Submissive behaviour is affected by group size in a social fish.

For social groups to form and be stable over time, animals must develop strategies to cope with conflict among group members. Animals may behave submissively either by fleeing from an aggressor, or by signalling submission. The use of these two submissive responses may vary depending on the social and ecological context. Group size is a key aspect of social context for group living animals, as individuals in smaller groups may respond to aggression differently than those from larger groups. Here, we examine the relationship between group size and submissive behaviour in a cooperatively breeding fish, the daffodil cichlid (Neolamprologus pulcher). We found that subordinate fish showed similar levels of submission signals in response to dominant aggression in larger and smaller groups, however, subordinates from larger groups were less likely to flee from dominant aggression than those in smaller groups. Subordinates in larger groups also showed more digging behaviour which may be also used to avoid conflict with the dominant group members. Our data show that social context affects submissive behaviour in a cooperatively breeding fish.

Proceedings of the Think Tank for Osteosarcoma Medical Advisory Board.

A "Think Tank for Osteosarcoma" medical advisory board meeting was held in Santa Monica, CA, USA on February 2-3, 2024. The goal was to develop a strategic approach to prevent recurrence of osteosarcoma. Osteosarcoma metabolism and the genomic instability of osteosarcoma, immunotherapy for osteosarcoma, CAR-T cell therapy, DeltaRex-G tumor-targeted gene therapy, repurposed drugs, alternative medicines, and personalized medicine were discussed. Only DeltaRex-G was voted on. The conclusions were the following: No intervention has been demonstrated to improve survival in a clinical trial. Additionally, the consensus (10/12 in favor) was that DeltaRex-G without immunotherapy may be administered for up to one year. Phase 2/3 randomized studies of DeltaRex-G should be performed to determine whether the incidence of recurrence could be reduced in high-risk individuals. Furthermore, a personalized approach using drugs with minimal toxicity could be attempted with the acknowledgement that there are no efficacy data to base this on. Repurposed drugs and alternative therapies should be tested in mouse models of osteosarcoma. Moreover, unmodified IL-2 primed Gamma Delta (NK) cell therapy may be used to prevent recurrence. Lastly, rapid development of CAR-T cell therapy is recommended, and an institute dedicated to the study of osteosarcoma is needed.

Fellowship of the fin: Fish empathy and oxytocin.

Zebrafish exhibit fear contagion, a basic form of empathy, and when observing social fellows that have been exposed to predation cues, will themselves exhibit similar distress behaviours. As in mammals, the nonapeptide hormone oxytocin is essential for this empathic response, and homologous areas of the brain are involved, suggesting that the mechanistic basis of empathy may be conserved among vertebrates.

Interacting effects of environmental enrichment across multiple generations on early life phenotypes in zebrafish.

The environment plays an important role in an individual's development during early life, however, parents may also influence offspring development through so called "parental effects." We examined the effects of environmental enrichment in zebrafish (Danio rerio) across two generations through the paternal lineage. Fathers and grandfathers were exposed to either standard or high levels of housing enrichment for 4-weeks during adulthood. First-generation (F1) and second-generation (F2) offspring were obtained from controlled breeding and tested as larvae for changes in morphology at hatching stage (72hpf), and in locomotor activity at larval stage (120hpf) in both generations. We found paternal experience of enrichment resulted in changes in trunk length of F1 offspring and changes in spine curvature and dorsal length of F2 offspring, while changes in snout morphology of F2 offspring seemed to be driven by whether grandpaternal and paternal experience of the environment was matched or not. We found that while paternal enrichment increased the frequency of spontaneous movement in F1 and F2 offspring, interacting effects of paternal and grandpaternal enrichment on movement distance were seen in F2 offspring, and that spontaneous movement and the distance that larvae swam are thus distinct phenotypes that were differentially affected by the experiences of previous paternal generations. Taken together, these findings suggest that the parental and grandparental environment influence zebrafish behavior and morphology. The nature of these effects and the design of this study mean that these phenotypes were likely the result of nongenetic transmission through the paternal germline.

Three year results of Blessed: Expanded access for DeltaRex-G for an intermediate size population with advanced pancreatic cancer and sarcoma (NCT04091295) and individual patient use of DeltaRex-G for solid malignancies (IND# 19130).

