Caenorhabditis elegans as a model system for studying drug induced mitochondrial toxicity

Today HIV-1 infection is recognized as a chronic disease with obligatory lifelong treatment to keep viral titers below detectable levels. The continuous intake of antiretroviral drugs however, leads to severe and even life-threatening side effects, supposedly by the deleterious impact of nucleoside-analogue type compounds on the functioning of the mitochondrial DNA polymerase. For detailed investigation of the yet partially understood underlying mechanisms, the availability of a versatile model system is crucial. We therefore set out to develop the use of Caenorhabditis elegansto study drug induced mitochondrial toxicity. Using a combination of molecular-biological and functional assays, combined with a quantitative analysis of mitochondrial network morphology, we conclude that anti-retroviral drugs with similar working mechanisms can be classified into distinct groups based on their effects on mitochondrial morphology and biochemistry. Additionally we show that mitochondrial toxicity of antiretroviral drugs cannot be exclusively attributed to interference with the mitochondrial DNA polymerase

Contribution of Amino Acid Region 659−663 of Factor Va Heavy Chain to the Activity of Factor Xa within Prothrombinase†,‡

ABSTRACT: Factor Va, the cofactor of prothrombinase, is composed of heavy and light chains associated noncovalently in the presence of divalent metal ions. The COOH-terminal region of the heavy chain contains acidic amino acid clusters that are important for cofactor activity. In this work, we have investigated the role of amino acid region 659-663, which contains five consecutive acidic amino acid residues, by site-directed mutagenesis. We have generated factor V molecules in which all residues were mutated to either lysine (factor V 5K) or alanine (factor V 5A). We have also constructed a mutant molecule with this region deleted (factor V Δ659-663). The recombinant molecules along with wild-type factor V (factor V WT) were transiently expressed in mammalian cells, purified, and assessed for cofactor activity. Two-stage clotting assays revealed that the mutant molecules had reduced clotting activities compared to that of factor Va WT. Kinetic analyses of prothrombinase assembled with the mutant molecules demonstrated diminished k cat values, while the affinity of all mutant molecules for factor Xa was similar to that for factor Va WT. Gel electrophoresis analyses of plasma-derived and recombinant mutant prothrombin activation demonstrated delayed cleavage of prothrombin at both Arg 320 and Arg 271 by prothrombinase assembled with the mutant molecules, resulting i

Haplotypes encoding the factor VIII 1241Glu variation, factor VIII levels and the risk of venous thrombosis \ud \ud

Levels of factorVIII (FVIII) are associated with the risk of venous thrombosis.The FVIII variation D1241E has been reported to be associated with decreased levels of FVIII. Our objective was to study whether D1241E is associated with levels of FVIII and the risk of venous thrombosis and whether this association is caused by D1241E or another linked variation.We analyzed the association of three FVIII gene haplotypes encoding 1241E (further denoted as HT1, HT3, and HT5) with FVIII levels and thrombosis risk. This analysis was performed in the Leiden Thrombophilia Study (LETS). The control populations of two case-controls studies on arterial thrombosis in men and women, respectively, were used to confirm the effects observed on FVIII:C in the LETS.In men,HT1 was associated with a 6% re- duction in FVIII:C and with a reduced risk of venous thrombosis [odds ratio 0.4 (CI95 0.2–0.8)]. Logistic regression showed that the risk reduction was only partially dependent of the reduction in FVIII levels. HT1 showed no effects in women on either FVIII:C or risk of thrombosis.The number of carriers of HT3 and HT5 was too low to make an accurate estimate of the risk of venous thrombosis. Neither HT3 nor HT5 showed effects on levels of FVIII:C.When we consider that all three haplotypes encoding 1241E show different effects on FVIII:C and thrombosis risk, it is possible that D1241E is not the functional variation. However, FVIII gene variations do contribute to both levels of FVIII and the risk of thrombosi

High-Frame-Rate Power Doppler Ultrasound Is More Sensitive than Conventional Power Doppler in Detecting Rheumatic Vascularisation

