Microfluidic-based 18F-labeling of biomolecules for immuno-positron emission tomography

Methods for tagging biomolecules with fluorine 18 as immuno-positron emission tomography (immunoPET) tracers require tedious optimization of radiolabeling conditions and can consume large amounts of scarce biomolecules. We describe an improved method using a digital microfluidic droplet generation (DMDG) chip, which provides computer-controlled metering and mixing of 18F tag, biomolecule, and buffer in defined ratios, allowing rapid scouting of reaction conditions in nanoliter volumes. The identified optimized conditions were then translated to bench-scale 18F labeling of a cancer-specific engineered antibody fragments, enabling microPET imaging of tumors in xenografted mice at 0.5 to 4 hours postinjection.     

Modulatory effects of ozone on THP-1 cells in response to SP-A stimulation

Ozone (O(3)), a major component of air pollution and a strong oxidizing agent, can lead to lung injury associated with edema, inflammation, and epithelial cell damage. The effects of O(3) on pulmonary immune cells have been studied in various in vivo and in vitro systems. We have shown previously that O(3) exposure of surfactant protein (SP)-A decreases its ability to modulate proinflammatory cytokine production by cells of monocyte/macrophage lineage (THP-1 cells). In this report, we exposed THP-1 cells and/or native SP-A obtained from bronchoalveolar lavage of patients with alveolar proteinosis to O(3) and studied cytokine production and NF-kappaB signaling. The results showed 1) exposure of THP-1 cells to O(3) significantly decreased their ability to express TNF-alpha in response to SP-A; TNF-alpha production, under these conditions, was still significantly higher than basal (unstimulated) levels in filtered air-exposed THP-1 cells; 2) exposure of both THP-1 cells and SP-A to O(3) did not result in any significant differences in TNF-alpha expression compared with basal levels; 3) O(3) exposure of SP-A resulted in a decreased ability of SP-A to activate the NF-kappaB pathway, as assessed by the lack of significant increase and decrease of the nuclear p65 subunit of NF-kappaB and cytoplasmic IkappaBalpha, respectively; and 4) O(3) exposure of THP-1 cells resulted in a decrease in SP-A-mediated THP-1 cell responsiveness, which did not seem to be mediated via the classic NF-kappaB pathway. These findings indicate that O(3) exposure may mediate its effect on macrophage function both directly and indirectly (via SP-A oxidation) and by involving different mechanisms.     

The permeability transition pore triggers Bax translocation to mitochondria during neuronal apoptosis

Cerebellar granule neurons (CGNs) require depolarization for their survival in culture. When deprived of this stimulus, CGNs die via an intrinsic apoptotic cascade involving Bim induction, Bax translocation, cytochrome c release, and caspase-9 and -3 activation. Opening of the mitochondrial permeability transition pore (mPTP) is an early event during intrinsic apoptosis; however, the precise role of mPTP opening in neuronal apoptosis is presently unclear. Here, we show that mPTP opening acts as an initiating event to stimulate Bax translocation to mitochondria. A C-terminal (alpha9 helix) GFP-Bax point mutant (T182A) that constitutively localizes to mitochondria circumvents the requirement for mPTP opening and is entirely sufficient to induce CGN apoptosis. Collectively, these data indicate that the major role of mPTP opening in CGN apoptosis is to trigger Bax translocation to mitochondria, ultimately leading to cytochrome c release and caspase activation.     

Contributions of the amygdala to emotion processing: from animal models to human behavior

Research on the neural systems underlying emotion in animal models over the past two decades has implicated the amygdala in fear and other emotional processes. This work stimulated interest in pursuing the brain mechanisms of emotion in humans. Here, we review research on the role of the amygdala in emotional processes in both animal models and humans. The review is not exhaustive, but it highlights five major research topics that illustrate parallel roles for the amygdala in humans and other animals, including implicit emotional learning and memory, emotional modulation of memory, emotional influences on attention and perception, emotion and social behavior, and emotion inhibition and regulation.     

Human CD69 associates with an N-terminal fragment of calreticulin at the cell surface

CD69 is thought to be a pluripotent signaling molecule expressed on the surface of a number of activated leukocytes including B, T, and NK cells, monocytes, neutrophils, and platelets. While some advances have been made regarding the mechanisms by which CD69 may participate in such diverse functions as cell aggregation, cellular cytotoxicity, and release of cytokines and inflammatory mediators, the most proximal links of signal initiation have not been identified. Our study has identified, by immunoprecipitation and direct protein sequencing (LC/MS/MS), binding of CD69 to an N-terminal protein fragment of calreticulin expressed on the cell surface of human PBMCs. Given the recently identified roles calreticulin plays in cell adhesion and angiogensis, the identification of CD69 binding directly to calreticulin may provide insights into mechanism(s) by which CD69 or other CD69 family members, i.e., LLT1 and AICL participates in such diverse functions.     

