Towards accurate in vivo spectroscopy of the human prostate

The recent interest in photodynamic therapy of human prostate cancer is accompanied by a need for techniques for in vivo monitoring of optical and physiological characteristics. We propose time‐of‐flight (TOF) spectroscopy in combination with Monte Carlo evaluation as a reliable optical technique for quantitative assessment of absorption, scattering, hemoglobin content and tissue oxygenation in the human prostate. For the first time, we demonstrate Monte Carlo‐based evaluation of in vivo TOF photon migration data. We show that this approach is crucial in order to avoid the large errors associated with the use of time‐resolved diffusion theory of light propagation in prostate‐like tissues. This progress also allows us to present the first in vivo scattering spectroscopy of human prostate tissue. Furthermore, TOF spectroscopy, in contrast to the more common steady‐state approach, is insensitive to bleedings, and has been found highly reliable (100% success rate). (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Modelling soil C sequestration in spruce forest ecosystems along a Swedish transect based on current conditions

The change of current pools of soil C in Norway spruce ecosystems in Sweden were studied using a process-based model (CoupModel). Simulations were conducted for four sites representing different regions covering most of the forested area in Sweden and representing annual mean temperatures from 0.7°C to 7.1°C. The development of both tree layer and field layer (understory) was simulated during a 100-year period using data on standing stock volumes from the Swedish Forest Inventory to calibrate tree growth using different assumptions regarding N supply to the plants. The model successfully described the general patterns of forest stand dynamics along the Swedish climatic transect, with decreasing tree growth rates and increasing field layer biomass from south to north. However, the current tree growth pattern for the northern parts of Sweden could not be explained without organic N uptake and/or enhanced mineralisation rates compared to the southern parts. Depending on the assumption made regarding N supply to the tree, different soil C sequestration rates were obtained. The approach to supply trees with both mineralised N and organic N, keeping the soil C:N ratio constant during the simulation period was found to be the most realistic alternative. With this approach the soils in the northern region of Sweden lost 5 g C m⁻² year⁻¹, the soils in the central region lost 2 g C m⁻² year⁻¹, and the soils in the two southern regions sequestered 9 and 23 g C m⁻² year⁻¹, respectively. In addition to climatic effects, the feedback between C and N turnover plays an important role that needs to be more clearly understood to improve estimates of C sequestration in boreal forest ecosystems.     

Super-resolution microscopy reveals that Na+/K+-ATPase signaling protects against glucose-induced apoptosis by deactivating Bad

Activation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na⁺,K⁺-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20 mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10 nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca²⁺/calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.Subject terms: Apoptosis, Super-resolution microscopy     

Interactions between the juxtamembrane domain of the EGFR and calmodulin measured by surface plasmon resonance

One early response to epidermal growth factor receptor (EGFR) activation is an increase in intracellular calcium. We have used surface plasmon resonance (SPR) to study real-time interactions between the intracellular juxtamembrane (JM) region of EGFR and calmodulin. The EGFR-JM (Met⁶⁴⁴–Phe⁶⁸⁸) was expressed as a GST fusion protein and immobilised on a sensor chip surface. Calmodulin specifically interacts with EGFR-JM in a calcium-dependent manner with a high on and high off rate. Chemical modification of EGFR-JM by using arginine-selective phenylglyoxal or deletion of the basic segment Arg⁶⁴⁵–Arg⁶⁵⁷ inhibits the interaction. Phosphorylation of EGFR-JM by protein kinase C (PKC) or glutamate substitution of Thr⁶⁵⁴ inhibits the interaction, suggesting that PKC phosphorylation electrostatically interferes with calmodulin binding to basic arginine residues. Calmodulin binding was also inhibited by suramin. Our results suggest that EGFR-JM is essential for epidermal growth factor (EGF)-mediated calcium–calmodulin signalling and for signal integration between other signalling pathways.     

Optimization and validation of an ion chromatographic method for the simultaneous determination of sodium, ammonium and potassium in exhaled breath condensate

An ion chromatographic method with conductivity detection for the simultaneous quantification of sodium, ammonium and potassium in exhaled breath condensate (EBC) was developed and validated. A factorial design was used to optimize the chromatographic conditions, which resulted in baseline separations of the cations within 6 min. The method requires no pre-treatment of EBC samples. The optimized method was used for the intra-individual screening of cations in EBC of 10 healthy volunteers. The LOQs were low (0.3, 0.1 and 0.2 microM for sodium, ammonium and potassium, respectively), compared with levels detected in healthy volunteers. The responses were linear with good precision, and samples could be stored for at least 10 weeks at refrigerating conditions.     

