Imprinted loci in domestic livestock species as epigenomic targets for artificial selection of complex traits.

The phenomenon of genomic imprinting, whereby a subset of mammalian genes display parent-of-origin-specific monoallelic expression, is one of the most active areas of epigenetics research. Over the past two decades, more than 100 imprinted mammalian genes have been identified, while considerable advances have been made in elucidating the molecular mechanisms governing imprinting. These studies have helped to unravel the epigenome--a separate layer of regulatory information contained in eukaryotic chromosomes that influences gene expression and phenotypes without involving changes to the underlying DNA sequence. Although most studies of genomic imprinting in mammals have focussed on mouse models or human biomedical disorders, there is burgeoning interest in the phenotypic effects of imprinted genes in domestic livestock species. In particular, research has focused on imprinted genes influencing foetal growth and development, which are associated with economically important production traits in cattle, sheep and pigs. These findings, when coupled with the data emerging from the various different livestock genome projects, have major implications for the future of animal breeding, health and management. Here, we review current scientific knowledge regarding genomic imprinting in livestock species and evaluate how this information can be used in modern livestock improvement programmes.

PHLDA2 is an imprinted gene in cattle.

Genomic imprinting is an epigenetic non-Mendelian phenomenon found predominantly in placental mammals. Imprinted genes display differential expression in the offspring depending on whether the gene is maternally or paternally inherited. Currently, some 100 imprinted genes have been reported in mammals, and while some of these genes are imprinted across most mammalian species, others have been shown to be imprinted in only a few species. The PHLDA2 gene that codes for a pleckstrin homology-like domain, family A (member 2), protein has to date been shown to be a maternally expressed imprinted gene in humans, mice and pigs. Genes subject to imprinting can have major effects on mammalian growth, development and disease. For instance, disruption of imprinted genes can lead to aberrant growth syndromes in cloned domestic mammals, and it has been demonstrated that PHLDA2 mRNA expression levels are aberrant in the placenta of somatic clones of cattle. In this study, we demonstrate that PHLDA2 is expressed across a range of cattle foetal tissues and stages and provide the first evidence that PHLDA2 is a monoallelically expressed imprinted gene in cattle foetal tissues, and also in the bovine placenta.

Single nucleotide polymorphisms in the imprinted bovine insulin-like growth factor 2 receptor gene (IGF2R) are associated with body size traits in Irish Holstein-Friesian cattle.

The regulation of the bioavailability of insulin-like growth factors (IGFs) is critical for normal mammalian growth and development. The imprinted insulin-like growth factor 2 receptor gene (IGF2R) encodes a transmembrane protein receptor that acts to sequester and degrade excess circulating insulin-like growth factor 2 (IGF-II) - a potent foetal mitogen - and is considered an important inhibitor of growth. Consequently, IGF2R may serve as a candidate gene underlying important growth- and body-related quantitative traits in domestic mammalian livestock. In this study, we have quantified genotype-phenotype associations between three previously validated intronic bovine IGF2R single nucleotide polymorphisms (SNPs) (IGF2R:g.64614T>C, IGF2R:g.65037T>C and IGF2R:g.86262C>T) and a range of performance traits in 848 progeny-tested Irish Holstein-Friesian artificial insemination sires. Notably, all three polymorphisms analysed were associated (P ≤ 0.05) with at least one of a number of performance traits related to animal body size: angularity, body depth, chest width, rump width, and animal stature. In addition, the C-to-T transition at the IGF2R:g.65037T>C polymorphism was positively associated with cow carcass weight and angularity. Correction for multiple testing resulted in the retention of two genotype-phenotype associations (animal stature and rump width). None of the SNPs analysed were associated with any of the milk traits examined. Analysis of pairwise r(2) measures of linkage disequilibrium between all three assayed SNPs ranged between 0.41 and 0.79, suggesting that some of the observed SNP associations with performance may be independent. To our knowledge, this is one of the first studies demonstrating associations between IGF2R polymorphisms and growth- and body-related traits in cattle. These results also support the increasing body of evidence that imprinted genes harbour polymorphisms that contribute to heritable variation in phenotypic traits in domestic livestock species.

