Trait Anxiety Mediated by Amygdala Serotonin Transporter in the Common Marmoset.

High trait anxiety is associated with altered activity across emotion regulation circuitry and a higher risk of developing anxiety disorders and depression. This circuitry is extensively modulated by serotonin. Here, to understand why some people may be more vulnerable to developing affective disorders, we investigated whether serotonin-related gene expression across the brain's emotion regulation circuitry may underlie individual differences in trait anxiety using the common marmoset (Callithrix jacchus, mixed sexes) as a model. First, we assessed the association of region-specific expression of the serotonin transporter (SLC6A4) and serotonin receptor (HTR1A, HTR2A, HTR2C) genes with anxiety-like behavior; and second, we investigated their causal role in two key features of the high trait anxious phenotype: high responsivity to anxiety-provoking stimuli and an exaggerated conditioned threat response. While the expression of the serotonin receptors did not show a significant relationship with anxiety-like behavior in any of the targeted brain regions, serotonin transporter expression, specifically within the right ventrolateral prefrontal cortex (vlPFC) and most strongly in the right amygdala, was associated positively with anxiety-like behavior. The causal relationship between amygdala serotonin levels and an animal's sensitivity to threat was confirmed via direct amygdala infusions of a selective serotonin reuptake inhibitor (SSRI), citalopram. Both anxiety-like behaviors, and conditioned threat-induced responses were reduced by the blockade of serotonin reuptake in the amygdala. Together, these findings provide evidence that high amygdala serotonin transporter expression contributes to the high trait anxious phenotype and suggest that reduction of threat reactivity by SSRIs may be mediated by their actions in the amygdala.SIGNIFICANCE STATEMENT Findings here contribute to our understanding of how the serotonin system underlies an individual's expression of threat-elicited negative emotions such as anxiety and fear within nonhuman primates. Exploration of serotonergic gene expression across brain regions implicated in emotion regulation revealed that serotonin transporter gene expression in the ventrolateral prefrontal cortex (vlPFC) and most strongly in the amygdala, but none of the serotonin receptor genes, were predictive of interindividual differences in anxiety-like behavior. Targeting of amygdala serotonin reuptake with selective serotonin reuptake inhibitors (SSRIs) confirmed the causal relationship between amygdala serotonin transporter and an animal's sensitivity to threat by reversing expression of two key features of the high trait-like anxiety phenotype: high responsivity to anxiety-provoking uncertain threat and responsivity to certain conditioned threat.

Microsatellite genotyping reveals a signature in breast cancer exomes.

Genomic instability at microsatellite loci is a hallmark of many cancers, including breast cancer. However, much of the genomic variation and many of the hereditary components responsible for breast cancer remain undetected. We hypothesized that variation at microsatellites could provide additional genomic markers for breast cancer risk assessment. A total of 1,345 germline and tumor DNA samples from individuals diagnosed with breast cancer, exome sequenced as part of The Cancer Genome Atlas, were analyzed for microsatellite variation. The comparison group for our analysis, representing healthy individuals, consisted of 249 females which were exome sequenced as part of the 1,000 Genomes Project. We applied our microsatellite-based genotyping pipeline to identify 55 microsatellite loci that can distinguish between the germline of individuals diagnosed with breast cancer and healthy individuals with a sensitivity of 88.4 % and a specificity of 77.1 %. Further, we identified additional microsatellite loci that are potentially useful for distinguishing between breast cancer subtypes, revealing a possible fifth subtype. These findings are of clinical interest as possible risk diagnostics and reveal genes that may be of potential therapeutic value, including genes previously not associated with breast cancer.

Population-scale analysis of human microsatellites reveals novel sources of exonic variation.

