Divergent Response to the SSRI Citalopram in Male and Female Three-Spine Sticklebacks (Gasterosteus aculeatus).

Selective serotonin reuptake inhibitors (SSRIs) are psychotropic pharmaceuticals used as antidepressants. SSRIs are commonly found in surface waters in populated areas across the globe. They exert their effect by blocking the serotonin re-uptake transporter in the presynaptic nerve ending. The present study examined whether behavioural effects to exposure to SSRI citalopram depend on personality and sex in the stickleback (Gasterosteus aculeatus). Three aspects of stickleback behaviour are examined: feeding behaviour, aggression, and boldness. We exposed sticklebacks to 350-380 ng/l citalopram for 3 weeks. Feeding and aggressive behaviour were recorded before and after exposure, whereas scototaxis behaviour was tested after exposure. The results show treatment effects in feeding and aggressive behaviour. Feeding is suppressed only in the male group (χ2 = 20.4, P < 0.001) but not in the females (χ2 = 0.91, P = 0.339). Aggressive behaviour was significantly affected by treatment (χ2 = 161.9, P < 0.001), sex (χ2 = 86.3, P < 0.001), and baseline value (χ2 = 58.8, P < 0.001). Aggressiveness was suppressed by citalopram treatment. In addition, the fish showed no change in aggression and feeding behaviour over time regardless of sex and treatment, which indicate personality traits. Only females are affected by treatment in the scototaxis test. The exposed females spent significantly (χ2 = 5.02, P = 0.050) less time in the white zone than the female controls.

The psychoactive drug Escitalopram affects swimming behaviour and increases boldness in zebrafish (Danio rerio).

Selective serotonin re-uptake inhibitors are pharmaceuticals used to treat a range of psychological disorders. They are frequently found in surface waters in populated areas. In recent years, they have been shown to affect the behaviour of various aquatic organisms in a way that can have ecological effects. In this study, we exposed zebrafish of both sexes to nominally 0.00, 0.15 and 1.50 µg L-1 Escitalopram in flow-through tanks for three weeks. Subsequently, ten swimming behaviour parameters were quantified using high-resolution video tracking. There were noticeable gender differences in the behaviour responses to Escitalopram. Female fish exposed to 1.50 µg L-1 Escitalopram had a lower maximum swimming velocity, stopped less often and exhibited increased boldness (reduced thigmotaxis) compared to controls. Male fish exposed to 1.50 µg L-1 had a lower maximum swimming velocity compared to control fish. At the end of exposures, both length and weight of the females exposed to 1.50 µg L-1 Escitalopram were significantly less than the group of control fish. In addition, males exposed to 1.50 µg L-1 Escitalopram were significantly shorter than control fish. The behaviour, weight and body length of the fish exposed to nominally 0.15 µg L-1 was not significantly different from control fish in either sex. The results of this study demonstrate that Escitalopram can affect subtle but ecologically important aspects of fish behaviour and lends further credibility to the assumption that Escitalopram is an environmentally active pharmaceutical.

Integrated process development-a robust, rapid method for inclusion body harvesting and processing at the microscale level.

Escherichia coli stores large amounts of highly pure product within inclusion bodies (IBs). To take advantage of this beneficial feature, after cell disintegration, the first step to optimal product recovery is efficient IB preparation. This step is also important in evaluating upstream optimization and process development, due to the potential impact of bioprocessing conditions on product quality and on the nanoscale properties of IBs. Proper IB preparation is often neglected, due to laboratory-scale methods requiring large amounts of materials and labor. Miniaturization and parallelization can accelerate analyses of individual processing steps and provide a deeper understanding of up- and downstream processing interdependencies. Consequently, reproducible, predictive microscale methods are in demand. In the present study, we complemented a recently established high-throughput cell disruption method with a microscale method for preparing purified IBs. This preparation provided results comparable to laboratory-scale IB processing, regarding impurity depletion, and product loss. Furthermore, with this method, we performed a "design of experiments" study to demonstrate the influence of fermentation conditions on the performance of subsequent downstream steps and product quality. We showed that this approach provided a 300-fold reduction in material consumption for each fermentation condition and a 24-fold reduction in processing time for 24 samples.

Assigning Co-Regulated Human Genes and Regulatory Gene Clusters.

