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1.  Microscopic Visualization of Metabotropic Glutamate Receptors on the Surface of Living Cells Using Bifunctional Magnetic Resonance Imaging Probes 
ACS Chemical Neuroscience  2013;5(2):128-137.
A series of bimodal metabotropic glutamate-receptor targeted MRI contrast agents has been developed and evaluated, based on established competitive metabotropic Glu receptor subtype 5 (mGluR5) antagonists. In order to directly visualize mGluR5 binding of these agents on the surface of live astrocytes, variations in the core structure were made. A set of gadolinium conjugates containing either a cyanine dye or a fluorescein moiety was accordingly prepared, to allow visualization by optical microscopy in cellulo. In each case, surface receptor binding was compromised and cell internalization observed. Another approach, examining the location of a terbium analogue via sensitized emission, also exhibited nonspecific cell uptake in neuronal cell line models. Finally, biotin derivatives of two lead compounds were prepared, and the specificity of binding to the mGluR5 cell surface receptors was demonstrated with the aid of their fluorescently labeled avidin conjugates, using both total internal reflection fluorescence (TIRF) and confocal microscopy.
PMCID: PMC3930992  PMID: 24251400
mGluR5; imaging agents; lanthanides; MRI; microscopy
2.  Micro-cost Analysis of ALK Rearrangement Testing by FISH to Determine Eligibility for Crizotinib Therapy in NSCLC: Implications for Cost Effectiveness of Testing and Treatment 
Break-apart fluorescence in situ hybridization (FISH) is the gold standard test for anaplastic lymphoma kinase (ALK) gene rearrangement. However, this methodology often is assumed to be expensive and potentially cost-prohibitive given the low prevalence of ALK-positive non-small cell lung cancer (NSCLC) cases. To more accurately estimate the cost of ALK testing by FISH, we developed a micro-cost model that accounts for all cost elements of the assay, including laboratory reagents, supplies, capital equipment, technical and pathologist labor, and the acquisition cost of the commercial test and associated reagent kits and controls. By applying a set of real-world base-case parameter values, we determined that the cost of a single ALK break-apart FISH test result is $278.01. Sensitivity analysis on the parameters of batch size, testing efficiency, and the cost of the commercial diagnostic testing products revealed that the cost per result is highly sensitive to batch size, but much less so to efficiency or product cost. This implies that ALK testing by FISH will be most cost effective when performed in high-volume centers. Our results indicate that testing cost may not be the primary determinant of crizotinib (Xalkori®) treatment cost effectiveness, and suggest that testing cost is an insufficient reason to limit the use of FISH testing for ALK rearrangement.
PMCID: PMC4260793  PMID: 25520569
micro-cost; ALK rearrangement; FISH; cost effectiveness; crizotinib; companion diagnostic
3.  Theoretical study on the role of dynamics on the unusual magnetic properties in MnBi 
Scientific Reports  2014;4:7222.
We study the electronic structure and lattice dynamics in the ferromagnet MnBi using first-principles calculations and a tight-binding model. The band structure around the Fermi level is dominated by Bi-p states which are the primary contributors to the magnetic anisotropy energy in the low temperature structure. A tight-binding model consisting of Mn-d and Bi-p states is developed and the parameters are determined from first-principles calculations. Phonon dispersions and elastic moduli exhibit several interesting features. The results imply that the magnetic interaction with the crystal lattice in MnBi is considerably more complex than previously thought and in particular that there is a rich interplay between phonons and magnetism involving both magnetoelastic and magnetostrictive coupling.
PMCID: PMC4245533  PMID: 25428000
4.  Ferromagnetism of Fe3Sn and Alloys 
Scientific Reports  2014;4:7024.
Hexagonal Fe3Sn has many of the desirable properties for a new permanent magnet phase with a Curie temperature of 725 K, a saturation moment of 1.18 MA/m. and anisotropy energy, K1 of 1.8 MJ/m3. However, contrary to earlier experimental reports, we found both experimentally and theoretically that the easy magnetic axis lies in the hexagonal plane, which is undesirable for a permanent magnet material. One possibility for changing the easy axis direction is through alloying. We used first principles calculations to investigate the effect of elemental substitutions. The calculations showed that substitution on the Sn site has the potential to switch the easy axis direction. However, transition metal substitutions with Co or Mn do not have this effect. We attempted synthesis of a number of these alloys and found results in accord with the theoretical predictions for those that were formed. However, the alloys that could be readily made all showed an in-plane easy axis. The electronic structure of Fe3Sn is reported, as are some are magnetic and structural properties for the Fe3Sn2, and Fe5Sn3 compounds, which could be prepared as mm-sized single crystals.
