Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na⁺] and [K⁺] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na⁺‐selective class I‐HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single‐nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near‐isogenic lines (NILs): 157‐14 (double homozygote for the cheesmaniae alleles) and 157‐17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157‐14 in comparison with 157‐17. A significant difference between NILs was also found for [K⁺] and the [Na⁺]/[K⁺] ratio in leaf and stem but not for [Na⁺] arising a disagreement with the corresponding RIL population. Their association with leaf [Na⁺] and salt tolerance in tomato is also discussed.     

Micrometeorological measurements over 3 years reveal differences in N2O emissions between annual and perennial crops

Perennial crops can deliver a wide range of ecosystem services compared to annual crops. Some of these benefits are achieved by lengthening the growing season, which increases the period of crop water and nutrient uptake, pointing to a potential role for perennial systems to mitigate soil nitrous oxide (N₂O) emissions. Employing a micrometeorological method, we tested this hypothesis in a 3‐year field experiment with a perennial grass‐legume mixture and an annual corn monoculture. Given that N₂O emissions are strongly dependent on the method of fertilizer application, two manure application options commonly used by farmers for each crop were studied: injection vs. broadcast application for the perennial; fall vs. spring application for the annual. Across the 3 years, lower N₂O emissions (P < 0.001) were measured for the perennial compared to the annual crop, even though annual N₂O emissions increased tenfold for the perennial after ploughing. The percentage of N₂O lost per unit of fertilizer applied was 3.7, 3.1 and 1.3 times higher for the annual for each consecutive year. Differences in soil organic matter due to the contrasting root systems of these crops are probably a major factor behind the N₂O reduction. We found that a specific manure management practice can lead to increases or reductions in annual N₂O emissions depending on environmental variables. The number of freeze‐thaw cycles during winter and the amount of rainfall after fertilization in spring were key factors. Therefore, general manure management recommendations should be avoided because interannual weather variability has the potential to determine if a specific practice is beneficial or detrimental. The lower N₂O emissions of perennial crops deserve further research attention and must be considered in future land‐use decisions. Increasing the proportion of perennial crops in agricultural landscapes may provide an overlooked opportunity to regulate N₂O emissions.     

Brief debrisoquin administration to assess central dopaminergic function in children

Central dopaminergic (DA) function in children was assessed by monitoring plasma-free homovanillic acid (pHVA) levels after brief (18 hour) administration with debrisoquin sulfate, a peripherally active antihypertensive agent that blocks peripheral, but not central, HVA production. Brief debrisoquin administration resulted in marked reductions in pHVA in each of six patients studied. In five of the six patients, post-debrisoquin pHVA levels remained relatively stable over the six-hour period of observation. No significant cardiovascular or behavioral side effects of debrisoquin were observed. The brief debrisoquin administration method appears to be a safe, simple, and potentially valid peripheral technique for evaluating aspects of central dopaminergic function in children with neuropsychiatric disorders. Additional work is needed to further establish this method's validity and reliability.     

Membrane Potential and Catecholamine Secretion by Bovine Adrenal Chromaffin Cells: Use of Tetraphenylphosphonium Distribution and Carbocyanine Dye Fluorescence

Changes in plasma membrane potential of isolated bovine adrenal chromaffin cells were measured independently by two chemical probe methods and related to corresponding effects on catecholamine secretion. The lipophilic cation tetraphenylphosphonium (TPP+) and the carbocyanine dye 3,3'-dipropylthiadicarbocyanine [DiS-C3-(5)] were used. The necessity of evaluating the subcellular distribution of TPP+ among cytoplasmic, mitochondrial, secretory granule, and bound compartments was demonstrated and the resting plasma membrane potential determined to be -55 mV. The relationship between membrane potential and catecholamine secretion was determined in response to variations in extracellular K+ and to the presence of several secretagogues including cholinergic receptor ligands, veratridine, and ionophores for Na+ and K+. The dependence of potential on K+ concentration fit the Goldman constant field equation with a Na/K permeability ratio of 0.1. The dependence of both K+- and veratridine-evoked catecholamine secretion on membrane potential exhibited a potential threshold of about -40 mV before a significant rise in secretion occurred. This is likely related to the threshold for opening of voltage-sensitive Ca2+ channels. Acetylcholine and nicotine evoked a large secretory response without a sufficiently sustained depolarization to be detectable by the relatively slow potential sensitive chemical probes. Decamethonium induced a detectable depolarization of the chromaffin cells. Veratridine and gramicidin evoked both membrane depolarization and catecholamine release. By contrast the K ionophore valinomycin evoked significant levels of secretion without any depolarization. This is consistent with its utilization of an intracellular source of Ca2+ and the independence of its measured secretory response on extracellular Ca2+.     