Using our microsatellite specific genotyping method, we analyzed tandem repeats, which are known to be highly variable with some recognized as biomarkers causative of disease, in over 500 individuals who were exon sequenced in a 1000 Genomes Project pilot study. We were able to genotype over 97% of the microsatellite loci in the targeted regions. A total of 25,115 variations were observed, including repeat length and single nucleotide polymorphisms, corresponding to an average of 45.6 variations per individual and a density of 1.1 variations per kilobase. Standard variant detection did not report 94.2% of the exonic repeat length variations in part because the alignment techniques are not ideal for repetitive regions. Additionally some standard variation detection tools rely on a database of known variations, making them less likely to call repeat length variations as only a small percent of these loci (~6000) have been accurately characterized. A subset of the hundreds of non-synonymous variations we identified was experimentally validated, indicating an accuracy of 96.5% for our microsatellite-based genotyping method, with some novel variants identified in genes associated with cancer. We propose that microsatellite-based genotyping be used as a part of large scale sequencing studies to identify novel variants.

Equity and efficiency: striving for a solution for the introduction of new medicines.

The aim of this study was to describe the evolution of mechanisms to manage the introduction of new medicines and their impact on the health-care system in Scotland. The study was a review of published and unpublished national audits and surveys on the introduction of new medicines in Scotland. Before the Scottish Medicines Consortium (SMC) was established, Area Drug and Therapeutics Committees (ADTCs) played a key role in advising National Health Service (NHS) Boards in Scotland on the use of new medicines. There was evidence of variation in the medicines evaluated and, in some cases, the evidence used leading to different decisions for the same medicine. After the SMC was established, ADTC decisions had become more consistent and comprehensive. The role of ADTCs evolved from evaluation of medicines to assessment of local implications and implementation of SMC advice. There was increased recognition of the importance of monitoring medicine use. This review demonstrated a clear evolution in the evaluation and implementation of new medicines by ADTCs across NHS Scotland. After the SMC was established, more medicines were considered by ADTCs and there was greater consistency in those considered for local implementation. ADTCs have moved from evaluation of new medicines to other aspects of medicine management, including assessment of local implications and implementation of SMC advice.

Evaluation of microsatellite variation in the 1000 Genomes Project pilot studies is indicative of the quality and utility of the raw data and alignments.

We performed an analysis of global microsatellite variation on the two kindreds sequenced at high depth (~20×-60×) in the 1000 Genomes Project pilot studies because alterations in these highly mutable repetitive sequences have been linked with many phenotypes and disease risks. The standard alignment technique performs poorly in microsatellite regions as a consequence of low effective coverage (~1×-5×) resulting in 79% of the informative loci exhibiting non-Mendelian inheritance patterns. We used a more stringent approach in computing robust allelotypes resulting in 94.4% of the 1095 informative repeats conforming to traditional inheritance. The high-confidence allelotypes were analyzed to obtain an estimate of the minimum polymorphism rate as a function of motif length, motif sequence, and distribution within the genome.

A long AAAG repeat allele in the 5' UTR of the ERR-γ gene is correlated with breast cancer predisposition and drives promoter activity in MCF-7 breast cancer cells.

We sequenced the 5' UTR of the estrogen-related receptor gamma gene (ERR-γ) in ~500 patient and volunteer samples and found that longer alleles of the (AAAG)(n) microsatellite were statistically and significantly more likely to exist in the germlines of breast cancer patients when compared to healthy volunteers. This microsatellite region contains multiple binding sites for a number of transcription factors, and we hypothesized that the polymorphic AAAG-containing sequence in the 5' UTR region of ERR-γ might modulate expression of ERR-γ. We found that the 369 bp PCR product containing the AAAG repeat drove expression of a reporter gene in estrogen receptor positive breast cancer cells. Our results support a role for the 5' UTR region in ERR-γ expression, which is potentially mediated via binding to the variable tandem AAAG repeat, the length of which correlates with breast cancer pre-disposition. Our study indicates that the AAAG tetranucleotide repeat polymorphism in ERR-γ gene 5' UTR region may be a new biomarker for genetic susceptibility to breast cancer.

Status asthmaticus complicated by cardiac arrest.

In the setting of severe acute asthma, electrocardiographic abnormalities are not uncommon and some patients will develop reversible systolic dysfunction. However acute myocardial infarction and potentially fatal arrhythmias are rare. We report the case of a middle-aged indigenous male who suffered an ST-elevation myocardial infarction and then pulseless ventricular tachycardia arrest while still in the acute phase of treatment for status asthmaticus.

Global microsatellite content distinguishes humans, primates, animals, and plants.

