Questions about quitting (Q2): design and methods of a Multiphase Optimization Strategy (MOST) randomized screening experiment for an online, motivational smoking cessation intervention.

Effective interventions are needed to improve smokers' motivation for quitting, treatment utilization, and abstinence rates. The Internet provides an ideal modality for delivering such interventions, given the low cost, broad reach, and capacity to individually tailor content, but important methodological questions remain about how to best design and deliver an online, motivational intervention to smokers. The current paper reports on the intervention, study design and research methods of a randomized trial (called Questions about Quitting) designed to address some of these questions. Using a Multi-phase Optimization Strategy (MOST) screening experiment, the trial has two key aims: to examine the impact of four experimental intervention factors (each evaluated on two levels) on smokers' subsequent treatment utilization and abstinence, and to examine select moderators of each sub-factor's effectiveness. The experimental factors of interest are: navigation autonomy (content viewing order is dictated based on stage of change or not), use of self-efficacy based testimonials (yes vs. no), proactive outreach (reminder emails vs. no emails), and decisional framework (prescriptive vs. motivational tone). To our knowledge, this is the first application of the MOST methodology to explore these factors or to explore the optimal design for a motivational intervention targeting smokers not actively trying to quit smoking. The rationale for the experimental factor choice, intervention design, and trial methods are discussed. Outcome data are currently being collected and are not presented, but recruitment data confirm the feasibility of enrolling smokers at varying stages of readiness to quit.

Using microscopy to assess chorion structural integrity and parasitoid oviposition sites on stink bug (Hemiptera: Pentatomidae) eggs.

Previous efficacy studies found that many insecticides used by growers could be having an adverse effect on egg parasitoids (Telenomus podisi) developing in the eggs of the brown stink bug (Euschistus servus), while unhatched stink bugs experienced lower levels of mortality. One plausible explanation for this was that insecticides might enter parasitized eggs more readily via oviposition wounds. Parasitized E. servus eggs, as well as nonparasitized stink bug (Acrosternum hilare, E. servus, Murgantia histrionica, and Podisus maculiventris) eggs, were examined using electron microscopy. Egg response to perforation by a tungsten probe served as a control. Microscopy images depicted the chorion surface as characterized by a matrix of ridges and micropylar processes in a ring around the margin of the operculum. Observations of oviposition sites showed a "scab" formed where the ovipositor penetrated the chorion, and at sites penetrated by the probe. These formations appeared to be the result of fluids from inside the egg leaking out, drying, and hardening after oviposition or probe perforation, suggesting that the response was not due to substances secreted by the parasitoid. Further, no open wounds or holes were seen to increase the possibility of insecticides entering parasitized eggs.

Active-edge planar radiation sensors.

Many systems in medicine, biology, high-energy physics, and astrophysics require large area radiation sensors. In most of these applications, minimizing the amount of dead area or dead material is crucial. We have developed a new type of silicon radiation sensor in which the device is active to within a few microns of the mechanical edge. Their perimeter is made by a plasma etcher rather than a diamond saw. Their edges can be defined and also passivated by growing, in an intermediate step, a field oxide on the side surfaces. In this paper, the basic architecture and results from a synchrotron beam test are presented.

Practice parameter: temporal lobe and localized neocortical resections for epilepsy: report of the Quality Standards Subcommittee of the American Academy of Neurology, in association with the American Epilepsy Society and the American Association of Neurological Surgeons.

OBJECTIVES/METHODS: To examine evidence for effectiveness of anteromesial temporal lobe and localized neocortical resections for disabling complex partial seizures by systematic review and analysis of the literature since 1990. RESULTS: One intention-to-treat Class I randomized, controlled trial of surgery for mesial temporal lobe epilepsy found that 58% of patients randomized to be evaluated for surgical therapy (64% of those who received surgery) were free of disabling seizures and 10 to 15% were unimproved at the end of 1 year, compared with 8% free of disabling seizures in the group randomized to continued medical therapy. There was a significant improvement in quantitative quality-of-life scores and a trend toward better social function at the end of 1 year for patients in the surgical group, no surgical mortality, and infrequent morbidity. Twenty-four Class IV series of temporal lobe resections yielded essentially identical results. There are similar Class IV results for localized neocortical resections; no Class I or II studies are available. CONCLUSIONS: A single Class I study and 24 Class IV studies indicate that the benefits of anteromesial temporal lobe resection for disabling complex partial seizures is greater than continued treatment with antiepileptic drugs, and the risks are at least comparable. For patients who are compromised by such seizures, referral to an epilepsy surgery center should be strongly considered. Further studies are needed to determine if neocortical seizures benefit from surgery, and whether early surgical intervention should be the treatment of choice for certain surgically remediable epileptic syndromes.