Evaluating flux of labeled albumin into the pulmonary interstitium of sheep lungs.

A noninvasive method was used to measure the movement of 131I-labeled albumin across the pulmonary microvascular barrier of a blood-perfused in situ sheep lung lymph preparation. After injection of labeled albumin into the blood, external measurements of gamma activity were taken for 2 h. The interstitial concentrations were calculated by applying the external activities and sampled lung lymph concentrations to a mass transport model. For the external activities and lymph activities to yield the same quantitative results, two modifications were necessary. First, lymph concentrations were corrected for transport delay from the lymphatic system. Second, externally detected radioactivity had to be corrected for the contribution of unbound nuclide. Application of a mathematical model to the data indicated the extravascular distribution volume for albumin was 79% of the pulmonary blood volume, and the extravascular distribution volume for radiolabeled iodide was 4.42 times greater than the pulmonary blood volume. The permeability-surface area product for iodide in the lung was estimated to be 0.274 ml.min-1.g blood-free dry lung wt-1. The transport delay in the lymphatic system was approximately 30-45 min and represented a volume of 1.44-2.80 ml.     

Lifelong reduction in complex IV induces tissue‐specific metabolic effects but does not reduce lifespan or healthspan in mice

Loss of SURF1, a Complex IV assembly protein, was reported to increase lifespan in mice despite dramatically lower cytochrome oxidase (COX) activity. Consistent with this, our previous studies found advantageous changes in metabolism (reduced adiposity, increased insulin sensitivity, and mitochondrial biogenesis) in Surf1^?/? mice. The lack of deleterious phenotypes in Surf1^?/? mice is contrary to the hypothesis that mitochondrial dysfunction contributes to aging. We found only a modest (nonsignificant) extension of lifespan (7% median, 16% maximum) and no change in healthspan indices in Surf1^?/? vs. Surf1^+/+ mice despite substantial decreases in COX activity (22%–87% across tissues). Dietary restriction (DR) increased median lifespan in both Surf1^+/+ and Surf1^?/? mice (36% and 19%, respectively). We measured gene expression, metabolites, and targeted expression of key metabolic proteins in adipose tissue, liver, and brain in Surf1^+/+ and Surf1^?/? mice. Gene expression was differentially regulated in a tissue‐specific manner. Many proteins and metabolites are downregulated in Surf1^?/? adipose tissue and reversed by DR, while in brain, most metabolites that changed were elevated in Surf1^?/? mice. Finally, mitochondrial unfolded protein response (UPR^mt)‐associated proteins were not uniformly altered by age or genotype, suggesting the UPR^mt is not a key player in aging or in response to reduced COX activity. While the changes in gene expression and metabolism may represent compensatory responses to mitochondrial stress, the important outcome of this study is that lifespan and healthspan are not compromised in Surf1^?/? mice, suggesting that not all mitochondrial deficiencies are a critical determinant of lifespan.     

Three dimensional typological studies using scanning electron microscopy for characterization of Termitomyces pellets obtained from submerged growth conditions

There are gaps in existing understanding of fungal pellet growth dynamics. We used scanning electron microscopy (SEM) for morphological characterization of the biomass organization of Termitomyces pellets for seven species: T. microcarpus (TMI1), T. albuminosus (TAL1, TAL2), T. striatus (TSTR), T. aurantiacus (TAUR), T. heimii (THE1, THE2), T. globulus (TGLO) and T. clypeatus (TCL1, TCL2, TCL3, TCL4, TCL5). We assessed the utility of SEM for morphological and structural characterization of Termitomyces spp. in three dimensional (3D) pellet form to identify ideal pellet morphology for industrial use. Typological classification of Termitomyces species was based on furrows, isotropy, total motifs and fractal dimensions. The pellets formed were entangled and exhibited highly compacted mycelial mass with microheterogeneity and microporosity. The mean density of furrows of Termitomyces species was between 10,000 and 11,300 cm/cm², percentage isotropy was 30?80 and total motifs varied from 300 to 2500. TGLO exhibited the highest furrow mean density, 11243 cm/cm², which indicated a compact, cerebroid structure with complex ridges and furrows, whereas TAL2 exhibited the lowest furrow density. TMI1a exhibited a high percentage isotropic value, 74.6, TSTR exhibited the lowest, 30.9. Total motif number also was used as a typological classification parameter. Fractal values were 2.64?2.78 for various submerged conditions of Termitomyces species. TAL1 exhibited the highest fractal dimension and TAL2 the lowest, which indicates the complexity of branching patterns. Three-dimensional SEM image analysis can provide insight into pellet micromorphology and is a powerful tool for exploring topographical details of pellets.     

