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Yi Zhen Tan,1 Lucy R Thomsen,1 Nensi Shrestha,1 Adalberto Camisasca,2 Silvia Giordani,2 Rhonda Rosengren1 1Department of Pharmacology and Toxicology, University of Otago, Dunedin, 9016, New Zealand; 2School of Chemical Sciences, Dublin City University, Glasnevin, Dublin, D09 NA55, IrelandCorrespondence: Rhonda Rosengren, Department of Pharmacology and Toxicology, 18 Frederick Street, Dunedin, 9016, New Zealand, Tel +64 3 479 9141, Fax +64 3 479 9140, Email rhonda.rosengren@otago.ac.nzBackground: A nanoscale drug carrier could have a variety of therapeutic and diagnostic uses provided that the carrier is biocompatible in vivo. Carbon nano-onions (CNOs) have shown promising results as a nanocarrier for drug delivery. However, the systemic effect of CNOs in rodents is unknown. Therefore, we investigated the toxicity of CNOs following intravenous administration in female BALB/c mice.Results: Single or repeated administration of oxi-CNOs (125, 250 or 500 μg) did not affect mouse behavior or organ weight and there was also no evidence of hepatotoxicity or nephrotoxicity. Histological examination of organ slices revealed a significant dose-dependent accumulation of CNO aggregates in the spleen, liver and lungs (p< 0.05, ANOVA), with a trace amount of aggregates appearing in the kidneys. However, CNO aggregates in the liver did not affect CYP450 enzymes, as total hepatic CYP450 as well as CYP3A catalytic activity, as meased by erythromycin N-demethylation, and protein levels showed no significant changes between the treatment groups compared to vehicle control. CNOs also failed to act as competitive inhibitors of CYP3A in vitro in both mouse and human liver microsomes. Furthermore, CNOs did not cause oxidative stress, as indicated by the unchanged malondialdehyde levels and superoxide dismutase activity in liver microsomes and organ homogenates.Conclusion: This study provides the first evidence that short-term intravenous administration of oxi-CNOs is non-toxic to female mice and thus could be a promising novel and safe drug carrier.Keywords: carbon nano-onions, drug carriers, intravenous administration, toxicity, accumulation, drug metabolism, oxidative stress

Background & Purpose: The constant emergence and broad toxicological effects of synthetic cannabinoids create a discernible public health threat. The synthetic cannabinoid AMB-FUBINACA (AMB-FUB) is a potent agonist at the CB 1 receptor and has been associated with numerous fatalities. Synthetic cannabinoids are commonly abused alongside other drugs and medications, including a "party pill" drug, para-fluorophenylpiperazine (pFPP), and the antipsychotic risperidone. This research aimed to investigate the mechanisms underpinning AMB-FUB toxicity and the impact of clinically relevant co-exposures in vivo., Experimental Approach: Male and female C57Bl/6 mice received a single dose of AMB-FUB (3 or 6 mg kg -1 ), pFPP (10 or 20 mg kg -1 ) or vehicle intraperitoneally. Mice were co-exposed to AMB-FUB (3 mg kg -1 ) and pFPP (10 mg kg -1 ) or risperidone (0.5 mg kg -1 ) to investigate these drug combinations. To study receptor-dependency and potential rescue of AMB-FUB toxicity, rimonabant (3 mg kg -1 ) was administered both pre- and post-AMB-FUB. Adverse effects caused by drug administration, including hypothermia and convulsions, were recorded., Key Results: AMB-FUB induced CB 1 -dependent hypothermia and convulsions in mice. The combination of AMB-FUB and pFPP significantly potentiated hypothermia, as did risperidone pre-treatment. Interestingly, risperidone provided significant protection from AMB-FUB-induced convulsions in female mice. Pre- and post-treatment with rimonabant was able to significantly attenuate both hypothermia and convulsions in mice administered AMB-FUB., Conclusion & Implications: Factors such as dose, CB 1 signalling, and substance co-exposure significantly contribute to the toxicity of AMB-FUBINACA. Mechanistic understanding of synthetic cannabinoid toxicity and fatality can help inform overdose treatment strategies and identify vulnerable populations of synthetic cannabinoid users., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA), which has been associated with substantial abuse and health harm since 2016 in many countries including New Zealand. A characteristic of AMB-FUBINACA use in New Zealand has included the observation that forensic samples (from autopsies) and drugs seized by police have often been found to contain para-fluorophenylpiperazine (pFPP), a relatively little-characterised piperazine analogue that has been suggested to act through 5HT1a serotonin receptors. In the current study, we aimed to characterise the interactions of these two agents in rat physiological endpoints using plethysmography and telemetry, and to examine whether pFPP altered the subjective effects of AMB-FUBINACA in mice trained to differentiate a cannabinoid (THC) from vehicle. Though pFPP did not alter the ability of AMB-FUBINACA to substitute for THC, it did appear to abate some of the physiological effects of AMB-FUBINACA in rats by delaying the onset