Quantitative Assessment of Myocardial Perfusion in the Detection of Significant Coronary Artery Disease : Cutoff Values and Diagnostic Accuracy of Quantitative [O]H2O PET Imaging

Background

Recent studies have demonstrated improved diagnostic accuracy for detecting coronary artery disease (CAD) when myocardial blood flow (MBF) is quantified in absolute terms, but there are no uniformly accepted cutoff values for hemodynamically significant CAD.

Objectives

The goal of this study was to determine cutoff values for absolute MBF and to evaluate the diagnostic accuracy of quantitative [¹⁵O]H₂O positron emission tomography (PET).

Methods

A total of 330 patients underwent both quantitative [¹⁵O]H₂O PET imaging and invasive coronary angiography in conjunction with fractional flow reserve measurements. A stenosis >90% and/or fractional flow reserve ≤0.80 was considered obstructive; a stenosis <30% and/or fractional flow reserve >0.80 was nonobstructive.

Results

Hemodynamically significant CAD was diagnosed in 116 (41%) of 281 patients who fulfilled study criteria for CAD. Resting perfusion was 1.00 ± 0.25 and 0.92 ± 0.23 ml/min/g in regions supplied by nonstenotic and significantly stenosed vessels, respectively (p < 0.001). During stress, perfusion increased to 3.26 ± 1.04 ml/min/g and 1.73 ± 0.67 ml/min/g, respectively (p < 0.001). The optimal cutoff values were 2.3 and 2.5 for hyperemic MBF and myocardial flow reserve, respectively. For MBF, these cutoff values showed a sensitivity, specificity, and accuracy for detecting significant CAD of 89%, 84%, and 86%, respectively, at a per-patient level and 87%, 85%, and 85% at a per-vessel level. The corresponding myocardial flow reserve values were 86%, 72%, and 78% (per patient) and 80%, 82%, and 81% (per vessel). Age and sex significantly affected diagnostic accuracy of quantitative PET.

Conclusions

Quantitative MBF measurements with the use of [¹⁵O]H₂O PET provided high diagnostic performance, but both sex and age should be taken into account.     

Molecular profiling of Mycobacterium tuberculosis identifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette–Guérin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosinyladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.With a mortality rate exceeding 1.5 million deaths annually, Mycobacterium tuberculosis remains one of the world''s most important pathogens (1). M. tuberculosis succeeds as a pathogen because of productive infection of the endosomal network of phagocytes. Its residence within the phagosome protects it from immune responses during its decades long infection cycle. However, intracellular survival depends on active inhibition of pH-dependent killing mechanisms, which occurs for M. tuberculosis but not species with low disease-causing potential (2). Intracellular survival is also enhanced by an unusually hydrophobic and multilayered protective cell envelope. Despite study of this pathogen for more than a century, the spectrum of natural lipids within M. tuberculosis membranes is not yet fully defined. For example, the products of many genes annotated as lipid synthases remain unknown (3), and mass spectrometry detects hundreds of ions that do not correspond to known lipids in the MycoMass and LipidDB databases (4, 5).To broadly compare the lipid profiles of virulent and avirulent mycobacteria, we took advantage of a recently validated metabolomics platform (4). This high performance liquid chromatography–mass spectrometry (HPLC-MS) system uses methods of extraction, chromatography, and databases that are specialized for mycobacteria. After extraction of total bacterial lipids into organic solvents, HPLC-MS enables massively parallel detection of thousands of ions corresponding to diverse lipids that range from apolar polyketides to polar phosphoglycolipids. Software-based (XCMS) ion finding algorithms report reproducibly detected ions as molecular features. Each feature is a 3D data point with linked mass, retention time, and intensity values from one detected molecule or isotope. All features with equivalent mass and retention time from two bacterial lipid extracts are aligned, allowing pairwise comparisons of MS signal intensity to enumerate molecules that are overproduced in one strain with a false-positive rate below 1% (4).This comparative lipidomics system allowed an unbiased, organism-wide analysis of lipids from M. tuberculosis and the attenuated vaccine strain, Mycobacterium bovis Bacillus Calmette–Guérin (BCG). BCG was chosen because of its worldwide use as a vaccine and its genetic similarity to M. tuberculosis (6). We reasoned that any features that are specifically detected in M. tuberculosis might be clinically useful as markers to distinguish tuberculosis-causing bacteria from vaccines. Furthermore, given the differing potential for productive infection by the two strains, any M. tuberculosis-specific compounds would be candidate virulence factors. Comparative genomics of M. tuberculosis and BCG successfully identified “regions of deletion” (RD) that encode genes that were subsequently proven to promote productive M. tuberculosis infection (7), including the 6-kDa early secreted antigenic target (ESAT-6) secretion system-1 (ESX-1) (8, 9). We reasoned that a metabolite-based screen might identify new virulence factors because not all functions of RD genes are known. Also, biologically important metabolites could emerge from complex biosynthetic pathways that cannot be predicted from single-gene analysis.Comparison of M. tuberculosis and BCG lipid profiles revealed more than 1,000 differences, among which we identified a previously unknown M. tuberculosis-specific diterpene-linked adenosine and showed that it is produced by the enzyme Rv3378c. Previously, Rv3378c was thought to generate free tuberculosinol and isotuberculosinol (10–12). This discovery revises the enzymatic function of Rv3378c, which acts as a virulence factor to inhibit phagolysosome fusion (13). Whereas current models of prenyl transferase function emphasize iterative lengthening of prenyl pyrophosphates using one binding pocket, the crystal structure of Rv3378c identifies two pockets in the catalytic site, establishing a mechanism for heterologous prenyl transfer to nonprenyl metabolites.     