Early recognition of joint inflammation will increase treatment efficacy in rheumatoid arthritis (RA). Yet, conventional power Doppler (PD) ultrasound might not be sufficiently sensitive to detect minor inflammation. We investigated the sensitivity of high-frame rate Doppler, combined with singular value decomposition technique, to suppress tissue signals, for microvascular flow in a flow phantom setup and in a proof-of-principle study in healthy controls and in RA patients with different disease activities. In the flow phantom, minimal detectable flow velocity was a factor 3 lower with high-frame-rate PD than with conventional PD ultrasound. In the proof-of-principle study we detected a positive PD signal in all volunteers, diseased or healthy, with high-frame-rate PD ultrasound. We saw a gradual increase in PD signal in RA patients depending on disease activity. In conclusion, high-frame rate Doppler is more sensitive in detecting vascularisation than conventional PD ultrasound

Cardiac Shear Wave Elastography Using a Clinical Ultrasound System

The propagation velocity of shear waves relates to tissue stiffness. We prove that a regular clinical cardiac ultrasound system can determine shear wave velocity with a conventional unmodified tissue Doppler imaging (TDI) application. The investigation was performed on five tissue phantoms with different stiffness using a research platform capable of inducing and tracking shear waves and a clinical cardiac system (Philips iE33, achieving frame rates of 400-700 Hz in TDI by tuning the normal system settings). We also tested the technique in vivo on a normal individual and on typical pathologies modifying the consistency of the left ventricular wall. The research platform scanner was used as reference. Shear wave velocities measured with TDI on the clinical cardiac system were very close to those measured by the research platform scanner. The mean difference between the clinical and research systems was 0.18 ± 0.22 m/s, and the limits of agreement, from -0.27 to +0.63 m/s. In vivo, the velocity of the wave induced by aortic valve closure in the interventricular septum increased in patients with expected increased wall stiffness

Laparoscopic sleeve gastrectomy with an extensive posterior mobilization: Technique and preliminary results

Background: Laparoscopic sleeve gastrectomy (LSG) is becoming increasingly popular as a stand-alone procedure for the treatment of morbidly obese patients. A direct posterior approach to the angle of His was developed at our department to improve visualization of the difficult dissection of the short gastric vessels and to facilitate proper mobilization of the stomach around the left crus enabling safe realization of a tight sleeve. The technique and its preliminary results are described. Methods: LSG by posterior approach was performed in a consecutive series of 445 (110 male/335 female, age 18-63 years, mean body mass index 46 kg/m2 (range 35-76)) patients between 2007 and 2010. Results: Weight loss defined as mean percent excess weight loss (%EWL) was 71% (±26%) at 1 year, 69% (±25%) at 2 years, and 55% (±27%) at 3 years. Sixteen patients (4%) developed postoperative intra-abdominal hematoma, 8 patients (2%) anastomotic leakage, and 6 patients intra-abdominal abscess (1%), requiring reoperation in 20 patients (4%). Five patients (1%) had pulmonary embolism. Thirty-day mortality rate was 0.2%. Conclusions: LSG by the posterior approach is a safe and effective procedure, enabling a tight sleeve formation leading to satisfactory %EWL results. Since long-term results of LSG are unknown, further studies are needed to define the exact place of the LSG as a stand-alone bariatric proc

Improved diagnosis of Trichomonas vaginalis infection by PCR using vaginal swabs and urine specimens compared to diagnosis by wet mount microscopy, culture, and fluorescent staining

Four vaginal cotton swab specimens were obtained from each of 804 women visiting the outpatient sexually transmitted disease clinic of the Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands, for validation of various forms of Trichomonas vaginalis diagnostic procedures. One swab specimen was immediately examined by wet mount microscopy, a second swab was placed in Kupferberg's Trichosel medium for cultivation, and two swabs were placed in phosphate-buffered saline (PBS), pH 7.2. The resulting PBS suspension was used for direct staining with acridine orange and fluorescence microscopy, inoculation of modified Diamond's culture medium, and a PCR specific for T. vaginalis. A total of 70 samples positive in one or more of the tests were identified: 31 (3.8%) infections were detected by wet mount microscopy, and 36 (4.4%) were

Long-term outcome of ruptured abdominal aortic aneurysm: Impact of treatment and age

Background: Despite advances in operative repair, ruptured abdominal aortic aneurysm (rAAA) remains associated with high mortality and morbidity rates, especially in elderly patients. The purpose of this study was to evaluate the outcomes of emergency endovascular aneurysm repair (eEVAR), conventional open repair (OPEN), and conservative treatment in elderly patients with rAAA.Methods: We conducted a retrospective study of all rAAA patients treated with OPEN or eEVAR between January 2005 and December 2011 in the vascular surgery departmen