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1.  Functional Interactions Between the Nucleus Tractus Solitarius (NTS) and Nucleus Accumbens Shell in Modulating Memory for Arousing Experiences 
The shell division of the nucleus accumbens receives noradrenergic input from neurons in the nucleus of the solitary tract (NTS) that transmit information regarding fluctuations in peripheral hormonal and autonomic activity. Accumbens shell neurons also receive converging inputs from limbic areas such as the hippocampus and amygdala that process newly acquired information. However, few studies have explored whether peripheral information regarding changes in emotional arousal contributes to memory processing in the accumbens. The beneficial effects on memory produced by emotional arousal and the corresponding activation of NTS neurons may be mediated through influences on neuronal activity in the accumbens shell during memory encoding. To explore this putative relationship, Experiment 1 examined interactions between the NTS and the accumbens shell in modulating memory for responses acquired after footshock training in a water-motivated inhibitory avoidance task. Memory for the noxious shock was significantly improved by posttraining excitation of noradrenergic NTS neurons. The enhanced retention produced by activating NTS neurons was attenuated by suppressing neuronal activity in the accumbens shell with bupivacaine (0.25%/0.5μl). Experiment 2 examined the direct involvement of accumbens shell noradrenergic activation in the modulation of memory for psychologically arousing events such as a reduction in perceived reward value. Noradrenergic activation of the accumbens shell with phenylephrine (1.0μg/0.5μl) produced an enhancement in memory for the frustrating experience relative to control injections as evidenced by runway performance on an extended seven-day retention test. These findings demonstrate a functional relationship between NTS neurons and the accumbens shell in modulating memory following physiological arousal and identifies a role of norepinephrine in modulating synaptic activity in the accumbens shell to facilitate this process.
PMCID: PMC2175480  PMID: 17964820
2.  Lipid-shelled vehicles: engineering for ultrasound molecular imaging and drug delivery 
Accounts of chemical research  2009;42(7):881-892.
Ultrasound pressure waves can map the location of lipid-stabilized gas microbubbles after their intravenous administration in the body, facilitating an estimate of vascular density and microvascular flow rate. Microbubbles are currently approved by the Food and Drug Administration as ultrasound contrast agents for visualizing opacification of the left ventricle in echocardiography. However, the interaction of ultrasound waves with intravenously injected lipid-shelled particles, including both liposomes and microbubbles, is a far richer field. Particles can be designed for molecular imaging and loaded with drugs or genes—the mechanical and thermal properties of ultrasound can then effect localized drug release.
In this Account, we provide an overview of the engineering of lipid-shelled microbubbles (typical diameter 1000–10,000 nm) and liposomes (typical diameter 65–120 nm) for ultrasound-based applications in molecular imaging and drug delivery. The chemistries of the shell and core can be optimized to enhance stability, circulation persistence, drug loading and release, targeting to and fusion with the cell membrane, and therapeutic biological effects. To assess the biodistribution and pharmacokinetics of these particles, we incorporated positron emission tomography (PET) radioisotopes on the shell. The radionuclide 18F (half life ~2 hours) was covalently coupled to a dipalmitoyl lipid, followed by integration of the labeled lipid into the shell, facilitating short-term analysis of particle pharmacokinetics and metabolism of the lipid molecule. Alternately, labeling a formed particle with 64Cu (half life 12.7 hours)—after prior covalent incorporation of a copper-chelating moiety onto the lipid shell—permits pharmacokinetic study of particles over several days.
Stability and persistence in circulation of both liposomes and microbubbles are enhanced by long acyl chains and a polyethylene glycol coating. Vascular targeting has been demonstrated with both nano- and micro-diameter particles. Targeting affinity of the microbubble can be modulated by burying the ligand within a polymer brush layer; the application of ultrasound then reveals the ligand, enabling specific targeting of only the insonified region.
Microbubbles and liposomes require different strategies for both drug loading and release. Microbubble loading is inhibited by the gas core and enhanced by layer-by-layer construction or conjugation of drug-entrapped particles to the surface. Liposome loading is typically internal and is enhanced by drug-specific loading techniques. Drug release from a microbubble results from the oscillation of the gas core diameter produced by the sound wave, whereas that from a liposome is enhanced by heat produced from the local absorption of acoustic energy within the tissue microenvironment. Biological effects induced by ultrasound, such as changes in cell membrane and vascular permeability, can enhance drug delivery. In particular, as microbubbles oscillate near a vessel wall, shock waves or liquid jets enhance drug transport. Mild heating induced by ultrasound, either before or after injection of the drug, facilitates the transport of liposomes from blood vessels to the tissue interstitium, thus increasing drug accumulation in the target region.
Lipid-shelled vehicles offer many opportunities for chemists and engineers; ultrasound-based applications beyond the few currently in common use will undoubtedly soon multiply as molecular construction techniques are further refined.
PMCID: PMC2727628  PMID: 19552457
3.  A cumulative shear mechanism for tissue damage initiation in shock-wave lithotripsy 
Ultrasound in medicine & biology  2007;33(9):1495-1503.