COX2 Effects on endometrial carcinomas progression.

Uterine corpus cancer is one of the most prevalent gynecologic malignancies, among which endometrial cancers (EC) represent about 90 %. Despite the proven predictive value of several immunohistochemical markers, there remains a need to identify new indicators of EC progression and exploit them for therapeutic purposes. Potential candidates with diagnostic and therapeutic efficacy include cyclooxygenases (COXs). We studied 50 EC cases: 30 endometrioid (EEC), 10 serous (SEC), 10 clear-cell endometrial carcinomas (CCEC) and 10 cases of normal endometrial tissues. We investigated the expression of COX2, ER, PR, Ki-67, EGFR, p53, Bcl-2, VEGF, MMP1, CD31, and CD163 immunohistochemically. COX2 levels in EC tissue are elevated compared to the normal endometrium and depend on tumour histological features and differentiation. Elevated COX2 leads to increased tumour cell proliferation, apoptosis inhibition, increased VEGF expression, microvessel density, and M2 macrophage infiltration, and inhibition of PR expression. ER, EGFR, and MMP1 levels are unaffected by COX2, whose levels are independent of patient age and FIGO stage.

How Useful is COVID-19 Antibody Testing - A Current Assessment for Oncologists.

The coronavirus disease 2019 (COVID-19) pandemic due to infection by a new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has seriously disrupted the provision of oncology services and their uptake. Antibody testing, both at an individual level and of populations, has been widely viewed to be a key activity for guiding the options for treatment of high-risk individuals, as well as the implementation of safe control of infection measures. Ideally, the detection of a specific antibody should signify that all individuals tested have been infected by SARS-CoV-2 and that in the case of specific IgG that they are immune to further infection. This would enable SARS-CoV-2-infected individuals to be appropriately managed and healthcare workers shown to be immune to return to work where they would no longer pose a risk to their patients or be at risk themselves. Unfortunately, this is not the case for COVID-19, where it has been shown that immunity may not be protective, and seroconversion delayed or absent. The variability in antibody test performance, particularly that of lateral flow assays, has caused confusion for the public and healthcare professions alike. Many antibody test devices have been made available without independent evaluations and these may lack both adequate sensitivity and specificity. This review seeks to educate healthcare workers, particularly those working in oncology, of the current benefits and limitations of SARS-CoV-2 antibody testing.

High concordance of bovine single nucleotide polymorphism genotypes generated using two independent genotyping strategies.

Single nucleotide polymorphisms (SNPs) represent the most common form of DNA sequence variation in mammalian livestock genomes. While the past decade has witnessed major advances in SNP genotyping technologies, genotyping errors caused, in part, by the biochemistry underlying the genotyping platform used, can occur. These errors can distort project results and conclusions and can result in incorrect decisions in animal management and breeding programs; hence, SNP genotype calls must be accurate and reliable. In this study, 263 Bos spp. samples were genotyped commercially for a total of 16 SNPs. Of the total possible 4,208 SNP genotypes, 4,179 SNP genotypes were generated, yielding a genotype call rate of 99.31% (standard deviation ± 0.93%). Between 110 and 263 samples were subsequently re-genotyped by us for all 16 markers using a custom-designed SNP genotyping platform, and of the possible 3,819 genotypes a total of 3,768 genotypes were generated (98.70% genotype call rate, SD ± 1.89%). A total of 3,744 duplicate genotypes were generated for both genotyping platforms, and comparison of the genotype calls for both methods revealed 3,741 concordant SNP genotype call rates (99.92% SNP genotype concordance rate). These data indicate that both genotyping methods used can provide livestock geneticists with reliable, reproducible SNP genotypic data for in-depth statistical analysis.

Making enhanced recovery the norm not the exception.