Background: Innovative treatments are urgently needed for metastatic cancer. DeltaRex-G, a tumor-targeted retrovector encoding a dominant-negative/cytocidal cyclin G1 (CCNG1 gene) inhibitor construct-has been tested in over 280 cancer patients worldwide in phase 1, phase 2 studies and compassionate use studies, demonstrating long term (>10 years) survivorship in patients with advanced cancers, including pancreatic cancer, osteosarcoma, malignant peripheral nerve sheath tumor, breast cancer, and B-cell lymphoma. Patient and Methods: Endpoints: Survival, response, treatment-related adverse events. Study one is entitled "Blessed: Expanded Access for DeltaRex-G for Advanced Pancreatic Cancer and Sarcoma (NCT04091295)". Study two is entitled "Individual Patient Use of DeltaRex-G for Solid Malignancies (Investigational New Drug#19130). In both studies, patients will receive DeltaRex-G at 1-3 x 10e11 cfu i.v. over 30-45 min, three x a week until significant disease progression or unacceptable toxicity or death occurs. Results: Seventeen patients were enrolled, nine sarcoma, two pancreatic adenocarcinoma, one non-small cell lung cancer, two breast carcinoma, one prostate cancer, one cholangiocarcinoma and one basal cell carcinoma and actinic keratosis. Three patients were enrolled in Study 1 and 14 patients were enrolled in Study 2. Twelve of 17 enrolled patients were treated with DeltaRex-G monotherapy or in combination with United States Food and Drug Administration-approved cancer therapies. Five patients died before receiving DeltaRex-G. Efficacy Analysis: Of the 12 treated patients, 5 (42%) are alive 15-36 months from DeltaRex-G treatment initiation. Two patients with early-stage HR + HER2+ positive or triple receptor negative invasive breast cancer who received DeltaRex-G as adjuvant/first line therapy are alive in complete remission 23 and 16 months after DeltaRex-G treatment initiation respectively; three patients with metastatic chordoma, chondrosarcoma and advanced basal cell carcinoma are alive 36, 31, and 15 months after DeltaRex-G treatment initiation respectively. Safety Analysis: There were no treatment-related adverse events reported. Conclusion: Taken together, the data suggest that 1) DeltaRex-G may evoke tumor growth stabilization after failing standard chemotherapy, 2) DeltaRex-G may act synergistically with standard chemotherapy/targeted therapies, and 3) Adjuvant/first line therapy with DeltaRex-G for early-stage invasive carcinoma of breast may be authorized by the USFDA when patients refuse to receive toxic chemotherapy.

Regulatory Framework for Academic Investigator-Sponsored Investigational New Drug Development of Cell and Gene Therapies in the USA.

PURPOSE OF REVIEW: The promise of cell and gene therapy (CGT) products for a multitude of diseases has revitalized investigators to advance novel CGT product candidates to first-in-human trials by pursuing the investigational new drug (IND) mechanism administered by the United States (US) Food and Drug Administration (FDA). This review is intended to familiarize academic investigators with the IND governing regulations set forth by the FDA. RECENT FINDINGS: CGT products are extraordinarily complex biologics and, therefore, early-stage evaluation programs must be customized to satisfactorily address their unique developmental challenges. The US FDA continues to foster the development of transformational technology that will facilitate the broad application of safe and effective gene therapy products that have the potential to alleviate many conditions previously out of reach of therapeutic intervention. FDA is committed to working with the scientific community and industry to facilitate the availability of these treatments to patients. SUMMARY: The pathway to meet regulatory compliance during early stage IND programs can be daunting to academic investigators interested in CGT product development that typically don't progress beyond phase 1/2. However, by keeping abreast of current regulatory framework and building upon FDA's supportive infrastructure, an investigator can be well-positioned to advance innovative scientific discoveries towards early stage clinical assessments.

Phase I/II Manufacture of Lentiviral Vectors Under GMP in an Academic Setting.

