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1.  Divalent regulation and intersubunit interactions of human Connexin26 (Cx26) hemichannels 
Channels  2013;8(1):1-4.
Control of plasma membrane connexin hemichannel opening is indispensable, and is achieved by physiological extracellular divalent ion concentrations. Here, we explore the differences between regulation by Ca2+ and Mg2+ of human connexin26 (hCx26) hemichannels and the role of a specific interaction in regulation by Ca2+. To effect hemichannel closure, the apparent affinity of Ca2+ (0.33 mM) is higher than for Mg2+ (1.8 mM). Hemichannel closure is accelerated by physiological Ca2+ concentrations, but non-physiological concentrations of extracellular Mg2+ are required for this effect. Our recent report provided evidence that extracellular Ca2+ facilitates hCx26 hemichannel closing by disrupting a salt bridge interaction between positions D50 and K61 that stabilizes the open state. New evidence from mutant cycle analysis indicates that D50 also interacts with Q48. We find that the D50-Q48 interaction contributes to stabilization of the open state, but that it is relatively insensitive to disruption by extracellular Ca2+ compared with the D50-K61 interaction.
doi:10.4161/chan.26789
PMCID: PMC4048337  PMID: 24126106
Connexin; gating; calcium; salt bridge; channelopathies
2.  Affective and Neuroendocrine Effects of Withdrawal from Chronic, Long-Acting Opiate Administration 
Brain research  2013;1538:73-82.
Although the long-acting opiate methadone is commonly used to treat drug addiction, relatively little is known about effects of withdrawal from this drug in preclinical models. The current study examined affective, neuroendocrine, and somatic signs of withdrawal from the longer-acting methadone derivative l-alpha-acetylmethydol (LAAM) in rats. Anxiety-like behavior during both spontaneous and antagonist-precipitated withdrawal was measured by potentiation of the startle reflex. Withdrawal elevated corticosterone and somatic signs and blunted circadian variations in baseline startle responding. In addition, fear to an explicit, Pavlovian conditioned stimulus (fear-potentiated startle) was enhanced. These data suggest that anxiety-like behavior as measured using potentiated startle responding does not emerge spontaneously during withdrawal from chronic opiate exposure – in contrast to withdrawal from acute drug exposure – but rather is manifested as exaggerated fear in response to explicit threat cues.
doi:10.1016/j.brainres.2013.09.026
PMCID: PMC4053187  PMID: 24076207
acoustic startle; opiate dependence; corticosterone; LAAM; fear-potentiated startle; opiate withdrawal
3.  Long-term Analyses of Innervation and Neuromuscular Integrity in the Trembler J Mouse Model of Charcot-Marie-Tooth Disease 
Journal of neuropathology and experimental neurology  2013;72(10):10.1097/NEN.0b013e3182a5f96e.
A large fraction of hereditary demyelinating neuropathies, classified as Charcot-Marie-Tooth disease type IA (CMT 1A), is associated with misexpression of peripheral myelin protein 22. In this study we characterized morphological and biochemical changes that occur with disease progression in neuromuscular tissue of Trembler J mice, a spontaneous rodent model of CMT 1A. Using age-matched, 2- and 10-month-old wild type and Trembler J mice, we observed neuromuscular deficits that progress from distal to proximal regions. The impairments in motor performance are underlined by degenerative events at distal nerve segments and structural alterations at nerve-muscle synapses. Furthermore, skeletal muscle of affected mice showed reduced myofiber diameter, increased expression of the muscle atrophy marker muscle ring-finger protein 1 and fiber type switching. A dietary intervention of intermittent fasting attenuated these progressive changes and supported distal nerve myelination and neuromuscular junction integrity. In addition to the well-characterized demyelination aspects of this model, our investigations identified distinct degenerative events in distal nerves and muscle of affected neuropathic mice. Therefore, therapeutic studies aimed at slowing or reversing the neuropathic features of these disorders should include the examination of muscle tissue, as well as neuromuscular contact sites.
