Genome-wide expression and response to exposure-based psychological therapy for anxiety disorders.

Exposure-based psychological treatments for anxiety have high efficacy. However, a substantial proportion of patients do not respond to therapy. Research examining the potential biological underpinnings of therapy response is still in its infancy, and most studies have focussed on candidate genes. To our knowledge, this study represents the first investigation of genome-wide expression profiles with respect to treatment outcome. Participants (n=102) with panic disorder or specific phobia received exposure-based cognitive behavioural therapy. Treatment outcome was defined as percentage reduction from baseline in clinician-rated severity of their primary anxiety diagnosis at post treatment and 6 month follow-up. Gene expression was determined from whole blood samples at three time points using the Illumina HT-12v4 BeadChip microarray. Linear regression models tested the association between treatment outcome and changes in gene expression from pre-treatment to post treatment, and pre-treatment to follow-up. Network analysis was conducted using weighted gene co-expression network analysis, and change in the detected modules from pre-treatment to post treatment and follow-up was tested for association with treatment outcome. No changes in gene expression were significantly associated with treatment outcomes when correcting for multiple testing (q<0.05), although a small number of genes showed a suggestive association with treatment outcome (q<0.5, n=20). Network analysis showed no association between treatment outcome and change in gene expression for any module. We report suggestive evidence for the role of a small number of genes in treatment outcome. Although preliminary, these findings contribute to a growing body of research suggesting that response to psychological therapies may be associated with changes at a biological level.

Serotonin transporter [corrected] methylation and response to cognitive behaviour therapy in children with anxiety disorders.

Anxiety disorders that are the most commonly occurring psychiatric disorders in childhood, are associated with a range of social and educational impairments and often continue into adulthood. Cognitive behaviour therapy (CBT) is an effective treatment option for the majority of cases, although up to 35-45% of children do not achieve remission. Recent research suggests that some genetic variants may be associated with a more beneficial response to psychological therapy. Epigenetic mechanisms such as DNA methylation work at the interface between genetic and environmental influences. Furthermore, epigenetic alterations at the serotonin transporter (SERT) promoter region have been associated with environmental influences such as stressful life experiences. In this study, we measured DNA methylation upstream of SERT in 116 children with an anxiety disorder, before and after receiving CBT. Change during treatment in percentage DNA methylation was significantly different in treatment responders vs nonresponders. This effect was driven by one CpG site in particular, at which responders increased in methylation, whereas nonresponders showed a decrease in DNA methylation. This is the first study to demonstrate differences in SERT methylation change in association with response to a purely psychological therapy. These findings confirm that biological changes occur alongside changes in symptomatology following a psychological therapy such as CBT.

Locating, quantifying and characterising radiation hazards in contaminated nuclear facilities using a novel passive non-electrical polymer based radiation imaging device.

This paper provides a summary of recent trials which took place at the US Department of Energy Oak Ridge National Laboratory (ORNL) during December 2010. The overall objective for the trials was to demonstrate that a newly developed technology could be used to locate, quantify and characterise the radiological hazards within two separate ORNL hot cells (B and C). The technology used, known as RadBall(®), is a novel, passive, non-electrical polymer based radiation detection device which provides a 3D visualisation of radiation from areas where effective measurements have not been previously possible due to lack of access. This is particularly useful in the nuclear industry prior to the decommissioning of facilities where the quantity, location and type of contamination are often unknown. For hot cell B, the primary objective of demonstrating that the technology could be used to locate, quantify and characterise three radiological sources was met with 100% success. Despite more challenging conditions in hot cell C, two sources were detected and accurately located. To summarise, the technology performed extremely well with regards to detecting and locating radiation sources and, despite the challenging conditions, moderately well when assessing the relative energy and intensity of those sources. Due to the technology's unique deployability, non-electrical nature and its directional awareness the technology shows significant promise for the future characterisation of radiation hazards prior to and during the decommissioning of contaminated nuclear facilities.

The embryology, classification, epidemiology, and genetics of facial clefting.

Facial clefting results from a variety of genetic and environmental causes. It occurs when developing facial processes fail to fuse, merge, or interact; the clefts range from mild to severe. The embryology and classification of these clefts have been characterized. Moreover, the epidemiology has been determined from population data. Unfortunately, complete understanding of the genetics of facial clefting has not been completely uncovered. Facial clefts exist within more than 300 syndromes with only a few being commented on in this article. As the human genome project continues, the understanding of facial clefting and its syndromes may continue to improve. Such knowledge could advance diagnosis and treatment of the patient and counseling of the affected family. Other articles within this issue address the management of these clefts.

