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Pseudomonas aeruginosa is among the most problematic opportunistic pathogens for adults with cystic fibrosis (CF), causing repeated and resilient infections in the lung and surrounding airways. Evidence suggests that long-term infections are associated with diversification into specialized types but the underlying cause of that diversification and the effect it has on the persistence of infections remains poorly understood. Here, we use evolve-and-resequence experiments to investigate the genetic changes accompanying rapid, de novo phenotypic diversification in lab environments designed to mimic two aspects of human lung ecology: spatial structure and complex nutritional content. After ∼220 generations of evolution, we find extensive genetic variation present in all environments, including those that most closely resemble the CF lung. We use the abundance and frequency of nonsynonymous and synonymous mutations to estimate the ratio of mutations that are selectively neutral (hitchhikers) to those that are under positive selection (drivers). A significantly lower proportion of driver mutations in spatially structured populations suggests that reduced dispersal generates subpopulations with reduced effective population size, decreasing the supply of beneficial mutations and causing more divergent evolutionary trajectories. In addition, we find mutations in a handful of genes typically associated with chronic infection in the CF lung, including one gene associated with antibiotic resistance. This demonstrates that many of the genetic changes considered to be hallmarks of CF lung adaptation can arise as a result of adaptation to a novel environment and do not necessarily require antimicrobial treatment, immune system suppression, or competition from other microbial species to occur., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Introduction: Cell line derived from fish has been established as a promising tool for studying many key issues of aquaculture covering fish growth, disease, reproduction, genetics, and biotechnology. In addition, fish cell lines are very useful in vitro models for toxicological, pathological, and immunological studies. The easier maintenance of fish cell lines in flexible temperature regimes and hypoxic conditions make them preferable in vitro tools over mammalian cell lines. Great excitement has been observed in establishing and characterizing new fish cell lines representing diverse fish species and tissue types. The well-characterized and authenticated cell lines are of utmost essential as these represent cellular functions very similar to in vivo state of an organism otherwise it would affect the reproducibility of scientific research., Conclusion: The fish cell lines have exhibited encouraging results in several key aspects of in vitro research in aquaculture including virology, nutrition and metabolism, production of vaccines, and transgenic fish production. The review paper reports the cell lines developed from fish, their characterization, and biobanking along with their potential applications and challenges in in vitro research., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Sediment DNA (sedDNA) analyses are rapidly emerging as powerful tools for the reconstruction of environmental and evolutionary change. While there are an increasing number of studies using molecular genetic approaches to track changes over time, few studies have compared the coherence between quantitative polymerase chain reaction (PCR) methods and metabarcoding techniques. Primer specificity, bioinformatic analyses, and PCR inhibitors in sediments could affect the quantitative data obtained from these approaches. We compared the performance of droplet digital polymerase chain reaction (ddPCR) and high-throughput sequencing (HTS) for the quantification of target genes of cyanobacteria in lake sediments and tested whether the two techniques similarly reveal expected patterns through time. Absolute concentrations of cyanobacterial 16S rRNA genes were compared between ddPCR and HTS using dated sediment cores collected from two experimental (Lake 227, fertilized since 1969 and Lake 223, acidified from 1976 to 1983) and two reference lakes (Lakes 224 and 442) in the Experimental Lakes Area (ELA), Canada. Relative abundances of Microcystis 16S rRNA (MICR) genes were also compared between the two methods. Moderate to strong positive correlations were found between the molecular approaches among all four cores but results from ddPCR were more consistent with the known history of lake manipulations. A 100-fold increase in ddPCR estimates of cyanobacterial gene abundance beginning in ~1968 occurred in Lake 227, in keeping with experimental addition of nutrients and increase in planktonic cyanobacteria. In contrast, no significant rise in cyanobacterial abundance associated with lake fertilization was observed with HTS. Relative abundances of Microcystis between the two techniques showed moderate to strong levels of coherence in top intervals of the sediment cores. Both ddPCR and HTS approaches are suitable for sedDNA analysis, but studies aiming to quantify absolute abundances from complex environments should consider using ddPCR due to its high tolerance to PCR inhibitors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mejbel, Dodsworth, Baud, Gregory-Eaves and Pick.)

In the present study, we investigated whether the immortalized chicken hepatocellular carcinoma cell line, leghorn male hepatoma (LMH), had a comparable aryl hydrocarbon receptor (AhR) response to primary chicken embryonic hepatocytes (CEHs) when used in a well-established assay for chemical screening and prioritization. The LMH cells were grown as 2-dimensional (2D) confluent cells and 3D spheroids to determine the optimal cell culture states for chemical screening. Cytochrome P450 1A4 and 1A5 (CYP1A) activity and gene expression were compared between CEHs and LMH cells grown in 2 culture states following exposure to the dioxin-like compound 3,3',4,4',5-pentachlorobiphenyl (PCB-126). The CYP1A activity was measured using the ethoxyresorufin-O-deethylase (EROD) assay, and changes in mRNA expression associated with the AhR pathway were determined using a custom-designed polymerase chain reaction array. Among LMH cell culture states (i.e., 2D vs 3D), EROD induction was observed only in 3D LMH spheroids. Similarly, 3D spheroids had the greatest number of changes in AhR-related genes compared with confluent cells. Overall, these results suggest that LMH cells grown as 3D spheroids have a metabolic and gene expression profile that is comparable to that of CEH, and may represent a suitable animal-free alternative for in vitro screening of chemicals. Environ Toxicol Chem 2020;39:1693-1701. © 2020 SETAC., (© 2020 SETAC.)

Background: Gilg. is a neotropical vine native to Central America, investigated as part of a targeted study of the plant family Marcgraviaceae. Our previous research showed that extract of Souroubea sympetala leaf and small branch extract had anxiolytic effects in animals and acts as an agonist for the GABA S. sympetala leaf and small branch extract had anxiolytic effects in animals and acts as an agonist for the GABA A and its constituents on reconsolidation have not been assessed. Reconsolidation, the process by which formed memories are rendered labile and susceptible to change, may offer a window of opportunity for pharmacological manipulation of learned fear. Here, we assessed the effects of S. sympetala crude extract and isolated phytochemicals (orally administered) on the reconsolidation of conditioned fear. In addition, we explored whether betulin (BE), a closely related molecule to betulinic acid (BA, an active principal component of S. sympetala crude extract and isolated phytochemicals (orally administered) on the reconsolidation of conditioned fear. In addition, we explored whether betulin (BE), a closely related molecule to betulinic acid (BA, an active principal component of S. sympetala Male Sprague-Dawley rats received six 1.0-mA continuous foot shocks (contextual training). Twenty-four hours later, rats were re-exposed to the context (but in the absence of foot shocks). Immediately following memory retrieval (recall), rats received oral administration of Method: extract at various doses (8-75 mg/kg) or diazepam (1 mg/kg). In separate experiments, we compared the effects of BA (2 mg/kg), BE (2 mg/kg), and BA + BE (2 mg/kg BA + 2 mg/kg BE). The freezing response was assessed either 24 h later (day 3) or 5 days later (day 7). Effects of phytochemicals on fear expression were also explored using the elevated plus maze paradigm. S. sympetala extract at various doses (8-75 mg/kg) or diazepam (1 mg/kg). In separate experiments, we compared the effects of BA (2 mg/kg), BE (2 mg/kg), and BA + BE (2 mg/kg BA + 2 mg/kg BE). The freezing response was assessed either 24 h later (day 3) or 5 days later (day 7). Effects of phytochemicals on fear expression were also explored using the elevated plus maze paradigm. Results: S. sympetala leaf extract significantly attenuated the reconsolidation of contextual fear at the 25- and 75-mg/kg doses, but not at the 8-mg/kg dose. Furthermore, BA + BE, but not BA or BE alone, attenuated the reconsolidation of learned fear and exerted an anxiolytic-like effect on fear expression., (Copyright © 2019 Murkar, Cayer, James, Durst, Arnason, Sanchez-Vindas, Otarola Rojas and Merali.)