Microsatellites are highly mutable, repetitive sequences commonly used as genetic markers, but they have never been studied en masse. Using a custom microarray to measure hybridization intensities of every possible repetitive nucleotide motif from 1-mers to 6-mers, we examined 25 genomes. Here, we show that global microsatellite content varies predictably by species, as measured by array hybridization signal intensities, correlating with established taxonomic relationships, and particular motifs are characteristic of one species versus another. For instance, hominid-specific microsatellite motifs were identified despite alignment of the human reference, Celera, and Venter genomic sequences indicating substantial variation (30-50%) among individuals. Differential microsatellite motifs were mainly associated with genes involved in developmental processes, whereas those found in intergenic regions exhibited no discernible pattern. This is the first description of a method for evaluating microsatellite content to classify individual genomes.

Supporting self-management for patients with complex medical needs: recommendations of a working group.

Increasing numbers of persons live with complex chronic medical needs and are at risk for poor health outcomes. These patients require unique self-management support, as they must manage many, often interacting, tasks. As part of a conference on Managing Complexity in Chronic Care sponsored by the Department of Veterans Affairs, a working group was convened to consider self-management issues specific to complex chronic care. In this paper, we assess gaps in current knowledge on self-management support relevant to this population, report on the recommendations of our working group, and discuss directions for future study. We conclude that this population requires specialized, multidimensional self-management support to achieve a range of patient-centred goals. New technologies and models of care delivery may provide opportunities to develop this support. Validation and quantification of these processes will require the development of performance measures that reflect the needs of this population, and research to prove effectiveness.

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase.

The structure of the Escherichia coli flavodoxin NADP(+) oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6 min(-1); R174A, 132.1 min(-1); R184A, 305.5 min(-1) versus wild type, 338.9 min(-1)) and increased K(m) for NADPH (R144A, 5.3 microM; R174A, 20.2 microM; R184A, 54.4 microM versus wild type, 3.9 microM). The k(cat) value for NADH-dependent cytochrome c reduction was increased for R174A (42.3 min(-1)) and R184A (50.4 min(-1)) compared with the wild type (33.0 min(-1)), consistent with roles for R174 and R184 in discriminating between NADPH/NADH by interaction with the adenosine ribose 2'-phosphate. Stopped-flow studies indicated that affinity (K(d)) for NADPH was markedly reduced in mutants R144A (635 microM) and R184A (2.3 mM) compared with the wild type (<5 microM). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH K(d) ratio >175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (k(red)=8.82 s(-1) versus 22.63 s(-1) for the wild type), which also exhibited tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C-terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosphates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2'-phosphate group, and R144 likely to interact with the nicotinamide ribose 5'-phosphate group.

Identification of the [Fe-S] cluster-binding residues of Escherichia coli biotin synthase.

The gene encoding Escherichia coli biotin synthase (bioB) has been expressed as a histidine fusion protein, and the protein was purified in a single step using immobilized metal affinity chromatography. The His(6)-tagged protein was fully functional in in vitro and in vivo biotin production assays. Analysis of all the published bioB sequences identified a number of conserved residues. Single point mutations, to either serine or threonine, were carried out on the four conserved (Cys-53, Cys-57, Cys-60, and Cys-188) and one non-conserved (Cys-288) cysteine residues, and the purified mutant proteins were tested both for ability to reconstitute the [2Fe-2S] clusters of the native (oxidized) dimer and enzymatic activity. The C188S mutant was insoluble. The wild-type and four of the mutant proteins were characterized by UV-visible spectroscopy, metal and sulfide analysis, and both in vitro and in vivo biotin production assays. The molecular masses of all proteins were verified using electrospray mass spectrometry. The results indicate that the His(6) tag and the C288T mutation have no effect on the activity of biotin synthase when compared with the wild-type protein. The C53S, C57S, and C60S mutant proteins, both as prepared and reconstituted, were unable to covert dethiobiotin to biotin in vitro and in vivo. We conclude that three of the conserved cysteine residues (Cys-53, Cys-57, and Cys-60), all of which lie in the highly conserved "cysteine box" motif, are crucial for [Fe-S] cluster binding, whereas Cys-188 plays a hitherto unknown structural role in biotin synthase.