Anti-epileptiform effects of audiogenic seizure priming on in vitro kindling in rat hippocampus.

The effect of priming for audiogenic seizures (AGS) on the development of epileptiform activity in the hippocampus was studied using in vitro kindling (IVK) in Long-Evans rats. AGS priming consists of intense auditory stimulation during a critical period of auditory development, resulting in sound-induced clonic convulsions upon subsequent testing. Between postnatal day (PND) 28 and 50, slices from subjects primed and sham-primed for AGS on PND 18 were used for recording responses in area CA1 of hippocampus following Schaffer collateral stimulation from stratum radiatum of area CA2/CA3. The developmental priming procedure, which enhances auditory brainstem excitability, resulted in fewer afterdischarges in slices from primed subjects across initial IVK stimulation sequences. These results suggest that changes in excitability that occur with acoustic priming can initially diminish selective epileptiform response characteristics in forebrain areas such as the hippocampus.

Effects of low atmospheric CO(2) on plants: more than a thing of the past.

In recent geological time, atmospheric CO(2) concentrations were 25-50% below the current level. Photosynthetic productivity of C(3) plants is substantially reduced at these low CO(2) levels, particularly at higher temperatures and during stress. Acclimation of photosynthesis to reduced CO(2) levels might compensate for some of this inhibition, but plants have a limited capacity to modulate Rubisco and other photosynthetic proteins following CO(2) reduction. Because of this, low CO(2) probably acted as a significant evolutionary agent, selecting plants adapted to CO(2) deficiency. Adaptations to low CO(2) might still exist in plants and might constrain responses to a rising CO(2) concentration.

Permeability measurement of macromolecules and assessment of mucosal antigen sampling using in vitro converted M cells.

INTRODUCTION: M cells are located in the epithelial layer covering the gut-associated lymphoid tissue and are responsible for delivery of macromolecules and microorganisms to the underlying lymphoid cells. It has been shown that the human colonic cell line Caco-2 can be converted to M cells in vitro following coculture with isolated lymphocytes from murine Peyer's patches. Studies were undertaken to evaluate and characterize the transepithelial transport of select macromolecules across these in vitro derived M cells. METHODS: Caco-2 cells were converted to M cells as reported previously. The morphology of Caco-2 cells and M cells was compared by transmission electron microscopy (TEM). The transport properties of macromolecules such as horseradish peroxidase, FITC-conjugated polystyrene beads, and radiolabeled dextrans were examined. The activation of murine antigen-specific T cells following transport of the antigen ovalbumin across the M-cell barrier was assessed by measuring cytokine production. RESULTS: M cells were shown to be irregular in shape and have fewer and shorter microvilli compared to the Caco-2 cell progenitors. These cells were still able to form tight junctions and monolayers on polycarbonate membranes. Time-course studies demonstrated that the transport of polystyrene beads and large-molecular-weight dextrans at physiological temperature across M-cell-containing monolayers was size dependent and more rapid than across Caco-2 cell monolayers. The transport of dextrans was also shown to be temperature and concentration dependent. Befitting the role of the M cell in mucosal defense, protein antigen could be delivered by these cells in order to be processed and presented to antigen-specific CD4+ T lymphocytes. DISCUSSION: The M-cell permeability model is a functional and practical system for evaluating the transport properties of macromolecules and assessing the potential for intestinal mucosal antigen sampling to elicit immunological responses.

Using continuous quality improvement techniques to determine the causes of hospital readmission.

This 6-month retrospective study provided our medical center with a profile of the population of patients readmitted within 31 days of discharge. We found that chronic illness and age greater than 65 years were the high risk factors related to patient readmissions. These 2 attributes are common in critical care patients. In addition, the study indicated that data input and collection procedures must be correctly followed in order to have accurate information on admission and readmission. Accurate information is important to management and external agencies. Accuracy is also important so that future problems with continuous quality improvement can be recognized and resources properly assigned. Recommendations for improving the data collection and reporting procedures and for improving the readmission rates were made to management, to the quality improvement coordinator, and to the quality improvement/risk management director.

beta-carbonic anhydrase from Pisum sativum: crystallization and preliminary X-ray analysis.

Recombinant beta-carbonic anhydrase from the garden pea, Pisum sativum, was purified to homogeneity and crystallized. Crystals belong to the orthorhombic space group C222, with unit-cell parameters a = 136.3, b = 142.5, c = 201.4 A, alpha = beta = gamma = 90 degrees. Crystals typically diffracted anisotropically, with a maximal resolution of 2.0 A in the strongest direction. The calculated Matthews parameter predicts approximately eight molecules in the asymmetric unit, consistent with previous reports of the molecule being an octamer. However, examination of the self-rotation function revealed no fourfold symmetry axis and multiple weak twofold axes perpendicular to the crystallographic c axis, indicating that the oligomerization arrangement is not that of a 422 octamer.

