Naive B-cell trafficking is shaped by local chemokine availability and LFA-1-independent stromal interactions.

It is not known how naive B cells compute divergent chemoattractant signals of the T-cell area and B-cell follicles during in vivo migration. Here, we used two-photon microscopy of peripheral lymph nodes (PLNs) to analyze the prototype G-protein-coupled receptors (GPCRs) CXCR4, CXCR5, and CCR7 during B-cell migration, as well as the integrin LFA-1 for stromal guidance. CXCR4 and CCR7 did not influence parenchymal B-cell motility and distribution, despite their role during B-cell arrest in venules. In contrast, CXCR5 played a nonredundant role in B-cell motility in follicles and in the T-cell area. B-cell migration in the T-cell area followed a random guided walk model, arguing against directed migration in vivo. LFA-1, but not α4 integrins, contributed to B-cell motility in PLNs. However, stromal network guidance was LFA-1 independent, uncoupling integrin-dependent migration from stromal attachment. Finally, we observed that despite a 20-fold reduction of chemokine expression in virus-challenged PLNs, CXCR5 remained essential for B-cell screening of antigen-presenting cells. Our data provide an overview of the contribution of prototype GPCRs and integrins during naive B-cell migration and shed light on the local chemokine availability that these cells compute.

Temperature and Resource Availability May Interactively Affect Over-Wintering Success of Juvenile Fish in a Changing Climate

The predicted global warming may affect freshwater systems at several organizational levels, from organism to ecosystem. Specifically, in temperate regions, the projected increase of winter temperatures may have important effects on the over-winter biology of a range of organisms and especially for fish and other ectothermic animals. However, temperature effects on organisms may be directed strongly by resource availability. Here, we investigated whether over-winter loss of biomass and lipid content of juvenile roach (Rutilus rutilus) was affected by the physiologically relatively small (2-5°C) changes of winter temperatures predicted by the Intergovernmental Panel on Climate Change (IPCC), under both natural and experimental conditions. This was investigated in combination with the effects of food availability. Finally, we explored the potential for a correlation between lake temperature and resource levels for planktivorous fish, i.e., zooplankton biomass, during five consecutive winters in a south Swedish lake. We show that small increases in temperature (+2°C) affected fish biomass loss in both presence and absence of food, but negatively and positively respectively. Temperature alone explained only a minor part of the variation when food availability was not taken into account. In contrast to other studies, lipid analyses of experimental fish suggest that critical somatic condition rather than critical lipid content determined starvation induced mortality. Our results illustrate the importance of considering not only changes in temperature when predicting organism response to climate change but also food-web interactions, such as resource availability and predation. However, as exemplified by our finding that zooplankton over-winter biomass in the lake was not related to over-winter temperature, this may not be a straightforward task.

Regulation of pyridoxal $5\prime$-phosphate biosynthesis in {\it Escherichia coli\/} K-12

Vitamin B$\sb6$ (or pyridoxal 5$\sp\prime$-phosphate, PLP) is an essential, ubiquitous coenzyme that affects many aspects of amino acid and cellular metabolism in all organisms. The goal of this thesis is to examine the regulation of PLP biosynthesis in Escherichia coli K-12. First, PdxH oxidase is a PLP biosynthetic enzyme, which uses molecular oxygen as an electron acceptor under aerobic assay conditions. To test if facultative anaerobic E. coli uses another enzyme to replace the function of PdxH oxidase anaerobically, suppressors of a pdxH null mutant were isolated anaerobically after 2-aminopurine or spontaneous mutagenesis. Only one specific bypass mutation in another PLP biosynthetic gene pdxJ was found, suggesting that PdxH oxidase is able to function anaerobically and PdxT utilizes D-1-deoxyxyulose as a substrate. Second, regulation of the serC (pdxF)-aroA operon, which is involved the biosynthesis of L-serine, PLP and aromatic compounds was examined. A serC (pdxF) single gene transcript and a serC (pdXf)-aroA cotranscript initiated at P$\sb{serC\ (pdxF)}$ upstream of serC (pdxF) were detected. The expression of the operon is activated by leucine responsive regulatory protein (LRP) and repressed by cAMP receptor protein-cAMP complex (CRP$\cdot$cAMP) at the transcriptional level. LRP activates the operon by directly binding to the upstream consensus box. Binding of CRP$\cdot$cAMP to the upstream CRP box diminishes the activation effect of LRP. However, deletion of the CRP box did not affect the repression of CRP$\cdot$cAMP, suggesting that CRP$\cdot$cAMP may repress the operon indirectly by stimulating the activity or level of an unidentified repressor. The overall effect of this regulation is to maximize the expression of the operon when the cells are growing in minimal-glucose medium. In addition, the binding and the transcription of P$\sb{serC\ (pdxF)}$ by RNA polymerase require a supercoiled circular DNA, indicating that DNA supercoiling affects the transcription of the operon. Third, regulation of another PLP biosynthetic gene gapB was also examined. gapB is activated by CRP$\cdot$cAMP and repressed by catabolic repressor activator protein (CRA). However, the activation of CRP$\cdot$cAMP is epistatic to the repression of CRA. Due to the CRA repression, gapB was expressed at a low level in all the media tested, suggesting that it may be the rate-limiting step of PLP biosynthesis. In summary, unlike genes in many biosynthetic pathways, PLP biosynthetic genes are regulated by global regulators that are important for carbon and amino acid metabolism, instead of the end product(s) of the pathway. ^

