Immune regulatory and neuroprotective properties of preimplantation factor: From newborn to adult.

Embryonic-maternal interaction from the earliest stages of gestation has a key, sustained role in neurologic development, persisting into adulthood. Early adverse events may be detrimental in adulthood. Protective factors present during gestation could significantly impact post-natal therapy. The role of PreImplantation Factor (PIF) within this context is herein examined. Secreted by viable early embryos, PIF establishes effective embryonic-maternal communication and exerts essential trophic and protective roles by reducing oxidative stress and protein misfolding and by blunting the nocive let-7 microRNA related pathway. PIF's effects on systemic immunity lead to comprehensive immune modulation, not immune suppression. We examine PIF's role in protecting embryos from adverse maternal environment, which can lead to neurological disorders that may only manifest post-nataly: Synthetic PIF successfully translates endogenous PIF features in both pregnant and non-pregnant clinically relevant models. Specifically PIF has neuroprotective effects in neonatal prematurity. In adult relapsing-remitting neuroinflammation, PIF reverses advanced paralysis while promoting neurogenesis. PIF reversed Mycobacterium smegmatis induced brain infection. In graft-vs.-host disease, PIF reduced skin ulceration, liver inflammation and colon ulceration while maintaining beneficial anti-cancer, graft-vs.-leukemia effect. Clinical-grade PIF has high-safety profile even at supraphysiological doses. The FDA awarded Fast-Track designation, and university-sponsored clinical trials for autoimmune disorder are ongoing. Altogether, PIF properties point to its determining regulatory role in immunity, inflammation and transplant acceptance. Specific plans for using PIF for the treatment of complex neurological disorders (ie. traumatic brain injury, progressive paralysis), including neuroprotection from newborn to adult, are presented.

The Epicardium in the Embryonic and Adult Zebrafish

The epicardium is the mesothelial outer layer of the vertebrate heart. It plays an important role during cardiac development by, among other functions, nourishing the underlying myocardium, contributing to cardiac fibroblasts and giving rise to the coronary vasculature. The epicardium also exerts key functions during injury responses in the adult and contributes to cardiac repair. In this article, we review current knowledge on the cellular and molecular mechanisms underlying epicardium formation in the zebrafish, a teleost fish, which is rapidly gaining status as an animal model in cardiovascular research, and compare it with the mechanisms described in other vertebrate models. We moreover describe the expression patterns of a subset of available zebrafish Wilms' tumor 1 transgenic reporter lines and discuss their specificity, applicability and limitations in the study of epicardium formation.

Building Steprelationships: Emerging Adult Stepchildrens' Perceptions and Experiences of their Stepparents

A grounded theory study was conducted with ten (7 female, 3 male) emerging-adults in stepfamilies to examine their perceptions and experiences of their stepparents, and what factors influence the development and maintenance of these steprelationships. Three primary categories emerged from the data: (1) Stepchildrens' perceptions of their stepparents presence in their lives, including both physical closeness and physical and emotional involvement (2) The perceived level of authenticity within the steprelationship, and (3) The level of clout stepchildren afforded to their stepparents' position within their lives and families. Additional factors found to influence the levels of presence, authenticity, and clout were stepchildrens' emotional maturity, cultural background, relationships with their biological parents, and feelings regarding the use and acquisition of money.

Physician adoption of a practice guideline: Effect on the cost of medical care for adult patients with diabetes

In the demanding environment of healthcare reform, reduction of unwanted physician practice variation is promoted, often through evidence-based guidelines. Guidelines represent innovations that direct change(s) in physician practice; however, compliance has been disappointing. Numerous studies have analyzed guideline development and dissemination, while few have evaluated the consequences of guideline adoption. The primary purpose of this study was to explore and analyze the relationship between physician adoption of the glycated hemoglobin test guideline for management of adult patients with diabetes, and the cost of medical care. The study also examined six personal and organizational characteristics of physicians and their association with innovativeness, or adoption of the guideline. ^ Cost was represented by approved charges from a managed care claims database. Total cost, and diabetes and related complications cost, first were compared for all patients of adopter physicians with those of non-adopter physicians. Then, data were analyzed controlling for disease severity based on insulin dependency, and for high cost cases. There was no statistically significant difference in any of eight cost categories analyzed. This study represented a twelve-month period, and did not reflect cost associated with future complications known to result from inadequate management of glycemia. Guideline compliance did not increase annual cost, which, combined with the future benefit of glycemic control, lends support to the cost effectiveness of the guideline in the long term. Physician adoption of the guideline was recommended to reduce the future personal and economic burden of this chronic disease. ^ Only half of physicians studied had adopted the glycated hemoglobin test guideline for at least 75% of their diabetic patients. No statistically significant relationship was found between any physician characteristic and guideline adoption. Instead, it was likely that the innovation-decision process and guideline dissemination methods were most influential. ^ A multidisciplinary, multi-faceted approach, including interventions for each stage of the innovation-decision process, was proposed to diffuse practice guidelines more effectively. Further, it was recommended that Organized Delivery Systems expand existing administrative databases to include clinical information, decision support systems, and reminder mechanisms, to promote and support physician compliance with this and other evidence-based guidelines. ^

