Gates,GAVI, the glorious global funds and more: all you ever wanted to know

Global immunization programmes have achieved some remarkable successes. In 1977, Frank Fenner's Commission declared smallpox to have been eradicated by an 11-year-long intensive campaign. The Expanded Programme on Immunization encompassed six important childhood vaccines and reached over three-quarters of the world's children. Polio eradication has gone remarkably well, with only 10 out of 200 countries reporting residual cases. But amidst all the good news, there is also bad news. Coverage is variable; infrastructure is crumbling; and newer vaccines are not being incorporated in many country programmes. The Bill and Melinda Gates Foundation has introduced a new dynamic here. From their initial gift of $100 million in December 1998, their commitment to date is US$1.5 billion - and rising. At the centre is a Global Children's Vaccine Fund which permitted the launch, in January 2000, of the Global Alliance for Vaccines and Immunization. This is targeted to the 74 poorest countries of the world and is designed to improve vaccination infrastructure, to purchase newer vaccines and to support research and development. Even before we know how successful this programme will be, it has had its imitators. The Global Fund to Fight AIDS, TB and Malaria borrowed many concepts from GAVI. The Global Alliance for Improved Nutrition announced in May 2002 does so as well, and is heavily supported by Gates. Highly effective parasite control programmes antedate all this but will be much strengthened. However, we still face a sizeable budgetary gap both for research and for bringing the best advances to all people who need them.     

Effects of temperature on the production of hydrogen peroxide and volatile halocarbons by brackish-water algae

Marine algae produce volatile halocarbons, which have an ozone-depleting potential. The formation of these compounds is thought to be related to oxidative stress, involving H2O2 and algal peroxidases. In our study we found strong correlations between the releases of H2O2 and brominated and some iodinated compounds to the seawater medium, but no such correlation was found for CHCl3, suggesting the involvement of other formation mechanisms as well. Little is known about the effects of environmental factors on the production of volatile halocarbons by algae and in the present study we focused on the influence of temperature. Algae were sampled in an area of the brackish Baltic Sea that receives thermal discharge, allowing us to collect specimens of the same species that were adapted to different field temperature regimes. We exposed six algal species (the diatom Pleurosira laevis, the brown alga Fucus vesiculosus and four filamentous green algae, Cladophora glomerata, Enteromorpha ahlneriana, E. flexuosa and E. intestinalis) to temperature changes of 0-11 degrees C under high irradiation to invoke oxidative stress. The production rates, as well as the quantitative composition of 16 volatile halocarbons, were strongly species-dependent and different types of responses to temperature were recorded. However, no response patterns to temperature change were found that were consistent for all species or for all halocarbons. We conclude that the production of certain halocarbons may increase with temperature in certain algal species, but that the amount and composition of the volatile halocarbons released by algal communities are probably more affected by temperature-associated species shifts. These results may have implications for climatic change scenarios.     

Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.     

Polarized localization of yeast Pbs2 depends on osmostress, the membrane protein Sho1 and Cdc42

In Saccharomyces cerevisiae cells, high external osmolarity leads to the activation of a p38-related mitogen-activated protein (MAP) kinase though Pbs2. Pbs2 tagged with green fluorescent protein (Pbs2-GFP) is evenly distributed in the cytoplasm but excluded from the nucleus before and after exposure to stress. Here we show that a catalytically inactive form of Pbs2 attains a highly polarised localization during osmostress. This phenomenon depends of the osmosensor Sho1 and on a functional Cdc42 GTPase. Cdc42, but not the actin cytoskeleton, influences Sho1-dependent activation of the MAP kinase. Sho1 itself accumulates at sites of polar growth, but independently of stress conditions and Cdc42. These observations allow us to define the sequence of events that occurs during propogation of osmostress signals.     

Pheromone-Dependent G1 Cell Cycle Arrest Requires Far1 Phosphorylation, but May Not Involve Inhibition of Cdc28-Cln2 Kinase, In Vivo

In yeast, the pheromone α-factor acts as an antiproliferative factor that induces G₁ arrest and cellular differentiation. Previous data have indicated that Far1, a factor dedicated to pheromone-induced cell cycle arrest, is under positive and negative posttranslational regulation. Phosphorylation by the pheromone-stimulated mitogen-activated protein (MAP) kinase Fus3 has been thought to enhance the binding of Far1 to G₁-specific cyclin-dependent kinase (Cdk) complexes, thereby inhibiting their catalytic activity. Cdk-dependent phosphorylation events were invoked to account for the high instability of Far1 outside early G₁ phase. To confirm any functional role of Far1 phosphorylation, we undertook a systematic mutational analysis of potential MAP kinase and Cdk recognition motifs. Two putative phosphorylation sites that strongly affect Far1 behavior were identified. A change of serine 87 to alanine prevents the cell cycle-dependent degradation of Far1, causing enhanced sensitivity to pheromone. In contrast, threonine 306 seems to be an important recipient of an activating modification, as substitutions at this position abolish the G₁ arrest function of Far1. Only the phosphorylated wild-type Far1 protein, not the T306-to-A substitution product, can be found in stable association with the Cdc28-Cln2 complex. Surprisingly, Far1-associated Cdc28-Cln2 complexes are at best moderately inhibited in immunoprecipitation kinase assays, suggesting unconventional inhibitory mechanisms of Far1.     