PMCID: PMC4228330  PMID: 25387850
5.  A cell-intrinsic requirement for NF-κB-inducing kinase (NIK) in CD4 and CD8 T cell memory1 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(7):10.4049/jimmunol.1301328.
NF-κB-inducing kinase (NIK, MAP3K14) is an essential kinase linking a subset of TNF receptor family members to the noncanonical NF-κB pathway. In order to assess the cell-intrinsic role of NIK in murine T cell function, we generated mixed bone marrow chimeras using bone marrow from NIK knockout (NIK KO) and wild type (WT) donor mice and infected the chimeras with lymphocytic choriomeningitis virus (LCMV). The chimeras possess an apparently normal immune system including a mixture of NIK KO and WT T cells, and the virus was cleared normally. Comparison of the NIK KO and WT CD4 and CD8 T cell responses at 8 days post-infection revealed modest but significant differences in the acute response. In both CD4 and CD8 compartments, there were relatively fewer activated (CD44hi) NIK KO T cells, but within the CD44hi population, a comparable percentage of the activated cells produced IFN-γ in response to ex vivo stimulation with antigenic LCMV peptides, although IL-7R expression was reduced in the NIK KO CD8 T cells. Assessment of the LCMV-specific memory at 65 days postinfection revealed many more LCMV-specific WT memory T cells than NIK KO memory T cells in both the CD4 and CD8 compartments, although the small number of surviving NIK KO memory T cells responded to secondary challenge with virus. These results demonstrate a cell-intrinsic requirement for NIK in the generation and/or maintenance of memory T cells in response to acute viral infection.
PMCID: PMC3815446  PMID: 24006459
6.  Overcoming Catastrophic Interference in Connectionist Networks Using Gram-Schmidt Orthogonalization 
PLoS ONE  2014;9(9):e105619.
Connectionist models of memory storage have been studied for many years, and aim to provide insight into potential mechanisms of memory storage by the brain. A problem faced by these systems is that as the number of items to be stored increases across a finite set of neurons/synapses, the cumulative changes in synaptic weight eventually lead to a sudden and dramatic loss of the stored information (catastrophic interference, CI) as the previous changes in synaptic weight are effectively lost. This effect does not occur in the brain, where information loss is gradual. Various attempts have been made to overcome the effects of CI, but these generally use schemes that impose restrictions on the system or its inputs rather than allowing the system to intrinsically cope with increasing storage demands. We show here that catastrophic interference occurs as a result of interference among patterns that lead to catastrophic effects when the number of patterns stored exceeds a critical limit. However, when Gram-Schmidt orthogonalization is combined with the Hebb-Hopfield model, the model attains the ability to eliminate CI. This approach differs from previous orthogonalisation schemes used in connectionist networks which essentially reflect sparse coding of the input. Here CI is avoided in a network of a fixed size without setting limits on the rate or number of patterns encoded, and without separating encoding and retrieval, thus offering the advantage of allowing associations between incoming and stored patterns.
PACS Nos.: 87.10.+e, 87.18.Bb, 87.18.Sn, 87.19.La
PMCID: PMC4152133  PMID: 25180550
7.  Peripheral CD4+ T-cell tolerance is induced in vivo by rare antigen-bearing B cells in follicular, marginal zone, and B-1 subsets 
European journal of immunology  2013;43(7):10.1002/eji.201242784.