Measuring health state preferences for hemophilia: Development of a disease-specific utility instrument

Research has shown that disease-specific health related quality of life (HRQoL) instruments are more responsive than generic instruments to particular disease conditions. However, only a few studies have used disease-specific instruments to measure HRQoL in hemophilia. The goal of this project was to develop a disease-specific utility instrument that measures patient preferences for various hemophilia health states. The visual analog scale (VAS), a ranking method, and the standard gamble (SG), a choice-based method incorporating risk, were used to measure patient preferences. Study participants (n = 128) were recruited from the UT/Gulf States Hemophilia and Thrombophilia Center and stratified by age: 0–18 years and 19+. ^ Test retest reliability was demonstrated for both VAS and SG instruments: overall within-subject correlation coefficients were 0.91 and 0.79, respectively. Results showed statistically significant differences in responses between pediatric and adult participants when using the SG (p = .045). However, no significant differences were shown between these groups when using the VAS (p = .636). When responses to VAS and SG instruments were compared, statistically significant differences in both pediatric (p < .0001) and adult (p < .0001) groups were observed. Data from this study also demonstrated that persons with hemophilia with varying severity of disease, as well as those who were HIV infected, were able to evaluate a range of health states for hemophilia. This has important implications for the study of quality of life in hemophilia and the development of disease-specific HRQoL instruments. ^ The utility measures obtained from this study can be applied in economic evaluations that analyze the cost/utility of alternative hemophilia treatments. Results derived from the SG indicate that age can influence patients' preferences regarding their state of health. This may have implications for considering treatment options based on the mean age of the population under consideration. Although both instruments independently demonstrated reliability and validity, results indicate that the two measures may not be interchangeable. ^

Expression of Imprinted Genes Is Aberrant in Deceased Newborn Cloned Calves and Relatively Normal in Surviving Adult Clones

Cattle are the species used most frequently for the development of assisted reproductive technologies, such as nuclear transfer. Cattle cloning can be performed by a large number of laboratories around the world, and the efficiency of nuclear transfer in cattle is the highest among all species in which successful cloning has been achieved. However, an understanding of the expression of imprinted genes in this important species is lacking. In the present study, real time reverse transcription polymerase chain reaction (RT-PCR) was utilized to quantify the expression of the bovine Igf2, Igf2r, and H19 genes in eight major organs (brain, bladder, heart, kidney, liver, lung, spleen, and thymus) of somatic cell cloned calves that died shortly after birth, in three tissues (skin, muscle, and liver) of healthy clones that survived to adulthood, and in corresponding tissues of control animals from natural reproduction. We found that, deceased bovine cloned calves exhibited abnormal expression of all three genes studied in various organs. Large variations in the expression levels of imprinted genes were also seen among these clones, which were produced from the same genetic donor. In surviving adult clones, however, the expression of these imprinted genes was largely normal, except for the expression of the Igf2 gene in muscle, which was highly variable. Our data showed disruptions of expression of imprinted genes in bovine clones, which is possibly due to incomplete reprogramming of donor cell nuclei during nuclear transfer, and these abnormalities may be associated with the high neonatal mortality in cloned animals; clones that survived to adulthood, however, are not only physically healthy but also relatively normal at the molecular level of those three imprinted genes.

