Role of Epigenetics in Testicular Cancer Cell Drug Response

Testicular cancer is highly curable with the chemotherapeutic cisplatin. However, 15-20% of patients are resistant and succumb to their disease. Previously we showed that cisplatin refractory testicular cancer is highly sensitive to the DNA methyltransferase inhibitor, 5-aza deoxycytidine (5-aza). The mechanisms for cisplatin sensitivity and resistance in testicular cancer is unclear. If we can understand why testicular cancer is so curable, this knowledge could be applied to other cancer types. To better understand the mechanism of chemotherapy sensitivity and resistance in testicular cancer cells our lab generated two series of cell models, one resistant to cisplatin and the other resistant to 5- aza. We noted a reciprocal relationship between cisplatin and 5-aza resistance, with cisplatin resistance associated with increased sensitivity to 5-aza and 5-aza resistance associated with increased sensitivity to cisplatin. Transcriptomics revealed downregulation of the H3K27me3-mediated polycomb pathway in cisplatin resistant cells and upregulation of this pathway with 5-Aza resistance. To explore possible mechanisms for this reciprocal epigenetic modeling, the expression of the gene family responsible for histone lysine demethylation, the KDM family, was assessed by qPCR. Many KDM genes, including those responsible for H3K27me3 demethylation, were upregulated in cisplatin resistant cells and downregulated in 5-Aza resistant cells. Changes in KDM gene expression could explain, in part, globally altered levels of H3K27 methylation. We are performing genetic and pharmacologic studies to further validate a role for the H3K27me3 polycomb pathway in chemotherapeutic resistance, with the goal of devising novel therapies for testicular and potentially other cancers.Ope

Hypermethylation and global remodelling of DNA methylation is associated with acquired cisplatin resistance in testicular germ cell tumours

Testicular germ cell tumours (TGCTs) respond well to cisplatin-based therapy. However, cisplatin resistance and poor outcomes do occur. It has been suggested that a shift towards DNA hypermethylation mediates cisplatin resistance in TGCT cells, although there is little direct evidence to support this claim. Here we utilized a series of isogenic cisplatin-resistant cell models and observed a strong association between cisplatin resistance in TGCT cells and a net increase in global CpG and non-CpG DNA methylation spanning regulatory, intergenic, genic and repeat elements. Hypermethylated loci were significantly enriched for repressive DNA segments, CTCF and RAD21 sites and lamina associated domains, suggesting that global nuclear reorganization of chromatin structure occurred in resistant cells. Hypomethylated CpG loci were significantly enriched for EZH2 and SUZ12 binding and H3K27me3 sites. Integrative transcriptome and methylome analyses showed a strong negative correlation between gene promoter and CpG island methylation and gene expression in resistant cells and a weaker positive correlation between gene body methylation and gene expression. A bidirectional shift between gene promoter and gene body DNA methylation occurred within multiple genes that was associated with upregulation of polycomb targets and downregulation of tumour suppressor genes. These data support the hypothesis that global remodelling of DNA methylation is a key factor in mediating cisplatin hypersensitivity and chemoresistance of TGCTs and furthers the rationale for hypomethylation therapy for refractory TGCT patients

Surface pretreatments for medical application of adhesion

Medical implants and prostheses (artificial hips, tendono- and ligament plasties) usually are multi-component systems that may be machined from one of three material classes: metals, plastics and ceramics. Typically, the body-sided bonding element is bone. The purpose of this contribution is to describe developments carried out to optimize the techniques , connecting prosthesis to bone, to be joined by an adhesive bone cement at their interface. Although bonding of organic polymers to inorganic or organic surfaces and to bone has a long history, there remains a serious obstacle in realizing long-term high-bonding strengths in the in vivo body environment of ever present high humidity. Therefore, different pretreatments, individually adapted to the actual combination of materials, are needed to assure long term adhesive strength and stability against hydrolysis. This pretreatment for metal alloys may be silica layering; for PE-plastics, a specific plasma activation; and for bone, amphiphilic layering systems such that the hydrophilic properties of bone become better adapted to the hydrophobic properties of the bone cement. Amphiphilic layering systems are related to those developed in dentistry for dentine bonding. Specific pretreatment can significantly increase bond strengths, particularly after long term immersion in water under conditions similar to those in the human body. The bond strength between bone and plastic for example can be increased by a factor approaching 50 (pealing work increasing from 30 N/m to 1500 N/m). This review article summarizes the multi-disciplined subject of adhesion and adhesives, considering the technology involved in the formation and mechanical performance of adhesives joints inside the human body

