<Emphasis Type="Italic">Miscanthus × Giganteus</Emphasis> Growth and Nutrient Export on 22 Producer Fields

On-farm assessments of Miscanthus × giganteus growth and nutrient export across a wide range of management and environmental conditions are needed to determine and model how this crop performs and where it should be placed on the landscape. Therefore, Miscanthus growth and nutrient concentration and nutrient export at harvest were monitored during 2014 and 2015 at several landscape positions within 22 commercial production fields in central and southwestern Missouri and northeast Arkansas. Miscanthus shoot density and/or yield were best when it was grown: (i) following pasture converted to annual row crops or following row crops, (ii) on soils with colluvium parent material, (iii) on north-facing backslopes or footslopes, (iv) on soils with medium to fine texture, and (v) on well-drained/high runoff/low available water soils. Factors influencing nutrient concentrations varied by nutrient, but all concentrations consistently decreased as stands matured and most were more influenced by weather than were yield or nutrient export. Most effects on nutrient export were similar to effects on yield, but some nutrient exports were also influenced by manure history and weather conditions. Overall, cropping history prior to Miscanthus, landscape position, and soil properties such as parent material, soil textural class, and drainage class had the largest influence on Miscanthus growth and nutrient concentrations and exports. Weather conditions and inferior soils did not strongly influence Miscanthus production, but excessive soil moisture caused by various soil and weather factors often limited its growth. Thus, Miscanthus may be especially well-suited following annual crops on erosion-prone soils that drain well and have slope. These results will assist with the strategic cultivation of Miscanthus on Midwest landscapes.     

Calmodulin-binding proteins: visualization by 125I-calmodulin overlay on blots quenched with Tween 20 or bovine serum albumin and poly(ethylene oxide)

To streamline detection of calmodulin-binding proteins, blotting techniques for the electrophoretic transfer of proteins onto nitrocellulose filters, followed by overlay with 125I-calmodulin, have been adapted. Autoradiography of the 125I-calmodulin-labeled blots allows the identification and quantitation of proteins that possess affinity for calmodulin. Five protocols for suppressing nonspecific binding and for enhancing specific interactions of 125I-calmodulin with electrophoretically separated proteins were investigated. Tween 20 and bovine serum albumin alone, as well as combinations of bovine serum albumin and poly(ethylene oxide) or hemoglobin and gelatin, were evaluated as quenching and enhancing agents. Tween 20 proved highly effective for quenching nonspecific binding and for enhancing specific 125I-calmodulin binding of a 61,000-Mr rat brain protein, which was only faintly observed on blots quenched with proteins alone. However, Tween 20 dissociated 50% of 68,000-Mr proteins and 80% of 21,000-Mr 125I-labeled protein standards from the nitrocellulose filter. An alternative, the combination of bovine serum albumin followed by incubation with 15,000- to 20,000-Mr poly(ethylene oxide), proved satisfactory for the recovery of 61,000-Mr calmodulin-binding activity and for the detection of calmodulin-binding peptides (50,000 to 14,000 Mr) produced by limited proteolysis of rat brain 51,000-Mr calmodulin-binding protein. These blotting procedures for detection of calmodulin-binding proteins are compatible with a variety of one-dimensional and two-dimensional electrophoresis systems, including a two-dimensional electrophoresis system utilizing urea and sodium dodecyl sulfate in the first dimension and nonurea sodium dodecyl sulfate electrophoresis in the second, a system which proved useful for resolving calmodulin-binding proteins displaying anomalous electrophoretic migration in the presence of urea.     

Growth inhibition of Streptococcus mutans and Leuconostoc mesenteroides by sodium fluoride and ionic tin.

Sodium fluoride caused inhibition of growth rate and growth levels of Streptococcus mutans with glucose as the primary energy and carbon source. Stannous fluoride increased growth lag nad caused a much greater inhibition of growth rate than did sodium fluoride. Neither compound was found to be bactericidal when culture viability was measured after 6 days of incubation. Leuconostoc mesenteroides, which lacks a phosphotransferase system for sugar transport, showed less inhibition of growth rate with both inhibitors than did S. mutans, which possesses a phosphotransferase system. Metabolism of glucose or lactose which requires enolase activity shoed sodium fluoride inhibition, whereas metabolism of arginine or pyruvate does not involve enolase activity and showed no inhibition of growth.     

