radical generating system. This entailed the reduction of tert-butylhydroperoxide to the tert-butoxyl radical ((t)BuO(.-)) by a membrane-permeable Cu(I) complex, which was generated in situ by reduction of the Cu(II) complex by ascorbate. To assist in the identification of the radicals detected, preliminary studies were performed in methanolic solution, where the major radical trapped was shown to be (.-)CH(2)OH, resulting from H-atom abstraction from the alcohol by (t)BuO(.-). This conclusion was shown to be in agreement with predictions based on chemical kinetics, which were then used to support the proposal that the primary species trapped in the lipid vesicles were radicals derived from membrane fatty acids. This molecule represents the first of a new generation of spin traps which, through modification, can be used to position the radical-trapping nitrone moiety at chosen depths within biological membranes.     

NESTED AUTOMATED RIBOSOMAL INTERGENIC SPACER ANALYSIS: A RAPID AND ACCURATE METHOD FOR COMPARISON OF BACTERIAL COMMUNITY COMPOSITION

Nested automated ribosomal intergenic spacer analysis (ARISA) was used to examine the community structure of epilithic biofilms in freshwater streams experiencing different levels of human impact. This molecular fingerprinting technique generated reproducible profiles of bacterial community structure that varied significantly between stream sites. Nested ARISA was determined to be a cost-effective, high-throughput approach to assess bacterial community composition from very small sample volumes, requiring little sampling effort and without the need for taxonomic identification of individual organisms. In combination with multidimensional scaling, nested ARISA provides a rapid and sensitive method to carry out complex analyses of bacterial community structure.

PRACTICAL APPLICATIONS

Nested automated ribosomal intergenic spacer analysis (ARISA) provides a high-throughput molecular method with which to screen large numbers of environmental samples for differences in microbial community structure. This sensitive approach benefits assessments from small sample volumes or environments exhibiting reduced microbial biomass (both aquatic and terrestrial). Differences in bacterial community structure (obtained from ARISA profiles) could be used to characterize the impact of anthropogenic disturbance on freshwater systems, analogous to the current use of macroinvertebrate indicators of freshwater ecological health.     

Weighting of environmental trade-offs in CCS—an LCA case study of electricity from a fossil gas power plant with post-combustion CO2 capture, transport and storage

Purpose

Carbon capture and storage (CCS) is increasingly acknowledged as a potent global warming abatement option. It is demonstrated that whilst the global warming potential (GWP) decreases, the other environmental impact category potentials often increase in a life cycle perspective. Despite this, only a few studies clearly address this trade-off or use weighting to compare the positive and negative effects of CCS. The present life cycle assessment (LCA) study focuses, therefore, on presenting several environmental indicators and on weighting the inventory results in order to ascertain which of the analysed systems is to be preferred.

Method

The case studied is a projected gas power plant at Tjeldbergodden (Norway), where it is proposed to include post-combustion CCS. Four main scenarios have been analysed, one without and three with CCS. The principal variation between the CCS scenarios is that the steam required for amine regeneration is produced in three different ways: in a separate gas fuelled steam boiler; in a separate biomass fuelled steam boiler; and delivered from the low-pressure steam turbine in the power plant. Design information and technical specifications have been available. The study has used LCA methodology based on the ISO standard 14044, SimaPro 7.3.2.4 software and the Ecoinvent 2.0 database. The functional unit is 1?TWh electricity delivered to the grid. The following environmental impact categories have been included: GWP, acidification potential, eutrophication potential, photochemical ozone creation potential (POCP) and cumulative energy demand (CED). Three weighting methods have been used to ascertain the robustness of the weighting results: ReCiPe, EPS 2000 and IMPACT 2002+.