Characterisation of flavodoxin NADP+ oxidoreductase and flavodoxin; key components of electron transfer in Escherichia coli.

The genes encoding the Escherichia coli flavodoxin NADP+ oxidoreductase (FLDR) and flavodoxin (FLD) have been overexpressed in E. coli as the major cell proteins (at least 13.5% and 11.4% of total soluble protein, respectively) and the gene products purified to homogeneity. The FLDR reduces potassium ferricyanide with a kcat of 1610.3 min(-1) and a Km of 23.6 microM, and cytochrome c with a kcat of 141.3 min(-1) and a Km of 17.6 microM. The cytochrome c reductase rate is increased sixfold by addition of FLD and an apparent Km of 6.84 microM was measured for the affinity of the two flavoproteins. The molecular masses of FLDR and FLD apoproteins were determined as 27648 Da and 19606 Da and the isoelectric points as 4.8 and 3.5, respectively. The mass of the FLDR is precisely that predicted from the atomic structure and indicates that residue 126 is arginine, not glutamine as predicted from the gene sequence. FLDR and FLD were covalently crosslinked using 1-ethyl-3(dimethylamino-propyl) carbodiimide to generate a catalytically active heterodimer. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of both FLDR (-308 mV and -268 mV, respectively) and FLD (-254 mV and -433 mV, respectively) were measured using redox potentiometry. This confirms the electron-transfer route as NADPH-->FLDR-->FLD. Binding of 2' adenosine monophosphate increases the midpoint reduction potentials for both FLDR couples. These data highlight the strong stabilisation of the flavodoxin semiquinone (absorption coefficient calculated as 4933 M(-1) cm(-1) at 583 nm) with respect to the hydroquinone state and indicate that FLD must act as a single electron shuttle from the semiquinone form in its support of cellular functions, and to facilitate catalytic activity of microsomal cytochromes P-450 heterologously expressed in E. coli. Kinetic studies of electron transfer from FLDR/FLD to the fatty acid oxidase P-450 BM3 support this conclusion, indicating a ping-pong mechanism. This is the first report of the potentiometric analysis of the full E. coli NAD(P)H/FLDR/FLD electron-transfer chain; a complex critical to the function of a large number of E. coli redox systems.

Family correlates of bulimic characteristics in college females.

This article provides a short review of the literature that relates family characteristics to anorexia nervosa and bulimia nervosa. In addition, a quantitative analysis of the relationship between family variables and level of eating disturbance was performed on self-reported responses of 175 normal weight females in an attempt to verify an expansion of the continuum hypothesis outlined by Kagan and Squires (1985). Consistent with the continuum hypothesis, moderate relationships were found between level of family dysfunction and bulimic symptomatology. Among several family variables, inconsistent expression of affection by the mother best predicted severity of eating disturbance.

Intergenerational influences on the parent-infant relationship in the transition to parenthood.

PIP: In this study, questions were addressed concerning the intergenerational transmission of parent-child relationships in couples going through the transition to parenthood. During the 2nd trimester of their 1st pregnancy, 38 couples provided information concerning experiences of parenting in their family of origin, then were reinterviewed and observed interacting with their infants at 3 months postpartum. It was expected that when these young adults had reported prenatally better parenting by their parents on specific parenting variables, they in turn would experience early parenthood more adaptively and would show better parenting with their own infants. The analyses suggest that women's reports in interviews of their delight in their child, their investment in their child, their sensitivity to the child's needs, and their acceptance of the child--construed as a factor to represent quality of adaptation to parenting--were significantly predicted by their perception of their own mother's intrusiveness, the support they received from their fathers during adolescence, the sensitivity of their fathers, and, lastly, the subject mother's level of psychological health. Although individual psychological health added some prediction of maternal adaptation to parenting over and above that provided by the family of origin variables, neither psychological health nor marital competence seemed to mediate the relationship between family of origin variables and mother's adaptation to parenthood. For men, adaptation to parenthood was predicted powerfully by the perception of their father's intrusiveness and further predicted by the quality of their current marriage. Thus the quality of their marriage added to but did not replace the predictiveness of the family of origin variable.^ieng