In clinical gene transfer applications, lentiviral vectors (LV) have rapidly become the primary means to achieve permanent and stable expression of a gene of interest or alteration of gene expression in target cells. This status can be attributed primarily to the ability of the LV to (1) transduce dividing as well as quiescent cells, (2) restrict or expand tropism through envelope pseudo-typing, and (3) regulate gene expression within different cell lineages through internal promoter selection. Recent progress in viral vector design such as the elimination of unnecessary viral elements, split packaging, and self-inactivating vectors has established a significant safety profile for these vectors. The level of GMP compliance required for the manufacture of LV is dependent upon their intended use, stage of drug product development, and country where the vector will be used as the different regulatory authorities who oversee the clinical usage of such products may have different requirements. As such, successful GMP manufacture of LV requires a combination of diverse factors including: regulatory expertise, compliant facilities, validated and calibrated equipments, starting materials of the highest quality, trained production personnel, scientifically robust production processes, and a quality by design approach. More importantly, oversight throughout manufacturing by an independent Quality Assurance Unit who has the authority to reject or approve the materials is required. We describe here the GMP manufacture of LV at our facility using a four plasmid system where 293T cells from an approved Master Cell Bank (MCB) are transiently transfected using polyethylenimine (PEI). Following transfection, the media is changed and Benzonase added to digest residual plasmid DNA. Two harvests of crude supernatant are collected and then clarified by filtration. The clarified supernatant is purified and concentrated by anion exchange chromatography and tangential flow filtration. The final product is then diafiltered directly into the sponsor defined final formulation buffer and aseptically filled.

A Scalable Lentiviral Vector Production and Purification Method Using Mustang Q Chromatography and Tangential Flow Filtration.

Lentiviral vectors have rapidly become a favorite tool for research and clinical gene transfer applications which seek to permanently introduce alterations in the genome. This status can be attributed primarily to their ability to transduce dividing as well as quiescent cells. When coupled with internal promotor selection to drive expression in one cell type but not another, the ease with which the vectors can be pseudotyped to either restrict or expand tropism offers unique opportunities previously unavailable to the researcher to manipulate the genome. Although LV can be produced from stable packaging cell lines and/or in suspension culture, by and far, most LV vectors are produced using adherent 293 T cells grown in plasticware and production plasmids transiently transfected with either PEI or Calcium Phosphate. The media is usually changed and un-concentrated vector supernatant collected between 24 and 48 h post-transfection. The supernatant may then be purified by Mustang Q chromatography, concentrated by Tangential Flow Filtration, and finally diafiltered into the final formulation buffer of choice. Here we describe a pilot scale method for the manufacture of a Lentiviral vector that purifies and concentrates approximately 6 L of un-concentrated LV supernatant to approximately 150 mL. Typical titers for most vector constructs range between 1 × 108 and 1 × 109 infectious particles per mL. This method may be performed reiteratively to increase total volume or can be further scaled up to increase yield.

A nematode that can manipulate the behaviour of slugs.

The ability of parasites to manipulate the behaviour of their hosts has evolved multiple times, and has a clear fitness benefit to the parasite in terms of facilitating growth, reproduction and transfer to suitable hosts. The mechanisms by which these behavioural changes are induced are poorly understood, but in many cases parasite manipulation of serotonergic signalling in the host brain is implicated. Here we report that Phasmarhabditis hermaphrodita, a parasite of terrestrial gastropod molluscs, can alter the behaviour of slugs. Uninfected slugs (Deroceras panormitanum, Arion subfuscus and Arion hortensis) avoid areas where P. hermaphrodita is present, but slugs infected with P. hermaphrodita are more likely to be found where the nematodes are present. This ability is specific to P. hermaphrodita and other nematodes (Steinernema carpocapsae and Heterorhabditis bacteriophora) do not induce this behavioural change. To investigate how P. hermaphrodita changes slug behaviour we exposed slugs to fluoxetine (a selective serotonin reuptake inhibitor) and cyproheptadine (a serotonin receptor antagonist). Uninfected slugs fed fluoxetine no longer avoided areas where P. hermaphrodita was present; and conversely, infected slugs fed cyproheptadine showed no increased attraction to areas with nematodes. These findings suggest that a possible mechanism by which P. hermaphrodita is able to manipulate parasite avoidance behaviour in host slugs is by manipulating serotonergic signalling in the brain, and that increased serotonin levels are potentially associated with a reduction in parasite avoidance.

Forebrain activation during social exposure in wild-type guppies.