B cells are efficient APCs when they internalize antigen via BCR-mediated uptake. Adoptively transferred antigen-presenting B cells can induce T-cell tolerance to foreign and self antigens; however, it is unknown whether endogenous B cells presenting self-peptides interact with naïve T cells and contribute to peripheral T-cell self-tolerance. Moreover, the relative abilities of mature B-cell subsets to induce T-cell tolerance have not been examined. To address these questions, we created a new mouse model wherein a very small fraction of B cells expresses an antigen transgene that cannot be transferred to other APCs. We limited antigen expression to follicular, marginal zone, or B-1 B-cell subsets and found that small numbers of each subset interacted with naïve antigen-specific T cells. Although antigen expressed by B-1 B cells induced the most T-cell division, divided T cells subsequently disappeared from secondary lymphoid tissues. Independent of which B-cell subset presented antigen, the remaining T cells were rendered hyporesponsive, and this effect was not associated with Foxp3 expression. Our data show that physiologically relevant proportions of B cells can mediate peripheral T-cell tolerance, and suggest that the mechanisms of tolerance induction might differ among follicular, marginal zone, and B-1 B-cell subsets.
PMCID: PMC3844680  PMID: 23532986
B-cell subsets; CD4+ T cells; tolerance
8.  B-Raf is required for positive selection and survival of DP cells, but not for negative selection of SP cells 
International Immunology  2013;25(4):259-269.
B-Raf affects ERK and controls thymocyte positive but not negative selection.
The duration of signaling through the MAP kinase (or ERK pathway) cascade has been implicated in thymic development, particularly positive and negative selection. In T cells, two isoforms of the MAP kinase kinase kinase Raf function to transmit signals from the T-cell receptor to ERK: C-Raf and B-Raf. In this study, we conditionally ablated B-Raf expression within thymocytes to assess the effects on ERK activation and thymocyte development. The complete loss of B-Raf is accompanied by a dramatic loss of ERK activation in both the double positive (DP) and single positive (SP) thymocytes, as well as peripheral splenocytes. There was a significant decrease in the cellularity of KO thymi, largely due to a loss of pre-selected DP cells, a decrease in DP cells undergoing positive selection, and a defect in SP maturation. B-Raf plays significant roles in survival of DP thymocytes and function of SP cells in the periphery. Surprisingly, we saw no effect of B-Raf deficiency on negative selection of autoreactive SP thymocytes, despite the greatly reduced ERK activation in these cells.
PMCID: PMC3597848  PMID: 23334952
B-Raf; ERK; MAPK; positive selection; thymic development
9.  Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators 
The Journal of Clinical Investigation  2014;124(5):2059-2070.
Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients.
PMCID: PMC4001542  PMID: 24691443
10.  Changes in functional properties and 5-HT modulation above and below a spinal transection in lamprey 
In addition to the disruption of neural function below spinal cord injuries (SCI), there also can be changes in neuronal properties above and below the lesion site. The relevance of these changes is generally unclear, but they must be understood if we are to provide rational interventions. Pharmacological approaches to improving locomotor function have been studied extensively, but it is still unclear what constitutes an optimal approach. Here, we have used the lamprey to compare the modulatory effects of 5-HT and lesion-induced changes in cellular and synaptic properties in unlesioned and lesioned animals. While analyses typically focus on the sub-lesion spinal cord, we have also examined effects above the lesion to see if there are changes here that could potentially contribute to the functional recovery. Cellular and synaptic properties differed in unlesioned and lesioned spinal cords and above and below the lesion site. The cellular and synaptic modulatory effects of 5-HT also differed in lesioned and unlesioned animals, again in region-specific ways above and below the lesion site. A role for 5-HT in promoting recovery was suggested by the potential for improvement in locomotor activity when 5-HT was applied to poorly recovered animals, and by the consistent failure of animals to recover when they were incubated in PCPA to deplete 5-HT. However, PCPA did not affect swimming in animals that had already recovered, suggesting a difference in 5-HT effects after lesioning. These results show changes in 5-HT modulation and cellular and synaptic properties after recovery from a spinal cord transection. Importantly, effects are not confined to the sub-lesion spinal cord but also occur above the lesion site. This suggests that the changes may not simply reflect compensatory responses to the loss of descending inputs, but reflect the need for co-ordinated changes above and below the lesion site. The changes in modulatory effects should be considered in pharmacological approaches to functional recovery, as assumptions based on effects in the unlesioned spinal cord may not be justified.
PMCID: PMC4299445  PMID: 25653594
spinal cord; neuromodulation; spinal cord injury; lamprey; 5-HT
11.  Itinerant magnetism in doped semiconducting β-FeSi2 and CrSi2 
Scientific Reports  2013;3:3517.