Crystallization and preliminary X-ray crystallographic analysis of FlhD/FlhC complex from Escherichia coli.

The heterotetrameric (C(2)D(2)) FlhD/FlhC complex was first discovered as a transcriptional activator of the flagellar genes in Escherichia coli. Recent studies now show that FlhD/FlhC also regulates several non-flagellar target genes in E. coli. The FlhD/FlhC complex also plays several important roles in other microorganisms. The molecular interactions between FlhD and FlhC, as well as the mechanisms by which the complex may vary its DNA-binding specificity, are not clear. Determination of the FlhD/FlhC crystal structure will provide insight into these protein-protein and protein-DNA interactions. The initial steps in this investigation are reported here: the overexpression, purification and crystallization of the FlhD/FlhC complex, the characterization of this crystal form and the recording and processing of an initial diffraction data set. The obtained crystal form of the FlhD/FlhC complex is hexagonal (space group P6(1), unit-cell parameters a = b = 150.5, c = 115.9 A). The crystal density is very low (V(M) = 5.5), with 81.7% of its volume occupied by solvent. A single C(2)D(2) tetramer is present in the crystallographic asymmetric unit. A complete native data set has been collected to 4.5 A resolution.

Crystal structure of the global regulator FlhD from Escherichia coli at 1.8 A resolution.

FlhD is a 13.3 kDa transcriptional activator protein of flagellar genes and a global regulator. FlhD activates the transcription of class II operons in the flagellar regulon when complexed with a second protein FlhC (21.5 kDa). FlhD also regulates other expression systems in Escherichia coli. We are seeking to understand this plasticity of FlhD's DNA-binding specificity and, to this end, we have determined the crystal structure of the isolated FlhD protein. The structure was solved by substituting seleno-methionine for natural sulphur-methionine in FlhD, crystallizing the protein and determining the structure factor phases by the method of multiple-energy anomalous dispersion (MAD). The FlhD protein is dimeric. The dimer is tightly coupled, with an intimate contact surface, implying that the dimer does not easily dissociate. The FlhD monomer is predominantly alpha-helical. The C-termini of both FlhD monomers (residues 83-116) are completely disrupted by crystal packing, implying that this region of FlhD is highly flexible. However, part of the C-terminus structure in chain A (residues 83-98) was modelled using a native FlhD crystal. What is seen in chain A suggests a classic DNA-binding, helix-turn-helix (HTH) motif. FlhD does not bind DNA by itself, so it may be that the DNA-binding HTH motif becomes rigidly defined only when FlhD forms a complex with some other protein, such as FlhC. If this were true, it might explain how FlhD exhibits plasticity in its DNA-binding specificity, as each partner protein with which it forms a complex could allosterically affect the binding specificity of its HTH motif. A disulphide bridge is seen between the unique cysteine residues (Cys-65) of FlhD native homodimers. Alanine substitution at Cys-65 does not affect FlhD transcription activator activity, suggesting that the disulphide bond is not necessary for either dimer stability or this function of FlhD. Electrostatic potential analysis indicates that dimeric FlhD has a negatively charged surface.

Differential signatures of bacterial and mammalian IMP dehydrogenase enzymes.

IMP dehydrogenase (IMPDH) is an essential enzyme of de novo guanine nucleotide synthesis. IMPDH inhibitors have clinical utility as antiviral, anticancer or immunosuppressive agents. The essential nature of this enzyme suggests its therapeutic applications may be extended to the development of antimicrobial agents. Bacterial IMPDH enzymes show biochemical and kinetic characteristics that are different than the mammalian IMPDH enzymes, suggesting IMPDH may be an attractive target for the development of antimicrobial agents. We suggest that the biochemical and kinetic differences between bacterial and mammalian enzymes are a consequence of the variance of specific, identifiable amino acid residues. Identification of these residues or combination of residues that impart this mammalian or bacterial enzyme signature is a prerequisite for the rational identification of agents that specifically target the bacterial enzyme. We used sequence alignments of IMPDH proteins to identify sequence signatures associated with bacterial or eukaryotic IMPDH enzymes. These selections were further refined to discern those likely to have a role in catalysis using information derived from the bacterial and mammalian IMPDH crystal structures and site-specific mutagenesis. Candidate bacterial sequence signatures identified by this process include regions involved in subunit interactions, the active site flap and the NAD binding region. Analysis of sequence alignments in these regions indicates a pattern of catalytic residues conserved in all enzymes and a secondary pattern of amino acid conservation associated with the major phylogenetic groups. Elucidation of the basis for this mammalian/bacterial IMPDH signature will provide insight into the catalytic mechanism of this enzyme and the foundation for the development of highly specific inhibitors.