doi:10.1097/NEN.0b013e3182a5f96e
PMCID: PMC3810994  PMID: 24042197
Charcot-Marie-Tooth; Demyelinating neuropathy; Intermittent fasting; Muscle atrophy; Neuromuscular junction; Peripheral myelin protein 22; Peripheral nerve
4.  Etanercept plus topical corticosteroids as initial therapy for grade 1 acute graft-versus-host disease after allogeneic hematopoietic cell transplant 
Clinical diagnosis of grade 1 acute graft-versus-host disease (GVHD) marks the beginning of a potentially progressive and fatal course of GVHD after hematopoietic stem cell transplantation (HSCT). However, interventional studies to treat early GVHD are lacking. We conducted a single-arm prospective phase II trial to test the hypothesis that treatment of newly-diagnosed grade 1 acute GVHD with etanercept and topical corticosteroids would reduce progression to grade 2–4 within 28 days. Study patients (n=34) had a median age of 51 years (range, 10–67 years) and had undergone unrelated (n=22) or related (n=12) donor HSCT. Study patients were treated with etancercept (0.4 mg/kg, maximum 25 mg/dose) twice weekly for 4–8 weeks. Ten of 34 patients (29%) progressed to grade 2–4 acute GVHD within 28 days. The cumulative incidence of grade 2–4 and grade 3–4 acute GVHD at 1-year were 41% and 3%, respectively. Non-relapse mortality was 19% and overall survival was 63% at 2-years. Among a contemporaneous control cohort of patients that were diagnosed with grade 1 acute GVHD and treated with topical corticosteroids but not etanercept during the study period, 12 of 28 patients (43%) progressed to grade 2–4 GVHD within 28 days, with 1-year incidence of grade 2–4 GVHD and grade 3–4 GVHD of 61% (41% vs 61%, p=0.08) and 18% (3% vs 18%, p=0.05), respectively. Patients treated with etanercept also experienced less increase in GVHD plasma biomarkers ST2 (p=0.06) and Reg3α (p=0.01) 28 days after grade 1 acute GVHD diagnosis compared to contemporaneous control patients. This study was terminated early due to poor accrual. Future prospective studies are needed to identify patients with grade 1 acute GVHD at risk of swift progression to more severe GVHD and to establish consensus for the treatment of grade 1 acute GVHD. This trial is registered with ClinicalTrials.gov, number NCT00726375.
doi:10.1016/j.bbmt.2014.05.023
PMCID: PMC4145722  PMID: 24892263
5.  Demonstration of a Semi-Autonomous Hybrid Brain-Machine Interface using Human Intracranial EEG, Eye Tracking, and Computer Vision to Control a Robotic Upper Limb Prosthetic 
To increase the ability of brain-machine interfaces (BMIs) to control advanced prostheses such as the modular prosthetic limb (MPL), we are developing a novel system: the Hybrid Augmented Reality Multimodal Operation Neural Integration Environment (HARMONIE). This system utilizes hybrid input, supervisory control, and intelligent robotics to allow users to identify an object (via eye tracking and computer vision) and initiate (via brain-control) a semi-autonomous reach-grasp-and-drop of the object by the MPL. Sequential iterations of HARMONIE were tested in two pilot subjects implanted with electrocorticographic (ECoG) and depth electrodes within motor areas. The subjects performed the complex task in 71.4% (20/28) and 67.7% (21/31) of trials after minimal training. Balanced accuracy for detecting movements was 91.1% and 92.9%, significantly greater than chance accuracies (p < 0.05). After BMI-based initiation, the MPL completed the entire task 100% (one object) and 70% (three objects) of the time. The MPL took approximately 12.2 seconds for task completion after system improvements implemented for the second subject. Our hybrid-BMI design prevented all but one baseline false positive from initiating the system. The novel approach demonstrated in this proof-of-principle study, using hybrid input, supervisory control, and intelligent robotics, addresses limitations of current BMIs.
doi:10.1109/TNSRE.2013.2294685
PMCID: PMC4057363  PMID: 24760914
Brain-computer interface (BCI); Brain-machine interface (BMI); Electrocorticography (ECoG); Hybrid BCI; Intelligent robotics; Intracranial EEG (iEEG)
6.  Intercellular Communication Amplifies Stressful Effects in High-Charge, High-Energy (HZE) Particle-Irradiated Human Cells 
Journal of radiation research  2011;52(4):408-414.
Understanding the mechanisms that underlay the biological effects of particulate radiations is essential for space exploration and for radiotherapy. Here, we investigated the role of gap junction intercellular communication (GJIC) in modulating harmful effects induced in confluent cultures wherein most cells are traversed by one or more radiation tracks. We focused on the effect of radiation quality (linear energy transfer; LET) on junctional propagation of DNA damage and cell death among the irradiated cells. Confluent normal human fibroblasts were exposed to graded doses of 1 GeV protons (LET ~0.2 keV/μm) or 1 GeV/u iron ions (LET ~151 keV/μm) and were assayed for clonogenic survival and for micronucleus formation, a reflection of DNA damage, shortly after irradiation and following longer incubation periods. Iron ions were ~2.7 fold more effective than protons at killing 90% of the cells in the exposed cultures when assayed within 5–10 minutes after irradiation. When cells were held in the confluent state for several hours after irradiation, substantial repair of potentially lethal damage (PLDR), coupled with a reduction in micronucleus formation, occurred in cells exposed to protons, but not in those exposed to iron ions. In fact, such confluent holding after exposure to a similarly toxic dose of iron ions enhanced the induced toxic effect. However, following iron ion irradiation, inhibition of GJIC by 18-α-glycyrrhetinic acid eliminated the enhanced toxicity and reduced micronucleus formation to levels below those detected in cells assayed shortly after irradiation. The data show that low LET radiation induces strong PLDR within hours, but that high LET radiation with similar immediate toxicity does not induce PLDR and its toxicity increases with time following irradiation. The results also show that GJIC among irradiated cells amplifies stressful effects following exposure to high, but not LET radiation, and that GJIC has only minimal effect on cellular recovery following low LET irradiation.