Drug dosing during pregnancy—opportunities for physiologically based pharmacokinetic models

Drugs can have harmful effects on the embryo or the fetus at any point during pregnancy. Not all the damaging effects of intrauterine exposure to drugs are obvious at birth, some may only manifest later in life. Thus, drugs should be prescribed in pregnancy only if the expected benefit to the mother is thought to be greater than the risk to the fetus. Dosing of drugs during pregnancy is often empirically determined and based upon evidence from studies of non-pregnant subjects, which may lead to suboptimal dosing, particularly during the third trimester. This review collates examples of drugs with known recommendations for dose adjustment during pregnancy, in addition to providing an example of the potential use of PBPK models in dose adjustment recommendation during pregnancy within the context of drug-drug interactions. For many drugs, such as antidepressants and antiretroviral drugs, dose adjustment has been recommended based on pharmacokinetic studies demonstrating a reduction in drug concentrations. However, there is relatively limited (and sometimes inconsistent) information regarding the clinical impact of these pharmacokinetic changes during pregnancy and the effect of subsequent dose adjustments. Examples of using pregnancy PBPK models to predict feto-maternal drug exposures and their applications to facilitate and guide dose assessment throughout gestation are discussed

Tyrannosaurus Osborn 1905

Questions have been raised about the methods used and conclusions reached in this Letter 1. In revisiting the work, we realized that we did not provide sufficient methodological details regarding the many steps that went into our growth curve analysis, although the main conclusions of the paper were not affected. we regret any misunder- standing that might have resulted. A detailed rationale is available in the Supplementary Methods and Discussion of this Corrigendum and the source data are provided as Supplementary Data. we thank N. Myhrvold for bringing these issues to our attention. In our reanalysis we found a minor translational mistake affect- ing the reported growth for Tyrannosaurus, which does not appear to have contributed to Myhrvold’s concerns (details can be found in the Supplementary Methods and Discussion to this Corrigendum.) The correct equation is Mass = (5,649/[1 +e −0.55(Age−16.2)]) + 5. This produces a maximal growth rate of 758 kg yr −1 using points closely bounding the inflection point and 774 kg yr −1 using the instantaneous equation. The reported value was 767 kg yr −1. This slight discrepancy (see the corrected Fig. 2 in the Supplementary Methods and Discussion to this Corrigendum) does not compromise our conclusion that Tyrannosaurus primarily achieved gigantism through evolutionary acceleration.Published as part of Gregory M. Erickson, Peter J. Makovicky, Philip J. Currie, Mark A. Norell, Scott A. Yerby & Christopher A. Brochu, 2016, Corrigendum: Gigantism and comparative life-history parameters of tyrannosaurid dinosaurs, pp. 538 in Nature 531 on page 1, DOI: 10.1038/nature16487, http://zenodo.org/record/373650