Molecular genetic analyses of extracellular signaling pathways during murine retinal development

Extracellular signaling pathways initiated by secreted proteins are important in the co-ordination of tissue interactions in multi-cellular organisms, particularly during embryonic development. These signaling cascades direct diverse cellular events, including proliferation, differentiation and migration, in both autocrine and paracrine modes. In adult animals, abnormal function of these proteins often results in degenerative and tumourigenic syndromes. In this study, I have focused on elucidating the role of Bone Morphogenetic Protein (Bmp) signal transduction during neuronal specification and differentiation in the vertebrate embryo, using the mouse retina as a model. Using tissue-specific conditional knock-out approaches, the consequences of genetic loss-of-function of this signaling pathway on retinal physiology were examined. Mutant mice lacking Bmp type I receptor function displayed a range of retinal phenotypes, each of which appeared to be regulated at a different threshold of Bmp receptor activity. Novel essential functions for Bmp signaling were uncovered for retinal neurogenesis, cell survival, and axonal pathfinding at the optic disc. Further, BmprIa and BmprIa exhibited genetic interactions suggestive of functional redundancy. To further characterize the underlying molecular bases for the pleiotropic effects of Bmp receptors, retina-specific loss-of-function mutants of the obligate Bmp-activated transcriptional mediator Smad4 were generated. A comparison of the retina-specific Smad4 mutant phenotypes with those of the Bmp receptor mutant retina revealed that only a subset of retinal phenotypes, namely optic disc axon pathfinding and axial patterning were common for both classes of mutant animals. Thus, these results suggest that, contrary to the classic scheme of Bmp signal transduction, Smad4-independent pathways may be operative downstream of the type I receptors. Indeed, such alternative intracellular signaling cascades may constitute a molecular basis for the multiple cellular responses elicited by Bmp signaling. Finally, I tested whether the potential Bmp pathway targets, the extracellular ligands Fgf9 and Fgf15, mediate essential cellular processes in the retina. The analyses of Fgf9 −/−; Fgf15−/− mutant mice posit a novel shared role for these genes in intra-retinal axon pathfinding. Collectively, these studies have elucidated part of the molecular machinery directing mammalian neuro-retinal development, and provided useful in vivo models to study visual function. ^

Analyzing the function of myogenin in adult satellite cells through the construction of a myogenin conditional allele

Although mechanisms regulating the formation of embryonic skeletal muscle are well characterized, less is known about muscle formation in postnatal life. This disparity is unfortunate because the largest increases in skeletal muscle mass occur after birth. Adult muscle stem cells (satellite cells) appear to recapitulate the events that occur in embryonic myoblasts. In particular, the myogenic basic helix-loop-helix factors, which have crucial functions in embryonic muscle development, are assumed to have similar roles in postnatal muscle formation. Here, I test this assumption by determining the role of the myogenic regulator myogenin in postnatal life. Myogenin-null mice die at birth, necessitating the generation of floxed alleles of myogenin and the use of cre-recombinase lines to delete myogenin. Removing myogenin before embryonic muscle development resulted in myofiber deficiencies identical to those observed in myogenin-null mice. However, mice in which myogenin was deleted following embryonic muscle development had normal skeletal muscle, except for modest alterations in MRF4 and MyoD expression. Notably, myogenin-deleted mice were 30% smaller than controls, suggesting that myogenin's absence disrupted general body growth. These results suggest that skeletal muscle growth in postnatal life is controlled by mechanisms distinct from those occurring in embryonic muscle development. ^

Development and formative evaluation of an obesity prevention intervention for the pediatric primary care setting: Helping HAND (Healthy Activity and Nutrition Directions)

As the obesity epidemic continues to increase, the pediatric primary care office setting remains a relatively unexplored arena to offer obesity prevention interventions for children. The increased risk for adult obesity among 10 to 14 year-old children who are overweight, suggests obesity prevention programs should be introduced just before this age or early in this age period. Research is also accumulating on the importance of targeting parents along with children, since parents are in charge of the home environment for children. Therefore, the aim of this project was to develop an obesity prevention program called Helping HAND (Healthy Activity and Nutrition Directions) based on Social Cognitive Theory and authoritative parenting techniques for the pediatric primary care setting and conduct one-on-one interviews with parents as the initial formative evaluation of the intervention material for the obesity prevention intervention. A secondary aim of the project was to determine the feasibility of identifying appropriate subjects for the intervention, and conducting qualitative evaluations of the materials through recruitment through pediatric primary care settings. ^

