Physiology, pathophysiology and (mal)adaptations to chronic apnoeic training: a state-of-the-art review.

Breath-hold diving is an activity that humans have engaged in since antiquity to forage for resources, provide sustenance and to support military campaigns. In modern times, breath-hold diving continues to gain popularity and recognition as both a competitive and recreational sport. The continued progression of world records is somewhat remarkable, particularly given the extreme hypoxaemic and hypercapnic conditions, and hydrostatic pressures these athletes endure. However, there is abundant literature to suggest a large inter-individual variation in the apnoeic capabilities that is thus far not fully understood. In this review, we explore developments in apnoea physiology and delineate the traits and mechanisms that potentially underpin this variation. In addition, we sought to highlight the physiological (mal)adaptations associated with consistent breath-hold training. Breath-hold divers (BHDs) are evidenced to exhibit a more pronounced diving-response than non-divers, while elite BHDs (EBHDs) also display beneficial adaptations in both blood and skeletal muscle. Importantly, these physiological characteristics are documented to be primarily influenced by training-induced stimuli. BHDs are exposed to unique physiological and environmental stressors, and as such possess an ability to withstand acute cerebrovascular and neuronal strains. Whether these characteristics are also a result of training-induced adaptations or genetic predisposition is less certain. Although the long-term effects of regular breath-hold diving activity are yet to be holistically established, preliminary evidence has posed considerations for cognitive, neurological, renal and bone health in BHDs. These areas should be explored further in longitudinal studies to more confidently ascertain the long-term health implications of extreme breath-holding activity.

Baseline morphometric, haematological and plasma biochemical parameters in free-ranging eastern water dragons (Intellagama lesueurii lesueurii).

OBJECTIVE: The aim of this study was to determine baseline reference data for morphometric measurements and haematological and plasma biochemical parameters in clinically healthy eastern water dragons (Intellagama lesueurii lesueurii), accounting for the variables of season and sex. The clinical objective was to provide clinicians and researchers with baseline reference intervals (RIs) in order to assess accurately the health of a population or individual animals. METHODS: The study group comprised 39 free-ranging eastern water dragons. To monitor seasonal changes in data two study periods were assigned: September-October 2009 (spring) and January-March 2010 (summer). The 18 males and 21 females were captured in the grounds of Taronga Zoo, Mosman (33°50'45'' S, 151°15'20'' E) in Sydney, New South Wales, Australia. Basic morphometric, haematological and plasma biochemical parameters were measured. RIs were generated in accordance with the American Society of Veterinary Clinical Pathology guidelines. Data were analysed for the effects of sex, season and sex-season interactions using restricted maximal likelihood modelling. RESULTS: Reference values were established in eastern water dragons for morphometric data and RIs were generated for haematological and plasma biochemical levels. The study showed seasonal variation in uric acid. Weight, total length, snout-vent length and basophil % showed significant differences according to sex. Sex-season interactions were detected for calcium, monocyte % and total protein (refractometry). CONCLUSION: Multiple parameters exhibited normal physiological variation between season and sex and an interaction between the two factors. These variables need to be considered in the clinical context when interpreting results of haematological and biochemical analyses in eastern water dragons. The RIs established from this population will provide a basis for further studies in both free-living and captive eastern water dragons, and for comparison in clinical cases.

Qualification of cardiac troponins for nonclinical use: a regulatory perspective.

The US Food and Drug Administration (FDA) Biomarker Qualification Review Team presents its perspective on the recent qualification of cardiac troponins for use in nonclinical safety assessment studies. The goal of this manuscript is to provide greater transparency into the qualification process and factors that were considered in reaching a regulatory decision. This manuscript includes an overview of the data that were submitted and a discussion of the strengths and shortcomings of these data supporting the qualification decision. The cardiac troponin submission is the first literature-based biomarker application to be reviewed by the FDA and insights gained from this experience may aid future submissions and help streamline the characterization and qualification of future biomarkers.

Novel biomarkers of acute kidney toxicity.

Novel biomarkers of kidney toxicity are powerful tools not only with respect to their clinical applications but also because of their impact on drug development. These biomarkers can influence the assessment of efficacy of new drugs for kidney diseases as well as the risk management for new drugs. The science behind these novel biomarkers reflects the evolution over the past decade of genomic and proteomic platforms that have transformed the discovery and development of new biomarkers for preclinical and clinical applications in drug development. Several of these biomarkers are in use as transcriptomic biomarkers in animal models as well as translational proteomic biomarkers in animal models and in humans. Their ability to detect kidney damage earlier than is possible with currently accessible biomarkers is being given qualification through regulatory biomarker-qualification programs, which will help establish consensus for their widespread use.