Novel or unusual magnetism is a subject of considerable interest, particularly in metals and degenerate semiconductors. In such materials the interplay of magnetism, transport and other Fermi liquid properties can lead to fascinating physical behavior. One example is in magnetic semiconductors, where spin polarized currents may be controlled and used. We report density functional calculations predicting magnetism in doped semiconducting β-FeSi2 and CrSi2 at relatively low doping levels particularly for n-type. In this case, there is a rapid cross-over to a half-metallic state as a function of doping level. The results are discussed in relation to the electronic structure and other properties of these compounds.
PMCID: PMC3865465  PMID: 24343332
13.  Importance of non-parabolic band effects in the thermoelectric properties of semiconductors 
Scientific Reports  2013;3:3168.
We present an analysis of the thermoelectric properties of of n-type GeTe and SnTe in relation to the lead chalcogenides PbTe and PbSe. We find that the singly degenerate conduction bands of semiconducting GeTe and SnTe are highly non-ellipsoidal, even very close to the band edges. This leads to isoenergy surfaces with a strongly corrugated shape that is clearly evident at carrier concentrations well below 0.005 e per formula unit (7–9 × 1019 cm−3 depending on material). Analysis within Boltzmann theory suggests that this corrugation may be favorable for the thermoelectric transport. Our calculations also indicate that values of the power factor for these two materials may well exceed those of PbTe and PbSe. As a result these materials may exhibit n-type performance exceeding that of the lead chalcogenides.
PMCID: PMC3819605  PMID: 24196778
14.  Is Femoral Component Rotation in a TKA Reliably Guided by the Functional Flexion Axis? 
The position of the femoral component in a TKA in the axial plane influences patellar tracking and flexion gap symmetry. Errors in femoral component rotation have been implicated in the need for early revision surgery. Methods of guiding femoral component rotation at the time of implantation typically are derived from the mean position of the flexion-extension axis across experimental subjects. The functional flexion axis (FFA) of the knee is kinematically derived and therefore a patient-specific reference axis that can be determined intraoperatively by a computer navigation system as an alternative method of guiding femoral component rotation. However, it is unclear whether the FFA is reliable and how it compares with traditional methods.
We asked if the FFA could be measured reproducibly at different stages of the operative procedure; (2) where it lies in relation to a CT-derived gold standard; and (3) how it compares with more traditional methods of judging femoral component rotation.
Thirty-seven patients undergoing elective TKAs were recruited to the study. Preoperative CT scans were obtained and the transepicondylar axis (TEA) was identified. The TKA then was performed using computer navigation. The FFA was derived before incision and again after the surgical approach and osseous registration. The navigation system was used to register the surgical TEA. The FFA and surgical TEA then were compared with the CT-derived TEA.
The mean preincision FFA was similar to the intraoperative FFA and therefore deemed reproducible. We observed no differences in variability between surgical TEA and preincision FFA. The FFA was different from the CT-TEA and judged similar in accuracy to the surgical TEA.
The reliability and accuracy of the FFA were similar to those of other intraoperative methods. Further evaluation is required to ascertain whether the FFA improves on currently available methods for determining the ideal rotation of the femoral component during TKA.
PMCID: PMC3462881  PMID: 22895689
15.  Sagittal placement of the femoral component in total knee arthroplasty predicts knee flexion contracture at one-year follow-up 
International Orthopaedics  2012;36(9):1835-1839.
Flexion contracture has been shown to impair function and reduce satisfaction following total knee arthroplasty (TKA). The aim of this study was to identify modifiable intra-operative variables that predict post-TKA knee extension.
Data was collected prospectively on 95 patients undergoing total knee arthroplasty, including pre-operative assessment, intra-operative computer assisted surgery (CAS) measurements and functional outcome including range of motion at one year. Patients were divided into two groups: those with mild flexion contracture (> 5°) at the one-year follow-up and those achieving full extension.
The sagittal orientation of the distal femoral cut differed significantly between groups at the one-year follow-up (p = 0.014). Sagittal alignment of greater than 3.5° from the mechanical axis was shown to increase the relative risk of a mild flexion contracture at one-year follow-up by 2.9 times, independent of other variables.
Increasing the sagittal alignment of the distal femoral cut more than 3.5° from the mechanical axis is an independent risk factor for clinically detectable flexion contracture one year from index procedure.
