Handling mammalian mitochondrial tRNAs and aminoacyl-tRNA synthetases for functional and structural characterization

The mammalian mitochondrial (mt) genome codes for only 13 proteins, which are essential components in the process of oxidative phosphorylation of ADP into ATP. Synthesis of these proteins relies on a proper mt translation machinery. While 22 tRNAs and 2 rRNAs are also coded by the mt genome, all other factors including the set of aminoacyl-tRNA synthetases (aaRSs) are encoded in the nucleus and imported. Investigation of mammalian mt aminoacylation systems (and mt translation in general) gains more and more interest not only in regard of evolutionary considerations but also with respect to the growing number of diseases linked to mutations in the genes of either mt-tRNAs, synthetases or other factors. Here we report on methodological approaches for biochemical, functional, and structural characterization of human/mammalian mt-tRNAs and aaRSs. Procedures for preparation of native and in vitro transcribed tRNAs are accompanied by recommendations for specific handling of tRNAs incline to structural instability and chemical fragility. Large-scale preparation of mg amounts of highly soluble recombinant synthetases is a prerequisite for structural investigations that requires particular optimizations. Successful examples leading to crystallization of four mt-aaRSs and high-resolution structures are recalled and limitations discussed. Finally, the need for and the state-of-the-art in setting up an in vitro mt translation system are emphasized. Biochemical characterization of a subset of mammalian aminoacylation systems has already revealed a number of unprecedented peculiarities of interest for the study of evolution and forensic research. Further efforts in this field will certainly be rewarded by many exciting discoveries.     

Genetic diversity of maize inbred lines within and among heterotic groups revealed by RFLPs

Summary The objectives of this study were (1) to investigate genetic diversity for RFLPs in a set of important maize inbreds commonly used in Italian breeding programs, (2) to compare genetic similarities between unrelated lines from the same and different heterotic groups, and (3) to examine the potential of RFLPs for assigning maize inbreds to heterotic groups. Forty inbreds were analyzed for RFLPs with two restriction enzymes (EcoRI and HindIII) and 82 DNA clones uniformly distributed over the maize genome. Seventy clone-enzyme combinations gave single-banded RFLP patterns, and 79 gave multiple-banded RFLP patterns. The average number of RFLP patterns detected per clone-enzyme combination across all inbreds was 5.8. RFLP data revealed a wide range of genetic diversity within the two heterotic groups assayed, Iowa Stiff Stalk Synthetic (BSSS) and Lancaster Sure Crop (LSC). Genetic similarity (GS) between lines was estimated from binary RFLP data according to the method of Nei and Li (1979). The mean GS for line combinations of type BSSS × LSC (0.498) was substantially smaller than for unrelated line combinations or type BSSS × BSSS (0.584) but almost as great as for un-related line combinations of type LSC × LSC (0.506). Principal coordinate and cluster analyses based on GS values resulted in the separate groupings of lines, which is consistent with known pedigree information. A comparison between both methods for multivariate analyses of RFLP data is presented.     

Prefledging Growth and Recruitment of Female Lesser Scaup

Annual variation in juvenile recruitment is an important component of duck population dynamics, yet little is known about the factors affecting the probability of surviving and breeding in the first year of life. Two hypothesized mechanisms to explain annual variability are indirect carry-over effects (COEs) from conditions experienced during the prefledging period and direct effects from climatic conditions during the postfledging period. We used Cormack-Jolly-Seber models to estimate apparent survival and detection rates of 643 juvenile female lesser scaup (Aythya affinis) marked just prior to fledging at Red Rock Lakes National Wildlife Refuge in southwestern Montana, USA, 2010–2018. We evaluated COEs from hatch date, a hatch date × spring phenology interaction, and conspecific duckling density in addition to a direct climatic effect of winter conditions (indexed by the El Niño Southern Oscillation [ENSO]) and spring habitat conditions on the study area. We used growth data from a subset (n = 190) of known-aged ducklings to estimate the influence of hatch date and conspecific density on prefledging growth to help identify mechanisms underlying COEs. Prefledging growth and juvenile apparent survival were negatively related to measures of conspecific duckling density. We found evidence that detection probability varied annually for juvenile (but not adult) scaup, possibly representing decisions to delay breeding and not return to or remain at the study site in their first year of life. Like with apparent survival, there was suggestive evidence that detection probability decreased with increasing duckling density in the previous year. Hatching date was weakly negatively related to detection probability, but unrelated to apparent survival, whereas neither vital rate was related to winter ENSO index. Our results are consistent with a process where density-dependent growth rates in the prefledging period carry over to influence fitness in subsequent life-cycle stages. If this pattern generalizes to other systems, this density COE may have important implications for our understanding of duck population dynamics and reaffirms the importance of maintaining abundant brood-rearing habitats in conservation and management of ducks. © 2021 The Wildlife Society.     