Dissociation of the trimeric gp41 ectodomain at the lipid–water interface suggests an active role in HIV-1 Env-mediated membrane fusion

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. The actual fusion process involves a switch from a homotrimeric prehairpin intermediate conformation, consisting of parallel coiled-coil helices, to a postfusion state where the ectodomains are arranged as a trimer of helical hairpins, adopting a six-helix bundle (6HB) state. Here, we show by solution NMR spectroscopy that a water-soluble 6HB gp41 ectodomain binds to zwitterionic detergents that contain phosphocholine or phosphatidylcholine head groups and phospholipid vesicles that mimic T-cell membrane composition. Binding results in the dissociation of the 6HB and the formation of a monomeric state, where its two α-helices, N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), become embedded in the lipid–water interface of the virus and host cell. The atomic structure of the gp41 ectodomain monomer, based on NOE distance restraints and residual dipolar couplings, shows that the NHR and CHR helices remain mostly intact, but they completely lose interhelical contacts. The high affinity of the ectodomain helices for phospholipid surfaces suggests that unzippering of the prehairpin intermediate leads to a state where the NHR and CHR helices become embedded in the host cell and viral membranes, respectively, thereby providing a physical force for bringing these membranes into close juxtaposition before actual fusion.The first step of HIV infection involves fusion of the viral and target cell membranes, a process mediated by the viral envelope glycoprotein Env, consisting of subunits gp120 and gp41 (1). The envelope proteins form a noncovalent complex on the viral surface with the trimerized gp41 transmembrane subunit sequestered by three gp120 surface subunits (2–5). Binding of gp120 to the cell surface receptors CD4 and chemokine receptors CXCR4 or CCR5 triggers a cascade of conformational changes that disrupt the interactions between gp41 and gp120 and result in an extended gp41 conformation (1, 6). In this extended prefusion state, the highly hydrophobic N-terminal fusion peptide (FP) of gp41 anchors in the host cell membrane, while being spatially remote from its transmembrane domain (TM), which traverses the viral membrane (7, 8). After the host cell and viral membranes have fused, the gp41 ectodomain, which links the FP and TM domains, has transitioned into a C3-symmetric six-helix bundle (6HB), with the FP in physical proximity to the TM domain (9). The refolding of gp41 trimers into the highly stable 6HB arrangement is believed to overcome the large free-energy barrier of membrane fusion. Several atomic resolution structures of the 6HB postfusion state have been solved by X-ray crystallography, confirming that the C-terminal heptad repeat (CHR) helices pack in an antiparallel manner into the conserved hydrophobic grooves formed at the surface of the central trimer of N-terminal heptad repeat (NHR) helices (10–12).Contrary to the postfusion state, structural features of the prehairpin intermediates of HIV-1 gp41 remain the subject of much debate. The functional requirement that gp41’s fusion peptide engages the membrane of spatially distant host cells dictates an extended conformation for the time point where FP engages the membrane of the host cell. Cartoon models commonly depict this prehairpin intermediate as an extended trimer of linear NHR and CHR helices (13–17). Recent cryo-EM studies provide more detailed insights into the relatively subtle rearrangement of the trimeric helical NHR core, which is associated with rearrangements of gp120 relative to gp41 on receptor activation of Env, that leads to the release of FP from its hydrophobic burial site at the gp41–gp120 interface (5, 18, 19). Subsequent dissociation of the gp120 subunits leaves the gp41 core in a state somewhat similar to the common cartoon models, lacking the trimer-stabilizing interactions supplied by gp120.Although it seems clear that, initially, gp41 directly engages the viral and host cell membranes only by means of its TM and FP domains, there is evidence that, subsequently, the NHR region also interacts directly with the membranes and actively participates in the fusion process. In particular, the NHR-derived peptide, N36, binds to both zwitterionic and negatively charged phospholipid vesicles (20), whereas the N70 peptide, which encompasses the FP and NHR domains, is four times more fusogenic than FP alone for negatively charged membranes (21). The latter result suggests that the NHR segment takes an active role in destabilizing membranes and works synergistically with FP to increase the efficiency of lipid mixing. In another elegant set of experiments, Wexler-Cohen and Shai (14) showed that NHR-mimicking peptides, designed to interfere with formation of gp41’s 6HB state by competing with gp41 NHR insertion into the 6HB, have strongly increased inhibitory activity when they carry a membrane-anchoring alkyl chain. Increased inhibition is seen regardless of whether the alkyl chain is attached at the N or C terminus of the NHR peptide, suggesting that the gp41 NHR domain is embedded in the membrane surface. 6HB oligomers formed by NHR- and CHR-derived synthetic peptides dissociate in the presence of either zwitterionic or negatively charged phospholipid vesicles (20, 22). This lipid binding property has been postulated to facilitate membrane fusion by introducing an additional destabilization of the viral and target cell membranes, thereby lowering the free-energy barrier for fusion (23).In the present study, we show that the 6HB complex formed by an ectodomain that contains large segments of the NHR and CHR helices, connected by a six-residue linker (Core^S), dissociates and forms stable monomers on binding to either dodecyl phosphocholine (DPC) micelles or phospholipid vesicles of a lipid composition that mimics the T-cell membrane. The transition from trimers to monomers is associated with a significant decrease in α-helicity and also observed for a longer ectodomain construct (Core^IL) that encompasses the native immunodominant loop (IL) connecting the NHR and CHR helices. The Core^S construct was chosen for detailed characterization of the structure and dynamics of the gp41 ectodomain monomer in the presence of DPC micelles. An atomic structure determination by NMR spectroscopy of the gp41 ectodomain monomer, based on residual dipolar coupling (RDC) and NOE restraints, reveals a monomeric, flexibly linked two-helical structure lying on the surface of the DPC micelle without any specific interaction between the stable and well-defined NHR and CHR helices. We propose that formation of this lipid-bound state, where CHR embeds in the viral membrane and NHR in the membrane of the host cell, provides the force for pulling the two membranes into close juxtaposition, thereby priming the system for membrane fusion. After fusion, close spatial proximity between the opposite ends of the ectodomain then permits their tight interaction, which is seen in 6HB crystal structures of the full-length gp41 ectodomain (9).     