PMCID: PMC3427457  PMID: 22638608
16.  A europium luminescence assay of lactate and citrate in biological fluids† 
Organic & biomolecular chemistry  2009;7(8):1525-1528.
Ratiometric methods of analysis have been developed for the selective determination of lactate or citrate in microlitre samples of human serum, urine or prostate fluids following comparison of anion binding affinities for a family of nine luminescent europium(III) complexes.
PMCID: PMC3724753  PMID: 19343236
17.  Trophic Transfer of Arsenic from an Aquatic Insect to Terrestrial Insect Predators 
PLoS ONE  2013;8(6):e67817.
The movement of energy and nutrients from aquatic to terrestrial ecosystems can be substantial, and emergent aquatic insects can serve as biovectors not only for nutrients, but also for contaminants present in the aquatic environment. The terrestrial predators Tenodera aridifolia sinensis (Mantodea: Mantidae) and Tidarren haemorrhoidale (Araneae: Theridiidae) and the aquatic predator Buenoa scimitra (Hemiptera: Notonectidae) were chosen to evaluate the efficacy of arsenic transfer between aquatic and terrestrial environments. Culex tarsalis larvae were reared in either control water or water containing 1000 µg l−1 arsenic. Adults that emerged from the control and arsenic treatments were fed to the terrestrial predators, and fourth instar larvae were fed to the aquatic predator reared in control or arsenic contaminated water. Tenodera a. sinensis fed arsenic-treated Cx. tarsalis accumulated 658±130 ng g−1 of arsenic. There was no significant difference between control and arsenic-fed T. haemorrhoidale (range 142–290 ng g−1). Buenoa scimitra accumulated 5120±406 ng g−1 of arsenic when exposed to arsenic-fed Cx. tarsalis and reared in water containing 1000 µg l−1 arsenic. There was no significant difference between controls or arsenic-fed B. scimitra that were not exposed to water-borne arsenic, indicating that for this species environmental exposure was more important in accumulation than strictly dietary arsenic. These results indicate that transfer to terrestrial predators may play an important role in arsenic cycling, which would be particularly true during periods of mass emergence of potential insect biovectors. Trophic transfer within the aquatic environment may still occur with secondary predation, or in predators with different feeding strategies.
PMCID: PMC3694899  PMID: 23826344
18.  Quantitative Analysis of Fragrance and Odorants Released from Fresh and Decaying Strawberries 
Sensors (Basel, Switzerland)  2013;13(6):7939-7978.
The classes and concentrations of volatile organic compounds (VOC) released from fresh and decaying strawberries were investigated and compared. In this study, a total of 147 strawberry volatiles were quantified before and after nine days of storage to explore differences in the aroma profile between fresh strawberries (storage days (SRD) of 0, 1, and 3) and those that had started to decay (SRD = 6 and 9). In terms of concentration, seven compounds dominated the aroma profile of fresh strawberries (relative composition (RC) up to 97.4% by mass, sum concentration): (1) ethyl acetate = 518 mg·m−3, (2) methyl acetate = 239 mg·m−3, (3) ethyl butyrate = 13.5 mg·m−3, (4) methyl butyrate = 11.1 mg·m−3, (5) acetaldehyde = 24.9 mg·m−3, (6) acetic acid = 15.2 mg·m−3, and (7) acetone = 13.9 mg·m−3. In contrast, two alcohols dominated the aroma profile of decayed samples (RC up to 98.6%): (1) ethyl alcohol = 94.2 mg·m−3 and (2) isobutyl alcohol = 289 mg·m−3. Alternatively; if the aroma profiles are re-evaluated by summing odor activity values (ΣOAV); four ester compounds ((1) ethyl butyrate (6,160); (2) ethyl hexanoate (3,608); (3) ethyl isovalerate (1,592); and (4) ethyl 2-methylbutyrate (942)) were identified as the key constituents of fresh strawberry aroma (SRD-0). As the strawberries began to decay; isobutyl alcohol recorded the maximum OAV of 114 (relative proportion (RP) (SRD = 6) = 58.3%). However, as the decay process continued, the total OAV dropped further by 3 to 4 orders of magnitude—decreasing to 196 on SRD = 6 to 7.37 on SRD = 9. The overall results of this study confirm dramatic changes in the aroma profile of strawberries over time, especially with the onset of decay.