Transcriptional and translational regulators of gene expression in germ cell development

Germ cell development is a highly coordinated process driven, in part, by regulatory mechanisms that control gene expression. Not only transcription, but also translation, is under regulatory control to direct proper germ cell development. In this dissertation, I have focused on two regulators of germ cell development. One is the homeobox protein RHOX10, which has the potential to be both a transcriptional and translational regulator in mouse male germ cell development. The other is the RNA-binding protein, Hermes, which functions as a translational regulator in Xenopus laevis female germ cell development. ^ Rhox10 is a member of reproductive homeobox gene X-(linked (Rhox) gene cluster, of which expression is developmentally regulated in developing mouse testes. To identify the cell types and developmental stages in which Rhox10 might function, I characterized its temporal and spatial expression pattern in mouse embryonic, neonatal, and adult tissues. Among other things, this analysis revealed that both the level and the subcellular localization of RHOX10 are regulated during germ cell development. To understand the role of Rhox10 in germ cell development, I generated transgenic mice expressing an artificial microRNA (miRNA) targeting Rhox10. While this artificial miRNA robustly downregulated RHOX10 protein expression in vitro, it did not significantly reduce RHOX10 expression in vivo. So I next elected to knockdown RHOX10 levels in spermatogonial stem cells (SSCs), which I found highly express both Rhox10 mRNA and RHOX10 protein. Using a recently developed in vitro culture system for SSCs combined with a short-hairpin RNA (shRNA) approach, I strongly depleted RHOX10 expression in SSCs. These RHOX10-depleted cells exhibited a defect in the ability to form stem cell clusters in vitro. Expression profiling analysis revealed many genes regulated by Rhox10, including many meiotic genes, which could be downstream of Rhox10 in a molecular pathway that controls SSC differentiation. ^ RNA recognition motif (RRM) containing protein, Hermes is localized in germ plasm, where dormant mRNAs are also located, of Xenopus oocytes, which implicates its role in translational regulator. To understand the function of Hermes in oocyte meiosis, I used a morpholino oligonucleotide (MO) based knockdown approach. Microinjection of Hermes MO into fully grown oocytes, which are arrested in meiotic prophase, caused acceleration of oocytes reentry into meiosis (i.e., maturation) upon progesterone induction. Using a candidate approach, I identified at least three targets of Hermes: Ringo/Spy, Xcat2, and Mos. Ringo/Spy and Mos are known to have functions in oocyte maturation, while Ringo/Spy, Xcat2 mRNA are localized in the germ plasm of oocytes, which drives germ cell specification after fertilization. This led me to propose that Hermes functions in both oocyte maturation and germ cell development through its ability to regulate 3 crucial target mRNAs. ^

Evaluation of risk factors for long term changes in refractive error among adult patients at a Dallas Texas private setting A retrospective chart-review study

Background. Over 39.9% of the adult population forty or older in the United States has refractive error, little is known about the etiology of this condition and associated risk factors and their entailed mechanism due to the paucity of data regarding the changes of refractive error for the adult population over time.^ Aim. To evaluate risk factors over a long term, 5-year period, in refractive error changes among persons 43 or older by testing the hypothesis that age, gender, systemic diseases, nuclear sclerosis and baseline refractive errors are all significantly associated with refractive errors changes in patients at a Dallas, Texas private optometric office.^ Methods. A retrospective chart review of subjective refraction, eye health, and self-report health history was done on patients at a private optometric office who were 43 or older in 2000 who had eye examinations both in 2000 and 2005. Aphakic and pseudophakic eyes were excluded as well as eyes with best corrected Snellen visual acuity of 20/40 and worse. After exclusions, refraction was obtained on 114 right eyes and 114 left eyes. Spherical equivalent (sum of sphere + ½ cylinder) was used as the measure of refractive error.^ Results. Similar changes in refractive error were observed for the two eyes. The 5-year change in spherical power was in a hyperopic direction for younger age groups and in a myopic direction for older subjects, P<0.0001. The gender-adjusted mean change in refractive error in right eyes of persons aged 43 to 54, 55 to 64, 65 to 74, and 75 or older at baseline was +0.43D, +0.46 D, -0.09 D, and -0.23D, respectively. Refractive change was strongly related to baseline nuclear cataract severity; grades 4 to 5 were associated with a myopic shift (-0.38 D, P< 0.0001). The mean age-adjusted change in refraction was +0.27 D for hyperopic eyes, +0.56 D for emmetropic eyes, and +0.26 D for myopic eyes.^ Conclusions. This report has documented refractive error changes in an older population and confirmed reported trends of a hyperopic shift before age 65 and a myopic shift thereafter associated with the development of nuclear cataract.^