Mixed metal cyanide complexes derived from the CpCo(CN)3 anion

Reaction of the salt KCpCo(CN)₃ (1) with zinc iodide leads to the formation of a highly insoluble Co(III)/Zn(II) polymeric chain. However, pyridine was found to disrupt the aggregate that is formed to yield a crystalline trimetallic complex consisting of two CpCo(CN)₃ anions bridging a Zn(py)₄ dication through the cyanide ligands, [CpCo(CN)₂(μ-CN)]₂Zn(py)₄ (2). Along these same lines, reaction of CpCo(CN)₂PPh₃ (3) with AlCl₃ or CoCl₂ leads to the formation of a highly crystalline Co(II)/Co(III) diamond, [CpCo(PPh₃)(μ-CN)₂CoCl₂]₂ (4). Molecular structures of 1, 2, 3 and 4 were determined by single-crystal X-ray crystallography.     

Topology scanning and putative three-dimensional structure of the extracellular binding domains of the apical sodium-dependent bile acid transporter (SLC10A2)

The apical sodium-dependent bile acid transporter (ASBT, SLC10A2) facilitates the enterohepatic circulation of bile salts and plays a key role in cholesterol metabolism. The membrane topology of ASBT was initially scanned using a consensus topography analysis that predominantly predicts a seven transmembrane (TM) domain configuration adhering to the "positive inside" rule. Membrane topology was further evaluated and confirmed by N-glycosylation-scanning mutagenesis, as reporter sites inserted in the putative extracellular loops 1 and 3 were glycosylated. On the basis of a 7TM topology, we built a three-dimensional model of ASBT using an approach of homology-modeling and remote-threading techniques for the extramembranous domains using bacteriorhodopsin as a scaffold for membrane attachment points; the model was refined using energy minimizations and molecular dynamics simulations. Ramachandran scores and other geometric indicators show that the model is comparable in quality to the crystal structures of similar proteins. Simulated annealing and docking of cholic acid, a natural substrate, onto the protein surface revealed four distinct binding sites. Subsequent site-directed mutagenesis of the predicted binding domain further validated the model. This model agrees further with available data for a pathological mutation (P290S) because the mutant model after in silico mutagenesis loses the ability to bind bile acids.     

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.     

GEOGRAPHIC VARIATION IN THE SONGS OF NEOTROPICAL SINGING MICE: TESTING THE RELATIVE IMPORTANCE OF DRIFT AND LOCAL ADAPTATION

Patterns of geographic variation in communication systems can provide insight into the processes that drive phenotypic evolution. Although work in birds, anurans, and insects demonstrates that acoustic signals are sensitive to diverse selective and stochastic forces, processes that shape variation in mammalian vocalizations are poorly understood. We quantified geographic variation in the advertisement songs of sister species of singing mice, montane rodents with a unique mode of vocal communication. We tested three hypotheses to explain spatial variation in the song of the lower altitude species, Scotinomys teguina: selection for species recognition in sympatry with congener, S. xerampelinus, acoustic adaptation to different environments, and stochastic divergence. Mice were sampled at seven sites in Costa Rica and Panamá; genetic distances were estimated from mitochondrial control region sequences, between‐site differences in acoustic environment were estimated from climatic data. Acoustic, genetic and geographic distances were all highly correlated in S. teguina, suggesting that population differentiation in song is largely shaped by genetic drift. Contrasts between interspecific genetic‐acoustic distances were significantly greater than expectations derived from intraspecific contrasts, indicating accelerated evolution of species‐specific song. We propose that, although much intraspecific acoustic variation is effectively neutral, selection has been important in shaping species differences in song.     

The Evolution Pathway of Ammonia-Oxidizing Archaea Shaped by Major Geological Events

Primordial nitrification processes have been studied extensively using geochemical approaches, but the biological origination of nitrification remains unclear. Ammonia-oxidizing archaea (AOA) are widely distributed nitrifiers and implement the rate-limiting step in nitrification. They are hypothesized to have been important players in the global nitrogen cycle in Earth’s early history. We performed systematic phylogenomic and marker gene analyses to elucidate the diversification timeline of AOA evolution. Our results suggested that the AOA ancestor experienced terrestrial geothermal environments at ∼1,165 Ma (1,928–880 Ma), and gradually evolved into mesophilic soil at ∼652 Ma (767–554 Ma) before diversifying into marine settings at ∼509 Ma (629–412 Ma) and later into shallow and deep oceans, respectively. Corroborated by geochemical evidence and modeling, the timing of key diversification nodes can be linked to the global magmatism and glaciation associated with the assembly and breakup of the supercontinent Rodinia, and the later oxygenation of the deep ocean. Results of this integrated study shed light on the geological forces that may have shaped the evolutionary pathways of the AOA, which played an important role in the ancient global nitrogen cycle.