PMCID: PMC3715231  PMID: 23787728
fresh and decaying strawberry; strawberry fragrances; mass concentration; threshold; odor activity value (OAV)
19.  Dynamic systems approaches and levels of analysis in the nervous system 
Various analyses are applied to physiological signals. While epistemological diversity is necessary to address effects at different levels, there is often a sense of competition between analyses rather than integration. This is evidenced by the differences in the criteria needed to claim understanding in different approaches. In the nervous system, neuronal analyses that attempt to explain network outputs in cellular and synaptic terms are rightly criticized as being insufficient to explain global effects, emergent or otherwise, while higher-level statistical and mathematical analyses can provide quantitative descriptions of outputs but can only hypothesize on their underlying mechanisms. The major gap in neuroscience is arguably our inability to translate what should be seen as complementary effects between levels. We thus ultimately need approaches that allow us to bridge between different spatial and temporal levels. Analytical approaches derived from critical phenomena in the physical sciences are increasingly being applied to physiological systems, including the nervous system, and claim to provide novel insight into physiological mechanisms and opportunities for their control. Analyses of criticality have suggested several important insights that should be considered in cellular analyses. However, there is a mismatch between lower-level neurophysiological approaches and statistical phenomenological analyses that assume that lower-level effects can be abstracted away, which means that these effects are unknown or inaccessible to experimentalists. As a result experimental designs often generate data that is insufficient for analyses of criticality. This review considers the relevance of insights from analyses of criticality to neuronal network analyses, and highlights that to move the analyses forward and close the gap between the theoretical and neurobiological levels, it is necessary to consider that effects at each level are complementary rather than in competition.
PMCID: PMC3564044  PMID: 23386835
neuronal network; criticality; spinal cord
21.  Engineering controllable bidirectional molecular motors based on myosin 
Nature nanotechnology  2012;7(4):252-256.
Cytoskeletal motors drive the transport of organelles and molecular cargoes within cells1, and have potential applications in molecular detection and diagnostic devices2,3. Engineering molecular motors with dynamically controllable properties will allow selective perturbation of mechanical processes in living cells, and yield optimized device components for complex tasks such as molecular sorting and directed assembly3. Biological motors have previously been modified by introducing activation/deactivation switches that respond to metal ions4,5 and other signals6. Here we show that myosin motors can be engineered to reversibly change their direction of motion in response to a calcium signal. Building on previous protein engineering studies7–11 and guided by a structural model12 for the redirected power stroke of myosin VI, we constructed bidirectional myosins through the rigid recombination of structural modules. The performance of the motors was confirmed using gliding filament assays and single fluorophore tracking. Our general strategy, in which external signals trigger changes in the geometry and mechanics of myosin lever arms, should enable spatiotemporal control over a range of motor properties including processivity, stride size13, and branchpoint turning14.
PMCID: PMC3332125  PMID: 22343382
22.  Cyclosporine-resistant, Rab27a-independent Mobilization of Intracellular Preformed CD40L Mediates Antigen-specific T Cell Help In Vitro 
CD40L is critically important for the initiation and maintenance of adaptive immune responses. It is generally thought that CD40L expression in CD4+ T cells is regulated transcriptionally and made from new mRNA following antigen recognition. However, recent studies with two-photon microscopy revealed that the majority of cognate interactions between effector CD4+ T cells and APCs are too short for de novo synthesis of CD40L. Given that effector and memory CD4+ T cells store preformed CD40L (pCD40L) in lysosomal compartments and that pCD40L comes to the cell surface within minutes of antigenic stimulation, we and others have proposed that pCD40L might mediate T cell-dependent activation of cognate APCs during brief encounters in vivo. However, it has not been shown that this relatively small amount of pCD40L is sufficient to activate APCs, owing to the difficulty of separating the effects of pCD40L from those of de novo CD40L and other cytokines in vitro. Here we show that pCD40L surface mobilization is resistant to cyclosporine or FK506 treatment, while de novo CD40L and cytokine expression are completely inhibited. These drugs thus provide a tool to dissect the role of pCD40L in APC activation. We find that pCD40L mediates selective activation of cognate but not bystander APCs in vitro and that mobilization of pCD40L does not depend on Rab27a, which is required for mobilization of lytic granules. Therefore, effector CD4+ T cells deliver pCD40L specifically to APCs on the same time scale as the lethal hit of CTLs but with distinct molecular machinery.
