Functional significance of recruitable collaterals during temporary coronary occlusion evaluated by 99mTc-sestamibi single-photon emission computerized tomography

AbstractOBJECTIVESThe present study evaluated the impact of recruitable collaterals on regional myocardial perfusion measured by 99mtechnetium (Tc)-sestamibi single-photon emission computerized tomography (SPECT) during temporary coronary occlusion and related these estimates to the coronary wedge pressure and electrocardiographic (ECG) ST-segment changes.BACKGROUNDClinical variables (angina and ECG changes) and intracoronary flow and pressure recordings have indicated a protective role of recruitable collaterals on myocardial perfusion during percutaneous transluminal coronary angioplasty (PTCA).METHODSThirty patients (mean age 55 years, SD 9; 20 men) with stable angina pectoris and proximal nonoccluding single-vessel left anterior descending coronary artery (LAD)-stenosis scheduled for PTCA were included. Visualization of recruitable collaterals by ipsilateral and contralateral contrast injection, registration of coronary wedge pressure and injection of 99mTc-sestamibi during 90-s LAD occlusions were undertaken. A rest perfusion study was performed within four days before PTCA. As an estimate of the severity of regional hypoperfusion during occlusion, an occlusion/rest count ratio was calculated (mean defect pixel count during occlusion divided by mean pixel count in identical regions at rest).RESULTSThe scintigraphic occlusion/rest count ratio was higher in patients with recruitable collaterals (n = 16), 67 ± 11%, compared to patients without collaterals (n = 14), 60 ± 6% (p < 0.05). The occlusion/rest count ratio correlated with the coronary wedge pressure (R2= 0.34; p < 0.001). The occlusion/rest count ratio was lower, 61 ± 6%, in patients with ST-segment elevation (n = 23) versus 74 ± 9% in patients without ST-segment elevation (n = 7) (p < 0.0001).CONCLUSIONSUsing 99mTc-sestamibi SPECT imaging during brief episodes of coronary occlusion, the severity of regional myocardial hypoperfusion was reduced by the presence of recruitable collaterals in a selected patient population with proximal LAD stenoses. Our results demonstrate a protective effect of recruitable collaterals on myocardial perfusion during temporary coronary occlusion

Redox signals at the ER-mitochondria interface control melanoma progression.

Reactive oxygen species (ROS) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum (ER)-mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX1 and TMX3 oxidoreductases, which promote ER-mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial- and NOX4-derived ROS. The TMX-knockdown-induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT1. Furthermore, we identified NFAT1-positive and NFAT1-negative melanoma subgroups, wherein NFAT1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial- and redox-related proteins are under NFAT1 control and indicated that TMX1, TMX3, and NFAT1 are associated with poor disease outcome. Our study unravels a novel redox-controlled ER-mitochondria-NFAT1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease

Mdm38 interacts with ribosomes and is a component of the mitochondrial protein export machinery

Saccharomyces cerevisiae Mdm38 and Ylh47 are homologues of human Letm1, a protein implicated in Wolf-Hirschhorn syndrome. We analyzed the function of Mdm38 and Ylh47 in yeast mitochondria to gain insight into the role of Letm1. We find that mdm38Δ mitochondria have reduced amounts of certain mitochondrially encoded proteins and low levels of complex III and IV and accumulate unassembled Atp6 of complex V of the respiratory chain. Mdm38 is especially required for efficient transport of Atp6 and cytochrome b across the inner membrane, whereas Ylh47 plays a minor role in this process. Both Mdm38 and Ylh47 form stable complexes with mitochondrial ribosomes, similar to what has been reported for Oxa1, a central component of the mitochondrial export machinery. Our results indicate that Mdm38 functions as a component of an Oxa1-independent insertion machinery in the inner membrane and that Mdm38 plays a critical role in the biogenesis of the respiratory chain by coupling ribosome function to protein transport across the inner membrane

An in vitro system to silence mitochondrial gene expression

The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mech- anisms of mitochondrial gene expression remain poorly understood due to a lack of experimental ap- proaches to analyze these processes. Here, we present an in vitro system to silence translation in purified mitochondria. In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression

Pharmacokinetics of the phosphatidylserine tracers 99m Tc-lactadherin and 99m Tc-annexin V in pigs