Reprint of "Seed availability in hay meadows: Land-use intensification promotes seed rain but not the persistent seed bank''

Intensification of land use in semi-natural hay meadows has resulted in a decrease in species diversity. This is often thought to be caused by the reduced establishment of plant species due to high competition for light under conditions of increased productivity. Sowing experiments in grasslands have found reliable evidence that diversity can also be constrained by seed availability, implying that processes influencing the production and persistence of seeds may be important for the functioning of ecosystems. So far, the effects of land-use intensification on the seed rain and the persistence of seeds in the soil have been unclear. We selected six pairs of extensively managed (Festuco-Brometea) and intensively managed (Arrhenatheretalia) grassland with traditional late cutting regimes across Switzerland and covering an annual productivity gradient in the range 176-1211 gm(-2). In each grassland community, we estimated seed rain and seed bank using eight pooled seed-trap or topsoil samples of 89 cm(2) in each of six plots representing an area of c. 150 m(2). The seed traps were established in spring 2010 and collected simultaneously with soil cores after an exposure of c. three months. We applied the emergence method in a cold frame over eight months to estimate density of viable seeds. With community productivity reflecting land-use intensification, the density and species richness in the seed rain increased, while mean seed size diminished and the proportions of persistent seeds and of species with persistent seeds in the topsoil declined. Stronger limitation of seeds in extensively managed semi-natural grasslands can explain the fact that such grasslands are not always richer in species than more intensively managed ones. (C) 2013 Elsevier B.V. All rights reserved.

Activated carbon may have undesired side effects for testing allelopathy in invasive plants

Activated carbon has become a widely used tool to investigate root-mediated allelopathy of plants, especially in plant invasion biology, because it adsorbs and thereby neutralizes root exudates. Allelopathy has been a controversially debated phenomenon for years, which revived in plant invasion biology as one possible reason for the success of invasive plants. Noxious plant exudates may harm other plants and provide an advantage to the allelopathic plant. However, root exudates are not always toxic, but may stimulate the microbial community and change nutrient availability in the rhizosphere. In a greenhouse experiment, we investigated the interacting effects of activated carbon, arbuscular mycorrhiza and plant competition between the invasive Senecio inaequidens and the native Artemisia vulgaris. Furthermore, we tested whether activated carbon showed any undesired effects by directly affecting mycorrhiza or soil chemistry. Contrary to the expectation, S. inaequidens was a weak competitor and we could not support the idea that allelopathy was involved in the competition. Activated carbon led to a considerable increase in the aboveground biomass production and reduced the infection with arbuscular mycorrhiza of both plant species. We expected that arbuscular mycorrhiza promotes plant growth by increasing nutrient availability, but we found the contrary when activated carbon was added. Chemical analyses of the substrate showed, that adding activated carbon resulted in a strong increase in plant available phosphate and in a decrease of the C(organic)/N(total) ration both of which suggest stimulated microbial activity. Thus, activated carbon not only reduced potential allelopathic effects, but substantially changed the chemistry of the substrate. These results show that activated carbon should be handled with great care in ecological experiments on allelopathy because of possible confounding effects on the soil community.

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Poly(ethylene oxide)-b-poly(3-sulfopropyl methacrylate) block copolymers for calcium phosphate mineralization and biofilm inhibition.

Poly(ethylene oxide) (PEO) has long been used as an additive in toothpaste, partly because it reduces biofilm formation on teeth. It does not, however, reduce the formation of dental calculus or support the remineralization of dental enamel or dentine. The present article describes the synthesis of new block copolymers on the basis of PEO and poly(3-sulfopropyl methacrylate) blocks using atom transfer radical polymerization. The polymers have very large molecular weights (over 10(6) g/mol) and are highly water-soluble. They delay the precipitation of calcium phosphate from aqueous solution but, upon precipitation, lead to relatively monodisperse hydroxyapatite (HAP) spheres. Moreover, the polymers inhibit the bacterial colonization of human enamel by Streptococcus gordonii, a pioneer bacterium in oral biofilm formation, in vitro. The formation of well-defined HAP spheres suggests that a polymer-induced liquid precursor phase could be involved in the precipitation process. Moreover, the inhibition of bacterial adhesion suggests that the polymers could be utilized in caries prevention.

Anesthesia in patients with glucose-6-phosphate dehydrogenase deficiency: case report and perioperative anesthesiologic management

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a frequent congenital human enzyme defect, is the most frequent cause of hemolytic anemia triggered by drugs or infectious diseases. Drugs which induce acute hemolysis in patients with G6PD deficiency are often used in anesthesia and perioperative pain therapy. Considering the fact that patients from geographic regions with a high prevalence of the disease are often treated in European hospitals, special attention should be paid to this problem. We report a case of a 30-year-old female patient with favism and review the disease and anesthesia-related implications.

Phosphate disorders: hyperphosphatemia or pseudohyperphosphatemia?

We report the case of a 79 year old woman presenting with progressive confusion and drowsiness. Renal insufficiency with hyperkalemia as well as hypercalcemia and severe hyperphosphatemia were diagnosed. Renal insufficiency improved with treatment. However, hyperphosphatemia persisted without apparent explanation. We discuss possible causes of hyper- and pseudohyperphosphatemia. Specifically, phosphate analysis may be disturbed by the paraproteins in patients with multiple myeloma, resulting in pseudohyperphosphatemia. We review the standard laboratory phosphate measurement and the mechanisms of interference with paraproteins.