Dendrochronological analysis of white oak growth patterns across a topographic moisture gradient in southern Ohio

Moisture availability is a key factor that influences white oak (Quercus alba L.) growth and wood production. In unglaciated eastern North America, available soil moisture varies greatly along topographic and edaphic gradients. This study was aimed at determining the effects of soil moisture variability and macroclimate on white oak growth in mixed-oak forests of southern Ohio. Using accurately dated and measured tree rings, we analyzed 119 white oaks growing across an integrated moisture index (IMI), a computer-generated GIS model that simultaneously combines topographic and edaphic features into a moisture index scale. Growth trends varied considerably across the IMI, with trees in mesic sites exhibiting patterns much different from those in either xeric or intermediate sites. BAI growth and biomass increments were higher for trees growing in the intermediate and mesic sites than those from the xeric sites. Correlation and response function analyses, and redundancy analysis revealed significant relations between ring-width indices and climate, with current year May–July PDSI, precipitation and temperature as the most important correlates of white oak growth. Additionally, climatic influences on growth rate were variable across the IMI; trees in xeric sites showed much greater coefficients relative to those from the intermediate and mesic sites. Despite these differences, xeric and intermediate trees exhibited similar growth patterns. The present results provide further evidence of the usefulness of the IMI for identifying and comparing white oak growth patterns across the complex, dissected landscape of southern Ohio.     

Online flow cytometry for monitoring apoptosis in mammalian cell cultures as an application for process analytical technology

Apoptosis is the main driver of cell death in bioreactor suspension cell cultures during the production of biopharmaceuticals from animal cell lines. It is known that apoptosis also has an effect on the quality and quantity of the expressed recombinant protein. This has raised the importance of studying apoptosis for implementing culture optimization strategies. The work here describes a novel approach to obtain near real time data on proportion of viable, early apoptotic, late apoptotic and necrotic cell populations in a suspension CHO culture using automated sample preparation in conjunction with flow cytometry. The resultant online flow cytometry data can track the progression of apoptotic events in culture, aligning with analogous manual methodologies and giving similar results. The obtained near-real time apoptosis data are a significant improvement in monitoring capabilities and can lead to improved control strategies and research data on complex biological systems in bioreactor cultures in both academic and industrial settings focused on process analytical technology applications.     

Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.     

Effects of T142 phosphorylation and mutation R145G on the interaction of the inhibitory region of human cardiac troponin I with the C-domain of human cardiac troponin C

Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex, and its interaction with cardiac troponin C (cTnC) plays a critical role in transmitting the Ca(2+) signal to the other myofilament proteins in heart muscle contraction. The switch between contraction and relaxation involves a movement of the inhibitory region of cTnI (cIp) from cTnC to actin-tropomyosin. This region of cTnI is prone to missense mutations in heart disease, and a specific mutation, R145G, has been associated with familial hypertrophic cardiomyopathy. It also contains the unique cardiac PKC phosphorylation site at residue T142. To determine the structural consequences of the mutation R145G and the T142 phosphorylation on the interaction of cIp with cTnC, we have utilized 2D [(1)H, (15)N]-HSQC NMR spectroscopy to monitor the binding of native cIp, cIp-R (R145G), and cIp-P (phosphorylated T142), respectively, to the Ca(2+)-saturated C-domain of cTnC (cCTnC.2Ca(2+)). We also report a strategy for cloning, expression, and purification of cTnI peptide, and both synthetic and recombinant peptides are used in this study. NMR chemical shift mapping indicates that the binding epitope of cIp on cCTnC.2Ca(2+) is not greatly affected, but the affinity is reduced by approximately 14-fold by the T142 phosphorylation and approximately 4-fold by the mutation R145G, respectively. This suggests that these modifications of cIp have an adverse effect on the binding of cIp to cCTnC.2Ca(2+). These perturbations may correlate with the impairment or loss of cTnI function in heart muscle contraction.     

A proteomic study of cMyc improvement of CHO culture

Background  

The biopharmaceutical industry requires cell lines to have an optimal proliferation rate and a high integral viable cell number resulting in a maximum volumetric recombinant protein product titre. Nutrient feeding has been shown to boost cell number and productivity in fed-batch culture, but cell line engineering is another route one may take to increase these parameters in the bioreactor. The use of CHO-K1 cells with a c-myc plasmid allowing for over-expressing c-Myc (designated cMycCHO) gives a higher integral viable cell number. In this study the differential protein expression in cMycCHO is investigated using two-dimensional gel electrophoresis (2-DE) followed by image analysis to determine the extent of the effect c-Myc has on the cell and the proteins involved to give the new phenotype.     