Development,initial content validation and reliability of Nigerian Composite Lifestyle CVD risk factors questionnaire for adolescents

Background

Cardiovascular disease risk (CVD) factors affect every age category including adolescents in developing nations. Prevention strategies are effective only when there are epidemiological data for the targeted populations. The collection of such data is only made easy with composite lifestyle CVD risk factors measures that are culturally sensitive and acceptable among the target populations.

Objective

The objective of the study was to develop a culturally sensitive and friendly composite lifestyle CVD risk factors questionnaire for adolescents in Nigeria

Methods

A systematic review was conducted to identify existing, published questionnaires from which items could be selected. Content and face validation were conducted using an expert panel and a sub-sample of the target population. Data was analyzed qualitatively and reliability was assessed using intra-class correlation and Kappa statistic.

Results

Based on the comments received from experts, the questions were restructured, simplified, clarified, formatted, some questions were added and expert reached a consensus. Kappa showed fair to moderate agreement in 65% of the questions and perfect agreement in one question.

Conclusion

The CVD risk factors questionnaire has acceptable content validity and reliability and should be used to assess CVD risk factors among adolescents in Nigeria     

Residual Stresses Measurements in Laser Powder Bed Fusion Using Barkhausen Noise Analysis

In recent years, the advancement of technology brought the laser powder bed fusion process to its industrialisation step. Despite all the advancements in process repeatability and general quality control, many challenges remain unsolved due to the intrinsic difficulties of the process, notably the residual stresses issue. This work aimed to assess the usability of Barkhausen noise analysis (BNA) for the residual stress in situ monitoring of laser powder bed fusion on Maraging steel 300 (18Ni-300/1.2709). After measuring the evolution of grain size distribution over process parameter changes, two series of experiments were designed. First, a setup with an external force allows to validate the working principle of BNA on the chosen material processed using LPBF. The second experiment uses on-plates samples with different residual stress states. The results show a good stability in microstructure, a prerequisite for BNA. In addition, the external load setup acknowledges that signal variation correlates with the induced stress state. Finally, the on-plate measurement shows a similar signal variation to what has been observed in the literature for residual stress variation. It is shown that BNA is a suitable method for qualitative residual stresses variation monitoring developed during the LPBF process and underlines that BNA is a promising candidate as an in situ measurement method.     

Brief Report: Quantitative detection of hydrogen peroxide in rain,air, exhaled breath,and biological fluids by NMR spectroscopy

Hydrogen peroxide (H₂O₂) plays a key role in environmental chemistry, biology, and medicine. H₂O₂ concentrations typically are 6 to 10 orders of magnitude lower than that of water, making its quantitative detection challenging. We demonstrate that optimized NMR spectroscopy allows direct, interference-free, quantitative measurements of H₂O₂ down to submicromolar levels in a wide range of fluids, ranging from exhaled breath and air condensate to rain, blood, urine, and saliva. NMR measurements confirm the previously reported spontaneous generation of H₂O₂ in microdroplets that form when condensing water vapor on a hydrophobic surface, which can interfere with atmospheric H₂O₂ measurements. Its antimicrobial activity and strong seasonal variation speculatively could be linked to the seasonality of respiratory viral diseases.