Avian species differences in susceptibility to noise exposure.

Previous studies of hair cell regeneration and hearing recovery in birds after acoustic overstimulation have involved relatively few species. Studies of the effects of acoustic overexposure typically report high variability. Though it is impossible to tell, the data so far also suggest there may be considerable species differences in the degree of damage and the time course and extent of recovery. To examine this issue, we exposed four species of birds (quail, budgerigars, canaries, and zebra finches) to identical conditions of acoustic overstimulation and systematically analyzed changes in hearing sensitivity, basilar papilla morphology, and hair cell number. Quail and budgerigars showed the greatest susceptibility to threshold shift and hair cell loss after overstimulation with either pure tone or bandpass noise, while identical types of overstimulation in canaries and zebra finches resulted in much less of a threshold shift and a smaller, more diffuse hair cell loss. All four species showed some recovery of threshold sensitivity and hair cell number over time. Canary and zebra finch hearing and hair cell number recovered to within normal limits while quail and budgerigars continued to have an approximately 20 dB threshold shift and incomplete recovery of hair cell number. In a final experiment, birds were exposed to identical wide-band noise overstimulation under conditions of artificial middle ear ventilation. Hair cell loss was substantially increased in both budgerigars and canaries suggesting that middle ear air pressure regulation and correlated changes in middle ear transfer function are one factor influencing susceptibility to acoustic overstimulation in small birds.

Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase.

IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the Km for NAD (1180 microM) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 A with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne's Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione beta-synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors.

Effects of columella removal on inner ear morphology in the chick.

We are currently removing the single middle ear bone (columella) in the domestic chick to introduce chemical agents directly into the inner ear. Since we are interested in the effect of these agents on neural structures within the avian basilar papilla (BP), we are concerned about any subtle changes that might result from the surgical procedure of columella removal alone. The purpose of this study was to use light and transmission electron microscopy to analyze morphological changes in the inner ear after columella removal. Fifteen-day-old chicks underwent a unilateral, bilateral or a sham removal of the columella. After columella removal, the oval window was either plugged with Gelfoam or Kimwipe (standard accepted procedure to prevent possible perilymph leak) or left uncovered. After a 5-day survival period, morphological changes were observed in the tegmentum vasculosum (TV) of all ears receiving a columella removal as compared to unoperated ears. Further, ears with Gelfoam plugging the oval window also had damage to the hair cells and support cells of the basilar papilla. In contrast, there were no observable differences in either auditory afferent or efferent nerve terminals on hair cells in the BP from any ears that had the columella removed compared to those from unoperated ears. These results suggest that columella removal alone may produce morphological changes to the TV within 5 days of surgery but not to structures within the BP. On the other hand, columella removal with a Gelfoam plug results in damage not only to the TV but also to cells within the basilar papilla during this same survival time. Despite damage to other structures within the inner ear, cochlear efferent and afferent terminals on surviving hair cells were unaffected by columella removal with or without plugging.

Morphological evidence of ototoxicity of the iron chelator deferoxamine.

Recent reports of the role of iron-catalyzed free radical formation in gentamicin ototoxicity and the successful attenuation of gentamicin ototoxicity by iron chelators led us to re-examine experimental material from a previously unpublished study of deferoxamine. Deferoxamine was injected i.m. into adult Japanese quail at either 300 or 750 mg/kg body weight for 30 days. Examination of sections from the basilar papilla at the light microscope level indicated that supporting cells were damaged after the lower drug dose, and that both supporting cells and hair cells were damaged after the higher drug dose. High, prolonged exposure to deferoxamine produced pathological changes similar to those seen in the basilar papilla after much lower, shorter doses of gentamicin. These results demonstrate that deferoxamine damages the quail inner ear and are consistent with the idea that the ototoxic actions of gentamicin may be mediated by iron chelation.

MAD analysis of FHIT, a putative human tumor suppressor from the HIT protein family.

