Single molecule methods with applications in living cells.

Our knowledge about dynamic processes in biological cells systems has been obtained roughly on two levels of detail; molecular level experiments with purified components in test tubes and system wide experiments with indirect readouts in living cells. However, with the development of single molecule methods for application in living cells, this partition has started to dissolve. It is now possible to perform detailed biophysical experiments at high temporal resolution and to directly observe processes at the level of molecules in living cells.

A prediction of the renal and cardiovascular efficacy of aliskiren in ALTITUDE using short-term changes in multiple risk markers.

Introduction: We recently developed and validated in existing trials a novel algorithm (PRE score) to predict long-term drug efficacy based on short-term (month-6) drug-induced changes in multiple risk markers. To show the value of the PRE score for ongoing and planned clinical trials, we here report the predicted long-term cardio-renal efficacy of aliskiren in type 2 diabetes, which was investigated in the ALTITUDE trial, but unknown at the time this study was conducted.

Methods: We established the relation between multiple risk markers and cardio-renal endpoints (as defined in ALTITUDE) using a background database from past clinical trials.

Extracting intracellular diffusive states and transition rates from single-molecule tracking data.

We provide an analytical tool based on a variational Bayesian treatment of hidden Markov models to combine the information from thousands of short single-molecule trajectories of intracellularly diffusing proteins. The method identifies the number of diffusive states and the state transition rates. Using this method we have created an objective interaction map for Hfq, a protein that mediates interactions between small regulatory RNAs and their mRNA targets.

Direct renin inhibition in chronic kidney disease.

For approximately 6 years, the only commercially available direct renin inhibitor, aliskiren, which inhibits the renin-angiotensin-aldosterone system at the initial rate limiting step, has been marketed for the treatment of hypertension. Concurrently, much attention has been given to the possibility that renin inhibition could hold potential for improved treatment in patients with chronic kidney disease, with diabetic nephropathy as an obvious group of patients to investigate, as the activity of the renin-angiotensin-aldosterone system is enhanced in these patients and as there is an unmet need for improved treatment and prognosis in these patients. Several short term studies have been performed in diabetic nephropathy, showing a consistent effect on the surrogate endpoint lowering of albuminuria, both as monotherapy and in combination with other blockers of the renin-angiotensin-aldosterone system.

Orientational correlations in confined DNA.

We study how the orientational correlations of DNA confined to nanochannels depend on the channel diameter D by means of Monte Carlo simulations and a mean-field theory. This theory describes DNA conformations in the experimentally relevant regime where the Flory-de Gennes theory does not apply. We show how local correlations determine the dependence of the end-to-end distance of the DNA molecule upon D.

Canine serum amyloid A (SAA) measured by automated latex agglutination turbidimetry is useful for routine sensitive and specific detection of systemic inflammation in a general clinical setting.

Canine serum amyloid A (SAA) is a useful diagnostic marker of systemic inflammation. A latex agglutination turbidimetric immunoassay (LAT) was validated for automated measurements. The aim of the study was to evaluate the clinical applicability of SAA measured by the LAT.

Arterial stiffness is associated with cardiovascular, renal, retinal, and autonomic disease in type 1 diabetes.

Objective: In patients with type 1 diabetes, we investigated the association between arterial stiffness and diabetes complications.

Research Design And Methods: This was a cross-sectional study including 676 Caucasian patients with type 1 diabetes (374 [55%] men, aged 54 ± 13 years [mean ± SD]) and 51 nondiabetic controls (28 [55%] men, aged 47 ± 13 years). Aortic pulse wave velocity (PWV) was measured with SphygmoCor (AtCor Medical, Sydney, Australia) for 635 patients and all 51 controls.

Cardiorenal end points in a trial of aliskiren for type 2 diabetes.

Background: This study was undertaken to determine whether use of the direct renin inhibitor aliskiren would reduce cardiovascular and renal events in patients with type 2 diabetes and chronic kidney disease, cardiovascular disease, or both.