Nonclinical aspects of biopharmaceutical development: discussion of case studies at a PhRMA-FDA workshop.

Robust assessments of the nonclinical safety profile of biopharmaceuticals are best developed on a scientifically justified, case-by-case basis, with consideration of the therapeutic molecule, molecular target, and differences/similarities between nonclinical species and humans (ICH S6). Significant experience has been gained in the 10 years ensuing since publication of the ICH S6 guidance. In a PhRMA-FDA-sponsored workshop, "Nonclinical Aspects of Biopharmaceutical Development," industry and US regulatory representatives engaged in exploration of current scientific and regulatory issues relating to the nonclinical development of biopharmaceuticals in order to share scientific learning and experience and to work towards establishing consistency in application of general principles and approaches. The proceedings and discussions of this workshop confirm general alignment of strategy and tactics in development of biopharmaceuticals with regard to such areas as species selection, selection of high doses in toxicology studies, selection of clinical doses, the conduct of developmental and reproductive toxicity (DART) studies, and assessment of carcinogenic potential. However, several important aspects, including, for example, appropriate use of homologues, nonhuman primates, and/or in vitro models in the assessment of risk for potential developmental and carcinogenic effects, were identified as requiring further scientific exploration and discussion.

Conflicts between courtship and thermoregulation: the thermal ecology of amorous male garter snakes (Thamnophis sirtalis parietalis, colubridae).

Thermoregulatory behavior is an important component of daily activities for many reptiles, especially for small heliothermic (sun-basking) species that inhabit cold climates. However, the relative costs and benefits of thermoregulation depend on numerous factors, such that reptiles may sometimes accord a low priority to precise control of body temperatures. We observed and radio tracked garter snakes (Thamnophis sirtalis parietalis) in central Manitoba during the mating season (spring). Previous studies on this species have documented precise behavioral regulation of body temperatures during summer. In contrast, the courting snakes that we studied in springtime spent little time in overt thermoregulatory behavior. Body temperatures were extremely variable (both in outdoor enclosures and in the field) despite abundant opportunities for more precise thermal control. These small elongate reptiles cool so quickly (relative to the time periods needed for effective courtship) that any benefit to higher body temperatures would be transitory at best. Experiments show that hotter males are no better at obtaining matings or at detecting predators. Thus, male garter snakes concentrate on courtship rather than on basking. In the face of conflicting priorities, reptiles may often forgo precise thermoregulation because its benefits are too low, and its costs too high, compared with alternative behaviors.

The transvestite serpent: why do male garter snakes court (some) other males?

In large mating aggregations of red-sided garter snakes, Thamnophis sirtalis parietalis, in Manitoba, male courtship is directed not only to females, but also to other males with female-like skin lipids ('she-males'). We show that 'she-maleness' is an intrinsic property of a male rather than an artefact of lipid transfer from females, and that male-male courtship is very common in the field. She-males were distinctive in terms of appearance (they were heavier than other males and more often covered with mud), behaviour (they were inactive and rarely courted females) and performance (they were slow crawlers, ineffective courters and easily outcompeted by other males in mating trials). 'She-maleness' was not a characteristic of a particular subset of males, as envisaged in previous work; instead, it was a transitory phase that most (perhaps all) male snakes passed through soon after they first emerged from the winter den. Recently emerged males spent their first day or two relatively inactive, while restoring physiological functions (including locomotor performance and courtship ability). Experimental application of female skin lipids on to males dramatically decreased courtship levels of the recipient snakes. Thus, recently emerged males may derive two kinds of benefit from mimicking female skin lipids. First, female mimicry 'switches off' the male's own (energetically expensive) courtship at a time when that courtship would be unproductive. Second, it may disadvantage his rivals by distracting them from females, and increasing their energy expenditure. Copyright 2000 The Association for the Study of Animal Behaviour.

Increasing the power of psychiatric court diversion: a new model of supra-district diversion centre.

The object was to develop and evaluate a new concentrated model of psychiatric diversion scheme at the magistrates' court, designed to maximize the potential of such interventions. A one-year prospective study was undertaken of a consecutive series of 264 referrals to the new diversion project at an Inner London magistrates' court, with concurrent examination of police station custody records, magistrates' court returns, hospital admission data and remand prison transfer records for an area with a population of 500,000. The results showed that this one scheme originated 12.8% of all the unrestricted hospital orders in England under section 37 of the Mental Health Act 1983, 4.2% of section 35 orders, and 6.4% of section 48 and 48/49 remand prisoner transfers. Of all arrests in the central London area, 0.46% were referred to the scheme, with 0.28% being admitted. The seriousness of the charge did not have a significant effect on whether or not admission was achieved (p = 0.5365). The new model is a powerful intervention in the assessment and diversion of mentally disordered offenders. Similar supra-district diversion centres may have a role to play in other areas, complementing other local diversion exercises, some of which might better be relocated to the police station.