Results and discussion

The characterisation results show that the CCS scenarios have reduced impacts only in the case of GWP. The weighting demonstrates that in the ReCiPe model, climate change is strongly in focus, whilst in EPS 2000, human health and depletion of reserves are dominant. Climate change is also an important factor in IMPACT 2002+, together with effects on human health (respiratory inorganics). The process integration scenario has, however, the best result for all three weighting models. This contrasts with the results from the impact analysis where four of the five analysed impact categories rated the CCS-3 scenario as worse than the reference scenario. One possible option for improving the biofuel boiler scenario is to capture the CO₂ from the combustion of biomass in the external steam boiler. This would not, in all probability, affect the acidification, eutrophication, POCP and CED to any significant degree, but the GWP, and hence the ReCiPe and the IMPACT 2002+, weighting results could be expected to improve.

Conclusions

The weighting exercise has identified toxicity as a concern with regard to the biofuel boiler scenarios (CCS-2) and human health issues as having importance for the CCS-3 scenario. It would seem that process integration is a better CCS option than that of CCS providing steam from a separate steam boiler (without CCS), even where this boiler is biomass-fuelled. Any future analysis should focus both on the process integration scenario and the biofuel boiler scenario with CCS of biological CO₂.     

The neuronal adaptor protein X11alpha interacts with the copper chaperone for SOD1 and regulates SOD1 activity

The neuronal adaptor protein X11alpha participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11alpha, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11alpha/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathione S-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11alpha and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11alpha inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11alpha binding to CCS is inhibitory to SOD1 activation. X11alpha also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein. Thus, X11alpha may participate in copper homeostasis within neurons.     

When Do We Really Need Coronary Calcium Scoring Prior to Contrast-Enhanced Coronary Computed Tomography Angiography? Analysis by Age,Gender and Coronary Risk Factors

Aims

To investigate the value of coronary calcium scoring (CCS) as a filter scan prior to coronary computed tomography angiography (CCTA).

Methods and Results

Between February 2008 and April 2011, 732 consecutive patients underwent clinically indicated CCTA. During this ‘control phase’, CCS was performed in all patients. In patients with CCS≥800, CCTA was not performed. During a subsequent ‘CCTA phase’ (May 2011–May 2012) another 200 consecutive patients underwent CCTA, and CCS was performed only in patients with increased probability for severe calcification according to age, gender and atherogenic risk factors. In patients where CCS was not performed, calcium scoring was performed in contrast-enhanced CCTA images. Significant associations were noted between CCS and age (r = 0.30, p<0.001) and coronary risk factors (χ² = 37.9; HR = 2.2; 95%CI = 1.7–2.9, p<0.001). Based on these associations, a ≤3% pre-test probability for CCS≥800 was observed for males <61 yrs. and females <79 yrs. According to these criteria, CCS was not performed in 106 of 200 (53%) patients during the ‘CCTA phase’, including 47 (42%) males and 59 (67%) females. This resulted in absolute radiation saving of ∼1 mSv in 75% of patients younger than 60 yrs. Of 106 patients where CCS was not performed, estimated calcium scoring was indeed <800 in 101 (95%) cases. Non-diagnostic image quality due to calcification was similar between the ‘control phase’ and the ‘CCTA’ group (0.25% versus 0.40%, p = NS).

Conclusion

The value of CCS as a filter for identification of a high calcium score is limited in younger patients with intermediate risk profile. Omitting CCS in such patients can contribute to further dose reduction with cardiac CT studies.     

Thermofluor-based high-throughput stability optimization of proteins for structural studies

Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins' probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as a high-throughput approach for identifying optimal protein formulation for crystallization. The method also allowed optimal stabilizing buffer compositions to be rapidly identified for each protein. Furthermore, the method allowed the identification of potential ligands, physiological or non-physiological, that can be used in subsequent crystallization trials. For this study, the thermally induced melting points were determined in different buffers as well as with additives for a total of 25 Escherichia coli proteins. Crystallization trials were set up together with stabilizing and destabilizing additives identified using thermofluor screening. A twofold increase in the number of crystallization leads was observed when the proteins were cocrystallized with stabilizing additives as compared with experiments without these additives. This suggests that thermofluor constitutes an efficient generic high-throughput method for identification of protein properties predictive of crystallizability.