Developmental and genetic audiogenic seizure models: behavior and biological substrates.

Audiogenic seizure (AGS) models of developmental or genetic origin manifest characteristic indices of generalized seizures such as clonus or tonus in rodents. Studies of seizure-resistant strains in which AGS is induced by intense sound exposure during postnatal development provide models in which other neural abnormalities are not introduced along with AGS susceptibility. A critical feature of all AGS models is the reduction of neural activity in the auditory pathways from deafness during development. The initiation and propagation of AGS activity relies upon hyperexcitability in the auditory system, particularly the inferior colliculus (IC) where bilateral lesions abolish AGS. GABAergic and glutaminergic mechanisms play crucial roles in AGS, as in temporal lobe models of epilepsy, and participate in AGS modulatory and efferent systems including the superior colliculus, substantia nigra, basal ganglia and structures of the reticular formation. Catecholamine and indolamine systems also influence AGS severity. AGS models are useful for elucidating the underlying mechanisms for formation and expression of generalized epileptic behaviors, and evaluating the efficacy of modern treatment strategies such as anticonvulsant medication and neural grafting.

Tectal graft modulation of audiogenic seizures in Long-Evans rat.

Audiogenic seizure (AGS) activity can be induced in the seizure-resistant Long-Evans rat by postnatal priming. This study examined the effects of unilateral lesions of the inferior colliculus (IC) and implantation of tectal grafts on AGS components. Animals were primed with a 10-kHz tone burst at 120 dB on postnatal day 14 and tested for AGS susceptibility on day 28, and then two groups were unilaterally lesioned including animals receiving embryonic day 16-17 grafts of caudal tectum. Subsequently, animals were repeatedly tested for wild running and clonic-tonic convulsion components of AGS. The results demonstrate that unilaterally grafted animals with partial IC lesions showed significant reduction in the incidence of clonus expression with greater terminal uniphasic wild running behavior. These effects were stronger than in animals with comparable unilateral lesions alone. Many neurons in graft cases were in direct contact with host tissues to provide a substrate for tissue interactions previously demonstrated to promote neuron survival and remediate IC functions.

Comparative histology and vibration of the vocal folds: implications for experimental studies in microlaryngeal surgery.

OBJECTIVES/HYPOTHESIS: To determine the most suitable animal model for experimental studies on vocal fold surgery and function by a histological comparison of the microflap surgical plane and laryngeal videostroboscopy (LVS) in different species of animals. A second goal was to determine how the layered vocal fold structure in humans and three different animal species affects surgical dissection within the lamina propria. STUDY DESIGN: Prospective laboratory. METHODS: Three larynges each from dogs, monkeys, and pigs were compared with three ex vivo human larynges. Microflap surgery was performed on one vocal fold from each larynx. Both the operated and nonoperated vocal folds were examined histologically using stains specific for elastin, mature collagen, and ground substance. Based on the histological results, LVS was performed on two dogs and two pigs after first performing a tracheotomy for ventilation and airflow through the glottis. Arytenoid adduction sutures were placed to facilitate vocal fold adduction. RESULTS: The distributions of the collagen and elastin fibers were found to differ among the species with concentrations varying within species. Unlike the human vocal fold, which has a higher elastin concentration in the deeper layers of the lamina propria, both the pig and the dog had a thin band of elastin concentrated just deep to the basement membrane zone in the superficial layer. Just deep to this thin band, the collagen and the elastin were less concentrated. The monkey vocal fold had a very thin mucosal layer with less elastin throughout the mucosa. The microflap dissections in each of the dog, pig, and human vocal folds were similar, being located within that portion of the superficial lamina propria where the elastin and mature collagen are less concentrated. The microflap plane in the monkey vocal fold was more deeply located near the vocalis fibers. Despite the differences in elastin concentration, the microflap plane in both the dog and the pig was found to be similar to that in humans. The dog anatomy was much more suitable for microsuspension laryngoscopy and stroboscopic examination. The dog vocal folds vibrated in a similar fashion to human vocal folds with mucosal waves and vertical phase differences, features not seen in the pig vocal folds. CONCLUSIONS: Based on both the histological and stroboscopic results, the dog was believed to be a more suitable animal model for studies on vocal fold surgery, acknowledging that no animal's laryngeal anatomy is identical to that of the human. The dog LVS model presented allows for longitudinal laryngeal studies requiring repeated examinations at multiple time periods with histological correlation applied at sacrifice.

Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality.

Notch family genes encode transmembrane proteins involved in cell-fate determination. Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain with the E. coli (beta)-galactosidase gene. The mutant Notch2 gene encodes a 380 kDa Notch2-(beta)-gal fusion protein with (beta)-galactosidase activity. Notch2 homozygous mutant mice die prior to embryonic day 11.5, whereas heterozygotes show no apparent abnormalities and are fully viable. Analysis of Notch2 expression patterns, revealed by X-gal staining, demonstrated that the Notch2 gene is expressed in a wide variety of tissues including neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arches, but not heart. Histological studies, including in situ nick end labeling procedures, showed earlier onset and higher incidence of apoptosis in homozygous mutant mice than in heterozygotes or wild type mice. Dying cells were particularly evident in neural tissues, where they were seen as early as embryonic day 9.5 in Notch2-deficient mice. Cells from Notch2 mutant mice attach and grow normally in culture, demonstrating that Notch2 deficiency does not interfere with cell proliferation and that expression of the Notch2-(beta)-gal fusion protein is not toxic per se. In contrast to Notch1-deficient mice, Notch2 mutant mice did not show disorganized somitogenesis, nor did they fail to properly regulate the expression of neurogenic genes such as Hes-5 or Mash1. In situ hybridization studies show no indication of altered Notch1 expression patterns in Notch2 mutant mice. The results indicate that Notch2 plays an essential role in postimplantation development in mice, probably in some aspect of cell specification and/or differentiation, and that the ankyrin repeats are indispensable for its function.

Spatial sound detection and the role of the inferior colliculus in the Long-Evans rat.

The ability of Long-Evans hooded rats (n = 10) to detect sounds presented from sources in the horizontal plane at 0 degrees elevation and the effects of bilateral lesions of the inferior colliculus on these abilities were examined. Rats were trained on a directional detection task which required animals to suppress licking responses in a conditioned avoidance paradigm when 100-ms noise bursts were presented at random from speakers at 45 degrees intervals beginning at azimuth (0 degrees). A task performance rate was determined by reducing the correct lick suppression rate for signal trials by the proportion of incorrect suppression responses on non-signal trials. Higher performance rates were observed for stimuli presented from 0-90 degrees than for stimuli presented in the caudal hemifield prior to surgical procedures. Bilateral lesions restricted to the inferior colliculus reduced detection performance (p < 0.05) and shifted the best performance rates from sounds presented at 0-45 degrees to stimuli emitted from a 90 degrees source (p < 0.05). These results demonstrate that pigmented rats show differential detection levels for noise bursts presented from different locations throughout the horizontal interaural plane, and suggest that the inferior colliculus is involved in this aspect of directional hearing.

Audiogenic seizures in the developmentally primed Long-Evans rat.

The Long-Evans rat is a hybrid rodent strain with little innate susceptibility to audiogenic seizures (AGS). The present study examines parameters of acoustic priming (induced susceptibility) and testing for AGS during postnatal development subsequent to auditory function, and identifies the effects of stimulus intensity, repeated testing, and gender upon AGS activity. Rats were exposed to 125-dB SPL 10-kHz tone bursts at 14-36 days of age and tested with white noise at 14 or 19 days following sound exposure. All priming/testing combinations yielded AGS susceptibility; animals primed at 18 days showed the highest incidence of clonic seizures when tested 14 days later. All subjects displayed clonus at testing intensities of 120 dB, although some seizure behaviors could be elicited at 100 dB. Repeated testing at 120 dB increased latency to clonus and clonus duration, and total wild running activity. Gender differences for AGS expression were minimal. These results demonstrate the viability of the seizure-resistant Long-Evans rat for study of AGS.

Estrogen treatment and age effects on auditory brainstem responses in the post-breeding Long-Evans rat.

The auditory brainstem response (ABR) was recorded from 20-month-old Long Evans hooded female rats to determine if latency reductions occur from estrogen replacement. The ABR in these post-breeding age rats was also examined for reductions in response latencies as a function of adult age. Tone pip stimuli (8 and 40 kHz) were presented at 21, 51, or 81 s(-1). Aging control and ovariectomized animals showed slower response latencies for waves Ib-VI than young adults for 8 and 40 kHz stimulation at 21 s(-1). Increased stimulus rate resulted in longer latencies for all waves at 20 months. In contrast to hormone treatment effects in young adults, ABR latencies in post-breeding age estrogen-treated animals were not reduced, consistent with a general decrease in CNS responsiveness to estrogen steroids associated with age. The results also suggest that sensorineural modifications in the auditory system which prolong ABR latencies can occur early in the aging process of adult female subjects.