Global warming may disproportionately affect larger adults in a predatory coral reef fish

Global warming is expected to reduce body sizes of ectothermic animals. Although the underlying mechanisms of size reductions remain poorly understood, effects appear stronger at latitudinal extremes (poles and tropics) and in aquatic rather than terrestrial systems. To shed light on this phenomenon, we examined the size dependence of critical thermal maxima (CTmax) and aerobic metabolism in a commercially important tropical reef fish, the leopard coral grouper (Plectropomus leopardus) following acclimation to current‐day (28.5 °C) vs. projected end‐of‐century (33 °C) summer temperatures for the northern Great Barrier Reef (GBR). CTmax declined from 38.3 to 37.5 °C with increasing body mass in adult fish (0.45–2.82 kg), indicating that larger individuals are more thermally sensitive than smaller conspecifics. This may be explained by a restricted capacity for large fish to increase mass‐specific maximum metabolic rate (MMR) at 33 °C compared with 28.5 °C. Indeed, temperature influenced the relationship between metabolism and body mass (0.02–2.38 kg), whereby the scaling exponent for MMR increased from 0.74 ± 0.02 at 28.5 °C to 0.79 ± 0.01 at 33 °C, and the corresponding exponents for standard metabolic rate (SMR) were 0.75 ± 0.04 and 0.80 ± 0.03. The increase in metabolic scaling exponents at higher temperatures suggests that energy budgets may be disproportionately impacted in larger fish and contribute to reduced maximum adult size. Such climate‐induced reductions in body size would have important ramifications for fisheries productivity, but are also likely to have knock‐on effects for trophodynamics and functioning of ecosystems.     

LCA and decision making: when and how to use consequential LCA; 62nd LCA forum,Swiss Federal Institute of Technology,Zürich, 9 September 2016

The 62nd life cycle assessment (LCA) forum was held on 9 September 2016 to discuss the state of research and application with regard to consequential life cycle assessment. This conference report presents the highlights of the LCA forum. The state of the art of consequential LCA was presented from different viewpoints. It was pointed out that consequential LCA is more than marginal mixes and avoided burdens and involves causal modelling. It was also said that social responsibility calls for consequential LCA. Currently, different models are used to support decision making. It was suggested to make use of the variety of models to check the conclusiveness of their results and thus the reliability of the LCAs. Current and future implementations of consequential LCI models in background databases and linking algorithms were presented. Several speakers presented consequential LCA case studies covering the sectors energy, transport, housing and mining. Some of the LCA models used in the case studies are complemented with general and partial computable equilibrium models and agent-based models and use environmentally extended input-output data or process-based LCA data. Some of the presentations focused on elements such as constrained production, marginal market mixes and technologies or recycling and system expansion. In three parallel workshops, the needs, contents and methodology, and implementation of consequential LCA approaches were discussed. The participants seemed to generally agree on the basic goal that LCA should be able to reflect the consequences of decisions. The inquiry among the participants showed that the demand for consequential LCA studies is hardly existent. The appropriate implementation of consequential modelling in LCA databases and on the appropriate model to be used in consequential LCA case studies was debated. It revealed a need for further and extensive discussions to be able to reach (minimum) consensus.     

Elucidating the CXCL12/CXCR4 Signaling Network in Chronic Lymphocytic Leukemia through Phosphoproteomics Analysis

Background

Chronic Lymphocytic Leukemia (CLL) pathogenesis has been linked to the prolonged survival and/or apoptotic resistance of leukemic B cells in vivo, and is thought to be due to enhanced survival signaling responses to environmental factors that protect CLL cells from spontaneous and chemotherapy-induced death. Although normally associated with cell migration, the chemokine, CXCL12, is one of the factors known to support the survival of CLL cells. Thus, the signaling pathways activated by CXCL12 and its receptor, CXCR4, were investigated as components of these pathways and may represent targets that if inhibited, could render resistant CLL cells more susceptible to chemotherapy.

Methodology/Principal Findings

To determine the downstream signaling targets that contribute to the survival effects of CXCL12 in CLL, we took a phosphoproteomics approach to identify and compare phosphopeptides in unstimulated and CXCL12-stimulated primary CLL cells. While some of the survival pathways activated by CXCL12 in CLL are known, including Akt and ERK1/2, this approach enabled the identification of additional signaling targets and novel phosphoproteins that could have implications in CLL disease and therapy. In addition to the phosphoproteomics results, we provide evidence from western blot validation that the tumor suppressor, programmed cell death factor 4 (PDCD4), is a previously unidentified phosphorylation target of CXCL12 signaling in all CLL cells probed. Additionally, heat shock protein 27 (HSP27), which mediates anti-apoptotic signaling and has previously been linked to chemotherapeutic resistance, was detected in a subset (∼25%) of CLL patients cells examined.

