Earlier Migration Timing,Decreasing Phenotypic Variation,and Biocomplexity in Multiple Salmonid Species

Climate-induced phenological shifts can influence population, evolutionary, and ecological dynamics, but our understanding of these phenomena is hampered by a lack of long-term demographic data. We use a multi-decade census of 5 salmonid species representing 14 life histories in a warming Alaskan stream to address the following key questions about climate change and phenology: How consistent are temporal patterns and drivers of phenology for similar species and alternative life histories? Are shifts in phenology associated with changes in phenotypic variation? How do phenological changes influence the availability of resource subsidies? For most salmonid species, life stages, and life histories, freshwater temperature influences migration timing – migration events are occurring earlier in time (mean = 1.7 days earlier per decade over the 3–5 decades), and the number of days over which migration events occur is decreasing (mean = 1.5 days per decade). Temporal trends in migration timing were not correlated with changes in intra-annual phenotypic variation, suggesting that these components of the phenotypic distribution have responded to environmental change independently. Despite commonalities across species and life histories, there was important biocomplexity in the form of disparate shifts in migration timing and variation in the environmental factors influencing migration timing for alternative life history strategies in the same population. Overall, adult populations have been stable during these phenotypic and environmental changes (λ ≈1.0), but the temporal availability of salmon as a resource in freshwater has decreased by nearly 30 days since 1971 due to changes in the median date of migration timing and decreases in intra-annual variation in migration timing. These novel observations advance our understanding of phenological change in response to climate warming, and indicate that climate change has influenced the ecology of salmon populations, which will have important consequences for the numerous species that depend on this resource.     

Structural studies of the polysaccharide antigen of Eubacterium saburreum, Strain L 32

The structure of the polysaccharide antigen produced by Eubacterium saburreum, strain L 32, has been investigated. The principal methods used were methylation analysis, graded hydrolysis with acid, and n.m.r. spectroscopy. The polysaccharide, which contains the unusual sugar 3,6-dideoxy-D-arabino-hexose (tyvelose, Tyv), is composed of trisaccharide repeating-units having the following structure:

     

Artifactual sulfation of silver-stained proteins: implications for the assignment of phosphorylation and sulfation sites

Sulfation and phosphorylation are post-translational modifications imparting an isobaric 80-Da addition on the side chain of serine, threonine, or tyrosine residues. These two post-translational modifications are often difficult to distinguish because of their similar MS fragmentation patterns. Targeted MS identification of these modifications in specific proteins commonly relies on their prior separation using gel electrophoresis and silver staining. In the present investigation, we report a potential pitfall in the interpretation of these modifications from silver-stained gels due to artifactual sulfation of serine, threonine, and tyrosine residues by sodium thiosulfate, a commonly used reagent that catalyzes the formation of metallic silver deposits onto proteins. Detailed MS analyses of gel-separated protein standards and Escherichia coli cell extracts indicated that several serine, threonine, and tyrosine residues were sulfated using silver staining protocols but not following Coomassie Blue staining. Sodium thiosulfate was identified as the reagent leading to this unexpected side reaction, and the degree of sulfation was correlated with increasing concentrations of thiosulfate up to 0.02%, which is typically used for silver staining. The significance of this artifact is discussed in the broader context of sulfation and phosphorylation site identification from in vivo and in vitro experiments.     

Localization of iron-reducing activity in paddy soilby profile studies

Profiles of iron speciations (porewaterFe(II) and Fe(III), solid-phase Fe(II) andFe(III)) have been studied to localize both ironreduction and oxidation in flooded paddy soil. Sulfateand nitrate were determined to analyze interactions ofredox reactions involved in the iron cycle with thoseof the sulfur and nitrogen cycle. The development ofthe iron(II) and iron(III) profiles was observed inmicroscale over a time period of 11 weeks. After 11weeks the profiles were stable and showed lowestconcentrations of solid-phase iron(II) on the soilsurface with increasing concentrations to a soil depthof 10 mm ( 100 µmol/cm³). Profilesof iron(III) showed a maximum of iron(III) at a depthof 2 to 4 mm ( 100--200 µmol/cm³).Porewater iron(II) concentrations were three orders ofmagnitude lower than extracted iron(II) and indicatedthat most iron(II) was adsorbed to the solid-phase orimmobilized as siderite and vivianite. Diffusive lossof iron from the soil was indicated by iron recovery(0.3 µmol gdw^–1) in the flooding water after12 weeks. The organic content of the soil influencedthe concentrations of solid-phase iron(II) in deepersoil layers (> 6 mm); higher Fe(II) concentrationsin soil with limiting amounts of electron donors mayindicate lower consumption of CO₂ by methanogenicbacteria and therefore a higher sideriteprecipitation. Soil planted with rice showed similariron(II) profiles of fresh paddy soil cores. However,maximal iron(III) concentrations ( 350µmol/cm³) were present in planted soil at adepth of 1 to 2.5 mm where oxygen is provided by a matof fine roots. Sulfate and nitrate concentrations inthe porewater were highest on the soil surface (10µM NO₃ ^–, 40 µM SO₄ ^2–) anddecreased with depth. Similar profiles were detectedfor malate, acetate, lactate, and propionate, theconcentrations decreased gradually from the surface toa depth of 4 mm. Profiles of oxygen showed highestconcentrations at the surface due to photosyntheticproduction and a depletion of oxygen below 3 mm depth.Methane production rates measured from soil layersincubated separately in closed vessels were zero atthe soil surface and increased with depth. In soildepths below 4 mm where iron(III) concentrationsdecreased higher methane production rates werefound.     