Determination of hepatitis delta virus ribozyme N(–1) nucleobase and functional group specificity using internal competition kinetics

Biological catalysis involves interactions distant from the site of chemistry that can position the substrate for reaction. Catalysis of RNA 2′-O-transphosphorylation by the hepatitis delta virus (HDV) ribozyme is sensitive to the identity of the N(–1) nucleotide flanking the reactive phosphoryl group. However, the interactions that affect the conformation of this position, and in turn the 2′O nucleophile, are unclear. Here, we describe the application of multiple substrate internal competition kinetic analyses to understand how the N(–1) nucleobase contributes to HDV catalysis and test the utility of this approach for RNA structure–function studies. Internal competition reactions containing all four substrate sequence variants at the N(–1) position in reactions using ribozyme active site mutations at A77 and A78 were used to test a proposed base-pairing interaction. Mutants A78U, A78G, and A79G retain significant catalytic activity but do not alter the specificity for the N(–1) nucleobase. Effects of nucleobase analog substitutions at N(–1) indicate that U is preferred due to the ability to donate an H-bond in the Watson–Crick face and avoid minor groove steric clash. The results provide information essential for evaluating models of the HDV active site and illustrate multiple substrate kinetic analyses as a practical approach for characterizing structure–function relationships in RNA reactions.     

Phylogeography, genetic structure, and gene flow in the endemic freshwater shrimp Palaemonetes suttkusi from Cuatro Ci��negas, Mexico

The valley of Cuatro Ciénegas in Mexico has the highest degree of local endemicity of any habitat in North America. A large number of endemic aquatic species occur both in the Cuatro Ciénegas basin and in the upper parts of the Río Salado de los Nadadores drainage, located immediately to the east of the valley. No natural surface flow occurs between these basins but artificial canals connect both areas. It is not clear whether the sharing of endemics between the Cuatro Ciénegas basin and the Río Salado is due to migration through canals. We conducted a phylogeographic study of mitochondrial haplotypes of the endemic shrimp Palaemonetes suttkusi to determine the evolutionary distinctiveness of the population found in Río Salado. We discovered that P. suttkusi likely has occurred naturally in both basins well into the distant past. Based on molecular clock analyses of the COI gene, the populations in the Río Salado and much of the Cuatro Ciénegas basin likely began diverging from each other between 1.9 and 11.2 Myr ago. The general levels of divergence are substantial but our results suggest there has also likely been recent gene flow between these basins. This is consistent with migration through human-made canals, but also consistent with the occurrence of natural gene flow during intermittent wet periods in the past million years. We also found significant differentiation of the Pozas Azules area from the rest of the Cuatro Ciénegas basin, a finding that is concordant with several phylogeographic studies on other aquatic endemics in Cuatro Ciénegas. We recommend that the upper parts of the Río Salado, the Pozas Azules area, and the rest of the Cuatro Ciénegas basin should each be considered independent evolutionarily significant units for conservation, and that migration of species through human-made canals should be monitored and controlled.     

Many-to-One Mapping of Form to Function: A General Principle in Organismal Design?

We introduce the concept of many-to-one mapping of form to functionand suggest that this emergent property of complex systems promotesthe evolution of physiological diversity. Our work has focusedon a 4-bar linkage found in labrid fish jaws that transmitsmuscular force and motion from the lower jaw to skeletal elementsin the upper jaws. Many different 4-bar shapes produce the sameamount of output rotation in the upper jaw per degree of lowerjaw rotation, a mechanical property termed Maxillary KT. Weillustrate three consequences of many-to-one mapping of 4-barshape to Maxillary KT. First, many-to-one mapping can partiallydecouple morphological and mechanical diversity within clades.We found with simulations of 4-bars evolving on phylogeniesof 500 taxa that morphological and mechanical diversity wereonly loosely correlated (R² = 0.25). Second, redundant mappingpermits the simultaneous optimization of more than one mechanicalproperty of the 4-bar. Labrid fishes have capitalized on thisflexibility, as illustrated by several species that have MaxillaryKT = 0.8 but have different values of a second property, NasalKT. Finally, many-to-one mapping may increase the influenceof historical factors in determining the evolution of morphology.Using a genetic model of 4-bar evolution we exerted convergentselection on three different starting 4-bar shapes and foundthat mechanical convergence only created morphological convergencein simulations where the starting forms were similar. Many-to-onemapping is widespread in physiological systems and operatesat levels ranging from the redundant mapping of genotypes tophenotypes, up to the morphological basis of whole-organismperformance. This phenomenon may be involved in the uneven distributionof functional diversity seen among animal lineages.