Latency alterations of the auditory brainstem response in audiogenic seizure-prone Long-Evans rats.

Audiogenic seizure susceptibility in the normally seizure-resistant Long-Evans rat may result from altered processing in the auditory pathway. Representative waveform latencies of the auditory brainstem responses (ABR) were recorded to examine generator alterations at different levels of the auditory neuraxis. Male Long-Evans rats primed for audiogenic seizures (AGS) on PND 14 with a 10 kHz pure tone at 120 dB SPL for 8 min were tested for AGS on PND 28 with 120 dB SPL continuous white noise. Primed subjects displayed wild running culminating in clonic convulsions. Following behavioral testing at 4-6 months, vertex recordings of ABR waves Ia-VI were made in anesthetized subjects using pure tone stimulus bursts. AGS subjects showed marginally elevated ABR thresholds. Shorter ABR wave latencies were elicited in AGS subjects for peripheral and central auditory components with stimulus intensities above 50 dB PeSPL at 8 and 40 kHz. Interpeak intervals were reduced for waves Ia-V and III-V in AGS subjects. These results reveal that intense sound stimulation during a sensitive period of development later reduces processing time at higher intensity levels.

Histomorphometric and laryngeal videostroboscopic analysis of the effects of corticosteroids on microflap healing in the dog larynx.

The histologic and functional effects of unilateral, layered corticosteroids on lateral microflap healing in 15 dogs were analyzed. Histologic sections of steroid-treated vocal folds (VFs) were studied with computer morphometry to examine differences in the tissue healing response. Paired analysis revealed increases in the inflammatory infiltrate around the microflap in the steroid-treated VFs at 2, 4, and 6 weeks (6.3%, 30.6%, and 34.9%, all with p < .02). The neovascular response in the steroid-treated VFs was less at 2 weeks (-20.9%, p < .005) but greater at 4 and 6 weeks (16.3% and 4.3%, p < .005). To better characterize the effect of steroids on the healing process, a normal, time-dependent distribution was applied to the histologic data and demonstrated a delay in the steroid-treated VF tissue response of 12 days for the inflammatory infiltrate and 21 days for the neovascular response. Qualitative and quantitative analysis of in vivo laryngeal videostroboscopy (LVS) samples taken preoperatively and at sacrifice could not identify significant differences in appearance, amplitude, mucosal wave, or suppleness between the 2 VFs. Therefore, although corticosteroids cause a delay in wound healing, LVS does not discern differences in microflap characteristics between healing steroid-treated and control VFs at 2, 4, or 6 weeks. If steroids are used, the surgeon should account for a probable delay in wound healing, but should not expect an overall difference in functional outcome.

Processing of endogenous pre-mRNAs in association with SC-35 domains is gene specific.

Analysis of six endogenous pre-mRNAs demonstrates that localization at the periphery or within splicing factor-rich (SC-35) domains is not restricted to a few unusually abundant pre-mRNAs, but is apparently a more common paradigm of many protein-coding genes. Different genes are preferentially transcribed and their RNAs processed in different compartments relative to SC-35 domains. These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space. The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors. This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not. Despite similar molar abundance in muscle fiber nuclei, nascent transcript "trees" of highly complex dystrophin RNA are cotranscriptionally spliced outside of SC-35 domains, whereas posttranscriptional "tracks" of more mature myosin heavy chain transcripts overlap domains. Further analyses supported that endogenous pre-mRNAs exhibit distinct structural organization that may reflect not only the expression and complexity of the gene, but also constraints of its chromosomal context and kinetics of its RNA metabolism.

Ethanol synthesis by genetic engineering in cyanobacteria.

Cyanobacteria are autotrophic prokaryotes which carry out oxygenic photosynthesis and accumulate glycogen as the major form of stored carbon. In this research, we introduced new genes into a cyanobacterium in order to create a novel pathway for fixed carbon utilization which results in the synthesis of ethanol. The coding sequences of pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh) from the bacterium Zymomonas mobilis were cloned into the shuttle vector pCB4 and then used to transform the cyanobacterium Synechococcus sp. strain PCC 7942. Under control of the promoter from the rbcLS operon encoding the cyanobacterial ribulose-1, 5-bisphosphate carboxylase/oxygenase, the pdc and adh genes were expressed at high levels, as demonstrated by Western blotting and enzyme activity analyses. The transformed cyanobacterium synthesized ethanol, which diffused from the cells into the culture medium. As cyanobacteria have simple growth requirements and use light, CO2, and inorganic elements efficiently, production of ethanol by cyanobacteria is a potential system for bioconversion of solar energy and CO2 into a valuable resource.