Evidence suggests that inertial cavitation plays an important role in the renal injury incurred during shock-wave lithotripsy. However, it is unclear how tissue damage is initiated, and significant injury typically occurs only after a sufficient dose of shock waves. While it has been suggested that shock-induced shearing might initiate injury, estimates indicate that individual shocks do not produce sufficient shear to do so. In this paper, we hypothesize that the cumulative shear of the many shocks is damaging. This mechanism depends upon whether there is sufficient time between shocks for tissue to relax to its unstrained state. We investigate the mechanism with a physics-based simulation model wherein the the basement membranes that define the tubules and vessels in the inner medulla are represented as elastic shells surrounded by viscous fluid. Material properties are estimated from in vitro tests of renal basement membranes and documented mechanical properties of cells and extracellular gels. Estimates for the net shear deformation from a typical lithotripter shock (~ 0.1%) are found from a separate dynamic shock simulation. The results suggest that the larger interstitial volume (~ 40%) near the papilla tip gives the tissue there a relaxation time comparable to clinical shock delivery rates (~ 1Hz), thus allowing shear to accumulate. Away from the papilla tip, where the interstitial volume is smaller (≲ 20%), the model tissue relaxes completely before the next shock would be delivered. Implications of the model are that slower delivery rates and broader focal zones should both decrease injury, consistent with some recent observations.
PMCID: PMC2020810  PMID: 17507147
Shock-wave lithotripsy; renal injury; tissue damage; numerical simulation
4.  Empyema, acute respiratory failure, and septic shock after aspiration of a soft-shelled turtle (Pelodiscus sinensis) bone by an adult 
SpringerPlus  2014;3:452.
The common late complications of foreign body aspiration include granulation formation, obstructive pneumonia, and atelectasis. However, a foreign body-induced pleural infection is very rare, and especially when it is not iatrogenic.
Case description
A 64-year-old Chinese man was admitted to our hospital with septic shock and acute respiratory failure requiring intubation and mechanical ventilation. Computed tomography revealed multiloculated pleural effusion on the whole right side and right lung atelectasis, with enhanced thickening and calcification of pleura and a foreign body in the right intermediate bronchus. The effusion appeared as a cloudy fluid consistent with pus. A bedside bronchoscopy revealed an irregular foreign body lodged in the right intermediate bronchus. The hard bone was removed and confirmed to be a soft-shelled turtle bone. A final diagnosis of foreign body-induced empyema, acute respiratory failure, and septic shock was confirmed. The patient showed good recovery after completing a course of broad-spectrum antibiotics and undergoing chest tube drainage.
Discussion and evaluation
Although empyema has been reported previously as a rare complication of long-term retention of an aspirated foreign body, no case has been observed that was as serious as our current patient. In addition, a foreign body aspiration by a soft-shelled turtle bone was never reported before.
For the first time, we describe the successful treatment of an adult patient presenting with empyema, accompanied by serious conditions of acute respiratory failure and septic shock induced by aspiration of a soft-shelled turtle bone. Clinicians should consider the possibility of non-iatrogenic foreign body-induced empyema with acute onset of respiratory failure, when a patient’s symptoms cannot be attributed to an alternative obvious cause.
PMCID: PMC4143537  PMID: 25161865
Soft-shelled turtle bone; Foreign body aspiration; Empyema; Acute respiratory failure; Septic shock; Adult
5.  Ocean Acidification at High Latitudes: Potential Effects on Functioning of the Antarctic Bivalve Laternula elliptica 
PLoS ONE  2011;6(1):e16069.
Ocean acidification is a well recognised threat to marine ecosystems. High latitude regions are predicted to be particularly affected due to cold waters and naturally low carbonate saturation levels. This is of concern for organisms utilising calcium carbonate (CaCO3) to generate shells or skeletons. Studies of potential effects of future levels of pCO2 on high latitude calcifiers are at present limited, and there is little understanding of their potential to acclimate to these changes. We describe a laboratory experiment to compare physiological and metabolic responses of a key benthic bivalve, Laternula elliptica, at pCO2 levels of their natural environment (430 µatm, pH 7.99; based on field measurements) with those predicted for 2100 (735 µatm, pH 7.78) and glacial levels (187 µatm, pH 8.32). Adult L. elliptica basal metabolism (oxygen consumption rates) and heat shock protein HSP70 gene expression levels increased in response both to lowering and elevation of pH. Expression of chitin synthase (CHS), a key enzyme involved in synthesis of bivalve shells, was significantly up-regulated in individuals at pH 7.78, indicating L. elliptica were working harder to calcify in seawater undersaturated in aragonite (ΩAr = 0.71), the CaCO3 polymorph of which their shells are comprised. The different response variables were influenced by pH in differing ways, highlighting the importance of assessing a variety of factors to determine the likely impact of pH change. In combination, the results indicate a negative effect of ocean acidification on whole-organism functioning of L. elliptica over relatively short terms (weeks-months) that may be energetically difficult to maintain over longer time periods. Importantly, however, the observed changes in L. elliptica CHS gene expression provides evidence for biological control over the shell formation process, which may enable some degree of adaptation or acclimation to future ocean acidification scenarios.
