Synchrotron radiation can facilitate novel radiation therapy modalities such as microbeam radiation therapy (MRT) and high dose-rate synchrotron broad-beam radiation therapy (SBBR). Both of these ...modalities have unique physical properties that could be exploited for an improved therapeutic effect. While pre-clinical studies report promising normal tissue sparing phenomena, systematic toxicity data are still required. Our objective was to characterise the toxicity of SBBR and MRT and to calculate equivalent doses of conventional radiation therapy (CRT). A dose-escalation study was performed on C57BLJ/6 mice using total body and partial body irradiations. Dose-response curves and TD
values were subsequently calculated using PROBIT analysis. For SBBR at dose-rates of 37 to 41 Gy/s, we found no evidence of a normal tissue sparing effect relative to CRT. Our findings also show that the MRT valley dose, rather than the peak dose, best correlates with CRT doses for acute toxicity. Importantly, longer-term weight tracking of irradiated animals revealed more pronounced growth impairment following MRT compared to both SBBR and CRT. Overall, this study provides the first in vivo dose-equivalence data between MRT, SBBR and CRT and presents systematic toxicity data for a range of organs that can be used as a reference point for future pre-clinical work.
Gene expression varies markedly across the menstrual cycle and expression levels for many genes are under genetic control. We analyzed gene expression and mapped expression quantitative trait loci ...(eQTLs) in endometrial tissue samples from 229 women and then analyzed the overlap of endometrial eQTL signals with genomic regions associated with endometriosis and other reproductive traits. We observed a total of 45,923 cis-eQTLs for 417 unique genes and 2,968 trans-eQTLs affecting 82 unique genes. Two eQTLs were located in known risk regions for endometriosis including LINC00339 on chromosome 1 and VEZT on chromosome 12 and there was evidence for eQTLs that may be target genes in genomic regions associated with other reproductive diseases. Dynamic changes in expression of individual genes across cycle include alterations in both mean expression and transcriptional silencing. Significant effects of cycle stage on mean expression levels were observed for (2,427/15,262) probes with detectable expression in at least 90% of samples and for (2,877/9,626) probes expressed in some, but not all samples. Pathway analysis supports similar biological control of both altered expression levels and transcriptional silencing. Taken together, these data identify strong genetic effects on genes with diverse functions in human endometrium and provide a platform for better understanding genetic effects on endometrial-related pathologies.
Endocannabinoids mediate cellular functions and their activity is controlled by a complex system of enzymes, membrane receptors and transport molecules. Endocannabinoids are present in endometrium, a ...cyclical regenerative tissue requiring tightly regulated cellular mechanisms for maturation. The objective of this study was to investigate the gene expression of key elements involved in the endocannabinoid system across the menstrual cycle. RNA was isolated from endometrial tissue and genome-wide gene expression datasets were generated using RNA-sequencing. An a priori set of 70 genes associated with endocannabinoid system were selected from published literature. Gene expression across the menstrual cycle was analyzed using a moderated t test, corrected for multiple testing with Bonferroni's method. A total of 40 of the 70 genes were present in > 90% of the samples, and significant differential gene expression identified for 29 genes. We identified 4 distinct regulation patterns for synthesizing enzymes, as well as a distinct regulation pattern for degradations and transporting enzymes. This study charts the expression of endometrial endocannabinoid system genes across the menstrual cycle. Altered expression of genes that control endocannabinoid may allow fine control over endocannabinoid concentrations and their influence on cellular function, maturation and differentiation as the endometrium matures through the menstrual cycle.
High-dose synchrotron microbeam radiation therapy (MRT) can be effective at destroying tumors in animal models while causing very little damage to normal tissues. The aim of this study was to ...investigate the cellular processes behind this observation of potential clinical importance.
MRT was performed using a lattice of 25 mum-wide, planar, polychromatic, kilovoltage X-ray microbeams, with 200-microm peak separation. Inoculated EMT-6.5 tumor and normal mouse skin tissues were harvested at defined intervals post-MRT. Immunohistochemical detection of gamma-H2AX allowed precise localization of irradiated cells, which were also assessed for proliferation and apoptosis.