Elucidating the role of genetic variation in the regulation of gene expression is key to understanding the pathobiology of complex diseases which, in consequence, is crucial in devising targeted treatment options. Expression quantitative trait locus (eQTL) analysis correlates a genetic variant with the strength of gene expression, thus defining thousands of regulated genes in a multitude of human cell types and tissues. Some eQTL may not act independently of each other but instead may be regulated in a coordinated fashion by seemingly independent genetic variants. To address this issue, we combined the approaches of eQTL analysis and colocalization studies. Gene expression was determined in datasets comprising 49 tissues from the Genotype-Tissue Expression (GTEx) project. From about 33,000 regulated genes, over 14,000 were found to be co-regulated in pairs and were assembled across all tissues to almost 15,000 unique clusters containing up to nine regulated genes affected by the same eQTL signal. The distance of co-regulated eGenes was, on average, 112 kilobase pairs. Of 713 genes known to express clinical symptoms upon haploinsufficiency, 231 (32.4%) are part of at least one of the identified clusters. This calls for caution should treatment approaches aim at an upregulation of a haploinsufficient gene. In conclusion, we present an unbiased approach to identifying co-regulated genes in and across multiple tissues. Knowledge of such common effects is crucial to appreciate implications on biological pathways involved, specifically when a treatment option targets a co-regulated disease gene.

Plant volatile-induced aphid resistance in barley cultivars is related to cultivar age.

Recent studies have shown that volatile chemical interaction between certain barley (Hordeum vulgare) cultivars can cause reduced host plant acceptance by the aphid Rhopalosiphum padi, and that certain cultivars can induce this effect while others can respond. In this study, we tested whether inducing and responding capabilities are linked to year of release in Swedish two-rowed spring barley. Eighteen cultivars released between 1897 and 1992 were tested in randomly selected subsets with pairwise combinations of volatile emitters and receivers. Significantly reduced aphid acceptance as a result of exposure to volatiles from plants of a different cultivar were found in 24% of the cultivar combinations. In general, older cultivars had a higher degree of aphid resistance after barley volatile treatment than did younger cultivars. The inducing effect of the emitter was also related to date of emitter cultivar release but the time relationship was reversed. Combinations with a younger volatile emitter and an older volatile receiver gave the strongest reduction in aphid acceptance of treated plants. Linear relationships between microsatellite diversity of emitting cultivars and their efficiency as inducers indicated that younger cultivars might have a more unique odour, whereas older cultivars may be more sensitive to induction.

Behavioral effects of citalopram, tramadol, and binary mixture in zebrafish (Danio rerio) larvae.

Pharmaceuticals are emerging as environmentally problematic compounds. As they are often not appropriately removed by sewage treatment plants, pharmaceutical compounds end up in surface water environments worldwide at concentrations in the ng to µg L-1 range. There is a need to further explore single compound and mixture effects using e.g. in vivo test model systems. We have investigated, for the first time, behavioral effects in larval zebrafish (Danio rerio) exposed to a binary mixture of an antidepressant drug (citalopram) and a synthetic opioid (tramadol). Citalopram and tramadol have a similar mode of action (serotonin reuptake inhibition) and are known to produce drug-drug interactional effects resulting in serotonin syndrome (SS) in humans. Zebrafish embryo-larvae were exposed to citalopram, tramadol and 1:1 binary mixture from fertilization until 144 h post-fertilization. No effects on heart rate, spontaneous tail coiling, or death/malformations were observed in any treatment at tested concentrations. Behavior (hypoactivity in dark periods) was on the other hand affected, with lowest observed effect concentrations (LOECs) of 373 µg L-1 for citalopram, 320 µg L-1 for tramadol, and 473 µg L-1 for the 1:1 mixture. Behavioral EC50 was calculated to be 471 µg L-1 for citalopram, 411 µg L-1 for tramadol, and 713 µg L-1 for the 1:1 mixture. The results of this study conclude that tramadol and citalopram produce hypoactivity in 144 hpf zebrafish larvae. Further, a 1:1 binary mixture of the two caused the same response, albeit at a higher concentration, possibly due to SS.

Altered non-reproductive behavior and feminization caused by developmental exposure to 17α-ethinylestradiol persist to adulthood in three-spined stickleback (Gasterosteus aculeatus).