PMCID: PMC3016332  PMID: 21245932
6.  Daily and seasonal changes in heat exposure and the Hsp70 level of individuals from a field population of Xeropicta derbentina (Krynicki 1836) (Pulmonata, Hygromiidae) in Southern France 
Cell Stress & Chaperones  2012;18(4):405-414.
The Mediterranean land snail Xeropicta derbentina forms huge populations in Southern France. In order to characterize heat exposure and the induction of the 70-kD heat shock protein (Hsp70) response system during the life cycle of this snail, a selected population from the Vaucluse area, Provence, was investigated encompassing the issues of morphological life cycle parameters (shell size and colouration), the daily courses of heat exposure at different heights above the ground, of shell temperature, and that of the individual Hsp70 levels. The study covered all four seasons of the year 2011. Snails were found to be annual, reaching their final size in August. The shell colouration pattern showed high variation in juveniles (spring) with a strong tendency towards becoming uniformly white at old age in autumn. In all seasons, ambient air temperature decreased with increasing distance from the ground surface during daytime while remaining constantly low in the night. Overall, the Hsp70 level of individuals followed the ambient temperature during diurnal and seasonal variations. Correlation analysis revealed a positive association of individual shell temperature and Hsp70 level for the most part of the life cycle of the snails until late summer, whereas a negative correlation was found for aged animals indicating senescence effects on the capacity of the stress response system.
PMCID: PMC3682011  PMID: 23250584
Heat shock response; Mediterranean land snail; Stress proteins; Temperature; Life cycle
7.  In vitro transcription of human pararotavirus. 
Journal of Virology  1986;57(1):183-190.
Purified human pararotavirus obtained from stool samples from a 6-month-old infant was characterized. Electron microscopy of the viral particles subjected to different treatments suggested that the protein shells differed from those described for rotavirus. Treatment with both EDTA or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the presence or absence of Mg2+ seemed to convert the virions into core particles by removal of both the outer and inner shells, and no particles equivalent to single-shelled rotavirus were observed. Different procedures were used to activate the human pararotavirus-associated RNA-dependent RNA polymerase. The enzyme was not activated by chelating agents or by thermal shock as in rotavirus. Activation by thermal shock occurred only in the presence of the four ribonucleoside triphosphates and Mg2+. However, the polymerase of pararotavirus was found to be similar to those described for rotaviruses. When in vitro transcripts were analyzed, 11 RNA species having a migration pattern similar to that of the original genomic RNA were detected.
PMCID: PMC252713  PMID: 3001343
8.  Nucleus accumbens shell and core involvement in drug context-induced reinstatement of cocaine seeking in rats 
Psychopharmacology  2008;200(4):545-556.
The nucleus accumbens (NAC) is a functionally heterogeneous brain region with respect to its involvement in cocaine-seeking behavior triggered by drug-associated explicit conditioned stimuli, foot shock stress, or cocaine itself in the reinstatement animal model of drug relapse. However, it is not known whether the NAC or its subregions are critical for reinstatement of cocaine-seeking behavior produced by re-exposure to a previously cocaine-paired environmental context.
The present study was designed to evaluate potentially unique contributions of the NAC core and shell to this behavior.
Materials and methods
Rats were trained to lever press for unsignaled cocaine infusions (0.15 mg/infusion, intravenous) in a distinct environmental context. Lever responding was then extinguished in a distinctly different environmental context (extinction context) during a minimum of seven daily training sessions. Subsequently, using a counterbalanced testing design, rats were re-exposed to the cocaine-paired context or the extinction context while cocaine seeking (i.e., responding on the previously cocaine-reinforced lever) was assessed. Before each test session, neural activity was inhibited selectively in the NAC core or shell using bilateral microinfusions of the γ-aminobutyric acid agonists, baclofen and muscimol (0/0 or 1.0/0.1 mM; 0.3 μl per hemisphere).
Neural inactivation of the NAC shell or core attenuated responding in the cocaine context and, interestingly, increased responding in the extinction context. Control experiments indicated no effects on general activity or food-reinforced instrumental behavior.
These findings suggest that both subregions of the NAC may promote context-induced reinstatement by facilitating drug context-induced motivation for cocaine and context discrimination.
PMCID: PMC2613506  PMID: 18597075
Self-administration; Extinction; Reinstatement; Context; cocaine; Muscimol; Baclofen; Nucleus accumbens
9.  Crystal Structure of an Activated Variant of Small Heat Shock Protein Hsp16.5 
Biochemistry  2012;51(25):5105-5112.