The Sound and the Fury—Bees Hiss when Expecting Danger

Honey bees are important model systems for the investigation of learning and memory and for a better understanding of the neuronal basics of brain function. Honey bees also possess a rich repertoire of tones and sounds, from queen piping and quacking to worker hissing and buzzing. In this study, we tested whether the worker bees’ sounds can be used as a measure of learning. We therefore conditioned honey bees aversively to odours in a walking arena and recorded both their sound production and their movement. Bees were presented with two odours, one of which was paired with an electric shock. Initially, the bees did not produce any sound upon odour presentation, but responded to the electric shock with a strong hissing response. After learning, many bees hissed at the presentation of the learned odour, while fewer bees hissed upon presentation of another odour. We also found that hissing and movement away from the conditioned odour are independent behaviours that can co-occur but do not necessarily do so. Our data suggest that hissing can be used as a readout for learning after olfactory conditioning, but that there are large individual differences between bees concerning their hissing reaction. The basis for this variability and the possible ecological relevance of the bees’ hissing remain to be investigated.     

Urinary Biomarkers at Early ADPKD Disease Stage

Background

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by a decline in renal function at late disease stage when the majority of functional renal parenchyma is replaced by cystic tissue. Thus, kidney function, assessed by estimated glomerular filtration rate (eGFR) does not well represent disease burden in early disease. Here, we investigated various urinary markers for tubular injury and their association with disease burden in ADPKD patients at early disease course.

Methods

ADPKD patients between 18 and 40 years with an eGFR greater or equal to 70 ml per min per 1.73m² were eligible for this cross-sectional study. Urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL), Kidney Injury Molecule-1 (KIM-1), and Uromodulin (UMOD) were investigated by Enzyme-Linked Immunosorbent Assay. Clara Cell Protein 16 (CC16) was investigated by Latex Immuno Assay. Cryoscopy was performed to assess urine osmolality and Urinary Albumin-to-Creatinine Ratio (UACR) was calculated. The association and the predictive properties of the markers on eGFR and height adjusted total kidney volume (htTKV) was evaluated using multiple regression analysis, incorporating different control variables for adjustment. Internal bootstrapping validated the obtained results.

Results

In 139 ADPKD patients (age 31 ±7 years, mean eGFR of 93 ± 19 ml per min per 1.73 m²) the total kidney volume was negatively correlated with eGFR and UMOD and positive associated with age, UACR, KIM-1 and urine osmolality after adjustment for possible confounders. Urine osmolality and htTKV were also associated with eGFR, whereas no association of CC16, NGAL and UMOD with eGFR or htTKV was found.

Conclusion

UACR and urinary KIM-1 are independently associated with kidney size but not with renal function in our study population. Urine osmolality was associated with eGFR and kidney volume following adjustment for multiple confounders. Despite statistical significance, the clinical value of our results is not yet conceivable. Further studies are needed to evaluate the property of the aforementioned biomarkers to assess disease state at early ADPKD stage.     

The Evolution of Collagen Fiber Orientation in Engineered Cardiovascular Tissues Visualized by Diffusion Tensor Imaging

The collagen architecture is the major determinant of the function and mechanical behavior of cardiovascular tissues. In order to engineer a functional and load-bearing cardiovascular tissue with a structure that mimics the native tissue to meet in vivo mechanical demands, a complete understanding of the collagen orientation mechanism is required. Several methods have been used to visualize collagen architecture in tissue-engineered (TE) constructs, but they either have a limited imaging depth or have a complicated set up. In this study, Diffusion Tensor Imaging (DTI) is explored as a fast and reliable method to visualize collagen arrangement, and Confocal Laser Scanning Microscopy (CLSM) was used as a validation technique. Uniaxially constrained TE strips were cultured for 2 days, 10 days, 3 and 6 weeks to investigate the evolution of the collagen orientation with time. Moreover, a comparison of the collagen orientation in high and low aspect ratio (length/width) TE constructs was made with both methods. Both methods showed similar fiber orientation in TE constructs. Collagen fibers in the high aspect ratio samples were mostly aligned in the constrained direction, while the collagen fibers in low aspect ratio strips were mainly oriented in the oblique direction. The orientation changed to the oblique direction by extending culture time and could also be visualized. DTI captured the collagen orientation differences between low and high aspect ratio samples and with time. Therefore, it can be used as a fast, non-destructive and reliable tool to study the evolution of the collagen orientation in TE constructs.     

Extensive expression of markers for acetylcholine synthesis and of M2 receptors in tenocytes in therapy-resistant chronic painful patellar tendon tendinosis - a pilot study

We have recently obtained evidence favoring the occurrence of an up-regulation of a non-neuronal cholinergic system in chronic painful patellar tendon tendinosis. It seems possible that this up-regulation to a certain degree may be involved in the manifestations of the disease. Today, there is a new, very successful, line of treatment of patellar tendinosis in the form of Doppler guided sclerosing injections. However, a few patients seem resistant to this therapy. Therefore, we have in this pilot study investigated biopsies from the patellar tendon of three such therapy-resistant patients, using immunohistochemistry. In situ hybridization was also applied. Comparisons were made with a material of specimens from both normal (n=16) and tendinosis (n=7) tendons, also previously examined. The study showed that there were extensive immunoreactions for choline acetyltransferase (ChAT) and vesicular acetylcholine transporter, as well as for the M(2) muscarinic acetylcholine receptor, in the overwhelming majority of the tenocytes. The immunoreactions were more pronounced than those generally obtained in the tendinosis tissue of the previously studied patients and clearly more pronounced than those of patellar tendon tissue of controls. Also, for the first time, we here present findings of mRNA for ChAT within tenocytes. In conclusion, it appears as if there is an excessive local acetylcholine (ACh) production and an occurrence of marked ACh effects in cases of severe tendinosis. An excessive production of local ACh might be related to pain sensation and the processes that occur in tendinosis development, such as cell proliferation. Thus, the results of this pilot study suggest that non-neuronal ACh is highly involved in the pathology of therapy-resistant patellar tendinosis.