PMCID: PMC4058820  PMID: 21905305
Gap junction intercellular communication; Cell killing; Potentially lethal damage repair; DNA damage; Linear energy transfer of space radiation
7.  Formation of adherens junctions leads to the emergence of a tissue-level tension in epithelial monolayers 
Journal of Cell Science  2014;127(11):2507-2517.
ABSTRACT
Adherens junctions and desmosomes integrate the cytoskeletons of adjacent cells into a mechanical syncitium. In doing so, intercellular junctions endow tissues with the strength needed to withstand the mechanical stresses encountered in normal physiology and to coordinate tension during morphogenesis. Though much is known about the biological mechanisms underlying junction formation, little is known about how tissue-scale mechanical properties are established. Here, we use deep atomic force microscopy (AFM) indentation to measure the apparent stiffness of epithelial monolayers reforming from dissociated cells and examine which cellular processes give rise to tissue-scale mechanics. We show that the formation of intercellular junctions coincided with an increase in the apparent stiffness of reforming monolayers that reflected the generation of a tissue-level tension. Tension rapidly increased, reaching a maximum after 150 min, before settling to a lower level over the next 3 h as monolayers established homeostasis. The emergence of tissue tension correlated with the formation of adherens junctions but not desmosomes. As a consequence, inhibition of any of the molecular mechanisms participating in adherens junction initiation, remodelling and maturation significantly impeded the emergence of tissue-level tension in monolayers.
doi:10.1242/jcs.142349
PMCID: PMC4043320  PMID: 24659804
Adherens junctions; Desmosomes; Morphogenesis; Tension; Atomic force microscopy
8.  Motifs in the permeation pathway of connexin channels mediate voltage and Ca2+ sensing 
Connexin channels mediate electrical coupling, intercellular molecular signaling, and extracellular release of signaling molecules. Connexin proteins assemble intracellularly as hexamers to form plasma membrane hemichannels. The docking of two hemichannels in apposed cells forms a gap junction channel that allows direct electrical and selective cytoplasmic communication between adjacent cells. Hemichannels and junctional channels are gated by voltage, but extracellular Ca2+ also gates unpaired plasma membrane hemichannels. Unlike other ion channels, connexin channels do not contain discrete voltage- or Ca2+–sensing modules linked to a separate pore-forming module. All studies to date indicate that voltage and Ca2+ sensing are predominantly mediated by motifs that lie within or are exposed to the pore lumen. The sensors appear to be integral components of the gates, imposing an obligatory structural linkage between sensing and gating not commonly present in other ion channels, in which the sensors are semi-independent domains distinct from the pore. Because of this, the structural and electrostatic features that define connexin channel gating also define pore permeability properties, and vice versa; analysis/mutagenesis of gating and of permeability properties are linked. This offers unique challenges and opportunities for elucidating mechanisms of ligand and voltage-driven gating.
doi:10.3389/fphys.2014.00113
PMCID: PMC3978323  PMID: 24744733
connexin; hemichannels; voltage gating; gap junction channels; permeation; Ca2+ regulation
9.  ST2 as a Marker for Risk of Therapy-Resistant Graft-versus-Host Disease and Death 
The New England journal of medicine  2013;369(6):529-539.
Background
No plasma biomarkers are associated with the response of acute graft-versus-host disease (GVHD) to therapy after allogeneic hematopoietic stem-cell transplantation.
Methods
We compared 12 biomarkers in plasma obtained a median of 16 days after therapy initiation from 10 patients with a complete response by day 28 after therapy initiation and in plasma obtained from 10 patients with progressive GVHD during therapy. The lead biomarker, suppression of tumorigenicity 2 (ST2), was measured at the beginning of treatment for GVHD in plasma from 381 patients and during the first month after transplantation in three independent sets totaling 673 patients to determine the association of this biomarker with treatment-resistant GVHD and 6-month mortality after treatment or transplantation.