The neural mechanisms regulating social behaviour have received extensive attention in recent years, with much focus on 'complex' forms of sociality. Comparatively little research has addressed fundamental social behaviour, such as grouping, which impacts multiple determinants of fitness, such as foraging and avoiding predation. We are interested in the degree to which brain areas that regulate other forms of sociality are also involved in grouping behaviour, and so we investigated shoal-elicited activation of the brain in the guppy (Poecilia reticulata). Guppies are small, social fish that live in the rivers of Trinidad and, like many social fish, exhibit preferences for larger shoals. We first confirmed that our study population of wild-type guppies preferred to join a larger shoal, and then investigated the activation of four brain regions proposed to be involved in social behaviour and reward (the preoptic area, the dorsal part of the ventral telencephalon, the ventral part of the ventral telencephalon, and the supracommissural part of the ventral pallium). Subjects were exposed to a large shoal, a small shoal, or to a tank empty of conspecifics, and we used immediate early gene expression (egr-1) to assess neuronal activation. We found increased activation in the preoptic area when fish were exposed to a large shoal compared to controls that had no social exposure. There were no significant differences in activation within the other brain areas examined, possibly because these brain areas are not key regulators of grouping behaviour or have only a secondary role. The higher activation of the preoptic area during social exposure suggests functional homology in this highly-conserved region across all vertebrates.

C-tactile afferents: Cutaneous mediators of oxytocin release during affiliative tactile interactions?

Low intensity, non-noxious, stimulation of cutaneous somatosensory nerves has been shown to trigger oxytocin release and is associated with increased social motivation, plus reduced physiological and behavioural reactivity to stressors. However, to date, little attention has been paid to the specific nature of the mechanosensory nerves which mediate these effects. In recent years, the neuroscientific study of human skin nerves (microneurography studies on single peripheral nerve fibres) has led to the identification and characterisation of a class of touch sensitive nerve fibres named C-tactile afferents. Neither itch nor pain receptive, these unmyelinated, low threshold mechanoreceptors, found only in hairy skin, respond optimally to low force/velocity stroking touch. Notably, the speed of stroking which C-tactile afferents fire most strongly to is also that which people perceive to be most pleasant. The social touch hypothesis posits that this system of nerves has evolved in mammals to signal the rewarding value of physical contact in nurturing and social interactions. In support of this hypothesis, we review the evidence that cutaneous stimulation directly targeted to optimally activate C-tactile afferents reduces physiological arousal, carries a positive affective value and, under healthy conditions, inhibits responses to painful stimuli. These effects mirror those, we also review, which have been reported following endogenous release and exogenous administration of oxytocin. Taken together this suggests C-tactile afferent stimulation may mediate oxytocin release during affiliative tactile interactions.

Preparation of intravenous cholesterol tracer using current good manufacturing practices.

Studies of human reverse cholesterol transport require intravenous infusion of cholesterol tracers. Because insoluble lipids may pose risk and because it is desirable to have consistent doses of defined composition available over many months, we investigated the manufacture of cholesterol tracer under current good manufacturing practice (CGMP) conditions appropriate for phase 1 investigation. Cholesterol tracer was prepared by sterile admixture of unlabeled cholesterol or cholesterol-d7 in ethanol with 20% Intralipid(®). The resulting material was filtered through a 1.2 micron particulate filter, stored at 4°C, and tested at time 0, 1.5, 3, 6, and 9 months for sterility, pyrogenicity, autoxidation, and particle size and aggregation. The limiting factor for stability was a rise in thiobarbituric acid-reacting substances of 9.6-fold over 9 months (P < 0.01). The emulsion was stable with the Z-average intensity-weighted mean droplet diameter remaining at 60 nm over 23 months. The zeta potential (a measure of negative surface charge protecting from aggregation) was unchanged at -36.2. Rapid cholesterol pool size was 25.3 ± 1.3 g. Intravenous cholesterol tracer was stable at 4°C for 9 months postproduction. CGMP manufacturing methods can be achieved in the academic setting and need to be considered for critical components of future metabolic studies.

Demasculinization of male guppies increases resistance to a common and harmful ectoparasite.