MRT significantly reduced tumor cell proliferation by 24 h post-irradiation (p = 0.002). An unexpected finding was that within 24 h of MRT, peak and valley irradiated zones were indistinguishable in tumors because of extensive cell migration between the zones. This was not seen in MRT-treated normal skin, which appeared to undergo a coordinated repair response. MRT elicited an increase in median survival times of EMT-6.5 and 67NR tumor-inoculated mice similar to that achieved with conventional radiotherapy, while causing markedly less normal tissue damage.
This study provides evidence of a differential response at a cellular level between normal and tumor tissues after synchrotron MRT.
Endometriosis, defined as estrogen-dependent lesions containing endometrial glands and stroma outside the uterus, is a chronic and often painful gynecological condition that affects 6% to 10% of ...reproductive age women. Endometriosis has estimated annual costs of US $12 419 per woman (approximately €9579), comprising one-third of the direct health care costs with two-thirds attributed to loss of productivity. Decreased quality of life is the most important predictor of direct health care and total costs. It has been estimated that there is a mean delay of 6.7 years between onset of symptoms and a surgical diagnosis of endometriosis, and each affected woman loses on average 10.8 hours of work weekly, mainly owing to reduced effectiveness while working. To encourage and facilitate research into this debilitating disease, a consensus workshop to define future directions for endometriosis research was held as part of the 11th World Congress on Endometriosis in September 2011 in Montpellier, France. The objective of this workshop was to review and update the endometriosis research priorities consensus statement developed following the 10th World Congress on Endometriosis in 2008.(1) A total of 56 recommendations for research have been developed, grouped under 6 subheadings: (1) diagnosis, (2) classification and prognosis, (3) clinical trials, treatment, and outcomes, (4) epidemiology, (5) pathophysiology, and (6) research policy. By producing this consensus international research priorities statement, it is the hope of the workshop participants that researchers will be encouraged to develop new interdisciplinary research proposals that will attract increased funding support for work on endometriosis.
Abstract
STUDY QUESTION
Do genetic effects regulate gene expression in human endometrium?
SUMMARY ANSWER
This study demonstrated strong genetic effects on endometrial gene expression and some ...evidence for genetic regulation of gene expression in a menstrual cycle stage-specific manner.
WHAT IS KNOWN ALREADY
Genetic effects on expression levels for many genes are tissue specific. Endometrial gene expression varies across menstrual cycle stages and between individuals, but there are limited data on genetic control of expression in endometrium.
STUDY DESIGN, SIZE, DURATION
We analysed genome-wide genotype and gene expression data to map cis expression quantitative trait loci (eQTL) in endometrium.
PARTICIPANTS/MATERIALS, SETTING, METHODS
We recruited 123 women of European ancestry. DNA samples from blood were genotyped on Illumina HumanCoreExome chips. Total RNA was extracted from endometrial tissues. Whole-transcriptome profiles were characterized using Illumina Human HT-12 v4.0 Expression Beadchips. We performed eQTL mapping with ~8 000 000 genotyped and imputed single nucleotide polymorphisms (SNPs) and 12 329 genes.
MAIN RESULTS AND THE ROLE OF CHANCE
We identified a total of 18 595 cis SNP-probe associations at a study-wide level of significance (P < 1 × 10−7), which correspond to independent eQTLs for 198 unique genes. The eQTLs with the largest effect in endometrial tissue were rs4902335 for CHURC1 (P = 1.05 × 10−32) and rs147253019 for ZP3 (P = 8.22 × 10−30). We further performed a context-specific eQTL analysis to investigate if genetic effects on gene expression regulation act in a menstrual cycle-specific manner. Interestingly, five cis-eQTLs were identified with a significant stage-by-genotype interaction. The strongest stage interaction was the eQTL for C10ORF33 (PYROXD2) with SNP rs2296438 (P = 2.0 × 10−4), where we observe a 2-fold difference in the average expression levels of heterozygous samples depending on the stage of the menstrual cycle.