BACKGROUND: The fragile histidine triad (FHIT) protein is a member of the large and ubiquitous histidine triad (HIT) family of proteins. It is expressed from a gene located at a fragile site on human chromosome 3, which is commonly disrupted in association with certain cancers. On the basis of the genetic evidence, it has been postulated that the FHIT protein may function as a tumor suppressor, implying a role for the FHIT protein in carcinogenesis. The FHIT protein has dinucleoside polyphosphate hydrolase activity in vitro, thus suggesting that its role in vivo may involve the hydrolysis of a phosphoanhydride bond. The structural analysis of FHIT will identify critical residues involved in substrate binding and catalysis, and will provide insights into the in vivo function of HIT proteins. RESULTS: The three-dimensional crystal structures of free and nucleoside complexed FHIT have been determined from multiwavelength anomalous diffraction (MAD) data, and they represent some of the first successful structures to be measured with undulator radiation at the Advanced Photon Source. The structures of FHIT reveal that this protein exists as an intimate homodimer, which is based on a core structure observed previously in another human HIT homolog, protein kinase C interacting protein (PKCI), but has distinctive elaborations at both the N and C termini. Conserved residues within the HIT family, which are involved in the interactions of the proteins with nucleoside and phosphate groups, appear to be relevant for the catalytic activity of this protein. CONCLUSIONS: The structure of FHIT, a divergent HIT protein family member, in complex with a nucleotide analog suggests a metal-independent catalytic mechanism for the HIT family of proteins. A structural comparison of FHIT with PKCI and galactose-1-phosphate uridylyltransferase (GaIT) reveals additional implications for the structural and functional evolution of the ubiquitous HIT family of proteins.

The 2.4 A crystal structure of cholera toxin B subunit pentamer: choleragenoid.

Cholera toxin, a heterohexameric AB5 enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding the GM1 gangliosides exposed on the luminal surface of intestinal epithelial cells. The crystal structure of choleragenoid has been independently solved and refined at 2.4 A resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin, choleragen, the heat-labile enterotoxin from Escherichia coli, and for a choleragenoid-GM1 pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of choleragenoid to the A subunit or to its receptor pentasaccharide modestly affects the local stereochemistry without perceptibly altering the subunit interface.

The three-dimensional crystal structure of cholera toxin.

The clinical manifestations of cholera are largely attributable to the actions of a secreted hexameric AB5 enterotoxin (choleragen). We have independently solved and refined the three-dimensional structure of choleragen at 2.5 A resolution. The structure of the crystalline toxin closely resembles that described for the heat-labile enterotoxin from Escherichia coli (LT) with which it shares 80% sequence homology. In both cases, the wedge-shaped A subunit is loosely held high above the plane of the pentameric B subunits by the tethering A2 chain. The most striking difference between the two toxins occurs at the carboxyl terminus of the A2 chain. Whereas the last 14 residues of the A2 chain of LT threading through the central pore of the B5 assembly form an extended chain with a terminal loop, the A2 chain of choleragen remains a nearly continuous alpha-helix throughout its length. The four carboxyl-terminal residues of the A2 chain (KDEL sequence), disordered in the crystal structure of LT, are clearly visible in choleragen's electron-density map. In the accompanying article we describe the three-dimensional structure of the isolated B pentamer of cholera toxin (choleragenoid). Comparison of the crystalline coordinates of choleragen, choleragenoid, and LT provides a solid three-dimensional foundation for further experimental investigation. These structures, along with those of related toxins from Shigella dysenteria and Bordetella pertussis, offer a first step towards the rational design of new vaccines and anti-microbial agents.

Crystallization and preliminary X-ray diffraction studies of the human adenovirus serotype 2 proteinase with peptide cofactor.

Recombinant human adenovirus serotype 2 proteinase (both native and selenomethionine-substituted) has been crystallized in the presence of the serotype 12, 11-residue peptide cofactor. The crystals (space group P3(1)21 or P3(2)21, one molecule per asymmetric unit, a = b = 41.3 angstrum, c = 197.0 angstrum) grew in solutions containing 20-40% 2-methyl-2,4-pentanediol (MPD), 0.1-0.2 M sodium citrate, and 0.1 M sodium HEPES, pH 5.0-7.5. Diffraction data (84% complete to 2.2 angstrum resolution with Rmerge of 0.0335) have been measured from cryopreserved native enzyme crystals with the Argonne blue (1,024 x 1,024 pixel array) charge-coupled device detector at beamline X8C at the National Synchrotron Light Source (operated by Argonne National Laboratory's Structural Biology Center). Additionally, diffraction data from selenomethionine-substituted proteinase, 65% complete to 2.0 angstrum resolution with Rmerge values ranging 0.05-0.07, have been collected at three X-ray energies at and near the selenium absorption edge. We have determined three of the six selenium sites and are initiating a structure solution by the method of multiwavelength anomalous diffraction phasing.