of AMB-FUBINACA-mediated hypothermia and shortening duration of bradycardia. In HEK cells stably expressing the CB1 cannabinoid receptor, 5HT1a, or both CB1 and 5HT1a, cAMP signalling was recorded using a BRET biosensor (CAMYEL) to assess possible direct receptor interactions. Although low potency pFPP agonism at 5HT1a was confirmed, little evidence for signalling interactions was detected in these assays: additive or synergistic effects on potency or efficacy were not detected between pFPP and AMB-FUBINACA-mediated cAMP inhibition. Experiments utilising higher potency, classical 5HT1a ligands (agonist 8OH-DPAT and antagonist WAY100635) also failed to reveal evidence for mutual CB1/5HT1a interactions or cross-antagonism. Finally, the ability of pFPP to alter the metabolism of AMB-FUBINACA in rat and human liver microsomes into its primary carboxylic acid metabolite via carboxylesterase-1 was assessed by HPLC; no inhibition was detected. Overall, the effects we have observed do not suggest that increased harm/toxicity would result from the combination of pFPP and AMB-FUBINACA., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S2 and for positively charged residues in S1. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S2 pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The archetypical venomous lizard species are the helodermatids, the gila monsters (Heloderma suspectum) and the beaded lizards (Heloderma horridum). In the present study, the gila monster venom proteome was characterized using 2D-gel electrophoresis and tandem mass spectrometry-based de novo peptide sequencing followed by protein identification based on sequence homology. A total of 39 different proteins were identified out of the 58 selected spots that represent the major constituents of venom. Of these proteins, 19 have not previously been identified in helodermatid venom. The data showed that helodermatid venom is complex and that this complexity is caused by genetic isoforms and post-translational modifications including proteolytic processing. In addition, the venom proteome analysis revealed that the major constituents of the gila monster venom are kallikrein-like serine proteinases (EC 3.4.21) and phospholipase A2 (type III) enzymes (EC 3.1.1.4). A neuroendocrine convertase 1 homolog that most likely converts the proforms of the previously identified bioactive exendins into the mature and active forms was identified suggesting that these peptide toxins are secreted as proforms that are activated by proteolytic cleavage following secretion as opposed to being activated intracellularly. The presented global protein identification-analysis provides the first overview of the helodermatid venom composition., Biological Significance: The helodermatid lizards are the classical venomous lizards, and the pharmacological potential of the venom from these species has been known for years; best illustrated by the identification of exendin-4, which is now used in the treatment of type 2 diabetes. Despite the potential, no global analyses of the protein components in the venom exist. A hindrance is the lack of a genome sequence because it prevents protein identification using a conventional approach where MS data are searched against predicted protein sequences based on the genome sequence. However, in the recent years the development of software tools for de novo sequencing and homology searches have improved significantly facilitating the first global analysis of the major protein components of helodermatid venom presented in this study. We have used a 2D-gel approach and determined the protein components in the 58 major spots resulting in the identification of 39 unique proteins. Of these, 19 have not previously been identified in helodermatid venom. The analysis provides results with impact on our understanding of the function and evolution of venom proteins, and serves as a basis for further unraveling of the pharmaceutical potential of the venom components., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Human HtrA1 (high-temperature requirement protein A1) belongs to a conserved family of serine proteases involved in protein quality control and cell fate. The homotrimeric ubiquitously expressed protease has chymotrypsin-like specificity and primarily targets hydrophobic stretches in selected or misfolded substrate proteins. In addition, the enzyme is capable of exerting autolytic activity by removing the N-terminal insulin-like growth factor binding protein (IGFBP)/Kazal-like tandem motif without affecting the protease activity. In this study, we have addressed the mechanism governing the autolytic activity and find that it depends on the integrity of the disulfide bonds in the N-terminal IGFBP/Kazal-like domain. The specificity of the autolytic cleavage reveals a strong preference for cysteine in the P1 position of HtrA1, explaining the lack of autolysis prior to disulfide reduction. Significantly, the disulfides were reduced by thioredoxin, suggesting that autolysis of HtrA1 in vivo is linked to the endogenous redox balance and that the N-terminal domain acts as a redox-sensing switch.