Results
Of the 12 markers, ST2 had the most significant association with resistance to GVHD therapy and subsequent death without relapse. As compared with patients with low ST2 values at therapy initiation, patients with high ST2 values were 2.3 times as likely to have treatment-resistant GVHD (95% confidence interval [CI], 1.5 to 3.6) and 3.7 times as likely to die within 6 months after therapy (95% CI, 2.3 to 5.9). Patients with low ST2 values had lower mortality without relapse than patients with high ST2 values, regardless of the GVHD grade (11% vs. 31% among patients with grade I or II GVHD and 14% vs. 67% among patients with grade III or IV GVHD, P<0.001 for both comparisons). Plasma ST2 values at day 14 after transplantation were associated with 6-month mortality without relapse, regardless of the intensity of the conditioning regimen.
Conclusions
ST2 levels measured at the initiation of therapy for GVHD and during the first month after transplantation improved risk stratification for treatment-resistant GVHD and death without relapse after transplantation. (Funded by the National Institutes of Health.)
doi:10.1056/NEJMoa1213299
PMCID: PMC3943357  PMID: 23924003
10.  The cytoplasm of living cells behaves as a poroelastic material 
Nature materials  2013;12(3):253-261.
The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described by a poroelastic model, in which the cytoplasm is treated as a biphasic material consisting of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. However, direct supporting evidence for the model is lacking. Here we directly validate the poroelastic model to explain cellular rheology at physiologically relevant timescales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of the cytoplasm (cytoskeleton and macromolecular crowders) plays a fundamental role in setting cellular rheology.
doi:10.1038/nmat3517
PMCID: PMC3925878  PMID: 23291707
Cell mechanics; poroelasticity; viscoelasticity; microstructure
11.  Advances in predicting acute GVHD 
British journal of haematology  2012;160(3):288-302.
Summary
Acute graft-versus-host disease (GVHD) is a leading cause of non-relapse mortality following allogeneic haematopoietic cell transplantation. Attempts to improve treatment response in clinically-established GVHD have not improved overall survival, often due to the increased risk of infectious complications. Alternative approaches to decrease GVHD-related morbidity and mortality have focused on the ability to predict GVHD prior to clinical manifestation in an effort to provide an opportunity to abort GVHD development, and to gain new insights into GVHD pathophysiology. This review outlines the research efforts to date that have identified clinical and laboratory-based factors that are predictive of acute GVHD and describes future directions in developing algorithms that will improve the ability to predict the development of clinically relevant GVHD.
doi:10.1111/bjh.12142
PMCID: PMC3552019  PMID: 23205489
BMT; GVHD; BMT Immunology; Biomarkers; Prediction
12.  Insights on the mechanisms of Ca2+ regulation of connexin26 hemichannels revealed by human pathogenic mutations (D50N/Y) 
Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca2+ concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca2+ regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca2+ concentration, indicating that Ca2+ facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca2+-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca2+ insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca2+, and (b) facilitating closing and stabilization of the closed state in the presence of Ca2+. To explore the role of a negatively charged residue at position 50 in regulation by Ca2+, this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)−. D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES− restores the wild-type Ca2+ regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca2+ regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca2+. Mutant cycle analysis supports a Ca2+-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca2+ destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca2+.
doi:10.1085/jgp.201210893
PMCID: PMC3691447  PMID: 23797420
13.  Multiwalled carbon nanotubes in alfalfa and wheat: toxicology and uptake 
Data on the bioavailability and toxicity of carbon nanotubes (CNTs) in the environment, and, in particular, on their interactions with vascular plants, are limited. We investigated the effects of industrial-grade multiwalled CNTs (75 wt% CNTs) and their impurities on alfalfa and wheat. Phytotoxicity assays were performed during both seed germination and seedling growth. The germinations of both species were tolerant of up to 2560 mg l−1 CNTs, and root elongation was enhanced in alfalfa and wheat seedlings exposed to CNTs. Remarkably, catalyst impurities also enhanced root elongation in alfalfa seedlings as well as wheat germination. Thus the impurities, not solely the CNTs, impacted the plants. CNT internalization by plants was investigated using electron microscopy and two-dimensional Raman mapping. The latter showed that CNTs were adsorbed onto the root surfaces of alfalfa and wheat without significant uptake or translocation. Electron microscopy investigations of internalization were inconclusive owing to poor contrast, so Fe3O4-functionalized CNTs were prepared and studied using energy-filter mapping of Fe3O4. CNTs bearing Fe3O4 nanoparticles were detected in the epidermis of one wheat root tip only, suggesting that internalization was possible but unusual. Thus, alfalfa and wheat tolerated high concentrations of industrial-grade multiwalled CNTs, which adsorbed onto their roots but were rarely taken up.