The low ionic strength crystal structure of horse cytochrome c at 2.1 A resolution and comparison with its high ionic strength counterpart.

BACKGROUND: Cytochrome c is an integral part of the mitochondrial respiratory chain. It is confined to the intermembrane space of mitochondria, and has the function of transferring electrons between its redox partners. Solution studies of cytochrome c indicate that the conformation of the molecule is sensitive to the ionic strength of the medium. RESULTS: The crystal structures of cytochromes c from several species have been solved at extremely high ionic strengths of near-saturated solutions of ammonium sulfate. Here we present the first crystal structure of ferricytochrome c at low ionic strength refined at 2.1 A resolution. In general, the structure has the same features as those determined earlier. However, there are some differences in both backbone and side-chain conformations in several areas. These areas coincide with those observed by NMR and resonance Raman spectroscopy to be sensitive to ionic strength. CONCLUSIONS: Neither ionic strength nor crystal-packing interactions have much influence on the conformation of horse cytochrome c. Nevertheless, some differences in the side-chain conformations at high and low ionic strengths may be important for understanding how the protein functions. Close examination of the gamma-turn (residues 27-29) conserved in cytochromes c leads us to propose the 'negative classical' gamma-turn to describe this unusual feature.

Recovery of noise-induced changes in the dark cells of the quail tegmentum vasculosum.

Morphologic changes in the tegmentum vasculosum (TV) of adult quail after high intensity sound exposure were studied. Quail were continuously exposed to 115 dB SPL, 1500 Hz pure tone in a sound field for 12 h and either sacrificed immediately (0 day), 1, 2, 3, 4, 6 or 10 days later. Serial sections through the basilar papilla at 100 micron intervals from base to apex were obtained for study with light microscopy and TEM. Significant morphologic changes were found within the TV of quail sacrificed on days 0-4. On a quantitative scale, the majority of recovery occurred within the first 24 h. After four days survival the tegmentum appeared nearly normal. This recovery correlates well with the temporal pattern of threshold shift recovery. These results demonstrate a temporal correlation between ultrastructural changes in the TV and functional recovery of hearing after intense sound exposure. A potential etiologic role of the TV in avian temporary threshold shift is suggested.

Crystallization of wild-type and mutant ferricytochromes c at low ionic strength: seeding technique and X-ray diffraction analysis.

Ferricytochromes c were crystallized at low ionic strength by macroseeding techniques. Large crystals were grown by seed-induced self-nucleation which occurred anywhere in the drop, regardless of the location of the seed crystal. This unusual crystal-seeding method worked reproducibly in our hands, and X-ray quality crystals have been prepared of several ferricytochromes c: horse, rat (recombinant wild type), and two site-directed mutants of the latter, tyrosine 67 to phenylalanine (Y67F) and asparagine 52 to isoleucine (N52I). Crystals of any one of these four proteins could be used as seeds for the crystallization of any one of the others. All the crystals are of the same crystal form, with space group P2(1)2(1)2(1). There are two protein molecules per asymmetric unit. The crystals are stable in the X-ray beam and diffract to at least 2.0 A, resolution. Full crystallographic data sets have been collected from single crystals of all four proteins.

TEM analysis of neural terminals on autoradiographically identified regenerated hair cells.

Regenerated tall and short hair cells identified by autoradiography ([3H]thymidine) were analyzed for their neural contacts using transmission electron microscopy. Ears from mature Coturnix quail (N = 5) exposed to pure tone overstimulation (1500 Hz, 115 dB, 12 h) and treated with [3H] thymidine for 10 days were fixed, embedded, sectioned serially in 100 mu intervals and prepared for autoradiography. At fifty percent length along the papilla, alternating semi-thick (1 micron) and thin (70 nm) sections were taken at 50 microns intervals. Semi-thick sections were analyzed at the light microscope level for autoradiographic labeling of [3H]thymidine over the hair cell nucleus. When an autoradiographically labelled hair cell was identified the corresponding serial thin sections were analyzed in the transmission electron microscope. Seven autoradiographically labeled hair cells in semi-thin sections were positively identified in immediately adjacent thin serial sections. Labeled hair cells were morphologically similar to adjacent cells with no label and generally appeared to receive similar innervation. Regenerated short hair cells showed large chalice shaped, efferent terminals, intermediate hair cells received both afferent and efferent innervation and tall hair cells were contacted by two to three afferent terminals with synaptic specializations. These results provide conclusive evidence of both efferent and afferent synaptic contacts on newly regenerated hair cells of all types 10 days following acoustic trauma.