Predation plays a major role in energy and nutrient flow in the biological food chain. Plant carnivory has attracted much interest since Darwin's time, but many fundamental properties of the carnivorous lifestyle are largely unexplored. In particular, the chain of events leading from prey perception to its digestive utilization remains to be elucidated. One of the first steps after the capture of animal prey, i.e. the enzymatic breakup of the insects' chitin-based shell, is reflected by considerable chitinase activity in the secreted digestive fluid in the carnivorous plant Venus flytrap. This study addresses the molecular nature, function, and regulation of the underlying enzyme, VF chitinase-I. Using mass spectrometry based de novo sequencing, VF chitinase-I was identified in the secreted fluid. As anticipated for one of the most prominent proteins in the flytrap's "green stomach" during prey digestion, transcription of VF chitinase-I is restricted to glands and enhanced by secretion-inducing stimuli. In their natural habitat, Venus flytrap is exposed to high temperatures. We expressed and purified recombinant VF chitinase-I and show that the enzyme exhibits the hallmark properties expected from an enzyme active in the hot and acidic digestive fluid of Dionaea muscipula. Structural modeling revealed a relative compact globular form of VF chitinase-I, which might contribute to its overall stability and resistance to proteolysis. These peculiar characteristics could well serve industrial purposes, especially because of the ability to hydrolyze both soluble and crystalline chitin substrates including the commercially important cleavage of α-chitin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We developed a novel delay discounting task to investigate outcome impulsivity in pigs. As impulsivity can affect aggression, and might also relate to proactive and reactive coping styles, eight proactive (HR) and eight reactive (LR) pigs identified in a manual restraint test ("Backtest", after Bolhuis et al., 2003) were weaned and mixed in four pens of four unfamiliar pigs, so that each pen had two HR and two LR pigs, and aggression was scored in the 9h after mixing. In the delay discounting task, each pig chose between two levers, one always delivering a small immediate reward, the other a large delayed reward with daily increasing delays, impulsive individuals being the ones discounting the value of the large reward quicker. Two novel strategies emerged: some pigs gradually switched their preference towards the small reward ('Switchers') as predicted, but others persistently preferred the large reward until they stopped making choices ('Omitters'). Outcome impulsivity itself was unrelated to these strategies, to urinary serotonin metabolite (5-HIAA) or dopamine metabolite (HVA) levels, aggression at weaning, or coping style. However, HVA was relatively higher in Omitters than Switchers, and positively correlated with behavioural measures of indecisiveness and frustration during choosing. The delay discounting task thus revealed two response strategies that seemed to be related to the activity of the dopamine system and might indicate a difference in execution, rather than outcome, impulsivity., (© 2013.)