Quantum dots (QDs) are engineered nanoparticles (NPs) of semiconductor structure that possess unique optical and electronic properties and are widely used in biomedical applications; however, their risks are not entirely understood. This study investigated the tissue distribution and toxic effects of cadmium telluride quantum dots (CdTe-QDs) in male BALB/c mice for up to 1 week after single-dose intravenous injections. CdTe-QDs were detected in the blood, lung, heart, liver, spleen, kidney, testis and brain. Most CdTe-QDs accumulated in the liver, followed by the spleen and kidney. At high doses, exposure to CdTe-QDs resulted in mild dehydration, lethargy, ruffled fur, hunched posture, and body weight loss. Histological analysis of the tissues, upon highest dose exposures, revealed hepatic hemorrhage and necrotic areas in the spleen. The sera of mice treated with high doses of CdTe-QDs showed significant increases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin levels, as well as a reduction in albumin. CdTe-QD exposure also led to a reduced number of platelets and elevated total white blood cell counts, including monocytes and neutrophils, serum amyloid A, and several pro-inflammatory cytokines. These results demonstrated that the liver is the main target of CdTe-QDs and that exposure to CdTe-QDs leads to hepatic and splenic injury, as well as systemic effects, in mice. By contrast, cadmium chloride (CdCl 2 ), at an equivalent concentration of cadmium, appeared to have a different pharmacokinetic pattern from that of CdTe-QDs, having minimal effects on the aforementioned parameters, suggesting that cadmium alone cannot fully explain the toxicity of CdTe-QDs.

Background : Δ9-Tetrahydrocannabinol (THC, a CB1 receptor agonist) and Cannabidiol (CBD, a non-competitive antagonist of endogenous CB1 and CB2 ligands) are two primary components of Cannabis species, and may modulate fear learning in mammals. The CB1 receptor is widely distributed throughout the cortex and some limbic regions typically associated with fear learning. Humans with posttraumatic disorder (PTSD) have widespread upregulation of CB1 receptor density and reduced availability of endogenous cannabinoid anandamide, suggesting a role for the endocannabinoid system in PTSD. Pharmacological blockade of memory reconsolidation following recall of a conditioned response modulates the expression of learned fear and may represent a viable target for the development of new treatments for PTSD. In this study, we focused on assessing the impact of the key compounds of the marijuana plant both singly and, more importantly, in concert on attenuation of learned fear. Specifically, we assessed the impact of THC, CBD, and/or the remaining plant materials (post-extraction; background material), on reconsolidation of learned fear. Method : Male Sprague-Dawley rats received six 1.0 mA continuous foot shocks (contextual training). Twenty-four hours later, rats were re-exposed to the context. Immediately following memory retrieval (recall) rats received oral administration of low dose THC, high dose THC, CBD, CBD + low THC, CBD + high THC [as isolated phytochemicals and, in separate experiments, in combination with plant background material (BM)]. Rodents were tested for freezing response context re-exposure at 24 h and 7 days following training. Results : CBD alone, but not THC alone, significantly attenuated fear memory reconsolidation when administered immediately after recall. The effect persisted for at least 7 days. A combination of CBD and THC also attenuated the fear response. Plant BM also significantly attenuated reconsolidation of learned fear both on its own and in combination with THC and CBD. Finally, THC attenuated reconsolidation of learned fear only when co-administered with CBD or plant BM. Conclusion : CBD may provide a novel treatment strategy for targeting fear-memories. Furthermore, plant BM also significantly attenuated the fear response. However, whereas THC alone had no significant effects, its effects were modulated by the addition of other compounds. Future research should investigate some of the other components present in the plant BM (such as terpenes) for their effects alone, or in combination with isolated pure cannabinoids, on fear learning.

Neural tube defects (NTDs), one of the most common birth defects in human, present a multifactorial etiology with a poorly defined genetic component. The Dlx5 and Dlx6 bigenic cluster encodes two evolutionary conserved homeodomain transcription factors, which are necessary for proper vertebrate development. It has been shown that Dlx5/6 genes are essential for anterior neural tube closure, however their role in the formation of the posterior structures has never been described. Here, we show that Dlx5/6 expression is required during vertebrate posterior axis formation. Dlx5 presents a similar expression pattern in neural plate border cells during posterior neurulation of zebrafish and mouse. Dlx5/6-inactivation in the mouse results in a phenotype reminiscent of NTDs characterized by open thoracic and lumbar vertebral arches and failure of epaxial muscle formation at the dorsal midline. The dlx5a/6a zebrafish morphants present posterior NTDs associated with abnormal delamination of neural crest cells showing altered expression of cell adhesion molecules and defects of motoneuronal development. Our findings provide new molecular leads to decipher the mechanisms of vertebrate posterior neurulation and might help to gather a better understanding of human congenital NTDs etiology., Competing Interests: The authors have declared that no competing interests exist.

Exposure to dioxin-like compounds is consistently associated with concentration-dependent induction of cytochrome P4501A (CYP1A) enzymes in primary cultures of avian hepatocytes. We have previously demonstrated that the median effective concentration (EC50) for induction of this response is predictive of in vivo sensitivity to dioxin-like compounds in birds. We investigated sources of interindividual variation in the CYP1A response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wild herring gulls and considered how this variation may complicate dioxin sensitivity estimates based on the CYP1A bioassay. Concentration-dependent effects of TCDD on CYP1A mRNA expression were characterized in 55 hepatocyte cultures prepared from individual herring gull embryos. A large degree of variability was observed among the hepatocyte culture preparations. For example, 1) basal CYP1A4 and CYP1A5 mRNA expression varied by 20- and 126-fold, respectively, among individuals, and 2) exposure to TCDD induced CYP1A4 mRNA expression by 57-fold in the most responsive sample but did not significantly induce CYP1A4 mRNA expression above baseline values in 42% of hepatocyte culture preparations. Environmental and genetic factors contributing to the observed variability are discussed. Despite the large amount of interindividual variation, we conclude that reproducible EC50-based estimates of species sensitivity can be obtained from the CYP1A cell culture bioassay when samples are collected from relatively uncontaminated colonies. Environ Toxicol Chem 2019;38:660-670. © 2019 SETAC., (© 2019 SETAC.)

