Real-time and Label-free Bio-sensing of Molecular Interactions by Surface Plasmon Resonance: A Laboratory Medicine Perspective

Radioactive, chromogenic, fluorescent and other labels have long provided the basis of detection systems for biomolecular interactions including immunoassays and receptor binding studies. However there has been unprecedented growth in a number of powerful label free biosensor technologies over the last decade. While largely at the proof-of-concept stage in terms of clinical applications, the development of more accessible platforms may see surface plasmon resonance (SPR) emerge as one of the most powerful optical detection platforms for the real-time monitoring of biomolecular interactions in a label-free environment.In this review, we provide an overview of SPR principles and current and future capabilities in a diagnostic context, including its application for monitoring a wide range of molecular markers of disease. The advantages and pitfalls of using SPR to study biomolecular interactions are discussed, with particular emphasis on its potential to differentiate subspecies of analytes and the inherent ability for quantitation through calibration-free concentration analysis (CFCA). In addition, recent advances in multiplex applications, high throughput arrays, miniaturisation, and enhancements using noble metal nanoparticles that promise unprecedented sensitivity to the level of single molecule detection, are discussed.In summary, while SPR is not a new technique, technological advances may see SPR quickly emerge as a highly powerful technology, enabling rapid and routine analysis of molecular interactions for a diverse range of targets, including those with clinical applicability. As the technology produces data quickly, in real-time and in a label-free environment, it may well have a significant presence in future developments in lab-on-a-chip technologies including point-of-care devices and personalised medicine.     

Gill morphometrics of the thresher sharks (Genus Alopias): Correlation of gill dimensions with aerobic demand and environmental oxygen

Gill morphometrics of the three thresher shark species (genus Alopias) were determined to examine how metabolism and habitat correlate with respiratory specialization for increased gas exchange. Thresher sharks have large gill surface areas, short water–blood barrier distances, and thin lamellae. Their large gill areas are derived from long total filament lengths and large lamellae, a morphometric configuration documented for other active elasmobranchs (i.e., lamnid sharks, Lamnidae) that augments respiratory surface area while limiting increases in branchial resistance to ventilatory flow. The bigeye thresher, Alopias superciliosus, which can experience prolonged exposure to hypoxia during diel vertical migrations, has the largest gill surface area documented for any elasmobranch species studied to date. The pelagic thresher shark, A. pelagicus, a warm‐water epi‐pelagic species, has a gill surface area comparable to that of the common thresher shark, A. vulpinus, despite the latter's expected higher aerobic requirements associated with regional endothermy. In addition, A. vulpinus has a significantly longer water–blood barrier distance than A. pelagicus and A. superciliosus, which likely reflects its cold, well‐oxygenated habitat relative to the two other Alopias species. In fast‐swimming fishes (such as A. vulpinus and A. pelagicus) cranial streamlining may impose morphological constraints on gill size. However, such constraints may be relaxed in hypoxia‐dwelling species (such as A. superciliosus) that are likely less dependent on streamlining and can therefore accommodate larger branchial chambers and gills. J. Morphol. 276:589–600, 2015. © 2015 Wiley Periodicals, Inc.     

Downregulation of mu opioid receptor by RNA interference in the ventral tegmental area reduces ethanol consumption in mice

Pharmacological and genetic studies have implicated the mu opioid receptor (MOR) in the regulation of ethanol intake in animal models and humans. Non-specific antagonists of opioid receptors have been shown to affect ethanol consumption when infused directly into the ventral tegmental area (VTA) of rats. However, administration of MOR-selective antagonists into the VTA has yielded mixed results. We used RNA interference (RNAi) to specifically decrease levels of MOR messenger RNA in the VTA of mice and examined the effect on ethanol consumption in a two-bottle choice paradigm. Mice were injected in the VTA with lentivirus expressing either a small hairpin RNA (shRNA) targeting MOR or a control shRNA. One week after virus injection, mice were examined for ethanol consumption in a two-bottle choice experiment with increasing concentrations of ethanol over the course of 1 month. Expression of an shRNA targeting MOR in the VTA led to a significant reduction in ethanol consumption. These results strengthen the hypothesis that MOR in the VTA is one of the key brain substrates mediating alcohol consumption. The RNAi combined with lentiviral delivery can be used successfully in brain to effect a sustained reduction in expression of specific genes for behavioral analysis.     