Tyrannosaurus rex

Stemming from more than a century of investigation, considerable understanding of tyrannosaurid osteology 4, myology 5, neurology 6, behaviour 7, 8, physiology 3, 9, physical capabilities 10, 11 and phylogeny 12, 13 have been gained. Lacking are empirical data on tyrannosaurid life history such as growth rates, longevity and somatic maturity (adult size) from which the developmental possibilities for how T. rex attained gigantism can be formally tested. Recent advances in techniques for determining the ages at death of dinosaurs by using skeletal growth line counts 3, 14, coupled with developmental size estimates 3, make quantitative growth-curve reconstructions for dinosaurs feasible. These methods have been used to study growth rates in two small theropods, a small and a large ornithischian and a medium-sized and a gigantic sauropodomorph 3. These data were used to derive a regression of body mass against growth rate and to generalize broadly about non-avian dinosaur growth 3. However, because of the phylogenetically disparate nature of these data (that is, none are close outgroups to one another) it has not been possible to use them to infer how specific cases of size change occurred within dinosaurian sub-clades such as the Tyrannosauridae. Such an understanding requires multi-species sampling at low taxonomic levels (that is, among closely related species) and access to growth series spanning juvenile through adult stages, a rarity among extinct dinosaurs 15. Furthermore, it requires the capacity to account for growth line losses due to medullar cavity hollowing and cortical remodelling 16, two processes that are pervasive in the major weight-bearing bones from large theropods such as tyrannosaurids. The sampling problem has been overcome in North American tyrannosaurids. A flurry of recent discoveries has greatly increased the number of substantially complete specimens representing various growth stages available for study. For example, more than 30 T. rex specimens are known 4, 17, compared with only 11 reported in 1993 (ref. 18; see Supplementary Information). Recent work has broadened the developmental representation of these animals by showing that several purported ‘dwarf’ tyrannosaur species are juveniles of larger, previously recognized forms such as T. rex 12, 13, 19, 20. Finally, preliminary analyses for this research revealed that several non-weight-bearing bones in tyrannosaurids (for example pubes, fibulae, ribs, gastralia and postorbitals) did not develop hollow medullar cavities and showed negligible intracortical remodelling during their entire life history (Fig. 1). Like major long bones, these elements are effective for assessing longevity in living reptiles (Fig. 1) 21, 22 and hence provide a viable alternative method for determining the age at death of extinct reptiles such as tyrannosaurids. Here we exploit these findings to determine the pattern of growth in T. rex and three of its close tyrannosaurid relatives. We then use character optimization methods 23 to infer how T. rex attained giant proportions among tyrannosaurids. Finally, this new evidence is used to further our understanding of tyrannosaurid biology. In performing these analyses, we sampled several amedullar bones from adolescent, juvenile, sub-adult and adult representatives of the North American Late Cretaceous tyrannosaurids Albertosaurus sarcophagus, Gorgosaurus libratus, Daspletosaurus torosus and T. rex. Longevity in each of the 20 specimens was assessed from line counts in histological sections by using polarizing, dissecting and reflected-light microscopy (Fig. 1) 3, 14. Conservative estimates of body mass (see Supplementary Information) were made by using femoral circumference measures 24. Longevity and size data were plotted and least-squares regression was used to determine the first empirical growth curves for tyrannosaurids 3. The length and timing of the various developmental stages and the maximal growth rates for each taxon were compared 25. The results were examined in an evolutionary context 23 by using two competing phylogenetic hypotheses for the Tyrannosauridae 12, 13. Sampled longevities for T. rex ranged from 2 to 28 years and corresponding body mass estimates ranged from 29.9 to 5,654 kg (Table 1). The transition to somatic maturity in this taxon seems to have begun at about 18.5 years of age (Fig. 2). At least one individual (exemplified by FMNH (The Field Museum) PR 2081), showed evidence for prolonged senescence in the form of conspicuously narrow pericortical growth-line spacing (Fig. 1). Maximal growth rates in T. rex were 2.07 kg d ‾ 1 and such exponential rates were maintained for about 4 years (Fig. 2). The longevity estimates for T. rex outgroups ranged from 2 to 24 years and corresponding body sizes spanned from 50.3 to 1,791 kg (Table 1). Somatic maturity occurred at between 14 and 16 years in these taxa (Fig. 2). Like T. rex, at least some exceptionally large individuals of A. sarcophagus and D. torosus showed narrow pericortical growth-line spacing indicative of the onset of senescence. The maximal growth rates for the three smaller tyrannosaurid taxa ranged from 0.31 to 0.48 kg d ‾ 1 ; such exponential stage rates were also maintained for about 4 years (Fig. 2). Optimization of growth rates onto the two current phylogenetic hypotheses of tyrannosaurid relationships suggests that a 1.5-fold acceleration in maximal growth rate might diagnose Tyrannosaurinae (the clade comprising Daspletosaurus and Tyrannosaurus 13, 19, Fig. 2). A second substantial increase in growth rate optimizes as a physiological autapomorphy of Tyrannosaurus irrespective