How does the sequence of a single Small Heat Shock Protein (sHSP) assemble into oligomers of different sizes? To gain insight into the underlying structural mechanism, we determined the crystal structure of an engineered variant of Methanocaldococcus jannaschii Hsp16.5 wherein a 14 amino acid peptide from human heat shock protein 27 (Hsp27) was inserted at the junction of the N-terminal region and the α-crystallin domain. In response to this insertion, the oligomer shell expands from 24 to 48 subunits while maintaining octahedral symmetry. Oligomer rearrangement does not alter the fold of the conserved α-crystallin domain nor does it disturb the interface holding the dimeric building block together. Rather, the flexible C-terminal tail of Hsp16.5 changes its orientation relative to the α-crystallin domain which enables alternative packing of dimers. This change in orientation preserves a peptide-in-groove interaction of the C-terminal tail with an adjacent β-sandwich thereby holding the assembly together. The interior of the expanded oligomer, where substrates presumably bind, retains its predominantly non-polar character relative to the outside surface. New large windows in the outer shell provide increased access to these substrate-binding regions, thus accounting for the higher affinity of this variant to substrates. Oligomer polydispersity regulates sHSPs chaperone activity in vitro and has been implicated in their physiological roles. The structural mechanism of Hsp16.5 oligomer flexibility revealed here, which is likely to be highly conserved across the sHSP superfamily, explains the relationship between oligomer expansion observed in disease-linked mutants and changes in chaperone activity.
PMCID: PMC3384710  PMID: 22670769
Small heat shock proteins; Hsp16.5; Chaperone; crystal structure
10.  ‘Shell shock’ Revisited: An Examination of the Case Records of the National Hospital in London 
Medical History  2014;58(4):519-545.
During the First World War the National Hospital for the Paralysed and Epileptic, in Queen Square, London, then Britain’s leading centre for neurology, took a key role in the treatment and understanding of shell shock. This paper explores the case notes of all 462 servicemen who were admitted with functional neurological disorders between 1914 and 1919. Many of these were severe or chronic cases referred to the National Hospital because of its acknowledged expertise and the resources it could call upon. Biographical data was collected together with accounts of the patient’s military experience, his symptoms, diagnostic interpretations and treatment outcomes. Analysis of the notes showed that motor syndromes (loss of function or hyperkinesias), often combined with somato-sensory loss, were common presentations. Anxiety and depression as well as vegetative symptoms such as sweating, dizziness and palpitations were also prevalent among this patient population. Conversely, psychogenic seizures were reported much less frequently than in comparable accounts from German tertiary referral centres. As the war unfolded the number of physicians who believed that shell shock was primarily an organic disorder fell as research failed to find a pathological basis for its symptoms. However, little agreement existed among the Queen Square doctors about the fundamental nature of the disorder and it was increasingly categorised as functional disorder or hysteria.
PMCID: PMC4176276  PMID: 25284893
First World War; Trauma; Psychopathology; Case histories; Neurology; Hysteria
11.  Oyster Shell Proteins Originate from Multiple Organs and Their Probable Transport Pathway to the Shell Formation Front 
PLoS ONE  2013;8(6):e66522.
Mollusk shell is one kind of potential biomaterial, but its vague mineralization mechanism hinders its further application. Mollusk shell matrix proteins are important functional components that are embedded in the shell, which play important roles in shell formation. The proteome of the oyster shell had been determined based on the oyster genome sequence by our group and gives the chance for further deep study in this area. The classical model of shell formation posits that the shell proteins are mantle-secreted. But, in this study, we further analyzed the shell proteome data in combination with organ transcriptome data and we found that the shell proteins may be produced by multiple organs though the mantle is still the most important organ for shell formation. To identify the transport pathways of these shell proteins not in classical model of shell formation, we conducted a shell damage experiment and we determined the shell-related gene set to identify the possible transport pathways from multiple organs to the shell formation front. We also found that there may exist a remodeling mechanism in the process of shell formation. Based on these results along with some published results, we proposed a new immature model, which will help us think about the mechanism of shell formation in a different way.
PMCID: PMC3686672  PMID: 23840499
12.  Association between shell morphology of micro-land snails (genus Plectostoma) and their predator’s predatory behaviour 
PeerJ  2014;2:e329.
Predator–prey interactions are among the main ecological interactions that shape the diversity of biological form. In many cases, the evolution of the mollusc shell form is presumably driven by predation. However, the adaptive significance of several uncommon, yet striking, shell traits of land snails are still poorly known. These include the distorted coiled “tuba” and the protruded radial ribs that can be found in micro-landsnails of the genus Plectostoma. Here, we experimentally tested whether these shell traits may act as defensive adaptations against predators. We characterised and quantified the possible anti-predation behaviour and shell traits of Plectostoma snails both in terms of their properties and efficiencies in defending against the Atopos slug predatory strategies, namely, shell-apertural entry and shell-drilling. The results showed that Atopos slugs would first attack the snail by shell-apertural entry, and, should this fail, shift to the energetically more costly shell-drilling strategy. We found that the shell tuba of Plectostoma snails is an effective defensive trait against shell-apertural entry attack. None of the snail traits, such as resting behaviour, shell thickness, shell tuba shape, shell rib density and intensity can fully protect the snail from the slug’s shell-drilling attack. However, these traits could increase the predation costs to the slug. Further analysis on the shell traits revealed that the lack of effectiveness in these anti-predation shell traits may be caused by a functional trade-off between shell traits under selection of two different predatory strategies.