Rapid evolution of outer egg membrane proteins in the Drosophila melanogaster subgroup: a case of ecologically driven evolution of female reproductive traits

Although sexual selection has been predominantly used to explain the rapid evolution of sexual traits, eggs of oviparous organisms directly face both the challenges of sexual selection as well as natural selection (environmental challenges, survival in niches, etc.). Being the outermost membrane in most insect eggs, the chorion layer is the interface between the embryo and the environment, thereby serving to protect the egg. Adaptive ecological radiations such as divergence in ovipositional substrate usage and host-plant specializations can therefore influence the evolution of eggshell proteins. We can hypothesize that proteins localized on the outer eggshell may be affected to a greater degree by ecological challenges compared with inner eggshell proteins, and therefore, proteins localized in the outer eggshell (chorion membrane) may evolve differently (faster) than proteins localized in the inner egg membrane (vitelline membrane). We compared the evolutionary divergence of vitelline with chorion membrane proteins in species of the melanogaster subgroup and found that chorion proteins as a group are indeed evolving faster than vitelline membrane proteins. At least one vitelline membrane protein (Vm32E), specifically localized on the outer eggshell, is also evolving faster than other vitelline membrane proteins suggesting that all proteins localized on the outer eggshell may be evolving rapidly. We also found evidence that specific codons in chorion proteins cp15 and cp16 are evolving under positive selection. Polymorphism surveys of cp16 revealed inflated levels of divergence relative to polymorphism in specific regions of the gene, indicating that these regions are under strong selection. At the morphological level, we found notable difference in eggshell surface morphologies between specialist (Drosophila sechellia and Drosophila erecta) and generalist species of Drosophila. We do not know if any of the chorion proteins actually interact with spermatozoids, therefore leaving the possibility of rapid evolution through gametic interaction wide open. At this point, however, our results support previous suggestions that divergences in ecology, particularly, ovipositional substrate divergences may be a strong force driving the evolution of eggshell proteins.     

Dynamics of leaf area and nitrogen in the canopy of an annual herb, <Emphasis Type="Italic">Xanthium canadense</Emphasis>

We studied leaf area and nitrogen dynamics in the canopy of stands of an annual herb Xanthium canadense, grown at a high (HN)- and a low-nitorgen (LN) availability. Standing leaf area increased continuously through the vegetative growth period in the LN stand, or leveled off in the later stage in the HN stand. When scaled against standing leaf area, both production and loss rates of leaf area increased but with different patterns: the production rate was retarded, while the loss rate was accelerated, implying an upper limit of standing leaf area of the canopy. The rate of leaf-area production was higher in the HN than in the LN stand, which was caused by the higher rate of leaf production per standing leaf area as well as the greater standing leaf area in the HN stand. Although the rate of leaf-area loss was higher in the HN than in the LN stand, it was not significantly different between the two stands when compared at a common standing leaf area, suggesting involvement of light climate in determination of the leaf-loss rate. On the other hand, the rate of leaf-area loss was positively correlated with nitrogen demand for leaf area development across the two stands, suggesting that leaf loss was caused by retranslocation of nitrogen for construction of new leaves. A simple simulation model of leaf and nitrogen dynamics in the canopy showed that, at steady state, where the rate of leaf-area loss becomes equal to the production rate, the standing leaf area was still greater in the HN than in the LN stand. Similarly, when the uptake and loss of nitrogen are equilibrated, the standing nitrogen was greater in the HN than in the LN stand. These results suggest that leaf-area production is strongly controlled by nitrogen availability, while both nitrogen and light climate determine leaf-loss rates in the canopy.     

Investigating the form-function interface in African apes: Relationships between principal moments of area and positional behaviors in femoral and humeral diaphyses

Investigations of cross-sectional geometry in nonhuman primate limb bones typically attribute shape ratios to qualitative behavioral characterizations, e.g., leaper, slow climber, brachiator, or terrestrial vs. arboreal quadruped. Quantitative positional behavioral data, however, have yet to be used in a rigorous evaluation of such shape-behavior connections. African apes represent an ideal population for such an investigation because their relatedness minimizes phylogenetic inertia, they exhibit diverse behavioral repertoires, and their locomotor behaviors are known from multiple studies. Cross-sectional data from femoral and humeral diaphyses were collected for 222 wild-shot specimens, encompassing Pan paniscus and all commonly recognized African ape subspecies. Digital representations of diaphyseal cross sections were acquired via computed tomography at three locations per diaphysis. Locomotor behaviors were pooled broadly into arboreal and terrestrial categories, then partitioned into quadrupedal walking, quadrumanous climbing, scrambling, and suspensory categories. Sex-specific taxonomic differences in ratios of principal moments of area (PMA) were statistically significant more often in the femoral diaphysis than the humeral diaphysis. While it appears difficult to relate a measure of shape (e.g., PMA ratio) to individual locomotor modes, general locomotor differences (e.g., percentage arboreal vs. terrestrial locomotion) are discerned more easily. As percentage of arboreal locomotion for a group increases, average cross sections appear more circular. Associations between PMA ratio and specific locomotor behaviors are less straightforward. Individual behaviors that integrate eccentric limb positions (e.g., arboreal scrambling) may not engender more circular cross sections than behaviors that incorporate repetitive sagittal movements (e.g., quadrupedal walking) in a straightforward manner.     