BACKGROUND: Enhanced Recovery After Surgery ("STAAR" in our system) is multimodal care focused on the reduction of physiological and psychological stress. While enhanced recovery is well established in colorectal surgery, and there is evidence for effectiveness in other surgical disciplines, to date widespread use is limited. METHOD: We implemented a Lean process that, within 12 months, expanded STAAR to 13 surgical services lines involving >130 surgeons, and impacting the care of >6000 surgical patients/year. RESULTS: Implementation involved educational and administrative meetings (279 in the first 6 months) and rounding. Use of STAAR was defined as >60% compliance. LOS was reduced up to 40%, mortality index and transfusion decreased 67% and 23% respectively. Case mix index increased 17%. Readmission rates, infections, ER visits were not increased. CONCLUSION: Using a Lean process focused on value, STAAR protocols became the standard rather than the exception. Time investment by senior surgical leadership was extensive.

Overcoming challenges of cancer treatment programmes in developing countries: a sustainable breast cancer initiative in Ethiopia.

The incidence of breast cancer is rising in many developing countries. Here we describe a programme to improve the support infrastructure for the management of patients with breast cancer in Addis Ababa, Ethiopia. Tamoxifen, a cheap, oral, yet effective, anti-cancer agent was made available freely to encourage staff and patients to follow well-defined, but achievable, protocols of care. Mammography, improved histopathological review, tissue hormone receptor assays, agreed treatment algorithms with a cycle of continuous audit of over 250 patients and cross-departmental patient management groups led to a considerable improvement in the management of breast cancer patients in a single institution. Aspects of this programme are now being extended to other regional hospitals in Ethiopia. Fairly limited investments in programmes for cancer can stimulate considerable improvements in the overall approach to malignant disease by encouraging a positive approach, even in very low resource environments.

Carbachol- and elevated Ca(2+)-induced translocation of functionally active protein kinase C to the brush border of rabbit ileal Na+ absorbing cells.

Protein kinase C is involved in mediating the effects of elevated Ca2+ in ileal villus Na+ absorbing cells to inhibit NaCl absorption. The present studies were undertaken to understand the mechanism by which this occurs. The effects of carbachol and the calcium ionophore A23187, agents which elevate intracellular Ca2+ and inhibit NaCl absorption in ileal villus cells, were studied. Carbachol treatment of villus cells caused a rapid decrease in protein kinase C activity in cytosol, with an accompanying increase in microvillus membrane C kinase. Exposure of the villus cells to calcium ionophore also caused a quantitatively similar decrease in cytosol C kinase and increase in C kinase activity in the microvillus membrane. This increase caused by carbachol and Ca2+ ionophore was specific for the microvillus membrane. In fact, 30 s and 10 min after exposure of the cells to carbachol, basolateral membrane protein kinase C decreased, in a time-dependent manner; whereas 10 min of Ca2+ ionophore exposure did not alter basolateral C kinase. Exposure of villus cells to Ca2+ ionophore or carbachol caused similar increases in microvillus membrane diacylglycerol content. As judged by the ability to inhibit Na+/H+ exchange measured in ileal villus cell brush border membrane vesicles, the protein kinase C which translocated to the microvillus membrane was functionally significant. Inhibition of Na+/H+ exchange required ATP and was reversed by the protein kinase C antagonist H-7. In conclusion, the effect of carbachol and Ca2+ ionophore in regulation of ileal NaCl absorption is associated with an increase in microvillus membrane diacylglycerol content and functionally active protein kinase C. The effects of both carbachol and Ca2+ ionophore are different on brush border and basolateral membrane distribution of protein kinase C.

Heavy metals effect on breast cancer progression.