The global prevalence of depression is high during childbearing. Due to the associated risks to the mother and baby, the selective serotonin reuptake inhibitor fluoxetine (FLX) is often the first line of treatment. Given that FLX readily crosses the placenta, a fetus may be susceptible to the disruptive effects of FLX during this highly plastic stage of development. Here, we demonstrate that a 6-day FLX exposure to a fetus-relevant concentration at a critical developmental stage suppresses cortisol levels in the adult zebrafish (F 0 ). This effect persists for three consecutive generations in the unexposed descendants (F 1 to F 3 ) without diminution and is more pronounced in males. We also show that the in vivo cortisol response of the interrenal (fish "adrenal") to an i.p. injection of adrenocorticotropic hormone was also reduced in the males from the F 0 and F 3 FLX lineages. Transcriptomic profiling of the whole kidney containing the interrenal cells revealed that early FLX exposure significantly modified numerous pathways closely associated with cortisol synthesis in the male adults from the F 0 and F 3 generations. We also show that the low cortisol levels are linked to significantly reduced exploratory behaviors in adult males from the F 0 to F 2 FLX lineages. This may be a cause for concern given the high prescription rates of FLX to pregnant women and the potential long-term negative impacts on humans exposed to these therapeutic drugs., Competing Interests: The authors declare no conflict of interest.

The liver is a key metabolic organ contributing significantly to both lipid and cholesterol homeostasis in vertebrates. This study examines whether the human pharmaceutical atorvastatin (ATV), which is designed to lower cholesterol biosynthesis, could disrupt lipid dynamics in fish. The study investigates the effects of ATV at a physiologically relevant exposure regimen (concentration and duration) on gene transcripts and the biosynthesis of cholesterol and other lipid and non-lipid molecules in primary rainbow trout hepatocytes. Trout hepatocytes exposed to ATV increased the transcript abundance of genes involved in lipid metabolism (HMGCR1, LDLR, PPARα, PPARγ, and SREBP1) and xenobiotic metabolism (CYP3A27), and reduced cholesterol synthesis. This study demonstrates that lipid metabolism in trout hepatocytes is sensitive to the effects of ATV, and changes in gene expression occur within 3-6h after exposure., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Recent evidence indicated that methylmercury (MeHg) contaminated rice can be a significant source of MeHg human exposure, but the health implications are not known. The objective of this study was to study the kinetics, speciation, and effects of MeHg contaminated rice using a rat model. Five groups of adult Sprague-Dawley rats (n=10 in each group) were fed control rice, low (10ng/g MeHg) and high (25ng/g MeHg) MeHg contaminated rice. Two groups of the positive control were fed control rice spiked with the same levels of MeHgCl. Short-term exposure to low level of spiked MeHgCl stimulated the growth of male rats while long-term exposure to spiked MeHgCl inhibited the growth in female rats. There was no temporal variation of total mercury (THg) concentrations in the rat fecal samples from each group, and the THg concentrations significantly correlated with the inorganic Hg concentrations in the feeding rice. There were significant differences in the accumulation of THg and MeHg among different groups and different organs. THg and MeHg concentrations in the kidney were the highest among the organs examined. The blood and brain had high percentages of THg as MeHg, which indicates that MeHg can easily pass through the blood-brain barrier and has a high affinity for brain tissue. Exposure to rice containing 25ng/g MeHg decreased antioxidant function and damaged the nervous system in rats, but no significant effects were found in the group fed with rice containing 10ng/g MeHg. MeHgCys in rice is less toxic than spiked MeHgCl to rats. The toxicity of MeHg both decided by its concentration and speciation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study provides the characterization and tissue distribution of a β2-AR in the female European eel during silvering, aiming to better understand the adrenergic system involvement in this critical maturation event. A putative β2-AR (ADRB2) mRNA was cloned and sequenced. Amino acid residues and motifs important for ligand binding are generally conserved across fish and between fish and mammals, although the occurrence of some sequence variabilities may explain the noted peculiarities of eel AR interaction with pharmacological ligands. The tissue distribution of the ADRB2 gene product was analyzed in five tissues of the eel at different silvering stages and compared with that of the ADRA1 mRNA encoding an α1-AR subtype. On the whole, data suggested that relative ADRA1/ADRB2 tissue expression across silvering is part of the preparatory (molecular) adjustments required to face changes in habitats and migration efforts., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Engineered nanomaterials (ENMs) are increasingly incorporated into a variety of commercial applications and consumer products; however, ENMs may possess cytotoxic properties due to their small size. This study assessed the effects of two commonly used ENMs, zinc oxide nanoparticles (ZnONPs) and silver nanoparticles (AgNPs), in the model eukaryote Saccharomyces cerevisiae. A collection of ≈4600 S. cerevisiae deletion mutant strains was used to deduce the genes, whose absence makes S. cerevisiae more prone to the cytotoxic effects of ZnONPs or AgNPs. We demonstrate that S. cerevisiae strains that lack genes involved in transmembrane and membrane transport, cellular ion homeostasis, and cell wall organization or biogenesis exhibited the highest sensitivity to ZnONPs. In contrast, strains that lack genes involved in transcription and RNA processing, cellular respiration, and endocytosis and vesicular transport exhibited the highest sensitivity to AgNPs. Secondary assays confirmed that ZnONPs affected cell wall function and integrity, whereas AgNPs exposure decreased transcription, reduced endocytosis, and led to a dysfunctional electron transport system. This study supports the use of S. cerevisiae Gene Deletion Array as an effective high-throughput technique to determine cellular targets of ENM toxicity.

Tetradecabromo-1,4-diphenoxybenzene (TeDB-DiPhOBz) and 2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether (BDE-209) are flame retardant chemicals that can undergo photolytic degradation. The present study compared the time-dependent photolyic degradation of TeDB-DiPhOBz and BDE-209, and dioxin-like product formation as a result of (UV) irradiation (I; irradiation time periods of 0, 1, 4, 15 and 40 days). Photo-degraded product fractions of UV-I-TeDB-DiPhOBz (nominal concentration: 1.9 μM) were administered to chicken embryonic hepatocytes (CEH), and significant induction of CYP1A4/5 mRNA expression was observed for fractions collected at the day 15 and 40 time points (fold change of 7.3/3.6 and 9.1/4.7, respectively). For the UV-I-BDE-209 fractions (nominal concentration: 10 μM), significant CYP1A4/5 up-regulation occurred at all time points, and the fraction collected on day 1 induced the greatest fold change of 510/86, followed by 410/68 (day 4) and 110/26 (day 15), respectively. For the UV-I-BDE-209 fraction collected at day 40, significant CEH cytotoxicity was observed. As a result, CYP1A4/5 expression was determined at a nominal concentration of 1 μM instead of 10 μM and CYP1A4/5 fold changes of 11/8.2 (day 40) were observed. Fractions eliciting the greatest CYP1A4/5 mRNA upregulation were further screened for transcriptomic effects using a PCR array comprising 27 dioxin-responsive genes. A total of 6 and 16 of the 27 target genes were up or down-regulated following UV-I-TeDB-DiPhOBz and UV-I-BDE-209 exposure, respectively. Overall, and regardless of the formation rate, these results raise concerns regarding the potential formation of dioxin-like compounds from flame retardants in products and materials such as plastics, and in natural sunlight irradiation situations in the environment (e.g. in landfill sites or electronic waste facilities)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Carbonic anhydrase VI (CA VI) has been characterized as a secretory isozyme in mammals. Our present study confirmed the occurrence of CA VI in pufferfish (Takifugu rubripes). In this study, genomic sequence information for the CA VI of pufferfish was used for molecular cloning. We cloned a 1821 bp cDNA sequence, which consisted of a complete coding sequence of 1623bp and a deduced amino acid sequence of 540 amino acids from the open reading frame. A BLAST search indicated that this protein exhibits 53%, 79%, and 67% identity with human, tilapia, and gar CA VI, respectively. It also shows 63%-77% identity with other fish CA VI-like sequences (zebrafish, Asian arowana, salmon, and large yellow croaker). Moreover, alignment of two or more sequences revealed that the protein sequence of pufferfish CA VI has 34%-37% identity with mammalian and fish CA II sequences. An NH 2 -terminal signal peptide of 18 amino acids in length was predicted in the pufferfish CA VI sequence. Three potential N-linked glycosylation sites and two cysteine residues (Cys-28 and Cys-209) that are likely to form one disulfide bond were present in pufferfish CA VI. In silico and phylogenetic analyses revealed that pufferfish CA VI is an extracellular secretory protein. Active site analysis indicated that this protein is a low-activity CA isozymes due to a characteristic Val/Ile substitution at position 207. Homology modeling of puffer CA VI was performed using the crystal structure of human carbonic anhydrase XIV as a template structure, based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR and in situ hybridization results revealed that, the pufferfish CA VI is highly expressed in liver tissue., (Copyright © 2017 Elsevier B.V. All rights reserved.)