Parasites are detrimental to host fitness and therefore should strongly select for host defence mechanisms. Yet, hosts vary considerably in their observed parasite loads. One notable source of inter-individual variation in parasitism is host sex. Such variation could be caused by the immunomodulatory effects of gonadal steroids. Here we assess the influence of gonadal steroids on the ability of guppies (Poecilia reticulata) to defend themselves against a common and deleterious parasite (Gyrodactylus turnbulli). Adult male guppies underwent 31 days of artificial demasculinization with the androgen receptor-antagonist flutamide, or feminization with a combination of flutamide and the synthetic oestrogen 17 ß-estradiol, and their parasite loads were compared over time to untreated males and females. Both demasculinized and feminized male guppies had lower G. turnbulli loads than the untreated males and females, but this effect appeared to be mainly the result of demasculinization, with feminization having no additional measurable effect. Furthermore, demasculinized males, feminized males and untreated females all suffered lower Gyrodactylus-induced mortality than untreated males. Together, these results suggest that androgens reduce the ability of guppies to control parasite loads, and modulate resistance to and survival from infection. We discuss the relevance of these findings for understanding constraints on the evolution of resistance in guppies and other vertebrates.

Behavioural responses of feral and domestic guppies (Poecilia reticulata) to predators and their cues.

Predation is an important factor during adaptation to novel environments, and the feralisation of introduced domestic species often involves responding appropriately to allopatric predators despite a background of domestication and inbreeding. Twenty years ago, domestic guppies were introduced to a semi-natural environment at Burgers' Zoo in the Netherlands, where they have since been exposed to avian predation. We compared predation-linked behaviours in this feral population and in domestic guppies akin to the original founders. We found that both populations responded to a novel predator and to conspecific alarm cues. However, shoaling, an important anti-predator behaviour, was higher among feral guppies both at baseline and when exposed to the novel predator. We did not observe a linked suite of anti-predator behaviours across shoaling, predator inspection, alarm substance sensitivity and boldness, suggesting that these responses may be decoupled from one another depending on local predation regimes. As we compared two populations, we cannot identify the causal factors determining population differences, however, our results do suggest that shoaling is either a particularly consequential anti-predator adaptation or the most labile of the behaviours we tested. Finally, the behavioural adaptability of domestic guppies may help to explain their success as an invasive species.

Sexual experience affects reproductive behavior and preoptic androgen receptors in male mice.

Reproductive behavior in male rodents is made up of anticipatory and consummatory elements which are regulated in the brain by sensory systems, reward circuits and hormone signaling. Gonadal steroids play a key role in the regulation of male sexual behavior via steroid receptors in the hypothalamus and preoptic area. Typical patterns of male reproductive behavior have been characterized, however these are not fixed but are modulated by adult experience. We assessed the effects of repeated sexual experience on male reproductive behavior of C57BL/6 mice; including measures of olfactory investigation of females, mounting, intromission and ejaculation. The effects of sexual experience on the number of cells expressing either androgen receptor (AR) or estrogen receptor alpha (ERα) in the primary brain nuclei regulating male sexual behavior was also measured. Sexually experienced male mice engaged in less sniffing of females before initiating sexual behavior and exhibited shorter latencies to mount and intromit, increased frequency of intromission, and increased duration of intromission relative to mounting. No changes in numbers of ERα-positive cells were observed, however sexually experienced males had increased numbers of AR-positive cells in the medial preoptic area (MPOA); the primary regulatory nucleus for male sexual behavior. These results indicate that sexual experience results in a qualitative change in male reproductive behavior in mice that is associated with increased testosterone sensitivity in the MPOA and that this nucleus may play a key integrative role in mediating the effects of sexual experience on male behavior.

Genomic imprinting and mammalian reproduction.

Among animals, genomic imprinting is a uniquely mammalian phenomenon in which certain genes are monoallelically expressed according to their parent of origin. This silencing of certain alleles often involves differential methylation at regulatory regions associated with imprinted genes and must be recapitulated at every generation with the erasure and reapplication of these epigenetic marks in the germline. Imprinted genes encode regulatory proteins that play key roles in fetal growth and development, but they also exert wider effects on mammalian reproduction. Genetic knockout experiments have shown that certain paternally expressed imprinted genes regulate post-natal behavior in offspring as well as reproductive behaviors in males and females. These deficits involve changes in hypothalamic function affecting multiple areas and different neurochemical pathways. Paternally expressed genes are highly expressed in the hypothalamus which regulates growth, metabolism and reproduction and so are well placed to influence all aspects of reproduction from adults to the resultant offspring. Coadaptation between offspring and mother appears to have played an important role in the evolution of some paternally expressed genes, but the influence of these genes on male reproductive behavior also suggests that they have evolved to regulate their own transmission to successive generations via the male germline.