doi:10.1098/rsif.2012.0535
PMCID: PMC3481593  PMID: 22977097
plants; carbon nanotubes; nanotube uptake; nanotube phytotoxicity; Raman mapping; electron microscopy
14.  Have we made progress in the treatment of GVHD? 
One reason for the lack of progress in the treatment of acute graft versus host disease (GVHD) is the lack of reliable biomarkers. GVHD of the gastrointestinal (GI) tract is closely associated with non-relapse mortality (NRM) following hematopoietic cell transplantation (HCT). Using an unbiased, large-scale, quantitative proteomic discovery approach, we identified candidate biomarkers that were increased in plasma from HCT patients with GI GVHD. We then validated the lead candidate, REG3α, by ELISA in samples from more than 1,000 HCT patients from three transplant centers. Plasma REG3α concentrations were 3-fold higher in patients at GI GVHD onset than in all other patients. REG3α concentrations correlated most closely with lower GI GVHD at GVHD onset and predicted response to therapy at 4 weeks, 1-year NRM, and 1-year survival (P≤0.001). Multivariate analysis showed that advanced clinical stage, severe histologic damage, and high REG3α concentrations at the diagnosis of GVHD independently predicted 1-year NRM, which progressively increased with higher numbers of onset risk factors present. We conclude that REG3α is a plasma biomarker of GI GVHD that can be combined with clinical stage and histologic grade to improve risk stratification of patients, perhaps providing a platform for advances in the treatment of high-risk GVHD.
doi:10.1016/j.beha.2012.10.010
PMCID: PMC3513691  PMID: 23200544
biomarker; gastrointestinal (GI); graft versus host disease (GVHD); hematopoietic cell transplantation (HCT); REG3α
15.  Delivery of nicotine in an extract of a smokeless tobacco product reduces its reinforcement-attenuating and discriminative stimulus effects in rats 
Psychopharmacology  2011;220(3):565-576.
Rationale
Animal models of tobacco addiction rely on administration of nicotine alone or nicotine combined with isolated constituents. Models using tobacco extracts derived from tobacco products and containing a range of tobacco constituents might more accurately simulate tobacco exposure in humans.
Objective
To compare the effects of nicotine alone and an aqueous smokeless tobacco extract in several addiction-related animal behavioral models.
Methods
Nicotine alone and nicotine dose-equivalent concentrations of extract were compared in terms of their acute effects on intracranial self-stimulation (ICSS) thresholds, discriminative stimulus effects, and effects on locomotor activity.
Results
Similar levels of nicotine and minor alkaloids were achieved using either artificial saliva or saline for extraction, supporting the clinical relevance of the saline extracts used in these studies. Extract produced reinforcement-enhancing (ICSS threshold-decreasing) effects similar to those of nicotine alone at low to moderate nicotine doses, but reduced reinforcement-attenuating (ICSS threshold-increasing) effects at a high nicotine dose. In rats trained to discriminate nicotine alone from saline, intermediate extract doses did not substitute for the training dose as well as nicotine alone. Locomotor stimulant effects and nicotine distribution to brain were similar following administration of extract or nicotine alone.
Conclusions
The reinforcement-attenuating and discriminative stimulus effects of nicotine delivered in an extract of a commercial smokeless tobacco product differed from those of nicotine alone. Extracts of tobacco products may be useful for evaluating the abuse liability of those products and understanding the role of non-nicotine constituents in tobacco addiction.
doi:10.1007/s00213-011-2514-y
PMCID: PMC3363290  PMID: 21960181
Nicotine; smokeless tobacco; non-nicotine tobacco constituents; extract; intracranial self-stimulation; nicotine discrimination; locomotor sensitization
16.  Correlates of individual differences in compensatory nicotine self-administration in rats following a decrease in nicotine unit dose 
Psychopharmacology  2009;205(4):599-611.
Rationale
The ability of tobacco harm reduction strategies to produce significant reductions in toxin exposure is limited by compensatory increases in smoking behavior. Characterizing factors contributing to the marked individual variability in compensation may be useful for understanding this phenomenon and assessing the feasibility of harm reduction interventions.
Objective
To use an animal model of human compensatory smoking that involves a decrease in unit dose supporting nicotine self-administration (NSA) to examine potential contributors to individual differences in compensation.
Methods
Rats were trained for NSA during daily 23 hr sessions at a unit dose of 0.06 mg/kg/inf until responding was stable. The unit dose was then reduced to 0.03 mg/kg/inf for at least 10 sessions. Following reacquisition of NSA at the training dose and extinction, single-dose nicotine pharmacokinetic parameters were determined.