We have previously shown that human extracellular superoxide dismutase (EC-SOD) exists as two variants with differences in their disulfide bridge patterns: one form is the active enzyme (aEC-SOD), and the other is inactive (iEC-SOD). The availability of both active and inactive folding variants significantly reduces the specific activity of EC-SOD in vivo. Both forms are produced during biosynthesis, but the underlying folding mechanisms remain unclear. To address this issue, we expressed EC-SOD in heterologous systems that do not endogenously express iEC-SOD. Rodents express only aEC-SOD because they lack Cys195 (human EC-SOD sequence numbering), which is essential for the formation of iEC-SOD. However, cultured hamster cells and transgenic mice expressing human EC-SOD were able to produce both human a- and iEC-SOD variants, which led us to hypothesize that the folding was sequence-dependent rather than a property of the expression system. To substantiate this hypothesis, we expressed murine EC-SOD in a human cell line, and as expected, only aEC-SOD was produced. Significantly, when Cys195 was introduced, both murine aEC-SOD and a novel murine iEC-SOD were generated, and the specific activity of the murine EC-SOD was significantly reduced by the mutation. Collectively, these data suggest that Cys195 actuates the formation of iEC-SOD, independent of the expression system or host. In addition, the dual-folding pathway most likely requires biosynthesis factors that are common to both humans and rodents.

The Venus flytrap (Dionaea muscipula) is one of the most well-known carnivorous plants because of its unique ability to capture small animals, usually insects or spiders, through a unique snap-trapping mechanism. The animals are subsequently killed and digested so that the plants can assimilate nutrients, as they grow in mineral-deficient soils. We deep sequenced the cDNA from Dionaea traps to obtain transcript libraries, which were used in the mass spectrometry-based identification of the proteins secreted during digestion. The identified proteins consisted of peroxidases, nucleases, phosphatases, phospholipases, a glucanase, chitinases, and proteolytic enzymes, including four cysteine proteases, two aspartic proteases, and a serine carboxypeptidase. The majority of the most abundant proteins were categorized as pathogenesis-related proteins, suggesting that the plant's digestive system evolved from defense-related processes. This in-depth characterization of a highly specialized secreted fluid from a carnivorous plant provides new information about the plant's prey digestion mechanism and the evolutionary processes driving its defense pathways and nutrient acquisition.

Tool use is rare amongst rodents and has never been recorded in connection with agonistic displays. We witnessed a behaviour, stick display (StD), involving tool use in free-living Eurasian beavers (Castor fiber) that we conclude is a display behaviour. Two beavers were the main performers of the signal that was observed in at least six beavers from three families. Beavers reacted to displays by increased evasive and agonistic behaviours compared with their usual behavioural patterns when at territory borders. The behaviour was almost exclusively seen between rivals at territory borders. We suggest that the display is used in agonistic encounters, mainly in a territorial context.

Mammalian scent marking is often associated with territorial defense. However, males and females may demonstrate different activity patterns and play different roles. Female mammals nurture the young during lactation, while males purportedly perform other tasks more frequently, such as territorial maintenance and defense. This paper investigates the contribution made by mated pairs of adult males and females to territorial scent-marking in the obligate monogamous Eurasian beavers (Castor fiber). We hypothesized that both sexes should show territorial behavior, and predicted that they deposit a higher proportion of scent marks at borders. We also hypothesized that a sexual dimorphism exists due to reproductive constraints on the females, and predicted that males should invest significantly more in scent-marking behavior than females during summer. We obtained behavioral data by radio tracking six mated pairs of Eurasian beavers during spring and summer 2000-2001 on two rivers in southeastern Norway. Our results showed that both males and females clustered their scent marks near territorial borders, but males deposited a larger number of scent marks than females and spent more time at borders. Males were also found to have a higher scent marking rate and scent marks per night than females during summer, but not during spring. Overall, scent marks per night were higher in males than females. We conclude that both males and females Eurasian beavers carry out territorial behavior by scent marking, but males carry a larger part of the territorial defense during summer when females lactate. Our results are discussed in the light of the codefense hypothesis.