The diagnostic performance of quantitative flow ratio and perfusion imaging in patients with prior coronary artery disease.

AIMS: In chronic coronary syndrome (CCS) patients with documented coronary artery disease (CAD), ischaemia detection by myocardial perfusion imaging (MPI) and an invasive approach are viable diagnostic strategies. We compared the diagnostic performance of quantitative flow ratio (QFR) with single-photon emission computed tomography (SPECT), positron emission tomography (PET), and cardiac magnetic resonance imaging (CMR) in patients with prior CAD [previous percutaneous coronary intervention (PCI) and/or myocardial infarction (MI)]. METHODS AND RESULTS: This PACIFIC-2 sub-study evaluated 189 CCS patients with prior CAD for inclusion. Patients underwent SPECT, PET, and CMR followed by invasive coronary angiography with fractional flow reserve (FFR) measurements of all major coronary arteries (N = 567), except for vessels with a sub-total or chronic total occlusion. Quantitative flow ratio computation was attempted in 488 (86%) vessels with measured FFR available (FFR ≤0.80 defined haemodynamically significant CAD). Quantitative flow ratio analysis was successful in 334 (68%) vessels among 166 patients and demonstrated a higher accuracy (84%) and sensitivity (72%) compared with SPECT (66%, P < 0.001 and 46%, P = 0.001), PET (65%, P < 0.001 and 58%, P = 0.032), and CMR (72%, P < 0.001 and 33%, P < 0.001). The specificity of QFR (87%) was similar to that of CMR (83%, P = 0.123) but higher than that of SPECT (71%, P < 0.001) and PET (67%, P < 0.001). Lastly, QFR exhibited a higher area under the receiver operating characteristic curve (0.89) than SPECT (0.57, P < 0.001), PET (0.66, P < 0.001), and CMR (0.60, P < 0.001). CONCLUSION: QFR correlated better with FFR in patients with prior CAD than MPI, as reflected in the higher diagnostic performance measures for detecting FFR-defined, vessel-specific, significant CAD.

Comparison Between the Performance of Quantitative Flow Ratio and Perfusion Imaging for Diagnosing Myocardial Ischemia.

OBJECTIVES: This study compared the performance of the quantitative flow ratio (QFR) with single-photon emission computed tomography (SPECT) and positron emission tomography (PET) myocardial perfusion imaging (MPI) for the diagnosis of fractional flow reserve (FFR)-defined coronary artery disease (CAD). BACKGROUND: QFR estimates FFR solely based on cine contrast images acquired during invasive coronary angiography (ICA). Head-to-head studies comparing QFR with noninvasive MPI are lacking. METHODS: A total of 208 (624 vessels) patients underwent technetium-99m tetrofosmin SPECT and [15O]H2O PET imaging before ICA in conjunction with FFR measurements. ICA was obtained without using a dedicated QFR acquisition protocol, and QFR computation was attempted in all vessels interrogated by FFR (552 vessels). RESULTS: QFR computation succeeded in 286 (52%) vessels. QFR correlated well with invasive FFR overall (R = 0.79; p < 0.001) and in the subset of vessels with an intermediate (30% to 90%) diameter stenosis (R = 0.76; p < 0.001). Overall, per-vessel analysis demonstrated QFR to exhibit a superior sensitivity (70%) in comparison with SPECT (29%; p < 0.001), whereas it was similar to PET (75%; p = 1.000). Specificity of QFR (93%) was higher than PET (79%; p < 0.001) and not different from SPECT (96%; p = 1.000). As such, the accuracy of QFR (88%) was superior to both SPECT (82%; p = 0.010) and PET (78%; p = 0.004). Lastly, the area under the receiver operating characteristics curve of QFR, in the overall sample (0.94) and among vessels with an intermediate lesion (0.90) was higher than SPECT (0.63 and 0.61; p < 0.001 for both) and PET (0.82; p < 0.001 and 0.77; p = 0.002), respectively. CONCLUSIONS: In this head-to-head comparative study, QFR exhibited a higher diagnostic value for detecting FFR-defined significant CAD compared with perfusion imaging by SPECT or PET.

Clinical validation of the new T- and Y-shape models for the quantitative analysis of coronary bifurcations: an interobserver variability study.

OBJECTIVES: This article presents the results of an interobserver validation study of our new T- and Y-shape bifurcation models including their edge segment analyses. BACKGROUND: Over the last years, the coronary artery intervention procedures have been developed more and more toward bifurcation stenting. Because traditional straight vessel quantitative coronary arteriography (QCA) is not sufficient for these measurements, the need has grown for new bifurcation analysis methods. METHODS: In this article, our two new bifurcation analysis models are presented, the Y-shape and T-shape model. These models were designed for the accurate measurement of the clinically relevant parameters of a coronary bifurcation, for different morphologies and intervention strategies and include an edge segment analysis, to accurately measure (drug-eluting) stent, stent edge, and ostial segment parameters. RESULTS: The results of an interobserver validation study of our T-shape and Y-shape analyses are presented, both containing the pre- and post-intervention analyses of each 10 cases. These results are associated with only small systematic and random errors, in the majority of the cases compliant with the QCA guidelines for straight analyses. The results for the edge segment analyses are also very good, with almost all the values within the margins that have been set by our brachytherapy directive. CONCLUSIONS: Our new bifurcation approaches including their edge segment analyses are very robust and reproducible, and therefore a great extension to the field of quantitative coronary angiography.

Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger.

Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase-glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation.

Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics.

The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing beta-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.

Isolation of two laccase genes from the white-rot fungus Pleurotus eryngii and heterologous expression of the pel3 encoded protein.

In this paper we report the cloning and nucleotide sequence analysis of two new laccase genes from the white-rot fungus Pleurotus eryngii, named pel3 and pel4. Comparison of the protein sequences deduced from these genes with laccases previously described in P. eryngii indicates that these genes codify for new laccases in this fungus. We described the expression of pel3 gene in two different Aspergillus niger strains. Both the laccase signal peptide and the glucoamylase preprosequence of A. niger were used to target the secretion of the active enzyme. The highest levels of laccase expression were obtained by combining the last construction with an A. niger strain deficient in extracellular proteases secretion. The characterization of catalytic properties of the recombinant enzyme, together with the setting-up of a heterologous expression system for pel3, will provide the basis to study the biotechnological applications of this enzyme.

Highly efficient gene targeting in the Aspergillus niger kusA mutant.

Gene targeting frequencies in Aspergillus niger are often very low and hamper efficient functional genomics in this biotechnologically important fungus. Deletion of the A. niger kusA gene encoding the ortholog of the Ku70 protein in other eukaryotes, dramatically improved homologous integration efficiency and reached more than 80% compared to 7% in the wild-type background, when 500bp homologous flanks were used. Furthermore, the use of the DeltakusA strain resulted in a high frequency of heterokaryon formation (70%) in primary transformants in the case disrupting an essential gene. Deletion of kusA had no obvious effect on the growth of the fungus, but renders the DeltakusA strain 10 times more sensitive to X-ray irradiation and two to three times more sensitive to UV exposure. The highly efficient gene targeting in combination with the A. niger genome sequence allows a systematic approach to generate gene knockouts and will help in improving the capacities of A. niger as producer of commercially interesting proteins and metabolites.

The KlPGS1 gene encoding phosphatidylglycerolphosphate synthase in Kluyveromyces lactis is essential and assigned to chromosome I.

The phosphatidylglycerolphosphate synthase (CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase, EC 2.7.8.5) is an essential enzyme in biosynthesis of cardiolipin. In this work we report the isolation, heterological cloning, molecular characterization and physical mapping of the Saccharomyces cerevisiae PEL1/PGS1 homologue from Kluyveromyces lactis. The pel1 mutant strain of S. cerevisiae was used to isolate this homologue by screening a K. lactis genomic library. The novel cloned gene was named KlPGS1. Its coding region was found to consist of 1623 bp. The corresponding protein exhibits 55% amino acid identity to its S. cerevisiae counterpart. The presence of the mitochondrial presequence indicates its mitochondrial localization. Sporulation and ascus dissection of diploids heterozygous for single-copy disruption of KlPGS1 revealed that the KlPGS1 gene, is essential in K. lactis. Using a DIG-dUTP-labeled DNA probe-originated from the KlPGS1 gene and Southern hybridization of contour-clamped homogeneous electric field (CHEF)-separated K. lactis chromosomal DNA, the KlPGS1 gene was assigned to chromosome I. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the Accession No. AY176328.

Efficient gene targeting in Kluyveromyces lactis.

Integration of a DNA fragment in a host genome requires the action of a double-strand break (DSB) repair mechanism. Homologous recombination (HR) is initiated by binding of Rad52p to DNA ends and results in targeted integration. Binding of the Ku heterodimer (Ku70p/Ku80p) results in random integration via non-homologous end joining (NHEJ). In contrast to Saccharomyces cerevisiae, the budding yeast Kluyveromyces lactis shows variable, but in general low, gene targeting efficiency. To study and to improve gene targeting efficiency, K. lactis has been used as a model. The KlRAD51, KlRAD52 and KlKU80 genes have been isolated and deletion mutants for these genes have been constructed. Efficiency of gene targeting was determined at the KlADE2 locus using targeting constructs with different lengths of homologous flanking sequences. In wild-type K. lactis, the gene targeting efficiency ranged from 0% with 50 to 88% with 600 bp flanks. The Klku80 mutant, however, showed >97% gene targeting efficiency independently of the size of the homologous flanks. These results demonstrate that deletion of the NHEJ mechanism results in a higher gene targeting efficiency. Furthermore, increased gene targeting efficiency was achieved by the transformation of wild-type K. lactis with the KlADE2 deletion construct in the presence of excess small DNA fragments. Using this method, PCR-generated deletion constructs containing only 50 bp of homologous flanking sequences resulted in efficient targeted gene replacement.