BACKGROUND: Breast cancer is the most frequent localization of malignant process in American women and women of European countries. To date it is not possible to control the morbidity growth due to lack of effective ways of primary prevention. Comparing the incidence of breast cancer in developed countries with the countries of Asia and Africa, there is the fact of population predominance lesion in more urbanized countries. This suggests that the environment along with other factors, occupies a significant place in the initiation and progression of breast neoplasia. The impressive rates of industrial development led to the pollution of soil, surface water and, as a consequence, food by heavy metal salts.The purposes of this paper are as follows: the chemical composition determination of neoplastic breast tissue, evaluation of the DNA methylation level, study of prognostic-important receptors expression in the breast cancer cells, establishing linkages between all the derived indicators. METHODS: In our study we used the following methods: studying of the chemical composition of breast cancer tissue by atomic absorption spectrophotometry and energy-dispersion spectrometer; іmmunohistochemical study of ER, PR, HER2/neu, p53, Ki-67, E-cadherin and MGMT receptors; DNA extraction and investigation by oscillating infrared spectroscopy method. RESULTS: The total amount of heavy metals in breast cancer tissue ranged from 51.21 × 10-3 to 84.86 × 10-3 µg/kg. We have got the following results: the growth of heavy metals in neoplastic tissue is accompanied with the increase of HER2/neu, p53, Ki-67, MGMT expression and decrease of ER and PR expression. The increment of pathological DNA methylation is accompanied with the increasing amount of heavy metals in tumor tissue. CONCLUSIONS: Heavy metals through different pathogenetic links stimulate the progression of breast cancer and reduce its sensitivity to treatment. DNA of tumor tissue has a different level of methylation which changes with the amount of heavy metals in cancer cells. This is displayed on the synthesis of prognostically important receptors in neoplastic tissue.

Measurements of blood flow and exchanging water space in breast tumors using positron emission tomography: a rapid and noninvasive dynamic method.

A noninvasive dynamic method for the measurement of blood flow, using 15O-labeled water and positron emission tomography, has been developed and used to study 20 patients with breast carcinoma. The mean tumor flow was 29.8 +/- 17.0 (SE) ml/dl/min of tissue, while normal breast flow was 5.6 +/- 1.4 ml/dl/min of tissue. The exchanging water space of tissue known as the volume of distribution of the tracer (Vd) was also derived. This is defined as the volume of water in tissue that exchanges with a unit volume of water in arterial blood during the period of the study (7 min). The mean tumor Vd was 0.56 +/- 0.15 ml/ml while normal breast Vd was 0.14 +/- 0.05 ml/ml. The low value in normal breast is partly due to the high fat content of the tissue. The mean flow per unit of exchangeable volume was similar in tumor (52.8 +/- 22.0) and normal breast tissue (45.2 +/- 20.0). This suggests that the major discrepancy seen in measured values of flow between breast tumors and normal breast principally reflects the different composition of the two tissues. This method is rapid and suited for studying the reactivity of human tumor vasculature, so extending studies are being performed on animal tumors.

Germ cell-like telomeric length homeostasis in nonseminomatous testicular germ cell tumors.

Telomere maintenance plays an important role in cell proliferation and tumor survival. Human male germ cells, which carry long telomeres and express telomerase, give rise to a highly heterogeneous group of malignant tumors. We compared telomeric length and telomerase activity between two major histological types of primary testicular germ cell tumors. Fifteen out of 16 seminoma samples revealed telomeric restriction fragment (TRF) length below 13 kb; the remaining seminoma showed a major TRF fraction of 18 kb and a distinct minor fraction of above 23 kb length. In contrast, all 13 samples from nonseminomas showed TRF length >/=23 kb, which is similar to that reported in human sperm. Nine out of 11 seminoma specimens and six out of seven nonseminomas studied showed moderate to high telomerase activity, the only telomerase-negative nonseminoma being pure mature teratoma. These results indicate to a major difference in telomeric length between seminomas and nonseminomas, which is apparently unrelated to the presence of telomerase activity, and suggest a germline-like homeostasis of telomeric length is preserved in human nonseminomas. Oncogene (2000) 19, 4075 - 4078.

Genetic prodrug activation therapy for breast cancer: A phase I clinical trial of erbB-2-directed suicide gene expression.

