Academic literature on the topic 'Molecular avian gender'

Background and Aim: Many avian species are considered sexually monomorphic. In monomorphic bird species, especially in young birds, sex is difficult to identify based on an analysis of their external morphology. Accurate sex identification is essential for avian captive breeding and evolutionary studies. Methods with varying degrees of invasiveness such as vent sexing, laparoscopic surgery, steroid sexing, and chromosome inspection (karyotyping) are used for sex identification in monomorphic birds. This study aimed to assess the utility of a non-invasive molecular marker for gender identification in a variety of captive monomorphic birds, as a strategy for conservation. Materials and Methods: DNA was isolated from feather samples from 52 individuals representing 16 species of 11 families indigenous to both Indonesia and elsewhere. We amplified the chromodomain helicase DNA-binding (CHD) gene using polymerase chain reaction with MP, NP, and PF primers to amplify introns with lengths that differ between the CHD-W and the CHD-Z genes, allowing sex discrimination because the W chromosome is exclusively present in females. Results: Molecular bird sexing confirmed 33 females and 19 males with 100% accuracy. We used sequencing followed by alignment on one protected bird species (Probosciger aterrimus). Conclusion: Sex identification may be accomplished noninvasively in birds, because males only have Z sex chromosomes, whereas females have both Z and W chromosomes. Consequently, the presence of a W-unique DNA sequence identifies an individual as female. Sexing of birds is vital for scientific research, and to increase the success rate of conservation breeding programs.

Use of sexed semen in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of predetermined sex. Here we evaluate a production scheme involving single and bilateral twin transfer of Holstein female embryos to beef cattle recipients. Holstein oocytes were fertilized with the X-bearing fraction of gender-sorted Holstein semen. Cumulus cells were removed with aid of a vortex or microfluidic device (μFD). Half of the vortexed embryos were cultured in KSOMaaBSA (control), as were all μFD embryos. The remaining vortexed embryos were cultured in control medium with 6% avian white yolk (WY). Embryo production and transfer occurred across five replicates. Cows (n = 475) were synchronized using an Ovsynch protocol. They were administered GnRH on Day −9, PGF on Day −2, and GnRH on Day 0. Half of the cows received a CIDR (1.38 g progesterone) with the 1st GnRH injection. The CIDR was removed at the time of PGF treatment. Day 7 Grade 1 blastocysts were transferred fresh 7 days after the 2nd GnRH injection. Control and WY embryos were transferred as ipsilateral singles and bilateral twins; μFD embryos were transferred singly. Pregnancy was diagnosed with ultrasound between 41–46 days and confirmed between 60–90 days; fetal sexing confirmed that 95% of fetuses were female. Effects on embryo survival were analyzed by logistic regression. Chi-square analysis was applied to survival rates. Replication affected embryo survival (P < 0.05). There was no effect of cumulus removal, medium, or CIDR use. Fetal loss between ultrasounds was greater for twin vs. single transfers (30% vs. 15%, respectively; P < 0.01). Probability of embryo survival was estimated to increase ∼0.006 with each increasing day postpartum. Five cases of hydrallantois were detected during the 5th month of gestation for 1 control twin, 1 WY single, and 3 WY twin transfers, originating from 3 replicates. On a production per transfer basis, the proportion of fetuses obtained for single and twin transfers was 30% and 55%, respectively (P < 0.001). Although there was greater embryonic loss for twin compared to single transfers, a higher percentage of cows receiving twins established and maintained pregnancy. Large-scale transfer of IVP Holstein heifer embryos to beef recipients is a feasible production scheme. Table 1. Embryo survival and pregnancy rates

