Yohimbine Depresses Excitatory Transmission in BNST and Impairs Extinction of Cocaine Place Preference Through Orexin-Dependent,Norepinephrine-Independent Processes

The alpha2 adrenergic receptor (α₂-AR) antagonist yohimbine is a widely used tool for the study of anxiogenesis and stress-induced drug-seeking behavior. We previously demonstrated that yohimbine paradoxically depresses excitatory transmission in the bed nucleus of the stria terminalis (BNST), a region critical to the integration of stress and reward pathways, and produces an impairment of extinction of cocaine-conditioned place preference (cocaine-CPP) independent of α₂-AR signaling. Recent studies show yohimbine-induced drug-seeking behavior is attenuated by orexin receptor 1 (OX₁R) antagonists. Moreover, yohimbine-induced cocaine-seeking behavior is BNST-dependent. Here, we investigated yohimbine-orexin interactions. Our results demonstrate yohimbine-induced depression of excitatory transmission in the BNST is unaffected by alpha1-AR and corticotropin-releasing factor receptor-1 (CRFR₁) antagonists, but is (1) blocked by OxR antagonists and (2) absent in brain slices from orexin knockout mice. Although the actions of yohimbine were not mimicked by the norepinephrine transporter blocker reboxetine, they were by exogenously applied orexin A. We find that, as with yohimbine, orexin A depression of excitatory transmission in BNST is OX₁R–dependent. Finally, we find these ex vivo effects are paralleled in vivo, as yohimbine-induced impairment of cocaine-CPP extinction is blocked by a systemically administered OX₁R antagonist. These data highlight a new mechanism for orexin on excitatory anxiety circuits and demonstrate that some of the actions of yohimbine may be directly dependent upon orexin signaling and independent of norepinephrine and CRF in the BNST.     

“Bareback” Pornography Consumption and Safe-Sex Intentions of Men Having Sex with Men

Men having sex with men (MSM) commonly consume “bareback” pornography, which includes scenes of unprotected anal intercourse. Prior research on human imitative behavior suggests that these media might counteract efforts to promote safe-sex behaviors. To date, no studies have demonstrated a causal link between bareback pornography consumption and reduced safe-sex intentions. Study 1 utilized a correlational design conducted as an online survey. Study 2 was set in an actual MSM sex club, using a 2 × 2 mixed-factorial design to compare type of pornography (unprotected vs. protected anal intercourse) and age of actors (younger vs. older). As the main dependent variable in both studies, participants self-reported their inclinations toward unprotected versus protected intercourse, using a 100-point sliding scale (1 = unprotected, 100 = protected). In Study 1, more attention to unprotected sex acts on actual DVD film covers predicted lower safe-sex intentions, as compared to other elements of the film cover. In Study 2, safe-sex intentions after viewing unprotected-sex films were lower than after viewing protected-sex films. The results provide novel and ecologically valid evidence that “bareback” pornography consumption impacts viewer’s inclinations toward sexual risk-taking by lowering their intentions to use protected sex measures. Suggestions are given as to how these findings can be utilized for purposes of intervention and prevention of STI and HIV infections.     

Rosuvastatin-regulated post-translational phosphoproteome in human umbilical vein endothelial cells

Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, are widely prescribed as cholesterol-lowering drugs. Statins have recently been found to have pleiotropic effects that are independent of their lipid-lowering properties. Phosphorylation of serine, threonine, and tyrosine residues of functional proteins are considered to be important in the endothelial signaling cascade. In this study, protein phosphorylation status in human umbilical vein endothelial cells (ECs) after rosuvastatin treatment was examined. The proteins were collected from rosuvastatin-treated ECs and then the phosphorylated peptides purified by a Fe³⁺-immobilized metal-affinity chromatography bead system were examined by liquid chromatography–tandem mass spectrometry analysis. Alterations of the phosphorylation status of proteins were noticed after rosuvastatin treatment. There were 277 and 530 phosphorylated proteins identified from the control and rosuvastatin-treated ECs, respectively. Among those proteins, T78, in addition to S156 of the Ras-GTPase-activating protein, was phosphorylated after rosuvastatin treatment. Rosuvastatin reduced the phosphorylation of Y455 in HSP90 protein. Decreased phosphorylation of T211 with a concurrent increase in the T291 phosphorylation of Akt1 was observed under rosuvastatin treatment. Increased S633 phosphorylation was detected in endothelial nitric oxide synthase. Western blot analysis further showed an earlier and greater S633 phosphorylation than that of S1177 in endothelial nitric oxide synthase after rosuvastatin treatment. Changes in the phosphorylation status of these proteins may alter the protein’s function and affect endothelial physiology. The current study provides new insights leading to a better understanding of the pleiotropic effects of statins on the vascular system.     