Results
Decreases in nicotine intake following dose reduction were proportionally less than the decrease in unit dose, indicating partial compensation. Compensatory increases in infusion rates were observed across the course of the 23 hr sessions. The magnitude of compensation differed considerably between rats. Rats exhibiting the highest baseline infusion rates exhibited the lowest levels of compensation. Nicotine pharmacokinetic parameters were not significantly correlated with compensation. Infusion rates immediately returned to pre-reduction levels when baseline conditions were restored.
Conclusions
These findings provide initial insights into correlates of individual differences in compensation following a reduction in nicotine unit dose. The present assay may be useful for characterizing mechanisms and potential consequences of the marked individual differences in compensatory smoking observed in humans.
doi:10.1007/s00213-009-1567-7
PMCID: PMC3601673  PMID: 19475400
Nicotine; Self-administration; Rat; Harm Reduction; Compensation
17.  A lack of association between severity of nicotine withdrawal and individual differences in compensatory nicotine self-administration in rats 
Psychopharmacology  2011;217(2):153-166.
Rationale
Compensatory smoking may represent an adverse consequence of smoking reduction or the use of reduced nicotine tobacco products. Factors contributing to individual variability in compensation are poorly understood.
Objective
To examine whether severity of nicotine withdrawal as measured by elevated intracranial self-stimulation (ICSS) thresholds is related to individual differences in compensatory nicotine self-administration (NSA) following unit dose reduction.
Methods
Rats were trained for ICSS and NSA (0.06 mg/kg/inf). After stabilization, effects of reducing the nicotine unit dose to 0.03 mg/kg/inf were examined. Following reacquisition of NSA (0.06 mg/kg/inf), effects of antagonist-precipitated withdrawal and saline extinction (spontaneous withdrawal) were examined.
Results
Reducing the NSA unit dose produced partial compensation as indicated by increased infusion rates but a 35% mean decrease in daily nicotine intake. Magnitude of compensation varied considerably among rats. Dose reduction did not elicit withdrawal in rats as a group, although there were substantial increases in ICSS thresholds in some animals. Intracranial self-stimulation thresholds were consistently elevated during precipitated and spontaneous withdrawal, confirming that rats were nicotine-dependent. Individual differences in compensation were not correlated with changes in ICSS thresholds during dose reduction, precipitated withdrawal, or spontaneous withdrawal. In a secondary analysis, greater precipitated withdrawal severity predicted greater initial nicotine-seeking during extinction.
Conclusions
Severity of nicotine withdrawal was not related to the degree of compensation in this protocol. These data do not support a role for nicotine withdrawal in individual differences in compensation during reduced nicotine exposure, but do suggest that withdrawal may contribute to nicotine-seeking during early abstinence.
doi:10.1007/s00213-011-2273-9
PMCID: PMC3601679  PMID: 21494791
Nicotine; Self-administration; Intracranial self-stimulation; Withdrawal; Tobacco harm reduction; Compensation
18.  Stimulation of Cortical Myosin Phosphorylation by p114RhoGEF Drives Cell Migration and Tumor Cell Invasion 
PLoS ONE  2012;7(11):e50188.
Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells.
doi:10.1371/journal.pone.0050188
PMCID: PMC3501466  PMID: 23185572
19.  Human cell responses to ionizing radiation are differentially affected by the expressed connexins 
Journal of Radiation Research  2012;54(2):251-259.
In multicellular organisms, intercellular communication is essential for homeostatic functions and has a major role in tissue responses to stress. Here, we describe the effects of expression of different connexins, which form gap junction channels with different permeabilities, on the responses of human cells to ionizing radiation. Exposure of confluent HeLa cell cultures to 137Cs γ rays, 3.7 MeV α particles, 1000 MeV protons or 1000 MeV/u iron ions resulted in distinct effects when the cells expressed gap junction channels composed of either connexin26 (Cx26) or connexin32 (Cx32). Irradiated HeLa cells expressing Cx26 generally showed decreased clonogenic survival and reduced metabolic activity relative to parental cells lacking gap junction communication. In contrast, irradiated HeLa cells expressing Cx32 generally showed enhanced survival and greater metabolic activity relative to the control cells. The effects on clonogenic survival correlated more strongly with effects on metabolic activity than with DNA damage as assessed by micronucleus formation. The data also showed that the ability of a connexin to affect clonogenic survival following ionizing radiation can depend on the specific type of radiation. Together, these findings show that specific types of connexin channels are targets that may be exploited to enhance radiotherapeutic efficacy and to formulate countermeasures to the harmful effects of specific types of ionizing radiation.
doi:10.1093/jrr/rrs099
PMCID: PMC3589937  PMID: 23139176
gap junction permeability; cohort effects; radiotherapy; stress response; linear energy transfer/radiation quality
20.  Combined active and passive immunization against nicotine: Minimizing monoclonal antibody requirements using a target antibody concentration strategy 
International immunopharmacology  2011;11(11):1809-1815.