LARGE SCALE DATA
The summary eQTL results are publicly available to browse or download.
LIMITATIONS, REASONS FOR CAUTION
A limitation of the present study was the relatively modest sample size. It was not powered to identify trans-eQTLs and larger sample sizes will also be needed to provide better power to detect cis-eQTLs and cycle stage-specific effects, given the substantial changes in expression across the menstrual cycle for many genes.
WIDER IMPLICATIONS OF THE FINDINGS
Identification of endometrial eQTLs provides a platform for better understanding genetic effects on endometriosis risk and other endometrial-related pathologies.
STUDY FUNDING/COMPETING INTEREST(S)
Funding for this work was provided by NHMRC Project Grants GNT1026033, GNT1049472, GNT1046880, GNT1050208, GNT1105321 and APP1083405. There are no competing interests.
High-dose synchrotron microbeam radiation therapy (MRT) has shown the potential to deliver improved outcomes over conventional broadbeam (BB) radiation therapy. To implement synchrotron MRT ...clinically for cancer treatment, it is necessary to undertake dose equivalence studies to identify MRT doses that give similar outcomes to BB treatments. To develop an in vitro approach to determine biological dose equivalence between MRT and BB using two different cell-based assays. The acute response of tumour and normal cell lines (EMT6.5, 4T1.2, NMuMG, EMT6.5ch, 4T1ch5, SaOS-2) to MRT (50-560 Gy) and BB (1.5-10 Gy) irradiation was investigated using clonogenic and real time cell impedance sensing (RT-CIS)/xCELLigence assays. MRT was performed using a lattice of 25 or 50 microm-wide planar, polychromatic kilovoltage X-ray microbeams with 200 microm peak separation. BB irradiations were performed using a Co.sup.60 teletherapy unit or a synchrotron radiation source. BB doses that would generate biological responses similar to MRT were calculated by data interpolation and verified by clonogenic and RT-CIS assays. For a given cell line, MRT equivalent BB doses identified by RT-CIS/xCELLigence were similar to those identified by clonogenic assays. Dose equivalence between MRT and BB were verified in vitro in two cell lines; EMT6.5ch and SaOS-2 by clonogenic assays and RT-CIS/xCELLigence. We found for example, that BB doses of 3.4±0.1 Gy and 4.40±0.04 Gy were radiobiologically equivalent to a peak, microbeam dose of 112 Gy using clonogenic and RT-CIS assays respectively on EMT6.5ch cells. Our data provides the first determination of biological dose equivalence between BB and MRT modalities for different cell lines and identifies RT-CIS/xCELLigence assays as a suitable substitute for clonogenic assays. These results will be useful for the safe selection of MRT doses for future veterinary and clinical trials.
Endometriosis is an estrogen-dependent disorder where endometrial tissue
forms lesions outside the uterus. Endometriosis affects an estimated 10% of women
in the reproductive-age group, rising to 30% ...to 50% in patients with infertility
and/or pain, with significant impact on their physical, mental, and social
well-being. There is no known cure, and most current medical treatments are not
suitable long term due to their side-effect profiles. Endometriosis has an
estimated annual cost in the United States of $18.8 to $22 billion (2002 figures).
Although endometriosis was first described more than 100 years ago, current
knowledge of its pathogenesis, spontaneous evolution, and the pathophysiology of
the related infertility and pelvic pain, remain unclear. A consensus workshop was
convened following the 10th World Congress on Endometriosis to establish
recommendations for priorities in endometriosis research. One major issue
identified as impacting on the capacity to undertake endometriosis research is the
need for multidisciplinary expertise. A total of 25 recommendations for research
have been developed, grouped under 5 subheadings: (1) diagnosis, (2)
classification and prognosis, (3) treatment and outcome, (4) epidemiology, and (5)
pathophysiology. Endometriosis research is underfunded relative to other diseases
with high health care burdens. This may be due to the practical difficulties of
developing competitive research proposals on a complex and poorly understood
disease, which affects only women. By producing this consensus international
research priorities statement it is the hope of the workshop participants that
researchers will be encouraged to develop new interdisciplinary research proposals
that will attract increased funding support for work on endometriosis.