Development and accumulation of ABA in fluridone-treated and drought-stressed Vicia faba plants under different light conditions

The effects of fluridone on guard cell morphology, chloroplast ultrastructure and accumulation of drought stress-induced abscisic acid (ABA) were studied in Vicia faba L. plants grown under different light conditions. Drought stress was induced by allowing the leaves to lose 12% of their fresh weight. The appearance of defective and undeveloped stomata, and chloroplasts with a destroyed thylakoid membrane system was found in fluridone-treated plants grown at a photosynthetic photon flux (PPF) of 600 μmol m^-2 s^-1. Plants grown at a PPF of 40 μmol m^-2 s^-1 had diminished levels of ABA after imposition of dehydration. Fluridone treatment reduced the level of ABA in both unstressed and dehydrated leaves. Accumulation of ABA in the control plants was considerably reduced when they were exposed to dark periods of 24, 48 and 72 h just before imposition of the stress. Twenty-four hours after the dark treatment dehydration of the leaves resulted in a 3-fold decrease in the level of stress-induced ABA, and 72 h after dark treatment the amount of stress-induced ABA approximated the prestressed values. Fluridone-treated plants failed to accumulate ABA under water stress. In addition to functionally active chloroplasts, well-developed and functional stomata are required for drought stress to elicit a rise in ABA.     

Sea urchin Hox genes: insights into the ancestral Hox cluster

We describe the Hox cluster in the radially symmetric sea urchin and compare our findings to what is known from clusters in bilaterally symmetric animals. Several Hox genes from the direct-developing sea urchin Heliocidaris erythrogramma are described. CHEF gel analysis shows that the Hox genes are clustered on a < or = 300 kilobase (kb) fragment of DNA, and only a single cluster is present, as in lower chordates and other nonvertebrate metazoans. Phylogenetic analyses of sea urchin, amphioxus, Drosophila, and selected vertebrate Hox genes confirm that the H. erythrogramma genes, and others previously cloned from other sea urchins, belong to anterior, central, and posterior groups. Despite their radial body plan and lack of cephalization, echinoderms retain at least one of the anterior group Hox genes, an orthologue of Hox3. The structure of the echinoderm Hox cluster suggests that the ancestral deuterostome had a Hox cluster more similar to the current chordate cluster than was expected Sea urchins have at least three Abd-B type genes, suggesting that Abd-B expansion began before the radiation of deuterostomes.      

An Evidenced-Based Scale of Disease Severity following Human Challenge with Enteroxigenic Escherichia coli

Background

Experimental human challenge models have played a major role in enhancing our understanding of infectious diseases. Primary outcomes have typically utilized overly simplistic outcomes that fail to entirely account for complex illness syndromes. We sought to characterize clinical outcomes associated with experimental infection with enterotoxigenic Escherichia coli (ETEC) and to develop a disease score.

Methods

Data were obtained from prior controlled human ETEC infection studies. Correlation and univariate regression across sign and symptom severity was performed. A multiple correspondence analysis was conducted. A 3-parameter disease score with construct validity was developed in an iterative fashion, compared to standard outcome definitions and applied to prior vaccine challenge trials.

Results

Data on 264 subjects receiving seven ETEC strains at doses from 1x10⁵ to 1x10¹⁰ cfu were used to construct a standardized dataset. The strongest observed correlation was between vomiting and nausea (r = 0.65); however, stool output was poorly correlated with subjective activity-impacting outcomes. Multiple correspondence analyses showed covariability in multiple signs and symptoms, with severity being the strongest factor corresponding across outcomes. The developed disease score performed well compared to standard outcome definitions and differentiated disease in vaccinated and unvaccinated subjects.

Conclusion

Frequency and volumetric definitions of diarrhea severity poorly characterize ETEC disease. These data support a disease severity score accounting for stool output and other clinical signs and symptoms. Such a score could serve as the basis for better field trial outcomes and gives an additional outcome measure to help select future vaccines that warrant expanded testing in pivotal pre-licensure trials.