Growth and development of body fat distribution from skinfold measurements: Longitudinal principal components

Longitudinal principal components analyses on a combination of four subcutaneous skinfolds (biceps, triceps, subscapular and suprailiac) were performed using data from the London Longitudinal Growth Study. The main objectives were to discover at what age during growth sex differences in body fat distribution occur and to see if there is continuity in body fatness and body fat distribution from childhood into the adult status (18 years). The analyses were done for four age sectors (3mon-3yrs, 3yrs-8yrs, 8yrs-18yrs and 3yrs-18yrs). Longitudinal principal component one (LPC1) for each age interval in both sexes represents the population mean fat curve. Component two (LPC2) is a velocity of fatness component. Component three (LPC3) in the 3mon-3yrs age sector represents infant fat wave in both sexes. In the next two age sectors component three in males represents peaks and shifts in fat growth (change in velocity), while in females it represents body fat distribution. Component four (LPC4) in the same two age sectors is a reversal in the sexes of the patterns seen for component three, i.e., in males it is body fat distribution and in females velocity shifts. Components five and above represent more complicated patterns of change (multiple increases and decreases across the age interval). In both sexes there is strong tracking in fatness from middle childhood to adolescence. In males only there is also a low to moderate tracking of infant fat with middle to late childhood fat. These data are strongly supported in the literature. Several factors are known to predict adult fatness among the most important being previous levels of fatness (at earlier ages) and the age at rebound. In addition we found that the velocity of fat change in middle childhood was highly predictive of later fatness (r $\approx -$0.7), even more so than age at rebound (r $\approx -$0.5). In contrast to fatness (LPC1), body fat distribution (LPC3-LPC4) did not track well even though significant components of body fat distribution occur at each age. Tracking of body fat distribution was higher in females than males. Sex differences in body fat distribution are non existent. Some sex differences are evident with the peripheral-to-central ratios after age 14 years. ^

Targeted Deletion Of Functionally Validated Enhancers Defines Their Role in Mouse Limb Development

Transcriptional enhancers are genomic DNA sequences that contain clustered transcription factor (TF) binding sites. When combinations of TFs bind to enhancer sequences they act together with basal transcriptional machinery to regulate the timing, location and quantity of gene transcription. Elucidating the genetic mechanisms responsible for differential gene expression, including the role of enhancers, during embryological and postnatal development is essential to an understanding of evolutionary processes and disease etiology. Numerous methods are in use to identify and characterize enhancers. Several high-throughput methods generate large datasets of enhancer sequences with putative roles in embryonic development. However, few enhancers have been deleted from the genome to determine their roles in the development of specific structures, such as the limb. Manipulation of enhancers at their endogenous loci, such as the deletion of such elements, leads to a better understanding of the regulatory interactions, rules and complexities that contribute to faithful and variant gene transcription – the molecular genetic substrate of evolution and disease. To understand the endogenous roles of two distinct enhancers known to be active in the mouse embryo limb bud we deleted them from the mouse genome. I hypothesized that deletion of these enhancers would lead to aberrant limb development. The enhancers were selected because of their association with p300, a protein associated with active transcription, and because the human enhancer sequences drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. To confirm that the orthologous mouse enhancers, mouse 280 and 1442 (M280 and M1442, respectively), regulate expression in the developing limb we generated stable transgenic lines, and examined lacZ expression. In M280-lacZ mice, expression was detected in E11.5 fore- and hindlimbs in a region that corresponds to digits II-IV. M1442-lacZ mice exhibited lacZ expression in posterior and anterior margins of the fore- and hindlimbs that overlapped with digits I and V and several wrist bones. We generated mice lacking the M280 and M1442 enhancers by gene targeting. Intercrosses between M280 -/+ and M1442 -/+, respectively, generated M280 and M1442 null mice, which are born at expected Mendelian ratios and manifest no gross limb malformations. Quantitative real-time PCR of mutant E11.5 limb buds indicated that significant changes in transcriptional output of enhancer-proximal genes accompanied the deletion of both M280 and M1442. In neonatal null mice we observed that all limb bones are present in their expected positions, an observation also confirmed by histology of E18.5 distal limbs. Fine-scale measurement of E18.5 digit bone lengths found no differences between mutant and control embryos. Furthermore, when the developmental progression of cartilaginous elements was analyzed in M280 and M1442 embryos from E13.5-E15.5, transient development defects were not detected. These results demonstrate that M280 and M1442 are not required for mouse limb development. Though M280 is not required for embryonic limb development it is required for the development and/or maintenance of body size – adult M280 mice are significantly smaller than control littermates. These studies highlight the importance of experiments that manipulate enhancers in situ to understand their contribution to development.