PMCID: PMC3976122  PMID: 24749008
Genus Opisthostoma; Limestone; Malaysia; Borneo; Sabah; Molluscs; Gastropoda; Rathousiidae; Diplommatinidae; 3D morphometrics
13.  Free-Operant Avoidance Behavior by Rats after Reinforcer Revaluation Using Opioid Agonists and d-Amphetamine 
The Journal of Neuroscience  2014;34(18):6286-6293.
The associative processes that support free-operant instrumental avoidance behavior are still unknown. We used a revaluation procedure to determine whether the performance of an avoidance response is sensitive to the current value of the aversive, negative reinforcer. Rats were trained on an unsignaled, free-operant lever press avoidance paradigm in which each response avoided or escaped shock and produced a 5 s feedback stimulus. The revaluation procedure consisted of noncontingent presentations of the shock in the absence of the lever either paired or unpaired with systemic morphine and in a different cohort with systemic d-amphetamine. Rats were then tested drug free during an extinction test. In both the d-amphetamine and morphine groups, pairing of the drug and shock decreased subsequent avoidance responding during the extinction test, suggesting that avoidance behavior was sensitive to the current incentive value of the aversive negative reinforcer. Experiment 2 used central infusions of D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), a mu-opioid receptor agonist, in the periacqueductal gray and nucleus accumbens shell to revalue the shock. Infusions of DAMGO in both regions replicated the effects seen with systemic morphine. These results are the first to demonstrate the impact of revaluation of an aversive reinforcer on avoidance behavior using pharmacological agents, thereby providing potential therapeutic targets for the treatment of avoidance behavior symptomatic of anxiety disorders.
PMCID: PMC4004814  PMID: 24790199
avoidance; d-amphetamine; morphine; nucleus accumbens shell; periacqueductal gray; revaluation
14.  An ancient process in a modern mollusc: early development of the shell in Lymnaea stagnalis 
The morphological variety displayed by the molluscan shell underlies much of the evolutionary success of this phylum. However, the broad diversity of shell forms, sizes, ornamentations and functions contrasts with a deep conservation of early cell movements associated with the initiation of shell construction. This process begins during early embryogenesis with a thickening of an ectodermal, ‘dorsal’ (opposite the blastopore) population of cells, which then invaginates into the blastocoel to form the shell gland. The shell gland evaginates to form the shell field, which then expands and further differentiates to eventually become the adult shell-secreting organ commonly known as the mantle. Despite the deep conservation of the early shell forming developmental program across molluscan classes, little is known about the fine-scale cellular or molecular processes that underlie molluscan shell development.
Using modern imaging techniques we provide here a description of the morphogenesis of a gastropod shell gland and shell field using the pulmonate gastropod Lymnaea stagnalis as a model. We find supporting evidence for a hypothesis of molluscan shell gland specification proposed over 60 years ago, and present histochemical assays that can be used to identify a variety of larval shell stages and distinct cell populations in whole mounts.
By providing a detailed spatial and temporal map of cell movements and differentiation events during early shell development in L. stagnalis we have established a platform for future work aimed at elucidation of the molecular mechanisms and regulatory networks that underlie the evo-devo of the molluscan shell.
PMCID: PMC3728215  PMID: 23845038
Shell; Mollusc; Biomineralisation; Evolution; Development; Specification; Mantle; Alkaline phosphatase; Peroxidase
15.  Differential shell strength of Cepaea nemoralis colour morphs—implications for their anti-predator defence 
Die Naturwissenschaften  2013;100(9):843-851.
One of the most spectacular evolutionary forces is predation, evidenced to stimulate polymorphism in many prey species. Shell colour polymorphism of the land snail Cepaea nemoralis is a well-known model in evolutionary research. Nevertheless, the knowledge on the ecological causes driving its evolution remains incomplete and proximal factors shaping predatory pressure on C. nemoralis morphs are unknown. We evaluated shell crushing resistance and thickness, constituting crucial snail anti-predator defences in two shell areas (the apex and labium) of eight C. nemoralis morphotypes differing in shell colour and banding pattern. A GLM showed a significant effect of shell colour, banding pattern and shell thickness on shell strength. Pink shells were stronger than yellow ones, and banded forms had stronger shells than unbanded snails. The labium (usually attacked by mice) was generally thicker and more resistant than the apex (usually crushed by birds). Thicker shells were more resistant to crushing, and the rate of shell strength increase per unit of shell thickness was greater in pink and banded individuals compared to yellow and unbanded ones. Yellow and unbanded morphs have been found to be preferred by mice in the previous studies, which suggests that shell strength may be an important trait used in prey selection by these shell-crushing predators. The differences in potential anti-predator defences among snail morphs, found in the present study, justify future research on direct effect of C. nemoralis morphs shell strength on predator selectivity.
PMCID: PMC3753478  PMID: 23921905
Polymorphism; Shell crushing resistance; Labium; Banding type
16.  Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase 
Nature chemical biology  2008;4(11):700-707.
Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low levels, and a defensive cytotoxin at higher levels. The high active-site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock, and cancer. Our structural and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a novel specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents towards remote specificity pockets, accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active-site conservation.