Abstract
STUDY QUESTION
Are genetic effects on endometrial gene expression tissue specific and/or associated with reproductive traits and diseases?
SUMMARY ANSWER
Analyses of RNA-sequence data and ...individual genotype data from the endometrium identified novel and disease associated, genetic mechanisms regulating gene expression in the endometrium and showed evidence that these mechanisms are shared across biologically similar tissues.
WHAT IS KNOWN ALREADY
The endometrium is a complex tissue vital for female reproduction and is a hypothesized source of cells initiating endometriosis. Understanding genetic regulation specific to, and shared between, tissue types can aid the identification of genes involved in complex genetic diseases.
STUDY DESIGN, SIZE, DURATION
RNA-sequence and genotype data from 206 individuals was analysed and results were compared with large publicly available datasets.
PARTICIPANTS/MATERIALS, SETTING, METHODS
RNA-sequencing and genotype data from 206 endometrial samples was used to identify the influence of genetic variants on gene expression, via expression quantitative trait loci (eQTL) analysis and to compare these endometrial eQTLs with those in other tissues. To investigate the association between endometrial gene expression regulation and reproductive traits and diseases, we conducted a tissue enrichment analysis, transcriptome-wide association study (TWAS) and summary data-based Mendelian randomisation (SMR) analyses. Transcriptomic data was used to test differential gene expression between women with and without endometriosis.
MAIN RESULTS AND THE ROLE OF CHANCE
A tissue enrichment analysis with endometriosis genome-wide association study summary statistics showed that genes surrounding endometriosis risk loci were significantly enriched in reproductive tissues. A total of 444 sentinel cis-eQTLs (P < 2.57 × 10−9) and 30 trans-eQTLs (P < 4.65 × 10−13) were detected, including 327 novel cis-eQTLs in endometrium. A large proportion (85%) of endometrial eQTLs are present in other tissues. Genetic effects on endometrial gene expression were highly correlated with the genetic effects on reproductive (e.g. uterus, ovary) and digestive tissues (e.g. salivary gland, stomach), supporting a shared genetic regulation of gene expression in biologically similar tissues. The TWAS analysis indicated that gene expression at 39 loci is associated with endometriosis, including five known endometriosis risk loci. SMR analyses identified potential target genes pleiotropically or causally associated with reproductive traits and diseases including endometriosis. However, without taking account of genetic variants, a direct comparison between women with and without endometriosis showed no significant difference in endometrial gene expression.
LARGE SCALE DATA
The eQTL dataset generated in this study is available at http://reproductivegenomics.com.au/shiny/endo_eqtl_rna/. Additional datasets supporting the conclusions of this article are included within the article and the supplementary information files, or are available on reasonable request.
LIMITATIONS, REASONS FOR CAUTION
Data are derived from fresh tissue samples and expression levels are an average of expression from different cell types within the endometrium. Subtle cell-specifc expression changes may not be detected and differences in cell composition between samples and across the menstrual cycle will contribute to sample variability. Power to detect tissue specific eQTLs and differences between women with and without endometriosis was limited by the sample size in this study. The statistical approaches used in this study identify the likely gene targets for specific genetic risk factors, but not the functional mechanism by which changes in gene expression may influence disease risk.
WIDER IMPLICATIONS OF THE FINDINGS
Our results identify novel genetic variants that regulate gene expression in endometrium and the majority of these are shared across tissues. This allows analysis with large publicly available datasets to identify targets for female reproductive traits and diseases. Much larger studies will be required to identify genetic regulation of gene expression that will be specific to endometrium.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by the National Health and Medical Research Council (NHMRC) under project grants GNT1026033, GNT1049472, GNT1046880, GNT1050208, GNT1105321, GNT1083405 and GNT1107258. G.W.M is supported by a NHMRC Fellowship (GNT1078399). J.Y is supported by an ARC Fellowship (FT180100186). There are no competing interests.
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