Myelin-associated glycoprotein-mediated signaling in central nervous system pathophysiology

The myelin-associated glycoprotein (MAG) is a type I membrane-spanning protein expressed exclusively in oligoden drocytes and Schwann cells. It has two generally known pathophysiological roles in the central nervous system (CNS): maintenance of myelin integrity and inhibition of CNS axonal regeneration. The subtle CNS phenotype resulting from genetic ablation of MAG expression has made mechanistic analysis of its functional role in these difficult. However, the past few years have brought some major revelations, particularly in terms of mechanisms of MAG signaling through the Nogo-66 receptor (NgR) complex. Although apparently converging through NgR, a readily noticeable fact is that the neuronal growth inhibitory effect of MAG differs from that of Nogo-66. This may result from the influence of coreceptors in the form of gangliosides or from MAG-specific neuronal receptors such as NgR2. MAG has several other neuronal binding partners, and some of these may modulate its interaction with the NgR complex or downstream signaling. This article discusses new findings in MAG-forward and-reverse signaling and its role in CNS pathophysiology.     

Synthesis of 2-alkoxy-8-hydroxyadenylpeptides: towards synthetic epitope-based vaccines

The preparation of three different 2-alkoxy-8-hydroxyadenylpeptide conjugates has been accomplished by solid-phase synthesis combined with 'on-resin' Cu(I) catalyzed Huisgen cycloaddition. The immunogenicity of the compounds has been evaluated in IL-12 production and antigen presentation assays.     

Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum

Background

The identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.

Results

Here we show that tiling microarrays can detect de novo a large proportion of the genetic changes that differentiate one genome from another. We show that we detect most single nucleotide polymorphisms or small insertion deletion events and all known copy number variations that distinguish three laboratory isolates using readily accessible methods. We used the approach to discover mutations that occur during the selection process after transfection. We also elucidated a mechanism by which parasites acquire resistance to the antimalarial fosmidomycin, which targets the parasite isoprenoid synthesis pathway. Our microarray-based approach allowed us to attribute in vitro derived fosmidomycin resistance to a copy number variation event in the pfdxr gene, which enables the parasite to overcome fosmidomycin-mediated inhibition of isoprenoid biosynthesis.

Conclusions

We show that newly emerged single nucleotide polymorphisms can readily be detected and that malaria parasites can rapidly acquire gene amplifications in response to in vitro drug pressure. The ability to define comprehensively genetic variability in P. falciparum with a single overnight hybridization creates new opportunities to study parasite evolution and improve the treatment and control of malaria.     

Family Characteristics as Risk Factors for Childhood Acute Lymphoblastic Leukemia: A Population-Based Case-Control Study

Background

To date, few risk factors for childhood acute lymphoblastic leukemia (ALL) have been confirmed and the scientific literature is full of controversial “evidence.” We examined if family characteristics, particularly maternal and paternal age and number of older siblings, were risk factors for childhood acute lymphoblastic leukemia (ALL).

Methodology/Principal Findings

In this population-based nationwide matched case-control study, patients 0–14 years of age with ALL diagnosed 1991–2006 and registered in the Swiss Childhood Cancer Registry were linked with their census records of 1990 and 2000. Eight controls per case were selected from the census. The association between family characteristics and ALL was analyzed by conditional logistic regressions. We found that increasing maternal age was associated with incidence of ALL in the offspring (OR per 5-year increase in maternal age 1.18, 95% CI 1.05–1.31; p = 0.004), remaining stable (trend OR 1.14, 95% CI 0.99–1.31; p = 0.060) after adjustment for other risk factors. The association with paternal age was weaker (OR per 5-year increase 1.14, 95% CI 1.01–1.28, p = 0.032) and disappeared after adjustments. Number of older siblings was not associated with risk of ALL in the overall group of children aged 0–14 years at diagnosis. However, we found a negative trend between number of older siblings and ALL diagnosed at age 0–4 years (OR per sibling 0.85, 95% CI 0.68–1.06; p = 0.141) and a positive trend for ALL diagnosed at age 5–9 (OR 1.34, 95% CI 1.05–1.72; p = 0.019), with some evidence for an effect modification (p-value for interaction  = 0.040).

Conclusions

As in other studies, increasing maternal, but not paternal age was associated with risk of ALL. We found only a weak association with the number of older siblings, suggesting a delay in disease manifestation rather than a decrease in incidence.     

The Ca2+-pump of sickle cell plasma membranes. Purification and reconstitution of the ATPase enzyme

The sickle cell (Hb SS) membrane-bound Ca²⁺-ATPase was found to have a V_max in the range of 30–100% of the V_max of the normal enzyme. In all sickle cell preparations, the Ca²⁺-ATPase could be stimulated at least 4-fold by calmodulin, but the stimulation factor varied considerably (4–26 fold) in the different preparations. The affinity of the ghost sickle cell Ca²⁺-ATPase for Ca²⁺, ATP and calmodulin was comparable to that of the normal enzyme. The sickle cell Ca²⁺-ATPase was solubilized from the membrane with Triton-X-100, and purified through a calmodulin sepharose-4B column, a technique by which the Ca²⁺-ATPase from normal ghosts has been successfully isolated in a functionally active and pure form (see V. Niggli, E.S. Adynyah, J.T. Penniston and E. Carafoli, 1981, J.Biol.Chem.256,. 395 – 401). The specific activity of the isolated sickle cell enzyme was significantly decreased (up to 80%) with respect to that of the normal enzyme, but the amount of protein isolated was comparable to normal. All other parameters of the ATPase (affinity for Ca²⁺, ATP and calmodulin) were comparable to those found for the normal enzyme. In SDS polyacrylamide gel electrophoresis, the purified enzyme appeared as a single band protein with a Mr comparable to that of the normal enzyme. In the absence of calmodulin the sickle cell enzyme could be activated by acidic phospholipids, as reported for the normal enzyme. After reconstitution into liposomes it transported Ca²⁺ with normal efficiency (about 1 $\text{Ca}{}^{\mathrm{2+}}\text{ATP}$ hydrolyzed). Therefore, the only difference between the purified normal and the sickle cell enzyme appears to be the lower specific activity of the latter.     