There are two types of dermal skeletons in jawed vertebrates: placoderms and osteichthyans carry large bony plates (macromery), whereas chondrichthyans and acanthodians are covered by small scales (micromery). Fin spines are one of the last large dermal structures found on micromeric taxa and offer a potential source of histology and morphology that can be compared to those found on macromeric groups. Dermal fin spines offer a variety of morphology but aspects of their growth modes and homology are unclear. Here, we provide detailed descriptions of the microstructure and growth of a dorsal ridge spine from the acanthothoracid placoderm, Romundina stellina, using virtual three-dimensional paleohistological datasets. From these data we identify several layers of dentine ornamentation covering the lateral surfaces of the spine and reconstructed their growth pattern. We show that this spine likely grew posteriorly and proximally from a narrow portion of bone located along the leading edge of the spine. The spine is similarly constructed to the scales with a few exceptions, including the absence of polarized fibers distributed throughout the bone and the presence of a thin layer of perichondral bone. The composition of the spine (semidentine odontodes, dermal bone, perichondral bone) is identical to that of the Romundina dermal plates. These results illustrate the similarities and differences between the dermal tissues in Romundina and indicate that the spine grew differently from the dentinous fin spines from extant and fossil chondrichthyans. The morphology and histology of Romundina is most similar to the fin spine of the probable stem osteichthyan Lophosteus, with a well-developed inner cellular bony base and star-shaped odontodes on the surface. Results from these studies will undoubtedly have impact on our understanding of fossil fin spine histology and evolution, contributing to the on-going revision of early gnathostome phylogeny.

Antibiotic resistance evolves rapidly in response to drug selection, but it can also persist at appreciable levels even after the removal of the antibiotic. This suggests that many resistant strains can both be resistant and have high fitness in the absence of antibiotics. To explore the conditions under which high-fitness, resistant strains evolve and the genetic changes responsible, we used a combination of experimental evolution and whole-genome sequencing to track the acquisition of ciprofloxacin resistance in the opportunistic pathogen Pseudomonas aeruginosa under conditions of constant and fluctuating antibiotic delivery patterns. We found that high-fitness, resistant strains evolved readily under fluctuating but not constant antibiotic conditions and that their evolution was underlain by a trade-off between resistance and fitness. Whole-genome sequencing of evolved isolates revealed that resistance was gained through mutations in known resistance genes and that second-site mutations generally compensated for costs associated with resistance in the fluctuating treatment, leading to the evolution of cost-free resistance. Our results suggest that current therapies involving intermittent administration of antibiotics are contributing to the maintenance of antibiotic resistance at high levels in clinical settings. IMPORTANCE Antibiotic resistance is a global problem that greatly impacts human health. How resistance persists, even in the absence of antibiotic treatment, is thus a public health problem of utmost importance. In this study, we explored the antibiotic treatment conditions under which cost-free resistance arises, using experimental evolution of the bacterium Pseudomonas aeruginosa and the quinolone antibiotic ciprofloxacin. We found that intermittent antibiotic treatment led to the evolution of cost-free resistance and demonstrate that compensatory evolution is the mechanism responsible for cost-free resistance. Our results suggest that discontinuous administration of antibiotic may be contributing to the high levels of antibiotic resistance currently found worldwide.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is the rate-limiting enzyme of the mevalonic acid pathway and is required for cholesterol biosynthesis and the synthesis of Coenzyme Q10 (CoQ10). Statins inhibit HMGCR, thus inhibiting the downstream products of this pathway including the biosynthesis of decaprenyl-pyrophosphate that is critical for the synthesis of Coenzyme Q10 (CoQ10). We show that zebrafish (Danio rerio) larvae treated in tank water with Atorvastatin (ATV; Lipitor) exhibited movement alterations and reduced whole body tissue metabolism. The ATV-inhibition of HMGCR function altered transcript abundance of muscle atrophy markers (atrogen-1, murf) and the mitochondrial biogenesis marker (pgc-1α). Furthermore, ATV-induced reduction in larval response to tactile stimuli was reversed with treatment of CoQ10. Together, the implication of our results contributes to the understanding of the mechanisms of action of the statin-induced damage in this model fish species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Historically, a morphological species concept has applied shape subjectively in the delimitation of diatom species. This has led to confusion between taxa within the benthic diatom genus Neidium. Samples from Ontario, Quebec, Nova Scotia, Newfoundland (Canada) and New York (USA) were examined for Neidium taxa under LM and SEM. Fourier shape analysis showed that shape as a taxonomic character was not able to discern all species. Isolated individuals from the samples were amplified and sequenced for three chloroplast molecular markers (rbcL, psbC, and psbA) and one nuclear ribosomal molecular marker (18S). Phylogenetic reconstructions were completed with the concatenated chloroplast and 18S dataset using Maximum Likelihood and Bayesian analyses. The concatenated chloroplast dataset exhibited a species-level resolution phylogeny of Neidium taxa. The 18S dataset had a lower level of sequence divergence and was unable to differentiate between Neidium taxa. We present emended species descriptions and sequence data for four previously described species: Neidium sacoense, N. longiceps, N. fossum, and N. affine. We describe three novel species (Neidium lowei, N. promontorium, and N. potapovae) and identify two forms with unique molecular signatures. The distinguishing features of N. lowei are its size, valve shape, and longitudinal canal structure. Distinguishing features of N. promontorium are its valve shape, longitudinal canal and apex formation, and surface depression along the axial area. Neidium potapovae is distinguished by its size, formation of valve and apices and single longitudinal canal. This paper demonstrates how future phylogenetic treatments using single cell multigene sequencing can help resolve taxonomic confusion within diatoms., (© 2017 Phycological Society of America.)