Abstract Background: Phosphatidylserine (PS) is a phospholipid normally located in the inner leaflet of the cell membrane. PS is translocated from the inner to the outer leaflet of the plasma membrane during the early stages of apoptosis and in necrosis. In cell and animal studies, reversible PS externalisation to the outer membrane leaflet has been observed in viable cells. Hence, PS markers have been proposed as markers of both reversibly and irreversibly damaged cells. The purpose of this experimental study in pigs was to investigate the kinetics of the newly introduced PS marker technetium-99m-labelled lactadherin ( 99m Tc-lactadherin) in comparison with the well-known PS trace

Crystal structures of NUDT15 variants enabled by a potent inhibitor reveal the structural basis for thiopurine sensitivity

The enzyme NUDT15 efficiently hydrolyzes the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in NUDT15 are strongly associated with thiopurine intolerance, such as leukopenia, and preemptive NUDT15 genotyping has been clinically implemented to personalize thiopurine dosing. However, understanding the molecular consequences of these variants has been difficult, as no structural information was available for NUDT15 proteins encoded by clinically actionable pharmacogenetic variants because of their inherent instability. Recently, the small molecule NUDT15 inhibitor TH1760 has been shown to sensitize cells to thiopurines, through enhanced accumulation of 6-thio-guanine in DNA. Building upon this, we herein report the development of the potent and specific NUDT15 inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement and 6-thioguanine potentiation compared with TH1760, while showing no cytotoxicity on its own. This potent inhibitor also stabilized NUDT15, enabling analysis by X-ray crystallography. We have determined high-resolution structures of the clinically relevant NUDT15 variants Arg139Cys, Arg139His, Val18Ile, and V18_V19insGlyVal. These structures provide clear insights into the structural basis for the thiopurine intolerance phenotype observed in patients carrying these pharmacogenetic variants. These findings will aid in predicting the effects of new NUDT15 sequence variations yet to be discovered in the clinic

Rounding of low serum creatinine levels and consequent impact on accuracy of bedside estimates of renal function in cancer patients

To compare glomerular filtration rate measured by technetium-99m ([Tc(99m)]) DTPA clearance with estimated creatinine clearance (CrCl) (Cockcroft and Gault (C&G) method) in patients with serum creatinine (Scr) levels 100 ml min(-1). This work indicates that when bedside estimates of renal function are calculated using the C&G formula actual Scr should be used first to estimate CrCl. If the resultant CrCl is </=100 ml min(-1), then the Scr should be rounded up to 0.06 mmol l(-1) and CrCl recalculated. Further assessment of this approach is warranted in a larger cohort of patients

How does study quality affect the results of a diagnostic meta-analysis?

Background: The use of systematic literature review to inform evidence based practice in diagnostics is rapidly expanding. Although the primary diagnostic literature is extensive, studies are often of low methodological quality or poorly reported. There has been no rigorously evaluated, evidence based tool to assess the methodological quality of diagnostic studies. The primary objective of this study was to determine the extent to which variations in the quality of primary studies impact the results of a diagnostic meta-analysis and whether this differs with diagnostic test type. A secondary objective was to contribute to the evaluation of QUADAS, an evidence-based tool for the assessment of quality in diagnostic accuracy studies. Methods: This study was conducted as part of large systematic review of tests used in the diagnosis and further investigation of urinary tract infection (UTI) in children. All studies included in this review were assessed using QUADAS, an evidence-based tool for the assessment of quality in systematic reviews of diagnostic accuracy studies. The impact of individual components of QUADAS on a summary measure of diagnostic accuracy was investigated using regression analysis. The review divided the diagnosis and further investigation of UTI into the following three clinical stages: diagnosis of UTI, localisation of infection, and further investigation of the UTI. Each stage used different types of diagnostic test, which were considered to involve different quality concerns. Results: Many of the studies included in our review were poorly reported. The proportion of QUADAS items fulfilled was similar for studies in different sections of the review. However, as might be expected, the individual items fulfilled differed between the three clinical stages. Regression analysis found that different items showed a strong association with test performance for the different tests evaluated. These differences were observed both within and between the three clinical stages assessed by the review. The results of regression analyses were also affected by whether or not a weighting (by sample size) was applied. Our analysis was severely limited by the completeness of reporting and the differences between the index tests evaluated and the reference standards used to confirm diagnoses in the primary studies. Few tests were evaluated by sufficient studies to allow meaningful use of meta-analytic pooling and investigation of heterogeneity. This meant that further analysis to investigate heterogeneity could only be undertaken using a subset of studies, and that the findings are open to various interpretations. Conclusion: Further work is needed to investigate the influence of methodological quality on the results of diagnostic meta-analyses. Large data sets of well-reported primary studies are needed to address this question. Without significant improvements in the completeness of reporting of primary studies, progress in this area will be limited