PURPOSE: This trial was designed to test the safety and efficacy of a tumor-specific genetic prodrug activation therapy targeted by use of the human erbB-2 gene promoter. The erbB-2 oncogene is overexpressed in approximately 20% of cases of breast cancer and is associated with poor prognosis. PATIENTS AND METHODS: Twelve breast cancer patients received transcriptionally targeted gene therapy in a phase I clinical trial using direct intratumoral injection of plasmid construct combined with systemic administration of prodrug. The genetic prodrug activation therapy is specifically targeted to erbB-2-overexpressing breast cancer cells by use of a therapeutic cassette that contains the Escherichia coli cytosine deaminase gene driven by the tumor-specific erbB-2 promoter, thus allowing activation of fluorocytosine to the active cytotoxic fluorouracil only within tumor cells that express the oncogene. RESULTS: The approach was shown to be safe and to result in targeted gene expression in up to 90% of cases. Using a number of different assays, we demonstrated that significant levels of expression of the suicide gene were specifically restricted to erbB-2-positive tumor cells, confirming the selectivity of the approach. CONCLUSION: The results of this study, the first targeted gene therapy for breast cancer and the first to use the cytosine deaminase system in human subjects, are encouraging for the development of genetic prodrug activation therapies that exploit the transcriptional profile of cancer cells.

Developing a global strategy for cancer.

Over the next 25 years there will be a dramatic increase in the number of people developing cancer. Globally, 10 million new cancer patients are diagnosed each year and this will be 20 million by the year 2020. Cancer is now the public's most feared disease. Billions of dollars are spent annually on cancer research by the drug industry, cancer charities and governments, but a cure for cancer appears elusive. And yet, we are in the midst of a revolution in our ability to image parts of the body, painlessly and in fine detail. We also now understand the intricate workings of the human genome-ultimately responsible for controlling all biological processes in health and disease. By the year 2003 the entire DNA sequence of the human genome will be determined. Powerful computer networks will allow detailed comparisons of genetic structure, so identifying new risk factors. Gene chips will detect minute code changes of considerable relevance. Novel screening technologies will allow us to detect just a few cancer cells in a patient. Robotically guided destructive processes will target abnormal cells in patients long before any cancer-related symptoms develop. And all this is likely by the first quarter of the next century. How are people, society and healthcare systems going to deal with these tremendous technological advances for cancer? Detailed information will be available in every home through easily understandable computer links. Choices now made by professionals will be equally understandable to all. Public education on health will be strengthened allowing a more critical and realistic assessment of media reports on new technologies. But as technology becomes more complex, the gap between the global rich and poor could widen. The export of unhealthy lifestyles--cigarette smoking, dietary habits and sedentary occupations will disproportionately increase cancer in many developing countries, which can least afford the treatment costs. The WHO Cancer Programme is developing a strategy to identify priorities in cancer prevention, detection and treatment in a wide range of epidemiological and economic settings.

Developing a global strategy for cancer.

Over the next 25 years there will be a dramatic increase in the number of people developing cancer. Globally, 10 million new cancer patients are diagnosed each year and this will be 20 million by the year 2020. Cancer is now the public's most feared disease. Billions of dollars are spent annually on cancer research by the drug industry, cancer charities and governments, but a cure for cancer appears elusive. And yet, we are in the midst of a revolution in our ability to image parts of the body, painlessly and in fine detail. We also now understand the intricate workings of the human genome--ultimately responsible for controlling all biological processes in health and disease. By the year 2003 the entire DNA sequence of the human genome will be determined. Powerful computer networks will allow detailed comparisons of genetic structure, so identifying new risk factors. Gene chips will detect minute code changes of considerable relevance. Novel screening technologies will allow us to detect just a few cancer cells in a patient. Robotically guided destructive processes will target abnormal cells in patients long before any cancer-related symptoms develop. And all this is likely by the first quarter of the next century. How are people, society and healthcare systems going to deal with these tremendous technological advances for cancer? Detailed information will be available in every home through easily understandable computer links. Choices now made by professionals will be equally understandable to all. Public education on health will be strengthened allowing a more critical and realistic assessment of media reports on new technologies. But as technology becomes more complex, the gap between the global rich and poor could widen. The export of unhealthy lifestyles--cigarette smoking, dietary habits and sedentary occupations will disproportionately increase cancer in many developing countries, which can least afford the treatment costs. The WHO Cancer Programme is developing a strategy to identify priorities in cancer prevention, detection and treatment in a wide range of epidemiological and economic settings.