Methods: In a double-blind fashion, we randomly assigned 8561 patients to aliskiren (300 mg daily) or placebo as an adjunct to an angiotensin-converting-enzyme inhibitor or an angiotensin-receptor blocker. The primary end point was a composite of the time to cardiovascular death or a first occurrence of cardiac arrest with resuscitation; nonfatal myocardial infarction; nonfatal stroke; unplanned hospitalization for heart failure; end-stage renal disease, death attributable to kidney failure, or the need for renal-replacement therapy with no dialysis or transplantation available or initiated; or doubling of the baseline serum creatinine level.

Levels of NT-proBNP, markers of low-grade inflammation, and endothelial dysfunction during spironolactone treatment in patients with diabetic kidney disease.

Unlabelled: Renin-angiotensin-aldosterone system (RAAS) blockade may reduce levels of biomarkers of chronic low-grade inflammation and endothelial dysfunction. We investigated the effect of spironolactone added to standard RAAS blockade on these biomarkers in an analysis of four original studies.

Materials And Methods: The studies were double-blind, randomised, placebo-controlled studies in 46 type 1 and 23 type 2 diabetic patients with micro- or macroalbuminuria treated with angiotensin-converting enzyme inhibitor (ACE inhibitor) or angiotensin receptor blocker (ARB), and randomised to additional treatment with spironolactone 25 mg and placebo daily for 60 days.

A urinary peptide biomarker set predicts worsening of albuminuria in type 2 diabetes mellitus.

Aims/hypothesis: Microalbuminuria is considered the first clinical sign of kidney dysfunction and is associated with a poor renal and cardiovascular prognosis in type 2 diabetes. Detection of patients who are prone to develop micro- or macroalbuminuria may represent an effective strategy to start or optimise therapeutic intervention. Here we assessed the value of a urinary proteomic-based risk score (classifier) in predicting the development and progression of microalbuminuria.

Discrepancy between tonometric ambulatory and cuff-based office blood pressure measurements in patients with type 1 diabetes.

The aim of the current study was to compare ambulatory blood pressure (ABP) with office blood pressure (OBP) in diagnosing hypertension (HTN) in type 1 diabetes. The cross-sectional study included 569 type 1 diabetes patients, with a mean ± standard deviation (SD) age of 55 ± 13 years and diabetes duration of 33 ± 16 years, and 315 (55%) men. Blood pressure ≥ 130/80 mm Hg defined HTN.

[Renal disease by type 2 diabetes].

Diabetic nephropathy is the leading cause of end stage renal disease in the Western world. 30-35% of the patients with type 2 diabetes will have micro- or macroalbuminuria, warranting yearly screening. Treatment to prevent diabetic nephropathy is multifactorial, adressing known progression promoters such as blood pressure, glycaemia, dyslipidaemia and smoking and includes antithrombotic prophylaxis.

Urinary proteomics for early diagnosis in diabetic nephropathy.

Diabetic nephropathy (DN) is a progressive kidney disease, a well-known complication of long-standing diabetes. DN is the most frequent reason for dialysis in many Western countries. Early detection may enable development of specific drugs and early initiation of therapy, thereby postponing/preventing the need for renal replacement therapy.

Spectroscopic measurement of cartilage thickness in arthroscopy: ex vivo validation in human knee condyles.

Purpose: To evaluate the accuracy of articular cartilage thickness measurement when implementing a new technology based on spectroscopic measurement into an arthroscopic camera.

Methods: Cartilage thickness was studied by ex vivo arthroscopy at a number of sites (N = 113) in human knee joint osteoarthritic femoral condyles and tibial plateaus, removed from 7 patients undergoing total knee replacement. The arthroscopic image spectral data at each site were used to estimate cartilage thickness.

Ambulatory tonometric blood pressure measurements in patients with diabetes.

Background: Arterial tonometry is a novel technique for measuring ambulatory blood pressure (AMBP). The watch-like device BPro(®) (HealthSTATS International, Singapore) captures radial pulsewave reflection and calculates brachial blood pressure (BP). In this study we investigate if arterial tonometry is applicable and reliable in patients with diabetes.

Implementation of proteomic biomarkers: making it work.