Conclusions/Significance

Since PDCD4 and HSP27 have previously been associated with cancer and regulation of cell growth and apoptosis, these proteins may have novel implications in CLL cell survival and represent potential therapeutic targets. PDCD4 also represents a previously unknown signaling target of chemokine receptors; therefore, these observations increase our understanding of alternative pathways to migration that may be activated or inhibited by chemokines in the context of cancer cell survival.     

High mobility group box protein 1: an endogenous signal for dendritic cell maturation and Th1 polarization

High mobility group box protein 1 (HMGB1), a DNA binding nuclear and cytosolic protein, is a proinflammatory cytokine released by monocytes and macrophages. This study addressed the hypothesis that HMGB1 is an immunostimulatory signal that induces dendritic cell (DC) maturation. We show that HMGB1, via its B box domain, induced phenotypic maturation of DCs, as evidenced by increased CD83, CD54, CD80, CD40, CD58, and MHC class II expression and decreased CD206 expression. The B box caused increased secretion of the proinflammatory cytokines IL-12, IL-6, IL-1alpha, IL-8, TNF-alpha, and RANTES. B box up-regulated CD83 expression as well as IL-6 secretion via a p38 MAPK-dependent pathway. In the MLR, B box-activated DCs acted as potent stimulators of allogeneic T cells, and the magnitude of the response was equivalent to DCs activated by exposure to LPS, nonmethylated CpG oligonucleotides, or CD40L. Furthermore, B box induced secretion of IL-12 from DCs as well as IL-2 and IFN-gamma secretion from allogeneic T cells, suggesting a Th1 bias. HMGB1 released by necrotic cells may be a signal of tissue or cellular injury that, when sensed by DCs, induces and/or enhances an immune reaction.     

Effects of climate change on coral grouper (<Emphasis Type="Italic">Plectropomus</Emphasis> spp.) and possible adaptation options

Global climate change is increasingly considered one of the major threats to tropical coastal fisheries, potentially undermining important revenue and food security provided by coral reef ecosystems. While there has been significant and increasing work on understanding specific effects of climate change on coral reef fishes, few studies have considered large-bodied fisheries target species, limiting understanding of the effects of climate change on tropical fisheries. This review focuses on coral grouper (Plectropomus spp., and mainly Plectropomus leopardus), which are heavily fished throughout the Indian and Pacific oceans, and represent an exemplar group to assess potential effects of climate change on coral reef fisheries. In experimental studies, P. leopardus appear to be extremely sensitive to increasing ocean temperature, exhibiting declines in survivorship, aerobic scope and activity with relatively moderate increases in temperature. As such, ongoing ocean warming may jeopardize the catchability of coral grouper and sustainability of reef-based fisheries, especially at low latitudes. Notably, a significant portion of wild stocks of P. leopardus are already exposed to temperatures (≥30 °C) that have been shown to compromise individual performance and body condition. While there are considerable knowledge gaps in predicting effects of global climate change on coral grouper, such as their capacity to avoid, acclimate or adapt to changes in local environmental conditions, current information suggests that there is cause for concern. As such, we take the formative steps to outline both ecological and socioeconomic adaptations that could reduce vulnerability of coral reef fisheries to climate impacts on stocks of coral grouper, using a linked socio-economic framework.     

An integrative genetic linkage map of winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

We constructed a genetic linkage map based on a cross between two Swiss winter wheat (Triticum aestivum L.) varieties, Arina and Forno. Two-hundred and forty F₅ single-seed descent (SSD)-derived lines were analysed with 112 restriction fragment length polymorphism (RFLP) anonymous probes, 18 wheat cDNA clones coding for putative stress or defence-related proteins and 179 simple-sequence repeat (SSR) primer-pairs. The 309 markers revealed 396 segregating loci. Linkage analysis defined 27 linkage groups that could all be assigned to chromosomes or chromosome arms. The resulting genetic map comprises 380 loci and spans 3,086 cM with 1,131 cM for the A genome, 920 cM for the B genome and 1,036 cM for the D genome. Seventeen percent of the loci showed a significant (P < 0.05) deviation from a 1:1 ratio, most of them in favour of the Arina alleles. This map enabled the mapping of QTLs for resistance against several fungal diseases such as Stagonospora glume blotch, leaf rust and Fusarium head blight. It will also be very useful for wheat genetic mapping, as it combines RFLP and SSR markers that were previously located on separate maps. S. Paillard and T. Schnurbusch contributed equally to the work