of phylogenetic hypothesis and optimization criterion. T. rex is notable for its great size, which is at least 15-fold greater than the largest living terrestrial carnivorous animals today and second only to Giganotosaurus 26 among theropod dinosaurs. How did it attain such great proportions within the Tyrannosauridae? From the two competing hypotheses of tyrannosaurid phylogeny it is most parsimonious to conclude that T. rex acquired the majority of its giant proportions after diverging from the common ancestor of itself and D. torosus, a species with an optimized body mass of about 1,800 kg. Direct comparison between the tyrannosaurid growth curves shows that the transition to the exponential and stationary phases of development occurred about 2–4 years later in T. rex (Fig. 2). However, such temporal post-displacement had little to do with the evolution of its gigantism because the exponential stage, during which most body size is accrued 25, was not extended beyond the ancestral, 4-year condition observed in other tyrannosaurids. Rather, the key developmental modification that propelled T. rex to giant proportions was primarily through evolutionary acceleration in the exponential stage growth rate and the transition zones bounding it. This is reflected in the regions of maximal slope on the growth curves depicted in Fig. 2 and holds true regardless of which evolutionary hypothesis is correct and how the maximum growth rates are optimized. Notably, this method of attaining gigantism contrasts with that in the largest crocodilians and lizards, where ancestral growth rates were retained and the exponential stages lengthened 27. How other dinosaurs attained gigantism within their respective sub-clades will serve as an interesting line of inquiry in the future. Does the same pattern of acceleratory growth seen here characterize the means by which all or most members of the Dinosauria attained great size? ................................................................................................................................................................................................................................................................................................................................................................... FMNH, The Field Museum; RTMP, Royal Tyrrell Museum of Palaeontology; ICM, Indianapolis Children’s Museum; LACM, Los Angeles County Museum; AMNH, American Museum of Natural History; USNM, United States National Museum. R, rib; G, gastralia; F, fibula; P, pubis; C, dermal skull bones; OLB, other long bones; est., estimated; EFS, external fundamental system 16. Besides revealing how the evolution of T. rex gigantism was obtained, the data garnered here provide for a more comprehensive understanding of tyrannosaurid biology. For instance the presence of thin, tightly packed growth lines late in development (Fig. 1) shows that these animals, like nearly all (if not all) dinosaurs, had determinate growth 3, 14. They would not have gained an appreciably greater size than the largest specimens studied here and could spend nearly 30% of their lives as full-grown adults (Fig. 2). In addition, the maximal growth rates for these tyrannosaurid species are only 33–52% of the rates expected for non-avian dinosaurs of their size when compared with the more broadly sampled data of Erickson et al. 3. This provides the first evidence of its kind pointing to major differences in whole body growth rates among a non-avian dinosaur sub-clade. Such findings are not unexpected because similar patterns (for example primates within Eutheria) occur within living vertebrate groups 28. Our findings also have a bearing on the biomechanical capacities of tyrannosaurids. T. rex ’s capacity for ‘fast running’ was biomechanially infeasible after a body mass of about 1,000 kg was attained 11. This corresponds to a juvenile-sized animal just 13 years of age on the basis of our longevity data and conservative estimates of body mass (Fig. 2). If we assume that the same relationship held true for the smaller tyrannosaurid species studied here, such locomotory limitations would not have emerged until these animals were much closer to adult size (Fig. 2). Finally, a glimpse into the potential population age structure for a dinosaur is also afforded from these data. Currie 7 has described a catastrophic death assemblage consisting of eight or nine A. sarcophagus specimens thought to represent an entire pack or a subset of one. On the basis of femoral lengths, the age and developmental stage of each animal can now be estimated. The group seems to have consisted of two or three older adults ~21 or more years of age, one ~17-year-old young adult, four ~12–17- year-old sub-adults that were undergoing exponential stage growth at the time of death, and one ~10-year-old juvenile that was beginning the transition to exponential stage growth. A reopening of the site has revealed at least one more specimen (RTMP (Royal Tyrrell Museum of Palaeontology) 2002.45.46) shown here to be only 2 years old (Table 1). This indicates that A. sarcophagus groups, whether temporary or permanent, might have been composed of individuals spanning the age spectrum from adolescents to very old, senescent adults, a finding consistent with trackway evidence for other theropod dinosaurs 7.Published as part of Gregory M. Erickson, Peter J. Makovicky, Philip J. Currie, Mark A. Norell, Scott A. Yerby & Christopher A. Brochu, 2004, Gigantism and comparative life-history parameters of tyrannosaurid dinosaurs, pp. 772-775 in Nature 430 on pages 772-774, DOI: 10.1038/nature02699, http://zenodo.org/record/373647