Functions of GCN5 and P /CAF acetyltransferases in mammalian development

Histone acetyltransferases are important chromatin modifiers that function as transcriptional co-activators. The identification of the transcriptional regulator GCN5 as the first nuclear histone acetyltransferase in yeast directly linked chromatin remodeling to transcriptional regulation. Although emerging evidence suggests that acetyltransferases participate in multiple cellular processes, their roles in mammalian development remain undefined. In this study, I have cloned and characterized the mouse homolog of GCN5 and a closely related protein P/CAF that interacts with p300/CBP. In contrast to yeast GCN5, but similar to P/CAF, mouse GCN5 possesses an additional N-terminal domain that confers the ability to acetylate nucleosomal histones. GCN5 and P/CAF exhibit identical substrate specificity and both interact with p300/CBP. Interestingly, expression levels of GCN5 and P/CAF display a complementary pattern in mouse embryos and in adult tissues, suggesting that they have distinct tissue or developmental stage specific roles. To define the in vivo function of GCN5 and P/CAF, I have generated mice that are nullizygous for GCN5 or P/CAF. P/CAF null mice are viable and fertile with no gross morphological defects, indicating that P/CAF is dispensable for development and p300/CBP function in vivo. In contrast, mice lacking GCN5 die between 10.5–11 days of gestation. GCN5 null mice are severely retarded but have anterior ectopic outgrowth. Molecular marker analyses reveal that early mesoderm is formed in GCN5 null mice but further differentiation into distinct mesodermal lineages is perturbed. While presomitic mesoderm and chodamesoderm are missing in GCN5 mutant mice, extraembryonic tissues and lateral mesoderm are unaffected. This is consistent with our finding that GCN5 expression is absent in the heart and extraembryonic tissues but is uniform throughout the rest of the embryo. Remarkably, GCN5 mutant mice exhibit an unusually high incidence of apoptosis in the embryonic ectoderm and mesoderm. Finally, mice doubly null for GCN5 and P/CAF die much earlier than mice harboring the GCN5 mutation alone, suggesting that P/CAF and GCN5 share some overlapping function during embryogenesis. This work is the first study to show that specific acetyltransferase is important for cell survival as well as mesoderm differentiation or maintenance during early mammalian development. ^

Dive track profile, intermandibular angel sensor profile and immobilisation data of adult female Weddell seals at Drescher Inlet from expedition DRE2003

Determination of when and where animals feed and how much they consume is fundamental to understand their ecology and role in ecosystems. However, the lack of reliable data on feeding habits of wild animals, and particularly in marine endotherms, attests to the difficulty in doing this. A promising recent development proposes using a Hall sensor-magnet System - the inter-mandibular angle sensor (IMASEN) attached to animals' jaws to elucidate feeding events. We conducted trials on captive pinnipeds by feeding IMASEN-equipped animals with prey to examine the utility of this system. Most feeding events were clearly distinguishable from other jaw movements; only small prey items might not be resolved adequately. Based on the results of this study we examined feeding events from free-ranging Weddell seals fitted with IMASENs and dead-reckoners during December 2003 at Drescher Inlet (Riiser Larsen Ice Shelf, eastern Weddell Sea coast), and present data on prey capture and ingestion in relation to the three-dimensionalmovement patterns of the seals. A total of 19 Weddell seals were immobilised by using a combination of ketamine, xylazine, and diazepam. Eight seals were drugged once, six two times, and two and three were drugged three and four times each, coming to a total of 38 immobilisation procedures. Narcoses were terminated with yohimbine (doi:10.1594/PANGAEA.438931).