PMCID: PMC2868503  PMID: 18849972
nitric oxide synthase; isozyme-specific inhibitor; induced-fit; x-ray crystallography; quinazoline; aminopyridine; drug design
17.  Crystal Structure of Butyrate Kinase 2 from Thermotoga maritima, a Member of the ASKHA Superfamily of Phosphotransferases▿  
Journal of Bacteriology  2009;191(8):2521-2529.
The enzymatic transfer of phosphoryl groups is central to the control of many cellular processes. One of the phosphoryl transfer mechanisms, that of acetate kinase, is not completely understood. Besides better understanding of the mechanism of acetate kinase, knowledge of the structure of butyrate kinase 2 (Buk2) will aid in the interpretation of active-site structure and provide information on the structural basis of substrate specificity. The gene buk2 from Thermotoga maritima encodes a member of the ASKHA (acetate and sugar kinases/heat shock cognate/actin) superfamily of phosphotransferases. The encoded protein Buk2 catalyzes the phosphorylation of butyrate and isobutyrate. We have determined the 2.5-Å crystal structure of Buk2 complexed with (β,γ-methylene) adenosine 5′-triphosphate. Buk2 folds like an open-shelled clam, with each of the two domains representing one of the two shells. In the open active-site cleft between the N- and C-terminal domains, the active-site residues consist of two histidines, two arginines, and a cluster of hydrophobic residues. The ATP binding region of Buk2 in the C-terminal domain consists of abundant glycines for nucleotide binding, and the ATP binding motif is similar to those of other members of the ASKHA superfamily. The enzyme exists as an octamer, in which four disulfide bonds form between intermolecular cysteines. Sequence alignment and structure superposition identify the simplicity of the monomeric Buk2 structure, a probable substrate binding site, the key residues in catalyzing phosphoryl transfer, and the substrate specificity differences among Buk2, acetate, and propionate kinases. The possible enzyme mechanisms are discussed.
PMCID: PMC2668392  PMID: 19201797
18.  Historical approaches to post-combat disorders 
Almost every major war in the last century involving western nations has seen combatants diagnosed with a form of post-combat disorder. Some took a psychological form (exhaustion, combat fatigue, combat stress reaction and post-traumatic stress disorder), while others were characterized by medically unexplained symptoms (soldier's heart, effort syndrome, shell shock, non-ulcer dyspepsia, effects of Agent Orange and Gulf War Syndrome). Although many of these disorders have common symptoms, the explanations attached to them showed considerable diversity often reflected in the labels themselves. These causal hypotheses ranged from the effects of climate, compressive forces released by shell explosions, side effects of vaccinations, changes in diet, toxic effects of organophosphates, oil-well fires or depleted-uranium munitions. Military history suggests that these disorders, which coexisted in the civilian population, reflected popular health fears and emerged in the gaps left by the advance of medical science. While the current Iraq conflict has yet to produce a syndrome typified by medically unexplained symptoms, it is unlikely that we have seen the last of post-combat disorders as past experience suggests that they have the capacity to catch both military planners and doctors by surprise.
PMCID: PMC1569621  PMID: 16687259
post-combat disorders; shell shock; post-traumatic stress disorder; disordered action of the heart; Gulf War Syndrome
19.  The small heat shock proteins from Acidithiobacillus ferrooxidans: gene expression, phylogenetic analysis, and structural modeling 
BMC Microbiology  2011;11:259.
Acidithiobacillus ferrooxidans is an acidophilic, chemolithoautotrophic bacterium that has been successfully used in metal bioleaching. In this study, an analysis of the A. ferrooxidans ATCC 23270 genome revealed the presence of three sHSP genes, Afe_1009, Afe_1437 and Afe_2172, that encode proteins from the HSP20 family, a class of intracellular multimers that is especially important in extremophile microorganisms.
The expression of the sHSP genes was investigated in A. ferrooxidans cells submitted to a heat shock at 40°C for 15, 30 and 60 minutes. After 60 minutes, the gene on locus Afe_1437 was about 20-fold more highly expressed than the gene on locus Afe_2172. Bioinformatic and phylogenetic analyses showed that the sHSPs from A. ferrooxidans are possible non-paralogous proteins, and are regulated by the σ32 factor, a common transcription factor of heat shock proteins. Structural studies using homology molecular modeling indicated that the proteins encoded by Afe_1009 and Afe_1437 have a conserved α-crystallin domain and share similar structural features with the sHSP from Methanococcus jannaschii, suggesting that their biological assembly involves 24 molecules and resembles a hollow spherical shell.
We conclude that the sHSPs encoded by the Afe_1437 and Afe_1009 genes are more likely to act as molecular chaperones in the A. ferrooxidans heat shock response. In addition, the three sHSPs from A. ferrooxidans are not recent paralogs, and the Afe_1437 and Afe_1009 genes could be inherited horizontally by A. ferrooxidans.
PMCID: PMC3252397  PMID: 22151959
20.  Active Cytomegalovirus Infection in Patients with Septic Shock 
Emerging Infectious Diseases  2006;12(10):1517-1522.
Cytomegalovirus reactivation occurred in one third of patients and was associated with prolonged ventilation and stay in an intensive care unit.