Sudden declines in intelligence in old age predict death and dropout from longitudinal studies

It is well known that approaching death accelerates cognitive decline. The converse issue, that is, the question of whether rapid declines in cognitive ability are risk factors for imminent death, has not been investigated. Every 4 years between 1983 and 2003, we gave 1,414 healthy community residents who were aged between 49 and 93 years the Heim AH4-1 test of fluid intelligence. A modified Andersen-Gill model evaluated AH4-1 scores at entry to the study and changes in scores between successive quadrennial test sessions as risk factors for death and dropout. Deaths, dropouts, age, gender, occupational categories, and recruitment cohorts were also taken into account. Participants with lower AH4-1 scores on entry were significantly more likely to die or to drop out. At all ages and levels of baseline intelligence, the risks of deaths and dropouts further increased if test scores fell by 10%, and again increased if they fell by 20% during 4-year intervals between successive assessments.     

Rare recombination events generate sequence diversity among balancer chromosomes in Drosophila melanogaster

Multiply inverted balancer chromosomes that suppress exchange with their homologs are an essential part of the Drosophila melanogaster genetic toolkit. Despite their widespread use, the organization of balancer chromosomes has not been characterized at the molecular level, and the degree of sequence variation among copies of balancer chromosomes is unknown. To map inversion breakpoints and study potential diversity in descendants of a structurally identical balancer chromosome, we sequenced a panel of laboratory stocks containing the most widely used X chromosome balancer, First Multiple 7 (FM7). We mapped the locations of FM7 breakpoints to precise euchromatic coordinates and identified the flanking sequence of breakpoints in heterochromatic regions. Analysis of SNP variation revealed megabase-scale blocks of sequence divergence among currently used FM7 stocks. We present evidence that this divergence arose through rare double-crossover events that replaced a female-sterile allele of the singed gene (sn^X2) on FM7c with a sequence from balanced chromosomes. We propose that although double-crossover events are rare in individual crosses, many FM7c chromosomes in the Bloomington Drosophila Stock Center have lost sn^X2 by this mechanism on a historical timescale. Finally, we characterize the original allele of the Bar gene (B¹) that is carried on FM7, and validate the hypothesis that the origin and subsequent reversion of the B¹ duplication are mediated by unequal exchange. Our results reject a simple nonrecombining, clonal mode for the laboratory evolution of balancer chromosomes and have implications for how balancer chromosomes should be used in the design and interpretation of genetic experiments in Drosophila.Balancer chromosomes are genetically engineered chromosomes that suppress crossing over with their homologs and are used for many purposes in genetics, including construction of complex genotypes, maintenance of stocks, and estimation of mutation rates. Balancers typically carry multiple inversions that suppress genetic exchange or result in the formation of abnormal meiotic products if crossing over does occur (Fig. 1A); for example, single crossovers inside the inverted segment create acentric or dicentric chromosomes that will fail to segregate properly during meiosis or large deletions or duplications that will likely result in inviable gametes (1, 2). Balancers also often carry recessive lethal or sterile mutations to prevent their propagation as homozygotes as well as dominant markers for easy identification. First developed for use in Drosophila melanogaster, balancer chromosomes remain some of the most powerful tools for genetic analysis in this species (3).Open in a separate windowFig. 1.Consequences of a single or double crossover between a WT X chromosome and an X chromosome carrying a single inversion, In(1)dl-49. Euchromatin is shown in blue, heterochromatin is shown in gray, and centromeres are depicted as circles. Thin white lines mark locations of inversion breakpoints, and yellow crosses/thin lines mark locations of crossover events. (A) A single crossover event within the inverted segment results in the formation of chromosomes with deletions and zero (acentric) centromeres or duplications and two (dicentric) centromeres, neither of which will segregate properly during meiosis. (B) A double crossover within an inverted segment results in intact chromosomes with one centromere that will segregate properly during meiosis.Despite their widespread use, very little is known about the organization of Drosophila balancer chromosomes at the molecular level. Since their original syntheses decades ago, balancers have undergone many manipulations, including the addition or removal of genetic markers. Moreover, rare recombination events can cause spontaneous loss of deleterious alleles on chromosomes kept over balancers in stock, as well as loss of marker alleles on balancer chromosomes themselves (3). Likewise, recent evidence has shown that sequence variants can be exchanged between balancer chromosomes and their wild type (WT) homologs via gene conversion during stock construction or maintenance (4, 5). Thus, substantial variation may exist among structurally identical balancer chromosomes owing to various types of sequence exchange.To gain insight into the structure and evolution of balancer chromosomes, we have undertaken a genomic analysis of the most commonly used X chromosome balancer in D. melanogaster, First Multiple 7 (FM7). We have focused on FM7 because this X chromosome balancer series lacks lethal mutations and thus can be easily sequenced in a hemizygous or homozygous state. In addition, the FM7 chromosome has been shown to pair normally along most of its axis with a standard X chromosome, providing a structural basis for possible exchange events (6). Moreover, although details of how early balancers in D. melanogaster were created are not fully recorded, the synthesis and cytology of the FM7 series is reasonably well documented (3).The earliest chromosome in the FM7 series, FM7a, was constructed using two progenitor X chromosome balancers, FM1 and FM6, to create a chromosome carrying three inversions—In(1)sc⁸, In(1)dl-49, and In(1)FM6—relative to the WT configuration (7, 8) (Fig. 2A). Subsequently, a female-sterile allele of singed (sn^X2) was introduced onto FM7a to create FM7c, which prevents the loss of balanced chromosomes carrying recessive lethal or female-sterile mutations (9). More recently, versions of FM7a and FM7c have been generated that carry transgene insertions that allow the determination of balancer genotypes in embryonic or pupal stages (10–14).Open in a separate windowFig. 2.Structure of the FM7 balancer chromosome. Euchromatin is shown in blue, and heterochromatin is shown in gray. (A) Schematic view of the organization of WT and FM7 X chromosomes. FM7 contains three inversions—In(1)sc⁸, In(1)dl-49, and In(1)FM6—relative to WT. The six breakpoint junctions for the three inversions are numbered 1–6 and are shown in detail in B. (B) Location and organization of inversion breakpoints in FM7. Each inversion has two breakpoints that can be represented as A/B and C/D in the standard WT arrangement and as A/C and B/D in the inverted FM7 arrangement, where A, B, C, and D represent the sequences on either side of the breakpoints. Locations of euchromatic breakpoints are on Release 5 genome coordinates, and the identity of the best BLAST match in FlyBase is shown for heterochromatic sequences. Primers used for PCR amplification are shown above each breakpoint; details are provided in Methods and Datasets S2 and S3. Forward and reverse primers are named with respect to the orientation of the assembled breakpoint contigs, not the orientation of the WT or FM7 X chromosome.To identify the inversion breakpoints in FM7 balancers and to study patterns of sequence variation that may have arisen since the origin of the FM7 series, we sequenced genomes of eight D. melanogaster stocks carrying the FM7 chromosome (four FM7a and four FM7c). We discovered several megabase-scale regions in which FM7c chromosomes differ from one another, which presumably arose via double-crossover (DCO) events from balanced chromosomes (Fig. 1B). These DCOs eliminate the female-sterile sn^X2 allele in the centrally located In(1)dl-49 inversion and are expected to confer a fitness advantage to sn⁺ chromosomes, either by allowing propagation of sn⁺ FM7 as homozygotes in females or by sn⁺ FM7 males outcompeting sn^X2 FM7 males in culture. We found that loss of the sn^X2 allele is common in FM7c chromosomes by screening other FM7c-carrying stocks at the Bloomington Drosophila Stock Center. We also identified the breakpoints of the B¹ duplication carried on FM7, and found direct molecular evidence for the role of unequal exchange in the origin and reversion of the B¹ allele (15–19). Our results provide clear evidence that the common assumption that balancers are fully nonrecombining chromosomes is incorrect on a historical timescale, and that substantial sequence variation exists among balancer chromosomes in circulation today.     