Nicotine vaccines have shown preliminary evidence of efficacy for enhancing smoking cessation rates, but the serum nicotine-specific antibody (NicAb) concentrations produced are highly variable and many subjects do not develop effective levels. As an alternative to vaccination, passive immunization with nicotine-specific monoclonal antibodies could produce more uniform serum NicAb concentrations, but its use is limited by their high cost and shorter elimination half-life. This study investigated supplementing vaccination with monoclonal antibodies in a targeted fashion to increase vaccine efficacy while minimizing the required monoclonal antibody dose. Rats were vaccinated and then given individualized supplemental doses of the nicotine-specific monoclonal antibody Nic311 to achieve a target total serum NicAb concentration known to be effective for blocking locomotor sensitization (LMS) to nicotine. Rats received vaccine, Nic311, both, or neither, followed by 0.3 mg/kg nicotine s.c. for 10 days to produce LMS. Combination immunotherapy completely blocked the development of LMS, while monotherapy with vaccine or Nic311 alone were only minimally effective. Lower brain nicotine levels were associated with reduced locomotor activity averaged over days 7-10. Despite its greater efficacy, combination immunotherapy did not reduce the variability in the resulting total serum NicAb concentrations. Variability in total serum NicAb concentrations was contributed to by both vaccine-generated antibody and by Nic311. These data show that combination immunotherapy, using a Nic311 dose that is by itself only minimally effective, can substantially enhance nicotine vaccine efficacy. However, variability in serum NicAb levels with combination immunotherapy may make translation of this approach challenging.
doi:10.1016/j.intimp.2011.07.009
PMCID: PMC3204179  PMID: 21802529
nicotine; immunotherapy; locomotor sensitization; vaccine; monoclonal antibody; pharmacokinetics
21.  High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease 
Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2Rα5, TNFR16, HGF7, IL-88, elafin2, and REG3α3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.
doi:10.3791/4247
PMCID: PMC3499072  PMID: 23149907
Medicine; Issue 68; ELISA; Sequential ELISA; Cytokine; Blood plasma; biomarkers; proteomics; graft-versus-host disease; Small sample; Quantification
22.  The Role of Gap Junction Communication and Oxidative Stress in the Propagation of Toxic Effects among High-Dose α-Particle-Irradiated Human Cells 
Radiation research  2011;175(3):347-357.
We investigated the roles of gap junction communication and oxidative stress in modulating potentially lethal damage repair in human fibroblast cultures exposed to doses of α particles or γ rays that targeted all cells in the cultures. As expected, α particles were more effective than γ rays at inducing cell killing; further, holding γ-irradiated cells in the confluent state for several hours after irradiation promoted increased survival and decreased chromosomal damage. However, maintaining α-particle-irradiated cells in the confluent state for various times prior to subculture resulted in increased rather than decreased lethality and was associated with persistent DNA damage and increased protein oxidation and lipid peroxidation. Inhibiting gap junction communication with 18-α-glycyrrhetinic acid or by knockdown of connexin43, a constitutive protein of junctional channels in these cells, protected against the toxic effects in α-particle-irradiated cell cultures during confluent holding. Upregulation of antioxidant defense by ectopic overexpression of glutathione peroxidase protected against cell killing by α particles when cells were analyzed shortly after exposure. However, it did not attenuate the decrease in survival during confluent holding. Together, these findings indicate that the damaging effect of α particles results in oxidative stress, and the toxic effects in the hours after irradiation are amplified by intercellular communication, but the communicated molecule(s) is unlikely to be a substrate of glutathione peroxidase.
doi:10.1667/RR2372.1
PMCID: PMC3139025  PMID: 21388278
23.  Molecular dynamics simulations of the Cx26 hemichannel: Evaluation of structural models with Brownian dynamics 
The Journal of General Physiology  2011;138(5):475-493.