Regulation of midline development by antagonism of lefty and nodal signaling.

The embryonic midline is crucial for the development of embryonic pattern including bilateral symmetry and left-right asymmetry. In zebrafish, lefty1 (lft1) and lefty2 (lft2) have distinct midline expression domains along the anteroposterior axis that overlap with the expression patterns of the nodal-related genes cyclops and squint. Altered expression patterns of lft1 and lft2 in zebrafish mutants that affect midline development suggests different upstream pathways regulate each expression domain. Ectopic expression analysis demonstrates that a balance of lefty and cyclops signaling is required for normal mesendoderm patterning and goosecoid, no tail and pitx2 expression. In late somite-stage embryos, lft1 and lft2 are expressed asymmetrically in the left diencephalon and left lateral plate respectively, suggesting an additional role in laterality development. A model is proposed by which the vertebrate midline, and thus bilateral symmetry, is established and maintained by antagonistic interactions among co-expressed members of the lefty and nodal subfamilies of TGF-beta signaling molecules.     

Studies of self-inactivation of bovine seminal fluid NAD glycohydrolase

Bovine seminal fluid NAD glycohydrolase (NADase) was observed to be rapidly inactivated during catalytic hydrolysis of the substrate NAD. The first-order rate constant for the self-inactivation process was independent of enzyme concentration. The enzyme self-inactivation was a turnover-related process and the number of moles of NAD hydrolyzed required for inactivation was proportional to the enzyme concentration. A number of dinucleotides serving as substrates for the enzyme also promoted self-inactivation. The self-inactivation was an irreversible process having a different rate-limiting step from NAD hydrolysis and was not related to the reversible binding of products and substrate-competitive inhibitors. Modification of arginine residues of the enzyme resulted in the loss of NAD hydrolase activity with no differential effect on the self-inactivation process.     

Mutational Profiling Can Establish Clonal or Independent Origin in Synchronous Bilateral Breast and Other Tumors

Background

Synchronous tumors can be independent primary tumors or a primary-metastatic (clonal) pair, which may have clinical implications. Mutational profiling of tumor DNA is increasingly common in the clinic. We investigated whether mutational profiling can distinguish independent from clonal tumors in breast and other cancers, using a carefully defined test based on the Clonal Likelihood Score (CLS = 100 x # shared high confidence (HC) mutations/ # total HC mutations).

Methods

Statistical properties of a formal test using the CLS were investigated. A high CLS is evidence in favor of clonality; the test is implemented as a one-sided binomial test of proportions. Test parameters were empirically determined using 16,422 independent breast tumor pairs and 15 primary-metastatic tumor pairs from 10 cancer types using The Cancer Genome Atlas.

Results

We validated performance of the test with its established parameters, using five published data sets comprising 15,758 known independent tumor pairs (maximum CLS = 4.1%, minimum p-value = 0.48) and 283 known tumor clonal pairs (minimum CLS 13%, maximum p-value <0.01), across renal cell, testicular, and colorectal cancer. The CLS test correctly classified all validation samples but one, which it appears may have been incorrectly classified in the published data. As proof-of-concept we then applied the CLS test to two new cases of invasive synchronous bilateral breast cancer at our institution, each with one hormone receptor positive (ER+/PR+/HER2-) lobular and one triple negative ductal carcinoma. High confidence mutations were identified by exome sequencing and results were validated using deep targeted sequencing. The first tumor pair had CLS of 81% (p-value < 10–15), supporting clonality. In the second pair, no common mutations of 184 variants were validated (p-value >0.99), supporting independence. A plausible molecular mechanism for the shift from hormone receptor positive to triple negative was identified in the clonal pair.

Conclusion

We have developed the statistical properties of a carefully defined Clonal Likelihood Score test from mutational profiling of tumor DNA. Under identified conditions, the test appears to reliably distinguish between synchronous tumors of clonal and of independent origin in several cancer types. This approach may have scientific and clinical utility.