The synthetic estrogen 17α-ethinylestradiol (EE2), ubiquitous in the aquatic environment and commonly detected in sewage effluents, interferes with the endocrine system in multiple ways. Exposure during sensitive windows of development causes persistent effects on fertility, reproductive and non-reproductive behavior in mammals and fish. In the present study, three-spined stickleback (Gasterosteus aculeatus) were exposed to nominal 0 and 20 ng/L EE2 from fertilization to 7 weeks post-hatch. After 8 months of remediation in clean water three non-reproductive behaviors, not previously analyzed in developmentally EE2-exposed progeny of wild-caught fish, were evaluated. Chemical analysis revealed that the nominal 0 and 20 ng/L exposure contained 5 and 30 ng/L EE2, respectively. Therefore, the use of control fish from previous experiments was necessary for comparisons. Fish exposed during development showed significant concentration-dependent reduction in anxiety-like behavior in the scototaxis (light/dark preference) test by means of shorter latency to first entrance to the white compartment, more visits in white, and longer total time in white compared to unexposed fish. In the novel tank test, developmental exposure significantly increased the number of transitions to the upper half of the aquaria. Exposure to EE2 during development did not alter shoal cohesion in the shoaling test compared with unexposed fish but fish exposed to 30 ng/L EE2 had significantly longer latency to leave the shoal and fewer transitions away from the shoal compared to fish exposed to 5 ng/L EE2. Skewed sex ratio with more females, sex reversal in genetic males as well as intersex in males was observed after exposure to 30, but not 5 ng/L EE2. In conclusion, EE2 exposure during development in three-spined stickleback resulted in persistent effects on anxiety-like behaviors. These long-term effects from developmental exposure are likely to be of higher relevance for natural populations than are short-term effects from adult exposure.

Combinatory effects of low concentrations of 17α-etinylestradiol and citalopram on non-reproductive behavior in adult zebrafish (Danio rerio).

Sewage effluents contain pharmaceuticals, personal care products and industrial chemicals, exposing aquatic organisms to complex mixtures. The consequences of exposure to combinations of different classes of drugs in fish are largely unknown. In this study, we exposed adult zebrafish (Danio rerio) males and females for two weeks to low, environmentally relevant concentrations of the endocrine disrupting chemical 17α-etinylestradiol (EE2) and the selective serotonin re-uptake inhibitor (SSRI) citalopram, alone and in combination, and analyzed behaviors of importance for population fitness, scototaxis (light/dark preference), the novel tank test and shoal cohesion. Control water contained 0.4ng/L EE2 and the measured exposure concentrations were 0.9ng/L EE2 (nominal 0.1) and 1ng/L EE2 (nominal 0.5). The measured concentrations of citalopram were 0.1 (nominal 0.1) and 0.4µg/L (nominal 0.5). Both EE2 exposures increased anxiety in males in the scototaxis test, with significantly longer latency periods before entering and fewer visits to the white zone of the tank. The combined exposures (0.9ng/L EE2+0.1µg/L citalopram and 1ng/L EE2+0.4µg/L citalopram) resulted in abolishment of effects of EE2, with shorter latency period and more transitions to white than for fish exposed to EE2 alone. In the novel tank test, the results surprisingly indicated lower anxiety after both EE2 and citalopram exposure. Significantly more transitions to the upper half of the tank observed in males exposed to 0.1µg/L citalopram alone compared to control males. Males exposed to EE2 (0.9ng/L) had shorter latency period to the upper half. Combination exposure resulted in a longer latency and fewer transitions to the upper half compared to both control, EE2- and citalopram-exposed males. Males exposed to the combination spent significantly less time in the upper half than males EE2 or citalopram-exposed males. Females exposed to 1ng/L EE2 had fewer transitions to the upper half than the control group and females exposed to 0.4µg/L citalopram. In the shoaling test, males exposed to 0.1µg/L citalopram+0.9ng/L EE2 showed more transitions away from peers than males exposed to 0.1µg/L citalopram alone. In conclusion, low concentrations of EE2, closely above the predicted no effect concentration (NOEC) of 0.1ng/L, created anxiety-like behavior in zebrafish males. Citalopram showed marginal effects at these low concentrations but in the combination exposure the behavioral effects of EE2 were abolished. This is an initial effort to understand the effects of cocktails of anthropogenic substances contaminating aquatic environments.