A 10-Year Trend in Statin Use Among Older Adults in Australia: an Analysis Using National Pharmacy Claims Data

Background

Statins have become standard of care in the prevention and treatment of atherosclerotic cardiovascular disease. The objective of this study was to examine the trends in statin use among Australians aged ≥?65 years for the period 2007–2016.

Methods

Data from the Pharmaceutical Benefits Scheme covering a 10% random sample of the Australian population were analysed. The 1-year prevalence and incidence of statin use were determined for each year, as were the percentage of statin dispensations according to statin type or intensity and the percentage of new users prescribed each statin type or intensity. To describe relative changes, age-sex adjusted rate ratios (RRs) and 95% confidence intervals (CIs) were determined via Poisson regression modelling using 2007 as the reference year.

Results

The 1-year prevalence of statin use increased consistently each year from 34.2% in 2007 to 44.1% in 2016 (RR 1.29, 95% CI 1.28–1.31). The 1-year incidence was 68.5 per 1000 in 2007 and 59.0 per 1000 in 2016 (RR 0.87, 95% CI 0.84–0.90). Women were 18% (age-adjusted rate ratio [aRR] 0.82, 95% CI 0.79–0.83) less likely than men to initiate statins across all years. The incidence of statin use was also highest among individuals aged 65–74 years, who were about 15% (sex-adjusted rate ratio [sRR] 1.15, 95% CI 1.13–1.16) and 45% (sRR 1.45, 95% CI 1.44–1.47) more likely to initiate statins than those aged 75–84 and ≥ 85 years, respectively. Atorvastatin was the most commonly dispensed statin across all years. The proportion of new users dispensed high-intensity statins increased year-on-year from 23.6% in 2007 to 30.5% in 2016 (RR 1.26, 95% CI 1.21–1.31).

Conclusion

The proportion of older adults in Australia using statins has increased over the last decade, although the incidence has declined. Atorvastatin is the most commonly dispensed statin and the use of high intensity statin has increased.