The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules

Every method used to quantify biomolecular interactions has its own strengths and limitations. To quantify protein‐DNA binding affinities, nitrocellulose filter binding assays with ³²P‐labeled DNA quantify K_d values from 10^?12 to 10^?8 M but have several technical limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors association and dissociation of a soluble macromolecule to an immobilized species; the ratio k_off/k_on determines K_d. However, for lactose repressor protein (LacI) and an engineered repressor protein (“LLhF”) binding immobilized DNA, complicated kinetic curves precluded this analysis. Thus, we determined whether the amplitude of the BLI signal at equilibrium related linearly to the fraction of protein bound to DNA. A key question was the effective concentration of immobilized DNA. Equilibrium titration experiments with DNA concentrations below K_d (equilibrium binding regime) must be analyzed differently than those with DNA near or above K_d (stoichiometric binding regime). For ForteBio streptavidin tips, the most frequent effective DNA concentration was ~2 × 10^?9 M. Although variation occurred among different lots of sensor tips, binding events with K_d ≥ 10^?8 M should reliably be in the equilibrium binding regime. We also observed effects from multi‐valent interactions: Tetrameric LacI bound two immobilized DNAs whereas dimeric LLhF did not. We next used BLI to quantify the amount of inducer sugars required to allosterically diminish protein‐DNA binding and to assess the affinity of fructose‐1‐kinase for the DNA‐LLhF complex. Overall, when experimental design corresponded with appropriate data interpretation, BLI was convenient and reliable for monitoring equilibrium titrations and thereby quantifying a variety of binding interactions.     

Mpdz expression in the caudolateral substantia nigra pars reticulata is crucially involved in alcohol withdrawal

Association studies implicate the multiple PDZ domain protein (MUPP1/MPDZ) gene in risk for alcoholism in humans and alcohol withdrawal in mice. Although manipulation of the Mpdz gene by homologous recombination and bacterial artificial chromosome transgenesis has suggested that its expression affects alcohol withdrawal risk, the potential confounding effects of linked genes and developmental compensation currently limit interpretation. Here, using RNA interference (RNAi), we directly test the impact of Mpdz expression on alcohol withdrawal severity and provide brain regional mechanistic information. Lentiviral‐mediated delivery of Mpdz short hairpin RNA (shRNA) to the caudolateral substantia nigra pars reticulata (clSNr) significantly reduces Mpdz expression and exacerbates alcohol withdrawal convulsions compared with control mice that delivered a scrambled shRNA. Neither baseline nor pentylenetetrazol‐enhanced convulsions differed between Mpdz shRNA and control animals, indicating Mpdz expression in the clSNr does not generally affect seizure susceptibility. To our knowledge, these represent the first in vivo Mpdz RNAi analyses, and provide the first direct evidence that Mpdz expression impacts behavior. Our results confirm that Mpdz is a quantitative trait gene for alcohol withdrawal and demonstrate that its expression in the clSNr is crucially involved in risk for alcohol withdrawal.     

Cloning and characterization of an adenosine kinase from Physcomitrella involved in cytokinin metabolism

Adenosine kinase (adk) from the moss Physcomitrella patens (Hedw.) B.S.G. was cloned from a cDNA library by functional complementation of an Escherichia coli purine auxotrophic strain. The length of the entire cDNA clone was 1175 bp with an open reading frame coding for a protein with a predicted molecular weight of 37.3 kDa. Southern analysis indicated the presence of a single adenosine kinase gene within the Physcomitrella genome. The deduced amino acid sequence had a 52% identity with the human adenosine kinase. The transfer of phosphate from ATP to adenosine resulting in AMP, as well as the phosphorylation of the cytokinin, isopentenyladenosine, to isopentenyladenosine monophosphate, was shown by in vitro enzyme assays using crude extracts from E. coli mutants expressing the adk cDNA clone and from Physcomitrella chloronemal tissue. Results from in vivo feeding of chloronemal tissue with tritiated isopentenyladenosine suggest that adenosine kinase plays an important role in the conversion of cytokinins towards their nucleotides in Physcomitrella.     

The metabolic requirements for transcellular movement and secretion of collagen.

Cultures of chick tendon fibroblasts were capable of normal ATP production and protein synthetic activity even though the normally high rate of glycolysis was markedly reduced by substitution of pyruvate for glucose. Iodoacetate and 2-deoxyglucose reduced ATP levels and protein synthesis even in the presence of pyruvate. Under these conditions, both inhibitors were shown to have effects on the energy metabolism of cells which were apparently unrelated to an inhibition of glycolysis. Selective inhibition of either glycolysis, by incubation in glucose-free medium, or of oxidative phosphorylation, by incubation with an uncoupler, was shown to have little effect on cellular ATP levels or intracellular transport and secretion of collagen. However, inhibition of both glycolysis and oxidative phosphorylation resulted in decreased cellular ATP levels and an inhibition of collagen secretion. This effect was not due to a requirement for continued protein synthesis, since inhibition of protein synthesis with cycloheximide or puromycin had little effect on collagen secretion. The ATP requirement for intracellular transport and secretion is discussed in relation to the secretory pathway for collagen.     