Cytomegalovirus (CMV) is a pathogen of emerging importance for patients with septic shock. In this prospective study, 25 immunocompetent CMV-seropositive patients with septic shock and an intensive care unit stay of >7 days were monitored by using quantitative pp65-antigenemia assay, shell vial culture, and virus isolation. Within 2 weeks, active CMV infection with low-level pp65-antigenemia (median 3 positive/5 × 105 leukocytes) developed in 8 (32%) patients. Infection was controlled within a few weeks (median 26 days) without use of antiviral therapy. Duration of intensive care and mechanical ventilation were significantly prolonged in patients with active CMV infection. CMV reactivation was associated with concomitant herpes simplex virus reactivation (p = 0.004). The association between active CMV infection and increased illness could open new therapeutic options for patients with septic shock. Future interventional studies are required.
PMCID: PMC3290950  PMID: 17176565
sepsis; reactivation; cytomegalovirus; herpes simplex virus; diagnostic assays
21.  Modeling of nonlinear viscous stress in encapsulating shells of lipid-coated contrast agent microbubbles 
Ultrasonics  2008;49(2):269-275.
A general theoretical approach to the development of zero-thickness encapsulation models for contrast microbubbles is proposed. The approach describes a procedure that allows one to recast available rheological laws from the bulk form to a surface form which is used in a modified Rayleigh-Plesset equation governing the radial dynamics of a contrast microbubble. By the use of the proposed procedure, the testing of different rheological laws for encapsulation can be carried out. Challenges of existing shell models for lipid-encapsulated microbubbles, such as the dependence of shell parameters on the initial bubble radius and the “compression-only” behavior, are discussed. Analysis of the rheological behavior of lipid encapsulation is made by using experimental radius-time curves for lipid-coated microbubbles with radii in the range 1.2 – 2.5 μm. The curves were acquired for a research phospholipid-coated contrast agent insonified with a 20-cycle, 3.0 MHz, 100 kPa acoustic pulse. The fitting of the experimental data by a model which treats the shell as a viscoelastic solid gives the values of the shell surface viscosity increasing from 0.30×10-8 kg/s to 2.63×10-8 kg/s for the range of bubble radii indicated above. The shell surface elastic modulus increases from 0.054 N/m to 0.37 N/m. It is proposed that this increase may be a result of the lipid coating possessing the properties of both a shear-thinning and a strain-softening material. We hypothesize that these complicated rheological properties do not allow the existing shell models to satisfactorily describe the dynamics of lipid encapsulation. In the existing shell models, the viscous and the elastic shell terms have the linear form which assumes that the viscous and the elastic stresses acting inside the lipid shell are proportional to the shell shear rate and the shell strain, respectively, with constant coefficients of proportionality. The analysis performed in the present paper suggests that a more general, nonlinear theory may be more appropriate. It is shown that the use of the nonlinear theory for shell viscosity allows one to model the “compression-only” behavior. As an example, the results of the simulation for a 2.03- μm-radius bubble insonified with a 6-cycle, 1.8 MHz, 100 kPa acoustic pulse are given. These parameters correspond to the acoustic conditions under which the “compression-only” behavior was observed by de Jong et al. [Ultrasound Med. Biol. 33 (2007) 653–656]. It is also shown that the use of the Cross law for the modeling of the shear-thinning behavior of shell viscosity reduces the variance of experimentally estimated values of the shell viscosity and its dependence on the initial bubble radius.
PMCID: PMC2826140  PMID: 18990417
Contrast agent; Shell model; Lipid coating; Compression-only behavior; Nonlinear viscosity
22.  Characterization of the pigmented shell-forming proteome of the common grove snail Cepaea nemoralis 
BMC Genomics  2014;15:249.
With a diversity of pigmented shell morphotypes governed by Mendelian patterns of inheritance, the common grove snail, Cepaea nemoralis, has served as a model for evolutionary biologists and population geneticists for decades. Surprisingly, the molecular mechanisms by which C. nemoralis generates this pigmented shelled diversity, and the degree of evolutionary conservation present between molluscan shell-forming proteomes, remain unknown.
Here, using next generation sequencing and high throughput proteomics, we identify and characterize the major proteinaceous components of the C. nemoralis shell, the first shell-proteome for a pulmonate mollusc. The recent availability of several marine molluscan shell-proteomes, and the dataset we report here, allow us to identify 59 evolutionarily conserved and novel shell-forming proteins. While the C. nemoralis dataset is dominated by proteins that share little to no similarity with proteins in public databases, almost half of it shares similarity with proteins present in other molluscan shells. In addition, we could not find any indication that a protein (or class of proteins) is directly associated with shell pigmentation in C. nemoralis. This is in contrast to the only other partially characterized molluscan-shell pigmentation mechanism employed by the tropical abalone Haliotis asinina.
The unique pulmonate shell-forming proteome that we report here reveals an abundance of both mollusc-specific and pulmonate-specific proteins, suggesting that novel coding sequences, and/or the extensive divergence of these sequences from ancestral sequences, supported the innovation of new shell types within the Conchifera. In addition, we report here the first evidence that molluscs use independently evolved mechanisms to pigment their shells. This proteome provides a solid foundation from which further studies aimed at the functional characterization of these shell-forming proteins can be conducted.