Isoxazolines as potent antagonists of the integrin alpha(v)beta(3).

Starting with lead compound 2, we sought to increase the selectivity for alpha(v)beta(3)-mediated cell adhesion by examining the effects of structural changes in both the guanidine mimetic and the substituent alpha to the carboxylate. To prepare some of the desired aminoimidazoles, a novel reductive amination utilizing a trityl-protected aminoimidazole was developed. It was found that guanidine mimetics with a wide range of pK(a)'s were potent antagonists of alpha(v)beta(3). In general, it appeared that an acylated 2-aminoimidazole guanidine mimetic imparted excellent selectivity for alpha(v)beta(3)-mediated adhesion versus alpha(IIb)beta(3)-mediated platelet aggregation, with selectivity of approximately 3 orders of magnitude observed for compounds 3g and 3h. It was also found in this series that the alpha-substituent was required for potent activity and that 2,6-disubstituted arylsulfonamides were optimal. In addition, the selective alpha(v)beta(3) antagonist 3h was found to be a potent inhibitor of alpha(v)beta(3)-mediated cell migration.

Disubstituted indazoles as potent antagonists of the integrin alpha(v)beta(3).

A new series of indazole-containing alpha(v)beta(3) integrin antagonists is described. Starting with lead compound 18a, variations in a number of structural features were explored with respect to inhibition of the binding of beta(3)-transfected 293 cells to fibrinogen and to selectivity for alpha(v)beta(3) over GPIIbIIIa, another RGD-binding integrin. Indazoles attached to a 2-aminopyridine or 2-aminoimidazole by a propylene linker at the indazole 1-position and to a diaminopropionate derivative via a 5-carboxylate amide provided the best potency with moderate selectivity. Several differences in the SAR of the diaminopropionate moiety were observed between this series and a series of isoxazoline-based selective GPIIbIIIa antagonists. Compound 34a (SM256) was a potent antagonist of alpha(v)beta(3) (IC(50) 2.3 nM) with 9-fold selectivity over GPIIbIIIa.

The characterization of potent novel warfarin analogs.

Racemic sodium warfarin, Coumadin, is widely used in the prevention of thromboembolic disease. The present study was undertaken to characterize three novel classes of warfarin analogs, and to compare them with the warfarin enantiomers. All three classes of compounds inhibit vitamin K epoxide reductase, the enzyme inhibited by racemic warfarin. The alcohol and the ester analogs have reduced protein binding compared with R-(+)-warfarin. The ester and the fluoro-derivatives have similar in vivo anticoagulant activity in the rat to that of S-(-)-warfarin. Thus, it is possible to synthesize novel warfarin analogs that differ from racemic warfarin or its enantiomers in certain selected properties.

Warfarin analog inhibition of human CYP2C9-catalyzed S-warfarin 7-hydroxylation.

Human metabolism of the S-warfarin enantiomer is catalyzed primarily by cytochrome P4502C9 (CYP2C9), which, because of the enzyme's broad drug substrate specificity, leads to drug-S-warfarin interactions. Several warfarin analogs have been synthesized and used to determine whether they exhibit diminished interactions with CYP2C9. The kinetics of the warfarin analogs' inhibition of human liver microsomal CYP2C9 catalyzed metabolism of S-warfarin to S-7-hydroxywarfarin have been investigated. R- and S-7-fluorowarfarin were both predominantly competitive inhibitors, whereas racemic 6-fluorowarfarin and racemic 6,7,8-trifluorowarfarin were predominantly mixed inhibitors with some competitive inhibition. For the alcohols produced by reductive methylation of the side chain of R- and S-warfarin, the R-enantiomer did not inhibit S-warfarin metabolism, whereas the S-enantiomer was primarily a competitive inhibitor. The fluorine substituted warfarins and the S-warfarin alcohol apparently bind with high affinity to CYP2C9. Thus their use clinically (if efficacious) would not prevent CYP2C9 associated warfarin-drug interactions. The R-warfarin alcohol did not inhibit CYP2C9 catalyzed metabolism of S-warfarin and is less likely than warfarin to participate in CYP2C9 associated warfarin-drug interactions.

Combinatorial regulation by promoter and intron 1 regions of the metallothionein SpMTA gene in the sea urchin embryo.

