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.     

Histology Verification Demonstrates That Biospectroscopy Analysis of Cervical Cytology Identifies Underlying Disease More Accurately than Conventional Screening: Removing the Confounder of Discordance

Background

Subjective visual assessment of cervical cytology is flawed, and this can manifest itself by inter- and intra-observer variability resulting ultimately in the degree of discordance in the grading categorisation of samples in screening vs. representative histology. Biospectroscopy methods have been suggested as sensor-based tools that can deliver objective assessments of cytology. However, studies to date have been apparently flawed by a corresponding lack of diagnostic efficiency when samples have previously been classed using cytology screening. This raises the question as to whether categorisation of cervical cytology based on imperfect conventional screening reduces the diagnostic accuracy of biospectroscopy approaches; are these latter methods more accurate and diagnose underlying disease? The purpose of this study was to compare the objective accuracy of infrared (IR) spectroscopy of cervical cytology samples using conventional cytology vs. histology-based categorisation.

Methods

Within a typical clinical setting, a total of n = 322 liquid-based cytology samples were collected immediately before biopsy. Of these, it was possible to acquire subsequent histology for n = 154. Cytology samples were categorised according to conventional screening methods and subsequently interrogated employing attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. IR spectra were pre-processed and analysed using linear discriminant analysis. Dunn’s test was applied to identify the differences in spectra. Within the diagnostic categories, histology allowed us to determine the comparative efficiency of conventional screening vs. biospectroscopy to correctly identify either true atypia or underlying disease.

Results

Conventional cytology-based screening results in poor sensitivity and specificity. IR spectra derived from cervical cytology do not appear to discriminate in a diagnostic fashion when categories were based on conventional screening. Scores plots of IR spectra exhibit marked crossover of spectral points between different cytological categories. Although, significant differences between spectral bands in different categories are noted, crossover samples point to the potential for poor specificity and hampers the development of biospectroscopy as a diagnostic tool. However, when histology-based categories are used to conduct analyses, the scores plot of IR spectra exhibit markedly better segregation.

Conclusions

Histology demonstrates that ATR-FTIR spectroscopy of liquid-based cytology identifies the presence of underlying atypia or disease missed in conventional cytology screening. This study points to an urgent need for a future biospectroscopy study where categories are based on such histology. It will allow for the validation of this approach as a screening tool.     

Controlled adhesion and growth of long term glial and neuronal cultures on Parylene-C

This paper explores the long term development of networks of glia and neurons on patterns of Parylene-C on a SiO(2) substrate. We harvested glia and neurons from the Sprague-Dawley (P1-P7) rat hippocampus and utilized an established cell patterning technique in order to investigate cellular migration, over the course of 3 weeks. This work demonstrates that uncontrolled glial mitosis gradually disrupts cellular patterns that are established early during culture. This effect is not attributed to a loss of protein from the Parylene-C surface, as nitrogen levels on the substrate remain stable over 3 weeks. The inclusion of the anti-mitotic cytarabine (Ara-C) in the culture medium moderates glial division and thus, adequately preserves initial glial and neuronal conformity to underlying patterns. Neuronal apoptosis, often associated with the use of Ara-C, is mitigated by the addition of brain derived neurotrophic factor (BDNF). We believe that with the right combination of glial inhibitors and neuronal promoters, the Parylene-C based cell patterning method can generate structured, active neural networks that can be sustained and investigated over extended periods of time. To our knowledge this is the first report on the concurrent application of Ara-C and BDNF on patterned cell cultures.     

The E-domain region of mechano-growth factor inhibits cellular apoptosis and preserves cardiac function during myocardial infarction

Insulin-like growth factor-1 (IGF-1) isoforms are expressed via alternative splicing. Expression of the minor isoform IGF-1Eb [also known as mechano-growth factor (MGF)] is responsive to cell stress. Since IGF-1 isoforms differ in their E-domain regions, we are interested in determining the biological function of the MGF E-domain. To do so, a synthetic peptide analog was used to gain mechanistic insight into the actions of the E-domain. Treatment of H9c2 cells indicated a rapid cellular uptake mechanism that did not involve IGF-1 receptor activation but resulted in a nuclear localization. Peptide treatment inhibited the intrinsic apoptotic pathway in H9c2 cells subjected to cell stress with sorbitol by preventing the collapse of the mitochondrial membrane potential and inhibition of caspase-3 activation. Therefore, we administered the peptide at the time of myocardial infarction (MI) in mice. At 2 weeks post-MI cardiac function, gene expression and cell death were assayed. A significant decline in both systolic and diastolic function was evident in untreated mice based on PV loop analysis. Delivery of the E-peptide ameliorated the decline in function and resulted in significant preservation of cardiac contractility. Associated with these changes were an inhibition of pathologic hypertrophy and significantly fewer apoptotic nuclei in the viable myocardium of E-peptide-treated mice post-MI. We conclude that administration of the MGF E-domain peptide may provide a means of modulating local tissue IGF-1 autocrine/paracrine actions to preserve cardiac function, prevent cell death, and pathologic remodeling in the heart.     

Structural and Functional Characterization of the Recombinant Death Domain from Death-Associated Protein Kinase

Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2) and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K _d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent ‘D-motif’ located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics assignment of the three-dimensional structure.     