Knowledge of the number and nature of genetic changes responsible for adaptation is essential for understanding and predicting evolutionary trajectories. Here, we study the genomic basis of compensatory adaptation to the fitness cost of fungicide resistance in experimentally evolved strains of the filamentous fungus Aspergillus nidulans The original selection experiment tracked the fitness recovery of lines founded by an ancestral strain that was resistant to fludioxonil, but paid a fitness cost in the absence of the fungicide. We obtained whole-genome sequence data for the ancestral A. nidulans strain and eight experimentally evolved strains. We find that fludioxonil resistance in the ancestor was likely conferred by a mutation in histidine kinase nikA , part of the two-component signal transduction system of the high-osmolarity glycerol (HOG) stress response pathway. To compensate for the pleiotropic negative effects of the resistance mutation, the subsequent fitness gains observed in the evolved lines were likely caused by secondary modification of HOG pathway activity. Candidate genes for the compensatory fitness increases were significantly overrepresented by stress response functions, and some were specifically associated with the HOG pathway itself. Parallel evolution at the gene level was rare among evolved lines. There was a positive relationship between the predicted number of adaptive steps, estimated from fitness data, and the number of genomic mutations, determined by whole-genome sequencing. However, the number of genomic mutations was, on average, 8.45 times greater than the number of adaptive steps inferred from fitness data. This research expands our understanding of the genetic basis of adaptation in multicellular eukaryotes and lays out a framework for future work on the genomics of compensatory adaptation in A. nidulans ., (Copyright © 2017 Dettman et al.)

Based on an analysis of sediment cores from Baptiste Lake (Alberta, Canada), we quantified century-scale trends in cyanobacteria and cyanotoxins, and identified possible drivers of toxigenic cyanobacteria. We measured concentrations of microcystins and pigments preserved in the sediment as proxies of toxigenic cyanobacteria and phytoplankton communities, respectively, while fossil diatom assemblages were used to infer past nutrient concentrations. Microcystins were detected in older sediments (ca. 1800s), pre-dating any significant alteration to the watershed. This demonstrates that toxigenic cyanobacteria may not be a recent phenomenon in eutrophic ecosystems. The dominant variants of microcystin throughout the sediment core were microcystin-LA and microcystin-LR. Other congeners including -LY, -7dmLR, -WR, -LF, -YR, and -LW (-RR was not detected) were mainly found in the upper layers of sediment (post 1980s). Starting in the 1990s, concentrations of microcystins both in the water column and in the sediment record increased in parallel. Total sediment microcystins were strongly correlated with historical nitrogen and phosphorus concentrations inferred from diatom assemblages (r=0.80-0.81, p<0.001, n=22); both nutrients increased over the past two decades coincident with the intensification of agriculture. Microcystins also tracked the rise in cyanobacterial pigments present throughout the core. In contrast, we found no relationship between climate-related variables and sediment microcystin concentrations, although such relationships were detected over the monitoring record with respect to water column concentrations. Overall, the rise in sediment microcystins was much greater than the rise in sediment cyanobacteria and diatom inferred nutrient concentrations. Furthermore, we demonstrate that the reconstruction of the microcystin sediment record can provide important insight for the development of realistic lake management goals. Applying this analytical approach to different lakes and regions of the world, where both natural and anthropogenic gradients vary, has the potential to markedly improve our understanding of long-term drivers of cyanotoxin production., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Radial glial cells (RGCs) in teleost brain are progenitor cells that express aromatase B and produce estrogens. Controversial data suggest that estrogens are critical for brain repair and neurogenesis in teleosts. Using a goldfish model for neurotoxin-induced Parkinson-like syndrome, we investigated the possible roles of RGCs, especially estrogen synthetic function, in the processes underlying dopamine neuron regeneration. The data indicate that dopamine neuron degeneration and aromatase activity inhibition could be respectively achieved in vivo with treatments with the neurotoxin 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) and fadrozole in female goldfish. The expression of genes in the telencephalon and hypothalamus related to RGC functions including gfap, gdnf and bdnf as well as genes related to mature dopamine neuron functions including th, slc6a3 and pitx3 are under modulation of estrogens. Together these results revealed that the activation of radial glial cells and dopamine neuron recovery after MPTP insult is aromatase-dependent. Findings in this study provide support for the hypothesis that endogenous estrogens are neuroprotective in goldfish. Future studies focus on the molecular pathways for enhancing protective functions of estrogens and understanding global effects of estrogens in the central nervous system., (Copyright © 2016 Elsevier Inc. All rights reserved.)

As human populations continue to expand, increases in coastal development have led to the alteration of much of the world's mangrove habitat, creating problems for the multitude of species that inhabit these unique ecosystems. Habitat alteration often leads to changes in habitat complexity and predation risk, which may serve as additional stressors for those species that rely on mangroves for protection from predators. However, few studies have been conducted to date to assess the effects of these specific stressors on glucocorticoid (GC) stress hormone levels in wild fish populations. Using the checkered puffer as a model, our study sought to examine the effects of physical habitat complexity and predator environment on baseline and acute stress-induced GC levels. This was accomplished by examining changes in glucose and cortisol concentrations of fish placed in artificial environments for short periods (several hours) where substrate type and the presence of mangrove roots and predator cues were manipulated. Our results suggest that baseline and stress-induced GC levels are not significantly influenced by changes in physical habitat complexity or the predator environment using the experimental protocol that we applied. Although more research is required, the current study suggests that checkered puffers may be capable of withstanding changes in habitat complexity and increases in predation risk without experiencing adverse GC-mediated physiological effects, possibly as a result of the puffers' unique morphological and chemical defenses that help them to avoid predation in the wild., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Lophosteus superbus is one of only a handful of probable stem-group osteichthyans known from the fossil record. First collected and described in the late 19th century from the upper Silurian Saaremaa Cliff locality in Estonia, it is known from a wealth of disarticulated scales, fin spines, and bone fragments. In this study we provide the first description of the morphology and paleohistology of a fin spine and scale from Lophosteus using virtual thin sections and 3D reconstructions that were segmented using phase-contrast synchrotron X-ray microtomography. These data reveal that both structures have fully or partially buried odontodes, which retain fine morphological details in older generations, including sharp nodes and serrated ridgelets. The vascular architecture of the fin spine tip, which is composed of several layers of longitudinally directed bone vascular canals, is much more complex compared to the bulbous horizontal canals within the scale, but they both have distinctive networks of ascending canals within each individual odontode. Other histological characteristics that can be observed from the data are cell spaces and Sharpey's fibers that, when combined with the vascularization, could help to provide insights into the growth of the structure. The 3D data of the scales from Lophosteus superbus is similar to comparable data from other fossil osteichthyans, and the morphology of the reconstructed buried odontodes from this species is identical to scale material of Lophosteus ohesaarensis , casting doubt on the validity of that species. The 3D data presented in this paper is the first for fossil fin spines and so comparable data is not yet available. However, the overall morphology and histology seems to be similar to the structure of placoderm dermal plates. The 3D datasets presented here provide show that microtomography is a powerful tool for investigating the three-dimensional microstructure of fossils, which is difficult to study using traditional histological methods. These results also increase the utility of fin spines and scales suggest that these data are a potentially rich source of morphological data that could be used for studying questions relating to early vertebrate growth and evolution., Competing Interests: The authors declare there are no competing interests.