Molecular properties of calcium-pumping ATPase from human erythrocytes

The Ca2+-pumping ATPase from human erythrocyte membranes, purified by the method previously reported [Niggli, V., Penniston, J. T., & Carafoli, E. (1979) J. Biol. Chem. 254, 9955-9958], was freed of minor impurities by extensive washing while bound to the calmodulin-Sepharose column. The pure enzyme showed a single band of Mr 138000, which contained no stainable carbohydrate. The enzyme retained calmodulin-stimulable ATPase activity; with appropriate assay conditions, an activity of 21.2 mumol/(mg x min) was obtained. Amino acid analysis showed that the ATPase had a larger proportion of polar amino acids than do other integral membrane proteins. Despite this, the ATPase showed a tendency to form dimers and higher aggregates even in the presence of sodium dodecyl sulfate and urea. The enzyme required Mg2+ but showed little activity unless a second ion was added. With regard to this second ion, the enzyme responded to alkaline earth metal ions in the order Ca2+ greater than Sr2+ much greater than Ba2+. It was highly specific for ATP and was stimulated by Na+ or K+; in all of these properties it resembled the enzyme in unfractionated membranes. Limited proteolysis using trypsin yielded, at short times, many fragments of various molecular weights; continued proteolysis resulted in two trypsin-resistant fragments of Mr 81000 and 33500. Analysis of the time course of proteolysis indicated that the ATPase existed in two or more conformations that had differing susceptibilities to proteolysis. It is suggested that these correspond to active and inactive conformers of the enzyme.     

Using systems thinking and open innovation to strengthen aquaculture policy for the United Nations Sustainable Development Goals

In a world of 9 billion people and a widening income gap between the rich and poor, it is time to rethink how aquaculture can strengthen its contribution to the second UN Sustainable Development Goal (SDG) of zero hunger in our generation. The disparity in the level of sustainable aquaculture development at present, between and within countries, especially regarding human access to farmed aquatic food remains highly variable across the globe. This paper offers a fresh look at the opportunities from using systems thinking and new open innovation measuring tools to grow sustainable aquaculture. Political will in many nations is the main constraint to aquaculture in realising its potential as an: accessible source of micronutrients and nutritious protein; aid to meeting conservation goals; economic prosperity generator where benefits extend to locals and provider of indirect social benefits such as access to education and well-being, among others. Resources to enable strong partnerships (SDG 17) between academia, civic society, government and industry should be prioritised by governments to build a sustainable aquatic food system, accessible to all, forever.     

Elemental Markers in Elasmobranchs: Effects of Environmental History and Growth on Vertebral Chemistry

Differences in the chemical composition of calcified skeletal structures (e.g. shells, otoliths) have proven useful for reconstructing the environmental history of many marine species. However, the extent to which ambient environmental conditions can be inferred from the elemental signatures within the vertebrae of elasmobranchs (sharks, skates, rays) has not been evaluated. To assess the relationship between water and vertebral elemental composition, we conducted two laboratory studies using round stingrays, Urobatis halleri, as a model species. First, we examined the effects of temperature (16°, 18°, 24°C) on vertebral elemental incorporation (Li/Ca, Mg/Ca, Mn/Ca, Zn/Ca, Sr/Ca, Ba/Ca). Second, we tested the relationship between water and subsequent vertebral elemental composition by manipulating dissolved barium concentrations (1x, 3x, 6x). We also evaluated the influence of natural variation in growth rate on elemental incorporation for both experiments. Finally, we examined the accuracy of classifying individuals to known environmental histories (temperature and barium treatments) using vertebral elemental composition. Temperature had strong, negative effects on the uptake of magnesium (D_Mg) and barium (D_Ba) and positively influenced manganese (D_Mn) incorporation. Temperature-dependent responses were not observed for lithium and strontium. Vertebral Ba/Ca was positively correlated with ambient Ba/Ca. Partition coefficients (D_Ba) revealed increased discrimination of barium in response to increased dissolved barium concentrations. There were no significant relationships between elemental incorporation and somatic growth or vertebral precipitation rates for any elements except Zn. Relationships between somatic growth rate and D_Zn were, however, inconsistent and inconclusive. Variation in the vertebral elemental signatures of U. halleri reliably distinguished individual rays from each treatment based on temperature (85%) and Ba exposure (96%) history. These results support the assumption that vertebral elemental composition reflects the environmental conditions during deposition and validates the use of vertebral elemental signatures as natural markers in an elasmobranch. Vertebral elemental analysis is a promising tool for the study of elasmobranch population structure, movement, and habitat use.     

Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit β by EPR Spectroscopy

Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site.