A Re-Analysis of the Cochrane Library Data: The Dangers of Unobserved Heterogeneity in Meta-Analyses

Background

Heterogeneity has a key role in meta-analysis methods and can greatly affect conclusions. However, true levels of heterogeneity are unknown and often researchers assume homogeneity. We aim to: a) investigate the prevalence of unobserved heterogeneity and the validity of the assumption of homogeneity; b) assess the performance of various meta-analysis methods; c) apply the findings to published meta-analyses.

Methods and Findings

We accessed 57,397 meta-analyses, available in the Cochrane Library in August 2012. Using simulated data we assessed the performance of various meta-analysis methods in different scenarios. The prevalence of a zero heterogeneity estimate in the simulated scenarios was compared with that in the Cochrane data, to estimate the degree of unobserved heterogeneity in the latter. We re-analysed all meta-analyses using all methods and assessed the sensitivity of the statistical conclusions. Levels of unobserved heterogeneity in the Cochrane data appeared to be high, especially for small meta-analyses. A bootstrapped version of the DerSimonian-Laird approach performed best in both detecting heterogeneity and in returning more accurate overall effect estimates. Re-analysing all meta-analyses with this new method we found that in cases where heterogeneity had originally been detected but ignored, 17–20% of the statistical conclusions changed. Rates were much lower where the original analysis did not detect heterogeneity or took it into account, between 1% and 3%.

Conclusions

When evidence for heterogeneity is lacking, standard practice is to assume homogeneity and apply a simpler fixed-effect meta-analysis. We find that assuming homogeneity often results in a misleading analysis, since heterogeneity is very likely present but undetected. Our new method represents a small improvement but the problem largely remains, especially for very small meta-analyses. One solution is to test the sensitivity of the meta-analysis conclusions to assumed moderate and large degrees of heterogeneity. Equally, whenever heterogeneity is detected, it should not be ignored.     

Increased Force Variability in Chronic Stroke: Contributions of Force Modulation below 1 Hz

Increased force variability constitutes a hallmark of arm disabilities following stroke. Force variability is related to the modulation of force below 1 Hz in healthy young and older adults. However, whether the increased force variability observed post stroke is related to the modulation of force below 1 Hz remains unknown. Thus, the purpose of this study was to compare force modulation below 1 Hz in chronic stroke and age-matched healthy individuals. Both stroke and control individuals (N = 26) performed an isometric grip task to submaximal force levels. Coefficient of variation quantified force variability, and power spectrum density of force quantified force modulation below 1 Hz with a high resolution (0.07 Hz). Analyses indicated that force variability was greater for the stroke group compared with to healthy controls and for the paretic hand compared with the non-paretic hand. Force modulation below 1 Hz differentiated the stroke individuals and healthy controls, as well as the paretic and non-paretic hands. Specifically, stroke individuals (paretic hand) exhibited greater power ∼0.2 Hz (0.07–0.35 Hz) and lesser power ∼0.6 Hz (0.49–0.77 Hz) compared to healthy controls (non-dominant hand). Similarly, the paretic hand exhibited greater power ∼0.2 Hz, and lesser power ∼0.6 Hz than the non-paretic hand. Moreover, variability of force was strongly predicted from the modulation of specific frequencies below 1 Hz (R ² = 0.80). Together, these findings indicate that the modulation of force below 1 Hz provides significant insight into changes in motor control after stroke.     

Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum

The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45°C but the enzyme became unstable at temperatures above 40°C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co(2+). The apparent K(m) for Co(2+) was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn(2+), Mg(2+), Ni(2+) and Ca(2+) but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 μmol mg(protein)(-1)min(-1); k(cat), 44.2 min(-1); K(m)(G1P), 0.10mM; K(m)(G1,6 diP), 1.03 μM; k(cat)/K(m)(G1P), 443 mM(-1)min(-1) and k(cat)/K(m)(G1,6 diP), 42,860 mM(-1)min(-1). The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.     

Fis1 and Bap31 bridge the mitochondria-ER interface to establish a platform for apoptosis induction

The mitochondria and the endoplasmic reticulum (ER) are two organelles that critically contribute to apoptosis induction. While it is established that they communicate, how cell death signals are transmitted from the mitochondria to the ER is unknown. Here, we show that the mitochondrial fission protein Fission 1 homologue (Fis1) conveys an apoptosis signal from the mitochondria to the ER by interacting with Bap31 at the ER and facilitating its cleavage into the pro-apoptotic p20Bap31. Exogenous apoptosis inducers likewise use this signalling route and induce the procession of Bap31. Moreover, we show that the recruitment of procaspase-8 to the Fis1-Bap31 platform is an early event during apoptosis induction. The association of procaspase-8 with the Fis1-Bap31 complex is dependent on the variant of death effector domain (vDED) in Bap31 and is required for the activation of procaspase-8. This signalling pathway establishes a feedback loop by releasing Ca(2+) from the ER that activates the mitochondria for apoptosis. Hence, the Fis1-Bap31 complex (ARCosome) that spans the mitochondria-ER interface serves as a platform to activate the initiator procaspase-8, and thereby bridges two critical organelles for apoptosis signalling.