Study of the electrical conductivity of biobased carbonaceous powder materials under moderate pressure for the application as electrode materials in energy storage technologies

This study focusses on the assessment of the electrical conductivity (EC) of biobased electrode materials for the application in energy storage devices and presents a simple and reproducible method to measure the EC of carbonaceous powders under moderate pressure (10–50 N). Based on the pyrolysis of corncob at three different temperatures (600, 800, and 900°C) and further treatments of the biochar obtained at 600°C, 11 different carbonaceous powder materials were produced including biochars, activated carbons, and composites. Composite materials were obtained by adding either metal oxide (RuO₂ or Fe₃O₄) in different proportions or additives which are commonly used in electrode production (5 wt% binder and 15 wt% conductive additive). Furthermore, one physically activated commercial AC based on peat with a known EC of 33 S/m was treated with additives and used as a reference. For all materials, an increase of applied pressure resulted in higher EC values due to closer particle contact. The comparison of two methods (with and without preload) showed that a prepelletization of the samples is not necessary to obtain reliable results. By analyzing the obtained EC values while taking mechanical and physicochemical properties into account, it could be shown that a high carbonization temperature and high specific surface area favor the increase of EC. Furthermore, certain proportions of metal oxides lead to an improvement of EC (40 wt% RuO₂, 10 wt% Fe₃O₄), while the treatment with additives leads to a decrease of EC. The EC values among all samples varied between 0.8 S/m (biochar) and 408 S/m (AC/RuO₂ composite) at the highest pressure level (637 kPa). Thus, promising biobased electrode materials for environmentally friendly energy storage technologies are presented with the aim of contributing to the establishment of a biobased resource and product platform for bioeconomy.     

On an attempt to treat primary and secondary osteoporosis with human growth hormone.

Three male patients with severe osteoporosis were treated with human growth hormone. One of them had a primary osteoporosis, the two others osteogenesis imperfecta. The duration of therapy was 8 to 15 months and average doses per day were 1.45 to 2.3 mg. While clinical and 47calcium kinetic data failed to prove marked influences of the treatment, histomorphometry of bone biopsies showed indisputable changes. There was an increase of periosteal new bone formation as well as of intracortical bone resorption, while at the same time the relative activity of osteoblasts on endosteal surfaces showed a significant increase.     

An Evaluation of Bilateral Breast Carcinoma: Simultaneous and Sequential Lesions

The detection of bilateral breast carcinoma, simultaneous or nonsimultaneous, appears to be increasing. Undoubtedly a major factor in this is the increased use of mammography and xeromammography. In a 21-year period at Saint John''s Hospital and Health Center, 1,189 cases of primary breast carcinoma were treated, of which 34 (2.9 percent) were bilateral. However, from 1972 to 1974 the incidence has risen from 1.9 to 10.8 percent, with a simultaneous rise in numbers of xeromammograms made. Xeromammography should be done routinely in patients in whom carcinoma of the breast is suspected. Its use should also be an integral part of the follow-up in patients in whom mastectomy has been carried out previously.     

Long‐lived larch clones may conserve adaptations that could restrict treeline migration in northern Siberia

The occurrence of refugia beyond the arctic treeline and genetic adaptation therein play a crucial role of largely unknown effect size. While refugia have potential for rapidly colonizing the tundra under global warming, the taxa may be maladapted to the new environmental conditions. Understanding the genetic composition and age of refugia is thus crucial for predicting any migration response. Here, we genotype 194 larch individuals from an ~1.8 km² area in northcentral Siberia on the southern Taimyr Peninsula by applying an assay of 16 nuclear microsatellite markers. For estimating the age of clonal individuals, we counted tree rings at sections along branches to establish a lateral growth rate that was then combined with geographic distance. Findings reveal that the predominant reproduction type is clonal (58.76%) by short distance spreading of ramets. One outlier of clones 1 km apart could have been dispersed by reindeer. In clonal groups and within individuals, we find that somatic mutations accumulate with geographic distance. Clonal groups of two or more individuals are observed. Clonal age estimates regularly suggest individuals as old as 2,200 years, which coincides with a major environmental change that forced a treeline retreat in the region. We conclude that individuals with clonal growth mode were naturally selected as it lowers the likely risk of extinction under a harsh environment. We discuss this legacy from the past that might now be a maladaptation and hinder expansion under currently strongly increasing temperatures.     

Formation and Degradation Pathways of Intermediate Products Formed during the Hydropyrolysis of Glucose as a Model Substance for Wet Biomass in a Tubular Reactor

In this study, glucose as a model substance for cellulose is pyrolyzed in supercritical water. The experiments are conducted in a continuously operated tubular reactor. From the usage of model substances, key information on the degradation pathway of biomass in supercritical water can be obtained. With this knowledge, it is tried to optimize a new method for gasification of wet biomass considering high yields of hydrogen and methane and also the suppressing of tar and char formation. The gaseous products mainly contain hydrogen, carbon dioxide, methane and a small amount of carbon monoxide. The effect of experimental conditions, such as pressure, temperature and reaction time, on the degradation of glucose is investigated in the experiments. The qualitative and quantitative composition of the gas and liquid phases formed are determined. The results show that only the amount of phenols increases with increasing temperature in the liquid phase. No complete gasification of glucose is achieved in the studied temperature range between 400 °C and 500 °C. The addition of alkali salts leads to a higher gas generation and to a decrease in carbon monoxide concentration via water‐gas‐shift reaction. A lower furfural concentration is obtained in the presence of KHCO₃. Furthermore, this study shows that there is a wide conformity between the results of real and model biomass. A simplified scheme for glucose degradation is also presented with the help of the results found.