Three unique base pair changes in a family with Gaucher disease

Summary Single-stranded cDNA was prepared from RNA obtained from a patient with type 1 Gaucher disease. The cDNA was amplified in vitro and analyzed by sequencing. Three base-pair changes were identified which included a G to C transversion at nucleotide 3119 of the active gene (Asp¹⁴⁰His), an A to C transversion at nucleotide 3170 (Lys¹⁵⁷Gln) and a G to A change at nucleotide 5309 (Glu³²⁶Lys). To study the mode of inheritance of the three different base-pair changes, genomic DNA was prepared from blood or skin fibroblasts of several family members. Genomic glucocerebrosidase DNA sequences were amplified and subjected to hybridization with allele-specific oligonucleotides (ASOs). The hybridization profiles demonstrated that two of the basepair changes originated from the mother and were transmitted to her two affected sons and to a grandchild, while the third base-pair change, originating from the father, was transmitted to his two affected sons, a carrier daughter and a second grandchild. Tests of other patients with Gaucher disease failed to disclose the presence of the three base-changes. This is a unique family with three base-pair changes tightly linked to Gaucher disease.     

Resistance to a DNA and a RNA virus in transgenic plants by using a single chimeric transgene construct

Tomato leaf curl Taiwan virus (ToLCTWV) and Tomato spotted wilt virus (TSWV) are two major tomato viruses that cause serious economic losses. In this study, a partial C2 gene from ToLCTWV and the middle half of the N gene of TSWV were fused as a chimeric transgene to develop multiple virus resistance in transgenic plants. This construct was introduced into Nicotiana benthamiana and tomato by Agrobacterium-mediated transformation. Several transgenic lines showed no symptom post agro-inoculation with ToLCTWV and displayed high resistance to TSWV. The detection of siRNAs indicated that the resistance was via RNA silencing. This study demonstrated that linkage of gene segments from two viruses with distinct genomic organization, one DNA and the other RNA, can confer multiple virus resistance in transgenic plants via gene silencing.     

Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.     

Enhanced activation of Akt and extracellular-regulated kinase pathways by simultaneous occupancy of Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT7A receptors in PC12 cells

The most commonly prescribed antidepressants, the serotonin (5-HT) selective reuptake inhibitors, increase 5-HT without targeting specific receptors. Yet, little is known about the interaction of multiple receptor subtypes expressed by individual neurons. Specifically, the effect of increases in cAMP induced by Gs-coupled 5-HT receptor subtypes on the signaling pathways modulated by other receptor subtypes has not been studied. We have, therefore, examined the activation of the extracellular-regulated kinase (ERK) and Akt pathways by Gs-coupled 5-HT7A receptors and Gq-coupled 5-HT2A receptors, which are co-expressed in discrete brain regions. Agonists for both receptors were found to activate ERK and Akt in transfected PC12 cells. 5-HT2A receptor-mediated activation of the two pathways was found to be Ca2+-dependent. In contrast, 5-HT7A receptor-mediated activation of Akt required increases in both [cAMP] and intracellular [Ca2+], while activation of ERK was inhibited by Ca2+. The activation of ERK and Akt stimulated by simultaneous treatment of cells with 5-HT2A and 5-HT7A receptor agonists was found to be at least additive. Cell-permeable cAMP analogs mimicked 5-HT7A receptor agonists in enhancing 5-HT2A receptor-mediated activation of ERK and Akt. A role was identified for the cAMP-guanine exchange factor, Epac, in this augmentation of ERK, but not Akt, activation. Our finding of enhanced activation of neuroprotective Akt and ERK pathways by simultaneous occupancy of 5-HT2A and 5-HT7A receptors may also be relevant to the interaction of other neuronally expressed Gq- and Gs-coupled receptors.     

Role of aromatic interactions in amyloid formation by peptides derived from human Amylin

Numerous polypeptides and proteins form amyloid deposits in vivo or in vitro. The mechanism of amyloid formation is not well-understood particularly in the case where unstructured polypeptides assemble to form amyloid. Aromatic-aromatic interactions are known to be important in globular proteins, and the possibility that they might play a key role in amyloid formation has been raised. The results of Ala-scanning experiments on short polypeptides derived from Amylin have suggested that aromatic interactions could be particularly important for this system. Here, we examine a set of Amylin-derived polypeptides in which the single aromatic residue has been substituted with a Leu and Ala. A peptide corresponding to residues 21-29 with a Phe-23 to Leu substitution, a free N terminus, and amidated C terminus readily forms amyloid. Shorter peptides derived from the putative minimal amyloid-forming segment of Amylin, residues 22-27, also form amyloid when Phe-23 is replaced by Leu. Amyloid formation is more facile when the N terminus is deprotonated and the peptide is uncharged. Substitution of the Phe with Ala results in a peptide that is noticeably less prone to form amyloid. A peptide corresponding to residues 10-19 of human Amylin with blocked termini and the sole aromatic residue, Phe-15, substituted by Leu readily forms amyloid. A Phe-15 to Ala substitution reduces significantly the ability to form amyloid. These results indicate that an aromatic residue is not required for amyloid formation in these systems and indicates that other factors such as size, beta-sheet propensity, and hydrophobicity of the side chain in question are also important.