地表火干扰时间序列上樟子松林竞争强度的变化

林火是樟子松林重要的干扰因子,通过空间代替时间对地表火干扰时间序列上的天然樟子松林进行全林木定位调查,以单木竞争模型分析林分不同组分的竞争强度,通过Kriging法估计了火后林下更新幼树的密度。结果表明,存活林木各组分的竞争强度均显著地降低;地表火干扰12 a后存活林木各组分的竞争强度均比火后1 a存活林木相应各组分的竞争强度显著地降低;另外,火后更新幼树也更多地趋于地表火干扰形成的林隙中。因此,地表火干扰时间序列上林木竞争强度显著的持续降低,火后存活个体将有更充足的可利用资源和环境,林火成为樟子松林发育演替的重要驱动力。     

Evaluating the efficacy of planktonic foraminifer calcite δO data for sea surface temperature reconstruction for the Late Miocene

This study examines the efficacy of published δ¹⁸O data from the calcite of Late Miocene surface dwelling planktonic foraminifer shells, for sea surface temperature estimates for the pre-Quaternary. The data are from 33 Late Miocene (Messinian) marine sites from a modern latitudinal gradient of 64°N to 48°S. They give estimates of SSTs in the tropics/subtropics (to 30°N and S) that are mostly cooler than present. Possible causes of this temperature discrepancy are ecological factors (e.g. calcification of shells at levels below the ocean mixed layer), taphonomic effects (e.g. diagenesis or dissolution), inaccurate estimation of Late Miocene seawater oxygen isotope composition, or a real Late Miocene cool climate. The scale of apparent cooling in the tropics suggests that the SST signal of the foraminifer calcite has been reset, at least in part, by early diagenetic calcite with higher δ¹⁸O, formed in the foraminifer shells in cool sea bottom pore waters, probably coupled with the effects of calcite formed below the mixed layer during the life of the foraminifera. This hypothesis is supported by the markedly cooler SST estimates from low latitudes—in some cases more than 9 °C cooler than present—where the gradients of temperature and the δ¹⁸O composition of seawater between sea surface and sea bottom are most marked, and where ocean surface stratification is high. At higher latitudes, particularly N and S of 30°, the temperature signal is still cooler, though maximum temperature estimates overlap with modern SSTs N and S of 40°. Comparison of SST estimates for the Late Miocene from alkenone unsaturation analysis from the eastern tropical Atlantic at Ocean Drilling Program (ODP) Site 958—which suggest a warmer sea surface by 2-4 °C, with estimates from oxygen isotopes at Deep Sea Drilling Project (DSDP) Site 366 and ODP Site 959, indicating cooler than present SSTs, also suggest a significant impact on the δ¹⁸O signal. Nevertheless, much of the original SST variation is clearly preserved in the primary calcite formed in the mixed layer, and records secular and temporal oceanographic changes at the sea surface, such as movement of the Antarctic Polar Front in the Southern Ocean. Cooler SSTs in the tropics and sub-tropics are also consistent with the Late Miocene latitude reduction in the coral reef belt and with interrupted reef growth on the Queensland Plateau of eastern Australia, though it is not possible to quantify absolute SSTs with the existing oxygen isotope data. Reconstruction of an accurate global SST dataset for Neogene time-slices from the existing published DSDP/ODP isotope data, for use in general circulation models, may require a detailed re-assessment of taphonomy at many sites.     

The role of the catalytic domain of E. coli GluRS in tRNA discrimination

Discrimination of tRNA^Gln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA^Gln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA^Gln. Our results demonstrate that in addition to the anticodon-binding domain, tRNA^Gln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.     

Docking of Antizyme to Ornithine Decarboxylase and Antizyme Inhibitor using Experimental Mutant and Double-Mutant Cycle Data

Antizyme (Az) is a highly conserved key regulatory protein bearing a major role in regulating polyamine levels in the cell. It has the ability to bind and inhibit ornithine decarboxylase (ODC), targeting it for degradation. Az inhibitor (AzI) impairs the activity of Az. In this study, we mapped the binding sites of ODC and AzI on Az using Ala scan mutagenesis and generated models of the two complexes by constrained computational docking. In order to scan a large number of mutants in a short time, we developed a workflow combining high-throughput mutagenesis, small-scale parallel partial purification of His-tagged proteins and their immobilization on a tris-nitrilotriacetic-acid-coated surface plasmon resonance chip. This combination of techniques resulted in a significant reduction in time for production and measurement of large numbers of mutant proteins. The data-driven docking results suggest that both proteins occupy the same binding site on Az, with Az binding within a large groove in AzI and ODC. However, single-mutant data provide information concerning the location of the binding sites only, not on their relative orientations. Therefore, we generated a large number of double-mutant cycles between residues on Az and ODC and used the resulting interaction energies to restrict docking. The model of the complex is well defined and accounts for the mutant data generated here, and previously determined biochemical data for this system. Insights on the structure and function of the complexes, as well as general aspects of the method, are discussed.