A simultaneous flow cytometric assay for c-myc oncoprotein and DNA in nuclei from paraffin embedded material.

A simultaneous flow cytometric assay for the c-myc oncoprotein and DNA in nuclei extracted from archival paraffin wax embedded clinical biopsies is presented. The nuclei were extracted by pepsin digestion after dewaxing 20 micron sections. The c-myc oncoprotein was probed with a mouse monoclonal antibody. This was raised against a synthetic peptide corresponding to a hydrophilic region of the protein predicted from the amino acid sequence. The technique is illustrated with biopsies from patients with testicular cancer and with benign and malignant neoplasms of the colon.

Monoclonal antibodies in oncology.

Monoclonal antibodies (MCAs) can be used to differentiate between normal and neoplastic cells and thus exploited for diagnostic and, ultimately, therapeutic gain. The evidence for the existence of human tumour antigens is reviewed. Several areas of diagnosis are already benefiting from the application of the monoclonal technology. Immunohistology can help the pathologist with difficult diagnostic problems. New classifications of lymphoma and leukaemia can be based on specific surface molecules. Similarly, the detection of shed tumour antigens is already established as part of the routine assessment of many patients with common solid tumours. Isotopically labelled monoclonal antibodies have been used to localise primary and metastatic tumours. The use of antibodies in this way is not only a promising diagnostic tool but also the first step in studying the possibility of arming antibodies to provide therapeutic agents. Such trials are currently in progress.

Oncogene expression in cholangiocarcinoma and in normal hepatic development.

The expression of the proteins encoded by the ras, myc, and erb B-2 oncogenes was examined in 63 paraffin-embedded human cholangiocarcinomas of Thai and English origin using immunohistochemistry. The observed distributions were compared with oncogene expression in a series of human hepatocellular carcinomas. In an attempt to relate expression of these three oncogenes to specific stages of normal tissue differentiation, tissue sections of normal fetal, infant, and adult human livers were also examined. Of 63 cholangiocarcinomas, 59 (95%) expressed p62 c-myc, 47 (75%) expressed p21 c-ras, and 46 (73%) expressed p190 c-erbB-2. The expression of c-myc and c-ras but not of c-erb B-2 correlated directly with tumor differentiation as judged by morphologic criteria. No difference was observed in oncogene expression between intrahepatic and extrahepatic cholangiocarcinomas. Twelve of 14 hepatocellular carcinomas (86%) stained positively for all three oncoproteins. During normal liver development, expression of c-myc and c-ras was shown to occur from 18 weeks' gestation until 5 years of age, but not thereafter. Expression of c-myc, c-ras, and c-erbB-2 oncogenes may be used as immunohistochemical markers to distinguish cholangiocarcinoma from nonneoplastic biliary tissues, and may provide useful information concerning the cell biology of tumor differentiation.

The partial purification and characterization of GnRH-like activity from prostatic biopsy specimens and prostatic cancer cell lines.

We have investigated the possibility of the secretion of gonadotrophin-releasing-hormone (GnRH)-like peptides by prostatic cancer cells in culture and their presence in cytosolic preparations from human prostatic biopsy specimens. A GnRH-specific radioimmunoassay showed GnRH-like activity in concentrated cytosolic preparations and conditioned media from DU 145, an androgen-insensitive human prostatic cell line and from LNCaP, an androgen-responsive prostatic cancer cell line. GnRH immunoreactivity in culture media correlated directly with cell numbers. HPLC demonstrated that this GnRH-like material co-migrated with synthetic GnRH. This homology between synthetic GnRH and partially purified prostatic GnRH was confirmed following V8 protease and trypsin digestion which resulted in similar alterations in HPLC characteristics. The mean content of GnRH-like activity/g specimen tissue was significantly more in malignant tissue (88.5 +/- 80.5 fmol) than in benign (29.6 +/- 22 fmol), though more specimens of benign tissue were positive (37/54) than malignant tissue (6/22). This observation, taken with an earlier finding of GnRH-specific receptors in a hormone-sensitive cell line and human cancer specimens provides supportive evidence for the autocrine hypothesis of cell regulation.