Imidazopyridine‐Based Inhibitors of Glycogen Synthase Kinase 3: Synthesis and Evaluation of Amide Isostere Replacements of the Carboxamide Scaffold

In this study, we explored the effect of bioisostere replacement in a series of glycogen synthase kinase 3 (GSK3) inhibitors based on the imidazopyridine core. The synthesis and biological evaluation of a number of novel sulfonamide, 1,2,4‐oxadiazole, and thiazole derivates as amide bioisosteres, as well as a computational rationalization of the obtained results are reported.     

Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses

Leukocyte migration into the epithelial compartment is an important feature in the active phase of mycobacterial infections. In this study, we used the Transwell model to investigate the mechanisms behind mycobacteria-induced leukocyte recruitment and investigated the role of TLR2 and TLR4 in this process. Infection of epithelial cells resulted in significantly increased secretion of the neutrophil chemotactic CXCL8 and IL-6, but no secretion of monocyte chemotactic CCL2 or TNF-α was observed. In contrast to epithelial response, mycobacteria-infected neutrophils and monocytes secreted all these cytokines. Corresponding with epithelial cytokine response, mycobacterial infection of the epithelial cells increased neutrophil diapedesis, but decreased monocyte recruitment. However, monocyte recruitment towards mycobacteria infected epithelial cells significantly increased following addition of neutrophil pre-conditioned medium. Mycobacterial infection also increases alveolar epithelial expression of TLR2, but not TLR4, as analyzed by flow cytometry, Western blotting and visualized by confocal microscopy. Blocking of TLR2 inhibited neutrophil recruitment and cytokine secretion, while blocking of TLR4 had a lesser effect. To summarize, we found that primary alveolar epithelial cells produced a selective TLR2-dependent cytokine secretion upon mycobacterial infection. Furthermore, we found that cooperation between cells of the innate immunity is required in mounting proper antimicrobial defence.     

Synthesis and evaluation of pyridylbenzofuran, pyridylbenzothiazole and pyridylbenzoxazole derivatives as ¹⁸F-PET imaging agents for β-amyloid plaques

The synthesis and SAR of new β-amyloid binding agents are reported. Evaluation of important properties for achieving good signal-to-background ratio is described. Compounds 27, 33, and 36 displayed desirable lipophilic and pharmacokinetic properties. Compound 27 was further evaluated with autoradiographic studies in vitro on human brain tissue and in vivo in Tg2576 mice. Compound 27 showed an increased signal-to-background ratio compared to flutemetamol 4, indicating its suitability as PET ligand for β-amyloid deposits in AD patients. The preparation of the corresponding (18)F-labeled PET radioligand of compound 27 is presented.     

The discovery of a novel series of glucokinase activators based on a pyrazolopyrimidine scaffold

Glucokinase is a key enzyme in glucose homeostasis since it phosphorylates glucose to give glucose-6-phosphate, which is the first step in glycolysis. GK activators have been proven to lower blood-glucose, and therefore have potential as treatments for type 2 diabetes. Here the discovery of pyrazolopyrimidine GKAs is reported. An original singleton hit from a high-throughput screen with micromolar levels of potency was optimised to give compounds with nanomolar activities. Key steps in this success were the introduction of an extra side-chain, which increased potency, and changing the linking functionality from a thioether to an ether, which led to improved potency and lipophilic ligand efficiency. This also led to more stable compounds with improved profiles in biological assays.     

Vicariance divergence and gene flow among islet populations of an endemic lizard

Allopatry and allopatric speciation can arise through two different mechanisms: vicariance or colonization through dispersal. Distinguishing between these different allopatric mechanisms is difficult and one of the major challenges in biogeographical research. Here, we address whether allopatric isolation in an endemic island lizard is the result of vicariance or dispersal. We estimated the amount and direction of gene flow during the divergence of isolated islet populations and subspecies of the endemic Skyros wall lizard Podarcis gaigeae, a phenotypically variable species that inhabits a major island and small islets in the Greek archipelago. We applied isolation-with-migration models to estimate population divergence times, population sizes and gene flow between islet-mainland population pairs. Divergence times were significantly correlated with independently estimated geological divergence times. This correlation strongly supports a vicariance scenario where islet populations have sequentially become isolated from the major island. We did not find evidence for significant gene flow within P. g. gaigeae. However, gene-flow estimates from the islet to the mainland populations were positively affected by islet area and negatively by distance between the islet and mainland. We also found evidence for gene flow from one subspecies (P. g. weigandi) into another (P. g. gaigeae), but not in the other direction. Ongoing gene flow between the subspecies suggests that even in this geographically allopatric scenario with the sea posing a strong barrier to dispersal, divergence with some gene flow is still feasible.