Letter to the Editor

Regarding Paper “Sample size determination in clinical trials with multiple co‐primary endpoints including mixed continuous and binary variables” by T. Sozu , T. Sugimoto , and T. Hamasaki Biometrical Journal (2012) 54 (5): 716–729 Article: http://dx.doi.org/10.1002/bimj.201100221 Authors' Reply: http://dx.doi.org/10.1002/bimj.201300032 This paper recently introduced a methodology for calculating the sample size in clinical trials with multiple mixed binary and continuous co‐primary endpoints modeled by the so‐called conditional grouped continuous model (CGCM). The purpose of this note is to clarify certain aspects of the methodology and propose an alternative approach based on latent means tests for the binary endpoints. We demonstrate that our approach is more powerful, yielding smaller sample sizes at powers comparable to those used in the paper.     

Protection of translation initiation factor eIF2 phosphorylation correlates with eIF2-associated glycoprotein p67 levels and requires the lysine-rich domain I of p67.

The rate of protein synthesis in mammals is largely regulated by phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2) that is modulated by the cellular glycoprotein, p67, due to its protection of eIF2alpha phosphorylation (POEP) activity. At the N-terminus of p67, there are three unique domains, and at the C-terminus there is a conserved amino acid sequence. To analyze the importance of these domains, C-terminal deletion mutants of rat p67 were expressed constitutively in KRC-7 cells. In these cells, the phosphorylation level of the alpha-subunit of eIF2 was determined, and it was found that expression of the 1-97 amino acid segment of rat p67 increases POEP activity in vivo, and induces the endogenous levels of p67. These cells also show increased growth rate, and efficient translation of chloramphenicol acetyltransferase and beta-galactosidase reporter genes. At the N-terminus of p67, there are two unique domains: a lysine-rich domain I with the sequence (36)KKKRRKKKK(44), and an acidic residue-rich domain with the sequence (77)EEKEKDDDDEDGDGD(91). Substitution of lysine-rich domain I with (36)NMKSGNKTQ(44) in rat recombinant p67 resulted in the inhibition of its POEP activity, and substitution of the acidic residue-rich domain with (77)QNIQKALEPEAGDGA(91), resulted in no inhibition of POEP activity in KRC-7 cells. Taken together, our data suggest that protection of translation initiation factor eIF2 phosphorylation correlates with eIF2-associated glycoprotein p67 levels and requires the lysine-rich domain I of p67.     

Role of the lipB gene product in the folding of the secreted lipase of Pseudomonas glumae

The LipB protein of Pseudomonas glumae is essential for the production of active extracellular lipase encoded by the lipA gene. When lipase is overproduced in P. glumae in the absence of a functional lipB gene, the enzyme accumulates intracellularly in an inactive conformation. Heterologous expression of the lipase in Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli indicated that LipB is not directly involved in the trans location of the lipase across the inner or outer membrane. However, the presence of LipB was essential for obtaining active lipase and had a profound influence on the stability of the protein to proteolytic degradation. Inactive iipase, produced in the absence of LipB could be activated in vitro by unfolding and refolding, which demonstrates that LipB activity is not responsible for an essential covalent modification of the enzyme. We propose that LipB is a lipase-specific foldase. Furthermore, proper folding of the lipase in the periplasm appears to be essential for Xcp-mediated translocation across the outer membrane.     

Seed- versus transplant-based eelgrass (Zostera marina L.) restoration success in a temperate marine lake

Despite active seagrass restoration gaining traction as a tool to halt and reverse worldwide seagrass losses, overall success remains limited. Restoration strategies, through seeding or transplantation, face different environmental bottlenecks that limit success. Choosing the most appropriate strategy of the two for a specific location, however, is hampered by lack of direct practical comparisons between strategies within a single system. To investigate potential life stage dependent bottlenecks, we compared seed-based and transplant-based restoration of Zostera marina in the subtidal saltwater Lake Grevelingen. Our results demonstrate that seedling recruitment was negatively impacted by bioturbation from the lugworm Arenicola marina and sediment movement due to hydrodynamic exposure. Transplant-based restoration was clearly more successful but surprisingly best predicted by leaf gluing by the ragworm Platynereis dumerilii. This previously undescribed interaction caused seagrass leaves to clump and reduce effective photosynthetic surface and leaf movement. We suggest that the observed behavior of these worms may result from a lack of foodweb interactions, illustrating the importance of  trophic control for seagrass restoration. Thus, in addition to recognizing life stage dependent environmental bottlenecks for restoration strategy selection, seagrass restoration may also require the active recovery of their associated food webs.     

SPATIAL AND TEMPORAL DEVELOPMENT OF IRON(III) REDUCTASE ACTIVITY IN ROOT SYSTEMS OF PISUM SATIVUM (FABACEAE) CHALLENGED WITH IRON-DEFICIENCY STRESS

The development of plasma membrane-associated iron(III) reductase activity was characterized in root systems of Pisum sativum during the first 2 wk of growth, as plants were challenged with iron-deficiency stress. Plants of a parental genotype (cv. Sparkle) and a functional iron-deficiency mutant genotype (E107) were grown hydroponically with or without supplemental iron. Iron(III) reductase activity was visualized by placing the roots in an agarose matrix containing 0.2 idm Fe(III)-ethylenediaminetetraacetic acid and 0.3 mM Na₂-bathophenanthrolinedisulfonic acid (BPDS). Red staining patterns, resulting from the formation of Fe(II)-BPDS, were used to identify iron(III)-reducing regions. Iron(III) reduction was extensive on roots of E107 as early as d 7, but not until d 11 for -Fe-treated Sparkle. Roots of +Fe-treated Sparkle showed limited regions of reductase activity throughout the period of study. For secondary lateral roots, iron(III) reduction was found for all growth types except + Fe-treated Sparkle. Treating Sparkle plants alternately to a cycle of iron deficiency, iron sufficiency, and iron deficiency revealed that reductase activity at a given root zone could be alternatively present, absent, and again present. Our results suggest that for Pisum roots grown under the present conditions, iron-deficiency stress induces the activation of iron(III) reductase capacity within 2 d.     

Characterization and prevalence of pigments in the muscle of hawkfish (Cheilodactylus bergi)

Melanin pigment was prevalent in skeletal muscle of 338 out of 1079 Cheilodactylus bergi sampled from the Argentine-Uruguay Common Fisheries Zone. A higher proportion of males than of females was pigmented, and only in males did that proportion increase with body length. No parasites were found in the muscle.