Paternal influence on female behavior: the role of Peg3 in exploration, olfaction, and neuroendocrine regulation of maternal behavior of female mice.

Genomic imprinting represents a mechanism through which parent-of-origin effects on offspring development may be mediated. However, investigation of the influence of imprinted genes on behavior has been limited. Here the authors investigate the role of the maternally imprinted/paternally expressed gene, Peg3, in several aspects of behavior using both 129Sv- and B6-Peg3 mutant female mice. Virgin Peg3 females on both genetic backgrounds were less exploratory and had higher rates of defecation with strain-dependent effects on activity levels and olfactory discrimination. Reproductive success, pup retrieval, and postnatal maternal care of pups were reduced in these females whereas indices of maternal aggression were higher among B6 Peg3-KO females. Differences in maternal care were apparent in females caring for biological or cross-fostered offspring and deficits in pup retrieval apparent beyond the immediate postpartum period. Oxytocin receptor binding in the MPOA and LS was reduced in Peg3-KO females. Thus, the authors demonstrate that disruptions to Peg3 influences aspects of female behavior that are critical for mediating maternal effects on offspring development, such as postpartum licking/grooming, and that effects of Peg3 are dependent on the maternal genetic background.

The meaning of weaning: influence of the weaning period on behavioral development in mice.

Maternal care during the first week postpartum has long-term consequences for offspring development in rodents. However, mother-infant interactions continue well beyond this period, with several physiological and behavioral changes occurring between days 18 and 28 PN. In the present study, we investigate the long-term effects on offspring behavior of being weaned at day 21 PN versus day 28 PN. We found that male and female offspring engage in higher initial levels of social interaction if weaned at day 28 PN, as well as sexually dimorphic changes in exploratory behavior. Females who were themselves weaned earlier also appeared to wean their own pups earlier. Sex-specific effects of weaning age were found on levels of oxytocin and vasopressin V1a receptor density in the hypothalamus, central nucleus of the amygdala and nucleus accumbens. These results indicate that altering weaning age in mice may be a useful model for investigating the development of sexual dimorphism in neurobiology and behavior.

The evolution of pheromonal communication.

Small-brained rodents have been the principle focus for pheromonal research and have provided comprehensive insights into the chemosensory mechanisms that underpin pheromonal communication and the hugely important roles that pheromones play in behavioural regulation. However, pheromonal communication does not start or end with the mouse and the rat, and work in amphibians reveals much about the likely evolutionary origins of the chemosensory systems that mediate pheromonal effects. The dual olfactory organs (the main olfactory epithelium and the vomeronasal organ), their receptors and their separate projection pathways appear to have ancient evolutionary origins, appearing in the aquatic ancestors of all tetrapods during the Devonian period and so pre-dating the transition to land. While the vomeronasal organ has long been considered an exclusively pheromonal organ, accumulating evidence indicates that it is not the sole channel for the transduction of pheromonal information and that both olfactory systems have been co-opted for the detection of different pheromone signals over the course of evolution. This has also led to great diversity in the vomeronasal and olfactory receptor families, with enormous levels of gene diversity and inactivation of genes in different species. Finally, the evolution of trichromacy as well as huge increases in social complexity have minimised the role of pheromones in the lives of primates, leading to the total inactivation of the vomeronasal system in catarrhine primates while the brain increased in size and behaviour became emancipated from hormonal regulation.

Design and production of retro- and lentiviral vectors for gene expression in hematopoietic cells.

Successful retroviral gene transfer into hematopoietic cells has been demonstrated in a number of small and large animal models and clinical trials. However, severe adverse events related to insertional muta-genesis in a recent clinical trial for X-linked severe combined immunodeficiency reinforced the need to develop novel retroviral vectors with improved biosafety. Improvements include the use of self-inactivating (SIN) vectors as well as improvements in vector design. This chapter describes the basic design of gamma-retroviral and lentiviral SIN vectors that utilize a split-packaging system and includes a description of the various cloning modules frequently used in the design of such vectors that impact biosafety, titer, and transgene expression. In addition, this chapter describes the methods used for high titer vector production using calcium phosphate transfection both at research scale and at large scale for clinical application using a closed system bioreactor.