PMCID: PMC4023409  PMID: 24684722
Biomineralization; Calcification; Mollusc; Pulmonate; Pigment; Shell; Protein; Evolution; Cepaea nemoralis
23.  Exogenous estradiol improves shell strength in laying hens at the end of the laying period 
Cracked shells, due to age related reduction of shell quality, are a costly problem for the industry. Parallel to reduced shell quality the skeleton becomes brittle resulting in bone fractures. Calcium, a main prerequisite for both eggshell and bone, is regulated by estrogen in a complex manner. The effects of estrogen, given in a low continuous dose, were studied regarding factors involved in age related changes in shell quality and bone strength of laying hens. A pellet containing 0.385 mg estradiol 3-benzoate (21-day-release) or placebo was inserted subcutaneously in 20 birds each of Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB) at 70 weeks of age. Eggs were collected before and during the experiment for shell quality measurements. Blood samples for analysis of total calcium were taken three days after the insertion and at sacrifice (72 weeks). Right femur was used for bone strength measurements and tissue samples from duodenum and shell gland were processed for morphology, immunohistochemical localization of estrogen receptors (ERα, ERβ), plasma membrane calcium ATPase (PMCA) and histochemical localization of carbonic anhydrase (CA).
Estrogen treatment increased shell thickness of both hybrids. In addition, shell weight and shell deformation improved in eggs from the brown hybrids. The more pronounced effect on eggs from the brown hybrid may be due to a change in sensitivity to estrogen, especially in surface epithelial cells of the shell gland, shown as an altered ratio between ERα and ERβ. A regulatory effect of estrogen on CA activity, but not PMCA, was seen in both duodenum and shell gland, and a possible connection to shell quality is discussed. Bone strength was unaffected by treatment, but femur was stronger in LSL birds suggesting that the hybrids differ in calcium allocation between shell and bone at the end of the laying period. Plasma calcium concentrations and egg production were unaffected.
A low continuous dose of estrogen improves shell strength but not bone strength in laying hens at the end of the laying period.
PMCID: PMC4067625  PMID: 24884886
Exogenous estradiol; Eggshell formation; Carbonic anhydrase; Estrogen receptors; Bone strength; Eggshell quality; Domestic hen
24.  Do freshwater mussel shells record road-salt pollution? 
Scientific Reports  2014;4:7168.
Road-salt pollution in streams in the Northeastern United States has become a major concern, but historical data are scarce. Freshwater bivalve shells have the ability to record past environmental information, and may act as archives of road-salt pollution. We sampled Elliptio complanata shells from four streams, as well as specimens collected in 1877. Average [Na/Ca]shell was highest in modern shells from the stream with the highest sodium concentrations, and low in shells collected from this same stream in 1877 as well as in the shells from other streams, suggesting that [Na/Ca]shell serves as a proxy for road-salt pollution. We expected higher [Na/Ca]shell in winter and spring. However, high-resolution [Na/Ca]shell analyses along the growth axis of one shell did not reveal any clear subannual patterns, which could be the result of shell growth cessation in winter and/or during periods of high stream sodium concentrations. Therefore, bulk [Na/Ca]shell analysis from multiple shells can be used as a proxy of large changes in stream sodium concentrations, but high-resolution variations in stream sodium concentrations do not seem to be recorded in the shells.
PMCID: PMC4241526  PMID: 25418687
25.  The dopamine response in the nucleus accumbens core-shell border differs from that in the core and shell during operant ethanol self-administration 
Ethanol self-administration has been shown to increase dopamine in the nucleus accumbens; however, dopamine levels in the accumbal subregions (core, shell, and core-shell border) have not yet been measured separately in this paradigm. The present study was designed to determine if dopamine responses during operant ethanol self-administration are similar in the core, core-shell border and shell, particularly during transfer from the home cage to the operant chamber and during consumption of the drinking solution.
Six groups of male Long-Evans rats were trained to lever-press for either 10% sucrose (10S) or 10% sucrose + 10% ethanol (10S10E) (with a guide cannula above the core, core-shell border, or shell of the accumbens). On experiment day, five-min microdialysis samples were collected from the core, core-shell border, or shell before, during, and after drinking. Dopamine and ethanol concentrations were analyzed in these samples.
A significant increase in dopamine occurred during transfer of the rats from the home-cage into the operant chamber in all six groups, with those trained to drink 10S10E exhibiting a significantly higher increase than those trained to drink 10S in the core and shell. No significant increases were observed during drinking of either solution in the core or shell. A significant increase in dopamine was observed during consumption of ethanol in the core-shell border.
We conclude that dopamine responses to operant ethanol self-administration are subregion specific. After operant training, accumbal dopamine responses in the core and shell occur when cues that predict ethanol availability are presented and not when the reinforcer is consumed. However, core-shell border dopamine responses occur at the time of the cue and consumption of the reinforcer.
PMCID: PMC3012159  PMID: 19413648
Dopamine; Microdialysis; Operant Ethanol Self-Administration; Core; Core-Shell Border; Shell; Nucleus Accumbens

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