The SpMTA metallothionein gene of the sea urchin Strongylocentrotus purpuratus is regulated developmentally, histospecifically, and by heavy-metal induction. The sequenced 5' flank of the gene can be divided into proximal, middle, and distal regions, each containing a pair of metal response elements (MREs). Canonical 7-bp core sequences are present in all except the middle-region MREs c and d, which contain 1-bp mismatches. Metal-induced expression in transgenic blastulae was increased with each consecutive addition of the middle and distal regions to a chimeric reporter gene construct containing the proximal SpMTA promoter region. Reduced metal induction through point mutation of the distal MREs e and f indicated that the MREs themselves were largely responsible for the transcriptional increase. These activities were further enhanced by SpMTA intron 1, but not when a specific interior region of the intron had been deleted. The atypical MREs c and d did not support induction by themselves, i.e., when present alone with mutated proximal MREs a and b. However, in the presence of intron 1, they were able to substitute for the nullified MREs a and b in the promotion of metal-induced expression. This capability suggests, furthermore, that these atypical MREs, in addition to responding to an intron 1 region, participate cooperatively with the canonical proximal MREs.

Maternal thermoregulation influences offspring viability in a viviparous lizard.

Gravid females of the viviparous scincid lizard Eulamprus heatwolei were maintained in the laboratory, with some females allowed to bask for 8 h/day and others for only 2 h/day. Maternal basking regimes influenced the gestation period, and significantly affected the body shapes, activity levels and running speeds of the offspring born to these females. Neonates from females with lesser basking opportunities were relatively short and fat, were very active, and were relatively fast runners. Effects of the embryos' thermal regime on the young lizards' morphology and running speed were still detectable two months after birth. Thus, much of the morphological and behavioral variation among neonatal reptiles may arise from phenotypically plastic responses to the thermal environments experienced during embryonic development, rather than from heritable genetic differences among individuals. Hence, selection on maternal thermoregulatory behavior may be an important avenue for adaptive modifications to neonatal phenotypes in reptiles.

Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression.

We have established the ability of the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) gene products to alter gene expression in human cells by using transient transfection assays. The cellular heat shock protein 70 (hsp70) promoter was transactivated following cotransfection with the HCMV IE regions in nonpermissive HeLa cells by UL36-38, US3, or IE1 and in permissive human diploid fibroblasts (HFF) by IE1 or IE2. Moreover, hsp70 expression was synergistically increased in HeLa cells cotransfected with US3 and UL36, with US3 and UL37, or with US3 and UL37x1. The synergistic transactivation of hsp70 expression by US3 and UL36-38 was not observed in HFF cells. Synergy was also not observed in HeLa cells between US3 and UL38, an early gene product encoded by the UL36-38 IE locus. Synergistic transactivation of hsp70 expression in HeLa cells required the syntheses of UL36-38 and US3 IE proteins, since nonsense mutants were not functional. hsp70 expression increased with increasing amounts of transfected US3 and UL37 DNA and occurred at the level of stable hsp70-promoted RNA. In contrast to the broad hsp70 response, promoters from the HCMV UL112 early gene and another cellular gene, brain creatine kinase, both responded strongly only to singly transfected IE2 in HeLa cells. Nevertheless, IE2 transactivation of the UL112 promoter was further stimulated by cotransfection of IE1 or of UL36-38 in both HeLa and HFF cells. Thus, different patterns of promoter transactivation and interactions between HCMV IE gene products in transactivation were found in HFF cells and in HeLa cells. These results establish the ability of the HCMV US3 and UL36-38 proteins to alter cellular and viral gene expression and are consistent with involvement of cellular transcription factors in HCMV IE regulation of gene expression.

Low protein catabolic rate and serum albumin correlate with increased mortality and abdominal complications in peritoneal dialysis patients.

We retrospectively reviewed 167 consecutive peritoneal dialysis patients with regard to serum albumin (Alb), mortality and abdominal complications. In addition, 25 patients were studied with serial measurements of urea kinetics. The patients were divided into four groups based on their dialysis index (DI) and normalized protein catabolic rate (NPCR) (Table I). 12/167 patients were identified with abdominal catastrophes. Before these complications occurred, the M Alb in this group was 2.67 + 0.24 (compared to age, sex and disease matched controls of 3.55 + .11 P < .05). Six of these patients died from abdominal complications. In the 26 patients with serial urea kinetic studies, 4/11 patients in group IV died (low NPCR and low DI) (P < .05 compared to Group I, II or III). We conclude that urea kinetic modeling is predictive of outcome in those patients with presumed poor nutrition and inadequate dialysis and that abdominal catastrophes are more common in those patients with poor nutrition. Prospective interventional studies should be designed in an attempt to improve the poor outcome in this group of patients.