Polychlorinated biphenyls (PCBs) are recognized physiological stressors to fish which over time may impair individual performance and perhaps fitness by inducing changes that could have population-level consequences. PCB-126 (3,3',4,4',5-pentachlorobiphenyl) accumulates in lipids and can subsequently be released into the bloodstream during periods of high activity that involve the mobilization of stored fuels to meet with increasing energy demands. The goal of this study was to determine if a sublethal exposure to PCB-126 altered the content of tissue energy supplies (carbohydrates, proteins, amino acids, triglycerides) and impaired swimming performance as well as oxygen consumption in rainbow trout (Oncorhynchus mykiss). Trout were injected intraperitoneally with a single Low (100μgkg(-1)) or High (400μgkg(-1)) dose of PCB-126 then swimming performance and metabolic rates from 1 to 9days post-injection were compared to Control (non-dosed) fish. Liver ethoxyresorufin-O-deethylase (EROD) activity was assessed as an indication of PCB-126 intoxication while plasma and white muscle tissue metabolites were analyzed as an index of physiological disturbance. Swimming performance, assessed using two successive modified critical swimming speed (Ucrit) tests, was highest for fish in the High PCB-126 treatment; however, their initial condition factor (K) was also higher, largely due to their greater body mass. Trout in the High and Low PCB-126 treatments exhibited impaired recovery following intense exercise as they swam comparatively poorly when provided a second challenge. PCB-exposed fish exhibited reduced spleen somatic indices as well as muscle glucose and glycogen contents; whereas plasma cortisol and glucose levels were elevated, indicating higher metabolic costs during recovery and muscle restoration. Overall, this research provides insights into the sublethal effects of a toxic organic compound on swimming performance in trout., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The fin-to-limb transition is an important evolutionary step in the colonization of land and diversification of all terrestrial vertebrates. We previously identified a gene family in zebrafish, termed actinodin, which codes for structural proteins crucial for the formation of actinotrichia, rigid fibrils of the teleost fin. Interestingly, this gene family is absent from all tetrapod genomes examined to date, suggesting that it was lost during limb evolution. To shed light on the disappearance of this gene family, and the consequences on fin-to-limb transition, we characterized actinodin regulatory elements. Using fluorescent reporters in transgenic zebrafish, we identified tissue-specific cis-acting regulatory elements responsible for actinodin1 (and1) expression in the ectodermal and mesenchymal cell populations of the fins, respectively. Mutagenesis of potential transcription factor binding sites led to the identification of one binding site crucial for and1 expression in ectodermal cells. We show that these regulatory elements are partially functional in mouse limb buds in a tissue-specific manner. Indeed, the zebrafish regulatory elements target expression to the dorsal and ventral ectoderm of mouse limb buds. Absence of expression in the apical ectodermal ridge is observed in both mouse and zebrafish. However, cells of the mouse limb bud mesoderm do not express the transgene, in contrast to zebrafish. Altogether these results hint for a change in regulation of and1 during evolution that led to the downregulation and eventual loss of this gene from tetrapod genomes., (Copyright © 2016. Published by Elsevier Inc.)

The commonly used lipid-lowering pharmaceuticals gemfibrozil (GEM) and atorvastatin (ATV) are detected in the aquatic environment; however, their potential effects on non-target fish species are yet to be fully understood. This study examined the effects of GEM and/or ATV on female and male adult zebrafish after a 30d dietary exposure. The exposure led to changes in several biochemical parameters, including reduction in cholesterol, triglycerides, cortisol, testosterone, and estradiol. Changes in cholesterol and triglycerides were also associated with changes in transcript levels of key genes involved with cholesterol and lipid regulation, including SREBP2, HMGCR1, PPARα, and SREBP1. We also noted higher CYP3A65 and atrogin1 mRNA levels in drug-treated male fish. Sex differences were apparent in some of the examined parameters at both biochemical and molecular levels. This study supports these drugs affecting cholesterol metabolism and steroid production in adult zebrafish. We conclude that the reduction in cortisol may impair the ability of these fish to mount a suitable stress response, whereas the reduction of sex steroids may negatively affect reproduction., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Propranolol (PROP) is a β-blocker prescribed mainly to treat human cardiovascular diseases and as a result of its wide usage and persistence, it is reported in aquatic environments. This study examined whether PROP alters developmental patterns and catecholamine (CA)-regulated processes in the zebrafish (Danio rerio) and if exposure during early life alters the stress response and behaviors of adults. The calculated 48h larva LC50 was 21.6mg/L, well above reported environmental levels (0.01-0.59μg/L). Stressed and PROP-exposed adult zebrafish had reduced testosterone and estradiol levels and exhibited behaviors indicating less anxiety than control fish. Furthermore, adults previously PROP-exposed as embryos/larvae had decreased growth in terms of body length and mass. Finally, these adults showed increased cholesterol and a dose-dependent decrease in testosterone levels compared with unexposed zebrafish. Thus PROP-exposure of zebrafish embryos/larvae alters developmental patterns and CA-regulated processes that may affect normal behaviors and responses to stressors, and at least some of these changes persist in the adult zebrafish., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Nestin is an intermediate filament protein involved in neurogenesis in fish, mice, and humans. In this study we used rapid amplification of cDNA ends PCR to isolate goldfish nestin (nes). PCR analysis and sequencing revealed three different nes transcripts of 4003, 2446, and 2126 nucleotides, which are predicted to generate proteins of 860, 274, and 344 amino acids in length. Sequence analysis suggests that these nes transcripts are likely a result of alternative splicing. We next applied a multiple-antigenic peptide strategy to generate a goldfish-specific nestin antibody. Western blotting with this antibody together with mass spectrometry verified the presence of major nestin protein isoforms with differing molecular weights (~70, 40 and 30kDa). We further examined expression patterns of these nestin protein isoforms in different parts of the goldfish brain and pituitary and found the telencephalon to express all three isoforms at a distinct level and abundance. We report that multiple nestin isoforms are present indicating another level of complexity for the regulation of intermediate filaments in comparison to mammals. Studying the differential roles and regulation of these nestins could lead to a better understanding of cellular remodeling during neurogenesis and the unparalleled regenerative abilities after damage in the teleost CNS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