While large numbers of proteomic biomarkers have been described, they are generally not implemented in medical practice. We have investigated the reasons for this shortcoming, focusing on hurdles downstream of biomarker verification, and describe major obstacles and possible solutions to ease valid biomarker implementation. Some of the problems lie in suboptimal biomarker discovery and validation, especially lack of validated platforms with well-described performance characteristics to support biomarker qualification.

Clinically significant copy number alterations and complex rearrangements of MYB and NFIB in head and neck adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) of the head and neck is a malignant tumor with poor long-term prognosis. Besides the recently identified MYB-NFIB fusion oncogene generated by a t(6;9) translocation, little is known about other genetic alterations in ACC. Using high-resolution, array-based comparative genomic hybridization, and massively paired-end sequencing, we explored genomic alterations in 40 frozen ACCs.

Lipid-based passivation in nanofluidics.

Stretching DNA in nanochannels is a useful tool for direct, visual studies of genomic DNA at the single molecule level. To facilitate the study of the interaction of linear DNA with proteins in nanochannels, we have implemented a highly effective passivation scheme based on lipid bilayers. We demonstrate virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents, including streptavidin-coated quantum dots, RecA proteins, and RecA-DNA complexes.

Impact of glycaemic control on the effect of direct renin inhibition in the AVOID study.

Introduction: Hyperglycaemia induces development and progression of microvascular complications in diabetes. A direct link between high glucose levels and intrarenal renin-angiotensin activation has been demonstrated. This post-hoc analysis assessed the influence of baseline glycaemic control on the reduction of albuminuria with aliskiren or placebo added to losartan in the Aliskiren in the EValuation of PrOteinuria In Diabetes (AVOID) study.

Baseline characteristics in the Aliskiren Trial in Type 2 Diabetes Using Cardio-Renal Endpoints (ALTITUDE).

Introduction: Patients with type 2 diabetes are at enhanced risk for macro- and microvascular complications. Albuminuria and/or reduced kidney function further enhances the vascular risk. We initiated the Aliskiren Trial in Type 2 Diabetes Using Cardio-Renal Endpoints (ALTITUDE).

Spironolactone diminishes urinary albumin excretion in patients with type 1 diabetes and microalbuminuria: a randomized placebo-controlled crossover study.

Aims: Adding aldosterone receptor blockade to standard renoprotective treatment may provide additional renoprotection in patients with overt nephropathy. We expected an impact of spironolactone in early diabetic nephropathy, and for this hypothesis we studied the effect on markers of glomerular and tubular damage in patients with Type 1 diabetes and persistent microalbuminuria.

Methods: A double-blind, randomized, placebo-controlled crossover study in 21 patients with Type 1 diabetes and microalbuminuria using spironolactone 25 mg or placebo once daily, for 60 days added to standard antihypertensive treatment.

Bandpass filtering of DNA elastic modes using confinement and tension.

During a variety of biological and technological processes, biopolymers are simultaneously subject to both confinement and external forces. Although significant efforts have gone into understanding the physics of polymers that are only confined, or only under tension, little work has been done to explore the effects of the interplay of force and confinement. Here, we study the combined effects of stretching and confinement on a polymer's configurational freedom.

High YKL-40 levels predict mortality in patients with type 2 diabetes.

Aims: We determined levels of the inflammatory marker YKL-40 in a population of patients with type 2 diabetes (T2D) and investigated the association with mortality.

Methods: In a prospective observational follow-up study, 290 patients with T2D, normoalbuminuria (n=177), microalbuminuria (n=71) and macroalbuminuria (n=42) were followed for a median (range) of 17.2 (0.

A single-step competitive binding assay for mapping of single DNA molecules.

Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification of pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy.

Fluorescence microscopy of nanochannel-confined DNA.

Stretching of DNA in nanoscale confinement allows for direct visualization of the genetic contents of the DNA on the single DNA molecule level. DNA stretched in nanoscale confinement also allows for studies of DNA-protein interactions and DNA polymer physics in confined environments. This chapter describes the basic steps to fabricate the nanostructures, to perform the experiments, and to analyze the data.