The recently published crystal structure of the Cx26 gap junction channel provides a unique opportunity for elucidation of the structure of the conductive connexin pore and the molecular determinants of its ion permeation properties (conductance, current–voltage [I-V] relations, and charge selectivity). However, the crystal structure was incomplete, most notably lacking the coordinates of the N-terminal methionine residue, which resides within the pore, and also lacking two cytosolic domains. To allow computational studies for comparison with the known channel properties, we completed the structure. Grand canonical Monte Carlo Brownian dynamics (GCMC/BD) simulations of the completed and the published Cx26 hemichannel crystal structure indicate that the pore is too narrow to permit significant ion flux. The GCMC/BD simulations predict marked inward current rectification and almost perfect anion selectivity, both inconsistent with known channel properties. The completed structure was refined by all-atom molecular dynamics (MD) simulations (220 ns total) in an explicit solvent and POPC membrane system. These MD simulations produced an equilibrated structure with a larger minimal pore diameter, which decreased the height of the permeation barrier formed by the N terminus. GCMC/BD simulations of the MD-equilibrated structure yielded more appropriate single-channel conductance and less anion/cation selectivity. However, the simulations much more closely matched experimentally determined I-V relations when the charge effects of specific co- and posttranslational modifications of Cx26 previously identified by mass spectrometry were incorporated. We conclude that the average equilibrated structure obtained after MD simulations more closely represents the open Cx26 hemichannel structure than does the crystal structure, and that co- and posttranslational modifications of Cx26 hemichannels are likely to play an important physiological role by defining the conductance and ion selectivity of Cx26 channels. Furthermore, the simulations and data suggest that experimentally observed heterogeneity in Cx26 I-V relations can be accounted for by variation in co- and posttranslational modifications.
doi:10.1085/jgp.201110679
PMCID: PMC3206306  PMID: 22006989
24.  Ribose Sugars Generate Interrnal Glycation Cross-links in Horse Heart Myoglobin 
Glycation of horse heart metmyoglobin with D-ribose 5-phosphate (R5P), D-2-deoxyribose 5-phosphate (dR5P), and D-ribose with inorganic phosphate at 37 °C generates an altered protein (Myo-X) with increased SDS PAGE mobility. The novel protein product has been observed only for reactions with the protein myoglobin and it is not evident with other common sugars reacted over a one week period. Myo-X is first observed at 1-2 days at 37 °C along with a second form that is consistent in mass with that of myoglobin attached to several sugars. MALDI mass spectrometry and other techniques show no evidence of the cleavage of a peptide from the myoglobin chain. Apomyoglobin in reaction with R5P also exhibited this protein form suggesting its occurrence was not heme-related. While significant amounts of O2− and H2O2 are generated during the R5P glycation reaction, they do not appear to play roles in the formation of the new form. The modification is likely due to an internal cross-link formed during a glycation reaction involving the N-terminus and an internal amine group; most likely the neighboring Lys133. The study shows the unique nature of these common pentose sugars in spontaneous glycation reactions with proteins.
doi:10.1016/j.bbrc.2011.02.138
PMCID: PMC3086664  PMID: 21376016
Glycation; ribose 5-phosphate; cross-linking; protein modification; heme degradation
25.  Mechanism for modulation of gating of connexin26-containing channels by taurine 
The Journal of General Physiology  2011;138(3):321-339.
The mechanisms of action of endogenous modulatory ligands of connexin channels are largely unknown. Previous work showed that protonated aminosulfonates (AS), notably taurine, directly and reversibly inhibit homomeric and heteromeric channels that contain Cx26, a widely distributed connexin, but not homomeric Cx32 channels. The present study investigated the molecular mechanisms of connexin channel modulation by taurine, using hemichannels and junctional channels composed of Cx26 (homomeric) and Cx26/Cx32 (heteromeric). The addition of a 28–amino acid “tag” to the carboxyl-terminal domain (CT) of Cx26 (Cx26T) eliminated taurine sensitivity of homomeric and heteromeric hemichannels in cells and liposomes. Cleavage of all but four residues of the tag (Cx26Tc) resulted in taurine-induced pore narrowing in homomeric hemichannels, and restored taurine inhibition of heteromeric hemichannels (Cx26Tc/Cx32). Taurine actions on junctional channels were fully consistent with those on hemichannels. Taurine-induced inhibition of Cx26/Cx32T and nontagged Cx26 junctional channels was blocked by extracellular HEPES, a blocker of the taurine transporter, confirming that the taurine-sensitive site of Cx26 is cytoplasmic. Nuclear magnetic resonance of peptides corresponding to Cx26 cytoplasmic domains showed that taurine binds to the cytoplasmic loop (CL) and not the CT, and that the CT and CL directly interact. ELISA showed that taurine disrupts a pH-dependent interaction between the CT and the CT-proximal half of the CL. These studies reveal that AS disrupt a pH-driven cytoplasmic interdomain interaction in Cx26-containing channels, causing closure, and that the Cx26CT has a modulatory role in Cx26 function.
doi:10.1085/jgp.201110634
PMCID: PMC3171079  PMID: 21844220

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