6-Formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are ligands of the aryl hydrocarbon receptor (AHR) and bind to the AHR with high affinity. Until recently, TCDD was considered to be the most potent AHR agonist, but several recent studies indicate that FICZ binds with greater affinity to the AHR than TCDD. To advance our understanding of the similarities and differences of the effects of FICZ and TCDD exposure in chicken embryo hepatocyte (CEH) cultures, we compared relative expression changes of 27 dioxin-responsive genes by the use of a chicken PCR array, porphyrin accumulation and ethoxyresorufin-O-deethylase (EROD) activity at different time points. In addition, an egg injection study was performed to assess the effects of FICZ on the developing chicken embryo. The results of the current study showed: (1) mean EROD-derived relative potency values for FICZ compared to TCDD changed as a function of time (i.e. 9, 0.004, 0.0008 and 0.00008 at 3, 8, 24, and 48h, respectively) in CEH cultures; (2) FICZ exposure did not result in porphyrin accumulation in CEH cultures; (3) concordance between gene expression profiles for FICZ and TCDD was time- and concentration-dependent, and (4) no mortality or morphological abnormalities were observed in chicken embryos injected with 0.87ng FICZ/g egg into the air cell. The results presented herein suggest that while FICZ and TCDD share similar molecular targets, transient versus sustained AHR activation by FICZ and TCDD result in differential transcriptomic responses. Moreover, rapid metabolism of FICZ in hepatocytes resulted in a significant decrease in the induction of EROD activity., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Radial glial cells (RGCs) are stem-like cells found in the developing and adult central nervous system. They function as both a scaffold to guide neuron migration and as progenitor cells that support neurogenesis. Our previous study revealed a close anatomical relationship between dopamine neurons and RGCs in the telencephalon of female goldfish. In this study, label-free proteomics was used to identify the proteins in a primary RGC culture and to determine the proteome response to the selective dopamine D1 receptor agonist SKF 38393 (10μM), in order to better understand dopaminergic regulation of RGCs. A total of 689 unique proteins were identified in the RGCs and these were classified into biological and pathological pathways. Proteins such as nucleolin (6.9-fold) and ependymin related protein 1 (4.9-fold) were increased in abundance while proteins triosephosphate isomerase (10-fold) and phosphoglycerate dehydrogenase (5-fold) were decreased in abundance. Pathway analysis revealed that proteins that consistently changed in abundance across biological replicates were related to small molecules such as ATP, lipids and steroids, hormones, glucose, cyclic AMP and Ca(2+). Sub-network enrichment analysis suggested that estrogen receptor signaling, among other transcription factors, is regulated by D1 receptor activation. This suggests that these signaling pathways are correlated to dopaminergic regulation of radial glial cell functions. Most proteins down-regulated by SKF 38393 were involved in cell cycle/proliferation, growth, death, and survival, which suggests that dopamine inhibits the progenitor-related processes of radial glial cells. Examples of differently expressed proteins including triosephosphate isomerase, nucleolin, phosphoglycerate dehydrogenase and capping protein (actin filament) muscle Z-line beta were validated by qPCR and western blot, which were consistent with MS/MS data in the direction of change. This is the first study to characterize the RGC proteome on a large scale in a vertebrate species. These data provide novel insight into glial protein networks that are associated with neuroendocrine function and neurogenesis in the teleost brain., Biological Significance: While the role of radial glial cells in organizing brain structure and neurogenesis has been well studied, protein profiling experiments in this unique cell type has not been conducted. This study is the first to profile the proteome of goldfish radial glial cells in culture and to study the regulation of progenitor functions of radial glial cells by the neurotransmitter dopamine. This study provides the foundation for molecular network analysis in fish radial glial cells, and identifies cellular processes and signaling pathways in these cells with roles in neurogenesis and neuroendocrine function. Lastly, this study begins to characterize signatures and biomarkers for specific neuroendocrine and neurogenesis disruptors., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Methylmercury (MeHg) is a known neurotoxicant affecting the central nervous system but effects on dopaminergic (DA) neurons are not well understood. Wild-type zebrafish (Danio rerio) and two transgenic lines: Tg(dat:eGFP) expressing enhanced green fluorescent protein (eGFP) in DA neuron clusters and Tg(dat:tom20 MLS-mCherry) expressing red fluorescence (mCherry) targeted to mitochondria of DA neurons were used to evaluate the effects of micromolar MeHg exposure on DA neuron and whole animal motor function during early development. Three-day-old larvae were exposed to micromolar concentrations of MeHg (0.03, 0.06, and 0.3μM) in system water. Exposure to 0.3μM MeHg caused mortality and significant morphological abnormalities including edema, curvature of the spine, and hemorrhages in zebrafish larvae after a 48h exposure period. At 0.06μM MeHg, the appearance of morphological abnormalities was delayed for 72h and far less severe, whereas 0.03μM MeHg did not cause any morphological defects or mortalities. A delayed but significant reduction in locomotor ability and mCherry fluorescence in specific brain regions in the 0.06μM MeHg exposed larvae suggests that DA neuron function rather than neuron numbers was compromised. Double immunolabeling with tyrosine hydroxylase and pan neural staining showed no effect of MeHg exposure. We have established Tg(dat:tom20 MLS-mCherry) zebrafish larvae as a model which can be used to assess MeHg neurotoxicity and that exposure to low dose MeHg (0.06μM) during development may predispose DA neurons to impairment caused by changes in mitochondrial dynamics., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Silver nanoparticles (Ag-NPs) are highly relevant for human and environmental exposure due to their widespread use in consumer and medical products and various applications. Thus, there is a need for evaluating potential toxicity of these NPs. The objective of this study was to investigate the toxic effects of the OECD (Organization for Economic Co-operation and Development) representative Ag-NPs, NM300K, in mouse macrophage J774A.1 and human colonic epithelial HT29 cells, using multiple endpoint assays. Exposure of test cells to different concentrations (1-250 μg/mL; total silver content) of NM300K for 24h showed a dose-dependent decrease in cell viability. At high doses, NM300K altered cell shape and induced the formation of vacuolar structures, as examined by confocal and electron microscopy. Moreover, NM300K induced inflammation as evidenced by the elevated levels of pro-inflammatory cytokines. Finally, high doses of NM300K led to increased production of reactive oxygen species and induction of oxidative stress, leading to oxidative DNA damage and apoptosis in test cells. At equivalent silver concentrations, NM300K were less cytotoxic than AgNO3. However, the similar patterns in the effects of NM300K and AgNO3 throughout the assessed toxicological endpoints suggest that Ag(+) released from these NPs by dissolution could be a primary contributor to toxicity. This study is among the first to characterize the potential toxicity of OECD representative AgNPs in vitro, and provides additional insight into the biological mechanisms associated with Ag-NP toxicity., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Influenza viruses evolve at such a high rate that vaccine recommendations need to be changed, but not quite on a regular basis. This observation suggests that the rate of evolution of these viruses is not constant through time, which begs the question as to when such rate changes occur, if they do so independently of the host in which they circulate and (or) independently of their subtype. To address these outstanding questions, we introduce a novel heuristics, Mclust*, that is based on a two-tier clustering approach in a phylogenetic context to estimate (i) absolute rates of evolution and (ii) when rate change occurs. We employ the novel approach to compare the two influenza surface proteins, hemagglutinin and neuraminidase, that circulated in avian, human, and swine hosts between 1960 and 2014 in two subtypes: H3N2 and H1N1. We show that the algorithm performs well in most conditions, accounting for phylogenetic uncertainty by means of bootstrapping and scales up to analyze very large data sets. Our results show that our approach is robust to the time-dependent artifact of rate estimation, and confirm pervasive punctuated evolution across hosts and subtypes. As such, the novel approach can potentially detect when vaccine composition needs to be updated.