PMCID: PMC3131475  PMID: 21677130
23.  Preformed CD40L Is Stored in Th1, Th2, Th17, and T Follicular Helper Cells as Well as CD4+8− Thymocytes and Invariant NKT Cells but Not in Treg Cells 
PLoS ONE  2012;7(2):e31296.
CD40L is essential for the development of adaptive immune responses. It is generally thought that CD40L expression in CD4+ T cells is regulated transcriptionally and made from new mRNA following antigen recognition. However, imaging studies show that the majority of cognate interactions between effector CD4+ T cells and APCs in vivo are too short to allow de novo CD40L synthesis. We previously showed that Th1 effector and memory cells store preformed CD40L (pCD40L) in lysosomal compartments and mobilize it onto the plasma membrane immediately after antigenic stimulation, suggesting that primed CD4+ T cells may use pCD40L to activate APCs during brief encounters. Indeed, our recent study showed that pCD40L is sufficient to mediate selective activation of cognate B cells and trigger DC activation in vitro. In this study, we show that pCD40L is present in Th1 and follicular helper T cells developed during infection with lymphocytic choriomeningitis virus, Th2 cells in the airway of asthmatic mice, and Th17 cells from the CNS of animals with experimental autoimmune encephalitis (EAE). pCD40L is nearly absent in both natural and induced Treg cells, even in the presence of intense inflammation such as occurs in EAE. We also found pCD40L expression in CD4 single positive thymocytes and invariant NKT cells. Together, these results suggest that pCD40L may function in T cell development as well as an unexpectedly broad spectrum of innate and adaptive immune responses, while its expression in Treg cells is repressed to avoid compromising their suppressive activity.
PMCID: PMC3283616  PMID: 22363608
24.  Design and analysis of DNA strand displacement devices using probabilistic model checking 
Designing correct, robust DNA devices is difficult because of the many possibilities for unwanted interference between molecules in the system. DNA strand displacement has been proposed as a design paradigm for DNA devices, and the DNA strand displacement (DSD) programming language has been developed as a means of formally programming and analysing these devices to check for unwanted interference. We demonstrate, for the first time, the use of probabilistic verification techniques to analyse the correctness, reliability and performance of DNA devices during the design phase. We use the probabilistic model checker prism, in combination with the DSD language, to design and debug DNA strand displacement components and to investigate their kinetics. We show how our techniques can be used to identify design flaws and to evaluate the merits of contrasting design decisions, even on devices comprising relatively few inputs. We then demonstrate the use of these components to construct a DNA strand displacement device for approximate majority voting. Finally, we discuss some of the challenges and possible directions for applying these methods to more complex designs.
PMCID: PMC3367817  PMID: 22219398
DNA computing; formal verification; probabilistic model checking; DNA strand displacement
25.  The Combined Application of Impinger System and Permeation Tube for the Generation of Volatile Organic Compound Standard Gas Mixtures at Varying Diluent Flow Rates 
Sensors (Basel, Switzerland)  2012;12(8):10964-10979.
Commercial standard gas generators are often complex and expensive devices. The objective of this research was to assess the performance of a simplified glass impinger system for standard gas generation from a permeation tube (PT) device. The performance of the impinger standard gas generation system was assessed for four aromatic VOCs (benzene, toluene, ethylbenzene, and m-xylene; BTEX) at varying flow rates (FR) of 50 to 800 mL·min−1. Because actual permeation rate (APR) values deviated from those computed by the manufacturer's formula (MPR), new empirical relationships were developed to derive the predicted PR (PPR) of the target components. Experimental results corrected by such a formula indicate that the compatibility between the APR and MPR generally increased with low FR, while the reproducibility was generally reduced with decreasing flow rate. Although compatibility between different PRs is at a relatively small and narrow FR range, the use of correction formula is recommendable for the accurate use of PT.
PMCID: PMC3472869  PMID: 23112641
impinge; permeation; benzene; toluene; ethylbenzene; xylene

Results 1-25 (63)