Due to their bioaccumulative properties, hydroxylated and methoxylated polybrominated diphenyl ethers (HO-/MeO-PBDEs) may pose ecological risks to wild life, including birds. However, their toxicity potencies in avian species are largely unknown. In the present study, an avian AHR1 luciferase reporter gene (LRG) assay with luciferase probes from chicken, pheasant and quail was used to test activations of avian aryl hydrocarbon receptor (AHR)-mediated pathways by 19 HO- or MeO-PBDEs in different avian species. Species-specific relative potencies (RePs) of HO-/MeO-PBDEs to tetrachlorodibenzo-p-dioxin (TCDD) and relative sensitivities of various species to each chemical were estimated. The results indicated that the ReP of the most potent HO-/MeO-PBDEs, 5-Cl-6-HO-BDE-47, was 7.8×10(-4) for chicken, 1.1×10(-2) for pheasant, and 1.7×10(-1) for quail comparing to TCDD. In addition, it was found that avian species with the greatest sensitivity to TCDD did not always have the greatest sensitivity to HO-/MeO-PBDEs and vice versa. This study contributed to filling in the knowledge gap regarding the dioxin-like activity of HO-/MeO-PBDEs in birds, and provided beneficial information for the prioritization of HO-/MeO-PBDEs for further research., Capsule Abstract: HO-/MeO-PBDEs activate avian AHR-mediated pathways in a congener- and species- specific manner. 5-Cl-6-HO-BDE-47 was the most potent among the nineteen HO-/MeO-PBDEs tested., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We report on two highly brominated polyphenyl ether flame retardants, tetradecabromo-1,4- diphenoxybenzene (TeDB-DiPhOBz) and 2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether (BDE-209), that formed photolytic degradation products in tetrahydrofuran (THF)/hexane solvent after 21 days of natural sunlight irradiation (SI). These degradation products of SI-TeDB-DiPhOBz and SI-BDE-209 included the numerous polybrominated homologue groups of polybenzofurans and dibenzofurans, respectively. Formation of similar polybenzofuran and dibenzofuran products was also observed following a 3 month exposure of the solid powder forms of TeDB-DiPhOBz and BDE-209 to natural SI. These resulting degradation product mixtures were administered to chicken embryonic hepatocytes (CEH) to determine effects on mRNA expression levels of 27 dioxin-responsive genes. For the solvent-based SI study, equivalent concentrations of 1 or 25 μM of SI-TeDB-DiPhOBz or 1 or 10 μM of SI-BDE-209 resulted in gene expression profiles that were similar to those of the most potent dioxin-like compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin. In addition, a concentration-dependent induction of CYP1A4 and CYP1A5 mRNA was observed following exposure to SI-TeDB-DiPhOBz and SI-BDE-209. Based on ECthreshold values for CYP1A4/5 mRNA expression, relative potency (ReP) values were 1 × 10(-6) and 1 × 10(-5) for SI-TeDB-DiPhOBz and SI-BDE-209, respectively. The SI TeDB-DiPhOBz and BDE-209 powder degradation product mixture also significantly induced CYP1A4 mRNA levels in CEH. Our findings clearly show that the environmental stability of TeDB-DiPhOBz and BDE-209, and possibly other highly brominated polyphenyl ethers, is of great concern from a dioxin-like degradation products and toxicity perspective.

Alterations to natural habitats are becoming more common due to changes in anthropogenic land use. As such, there is increasing interest in determining how wild animals adapt and respond to environmental stressors. The glucocorticoid (GC) stress response enables animals to react appropriately to environmental challenges but can be affected by many factors, two of which are habitat quality and time of year (i.e., season). This study tested whether baseline and maximum (stress-induced) whole-body cortisol concentrations varied in relation to habitat quality and season using wild central mudminnows (Umbra limi) collected from two connected streams differing in habitat quality in each of four seasons. Overall, baseline and maximum cortisol levels did not differ significantly between the two systems but there was evidence of a seasonal effect. Baseline cortisol levels in the fall and summer were significantly (P<0.01) lower than those in winter and spring and maximum cortisol levels in the summer were significantly lower (P<0.01) than those in the spring. Inconsistent with the prevailing paradigm, our results indicate that habitat quality does not always influence baseline GCs or the stress response. In contrast, baseline and maximum GCs in this species do vary seasonally. As such, seasonality should be considered in the interpretation of stress response data especially when using small-bodied stream fish as biological indicators., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aromatase cytochrome P450arom (cyp19) is the only enzyme that has the ability to convert androgens into estrogens. Estrogens, which are produced locally in the vertebrate brain play many fundamental roles in neuroendocrine functions, reproductive functions, socio-sexual behaviors, and neurogenesis. Radial glial cells (RGCs) are neuronal progenitor cells that are abundant in fish brains and are the exclusive site of aromatase B expression and neuroestrogen synthesis. Using a novel in vitro RGC culture preparation we studied the regulation of aromatase B by 17β-estradiol (E2) and dopamine (DA). We have established that activation of the dopamine D1 receptor (D1R) by SKF 38393 up-regulates aromatase B gene expression most likely through the phosphorylation of cyclic AMP response element binding protein (CREB). This up-regulation can be enhanced by low concentration of E2 (100 nM) through increasing the expression of D1R and the level of p-CREB protein. However, a high concentration of E2 (1 μM) and D1R agonist together failed to up-regulate aromatase B, potentially due to attenuation of esr2b expression and p-CREB levels. Furthermore, we found the up-regulation of aromatase B by E2 and DA both requires the involvement of esr1 and esr2a. The combined effect of E2 and DA agonist indicates that aromatase B in the adult teleost brain is under tight control by both steroids and neurotransmitters to precisely regulate neuroestrogen levels.

Background: Natural genetic variation ultimately arises from the process of mutation. Knowledge of how the raw material for evolution is produced is necessary for a full understanding of several fundamental evolutionary concepts. We performed a mutation accumulation experiment with wild-type and mismatch-repair deficient, mutator lines of the pathogenic bacterium Pseudomonas aeruginosa, and used whole-genome sequencing to reveal the genome-wide rate, spectrum, distribution, leading/lagging bias, and context-dependency of spontaneous mutations., Results: Wild-type base-pair mutation and indel rates were ~10(-10) and ~10(-11) per nucleotide per generation, respectively, and deficiencies in the mismatch-repair system caused rates to increase by over two orders of magnitude. A universal bias towards AT was observed in wild-type lines, but was reversed in mutator lines to a bias towards GC. Biases for which types of mutations occurred during replication of the leading versus lagging strand were detected reciprocally in both replichores. The distribution of mutations along the chromosome was non-random, with peaks near the terminus of replication and at positions intermediate to the replication origin and terminus. A similar distribution bias was observed along the chromosome in natural populations of P. aeruginosa. Site-specific mutation rates were higher when the focal nucleotide was immediately flanked by C:G pairings., Conclusions: Whole-genome sequencing of mutation accumulation lines allowed the comprehensive identification of mutations and revealed what factors of molecular and genomic architecture affect the mutational process. Our study provides a more complete view of how several mechanisms of mutation, mutation repair, and bias act simultaneously to produce the raw material for evolution.

The steroidogenic acute regulatory (StAR) protein is responsible for the movement of cholesterol across mitochondrial membranes and is therefore a key factor in regulating the timing and rate of steroidogenesis. In this study, we characterized the coding region of the star gene in the ranid Lithobates sylvaticus and studied star mRNA levels in steroidogenic tissues during development and under natural conditions. Our results support previous research showing that the StAR sequence is well conserved. We determined that star is expressed in both the interrenal and gonadal tissues of adults and in the tadpole gonad-mesonephros complex (GMC). The mRNA levels of star in the GMC were found to increase during tadpole development, reaching a maximum between Gosner stages (Gs) 32 and 38. We observed a significant drop in star mRNA levels at the end of prometamorphosis (Gs40-41), just before the start of the metamorphic climax. Significant differences in star levels between females and males, with males presenting higher levels than females, were detected at Gs36-38. To our knowledge, this is the first study that reports transitory star sex differences in tadpoles' developing GMC. Our results suggest an involvement of StAR in anuran late male GMC formation and development that requires further investigation., (© 2016 S. Karger AG, Basel.)