Abstract Abstract 3801 Background Aberrant DNA methylation at promoter CpG islands is recognized as one of the hallmarks driving the pathogenesis of myeloid malignancies, especially myelodysplastic syndromes (MDS). Shen et al. recently characterized an algorithm of 10 aberrantly methylated gene loci which was predictive for overall survival and progression-free survival in a large cohort of MDS patients. A particular cytogenetic subgroup of MDS patients with a deletion on the long arm of chromosome 5 (5q-syndrome) has been shown to benefit from treatment with lenalidomide. However, the exact underlying molecular mechanism of MDS with isolated deletion (5q) is still not understood. To further elucidate on the role of deregulated DNA methylation we analyzed DNA-methylation profiles of bone marrow cells from patients with MDS with an isolated deletion (5q). Methods All patients were diagnosed and treated within a German multicenter trial investigating the safety of Lenalidomide in patients with low risk myelodysplastic syndromes and an isolated deletion (5q) after informed consent and according to the declaration of Helsinki. Bone marrow cells of 47 MDS patients with deletion (5q) at initial diagnosis were analyzed (median age 70 years, range 41 – 88 years, IPSS score: low n= 22; intermediate-1 n = 25). DNA was extracted using the QIAGEN Allprep Kit® (Qiagen, Hilden, Germany). Genome wide DNA methylation analysis was performed using the HumanMethylation450 BeadChip (Illumina, San Diego, USA). Differential methylation of CpGs was defined by a minimum mean methylation difference of 15% as expressed by the beta-value of the array data and statistical significance set at q ≤ 0.01 according to the Benjamini-Hochberg-method for multiple significance testing. Analysis of array data was performed using Genome-Studio Software® (Illumina, San Diego, USA), Qlucore Omics explorer 2.3 (Qlucore software. Lund, Sweden) and Microsoft Excel 10.1® (Microsoft Software, Redmond, USA). Gene ontology analysis was performed using GATHER (http://http://gather.genome.duke.edu/). Results Using a q-value of ≤ 0.05 for the beta-value of the array and excluding gender-specific chromosomal CpGs, 473,929 CpGs were evaluable for analysis. Gene Ontology analysis using the GATHER Tool showed a significant enrichment of genes mapped to 5q31 (p<0.0001). Highly significant differential methylation profiles between MDS patients with isolated (5q) were found between patients with low and intermediate-1 IPSS score. CpGs differentially hypermethylated in intermediate-1 risk versus lows risk patients affected the coding regions of interesting candidate genes such as platelet-derived growth factor receptor, beta polypeptide (PDGFRB), clathrin interactor 1 (CLINT1), both located at 5q33 and suspected to be involved in the pathogenesis of 5q deleted MDS. Furthermore, transcriptional regulators such as proline, glutamate and leucine rich protein 1 (PELP1), v-myb myeloblastosis viral oncogene homolog (avian) (MYB), genes known to be involved in cancer like trichorhinophalangeal 1(TRPS1), and tumor suppressors like forkhead box P1 (FOXP1) and genes thought to be involved in the pathogenesis of MDS like Minichromosome maintenance protein2(MCM2) did show differentially DNA-methylation according to our selection criteria. Conclusions We present a comprehensive genome wide methylation analysis of MDS patients with an isolated deletion (5q) with low and intermediate-1 risk according to IPSS. Thereby we detected sets of significantly differentially methylated CpGs between both risk groups. Correlation of these data to clinical parameters might help to further elucidate the contribution of aberrant methylation to the phenotype of MDS with isolated deletion (5q) and could possibly help establishing novel prognostic markers based on differential methylation. Moreover, unraveling the role of aberrant methylation patterns might result in new therapeutic treatment approaches at least in a subset of patients. Disclosures: Nowak: Celgene: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ottmann:Celgene: Clinical trial participation Other. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Schlenk:Celgene: Research Funding. Ganser:Celgene: Research Funding. Germing:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hofmann:Celgene: Honoraria, Research Funding. Nolte:Celgene: Research Funding.

Abstract Chronic myeloid leukemia is a clonal myeloproliferative neoplasm characterized by the occurrence of the Ph1 chromosome in a primitive hematopoietic stem cell with a major amplification of myeloid compartment. Despite the major progress obtained by the use of tyrosine kinase inhibitors (TKI), resistances to these drugs occur with progression towards blast crisis (BC) during which there is an increase of BCR-ABL expression. To evaluate the potential targets of BCR-ABL in this context, a gene profiling analysis of the UT7-11 cell line overexpressing BCR-ABL fusion protein was performed using a whole transcriptome microarray. One of the highly upregulated genes was ETS1 with a fold change of +35.86. ETS1 proto-oncogene, the founder member of the ETS-domain family of transcription factors such as TEL and FLI1, is the human homolog of the avian erythroblastosis virus E26. They play a crucial role in stem cell biology, tumorigenesis and ETS1 has been shown to be involved in the regulation of granulocytic differentiation. Its role in CML pathophysiology has not been studied so far. We first showed by Western blots that ETS1 protein was highly increased in UT7-11 cells as compared to parental UT7 cells. Using a DOX-Inducible BCR-ABL system, we have shown that ETS1 expression was downregulated upon inhibition of BCR-ABL expression. ETS1 expression was a tyrosine kinase dependent event as its expression was reduced in the presence of TKI. To determine if ETS1 expression is upregulated in primary leukemic cells, we have analyzed leukemic cells of CML patients at diagnosis (n= 40) as compared to healthy controls (n= 30) showing an increase of ETS1 mRNA expression in primary CML cells in a highly significant manner (p < 0.0007). We then analyzed ETS1 targets using chip-sequencing in K562 cells, performed on HG19 human genome allowing to predict 3209 proximal promoters (-3000 upstream pb, + 50pb around Transcription Starting Sites) which represents a promoter enrichment of +33.68 (p-value=4.9E-324) as compared to the distribution of peaks in the whole genome. Chromosomes 19, 16, 17, 11, 12 and 1 were found over-represented with ETS1 bound events in Chip-sequencing. Integration of BCR-ABL transcriptome with ETS1 promoters identified by Chip-Sequencing led to the identification of 130 ETS1-targets activated by BCR-ABL, allowing reclassification of BCR-ABL transfected samples with transcriptome data by unsupervised classification. ETS1 transcriptional program regulated by BCR-ABL analysis performed on CD34+ from CML patients during progression of the disease (GSE47927) allowed discrimination of the different states of the progression (from the chronic phase to accelerated phase and BC p-value=1.63E-33). This analysis was also found significant by Gene Set Enrichment Analysis ETS1_BC specific geneset (NES=+1.52, p-value<0.0001). Among this ETS1 program specific of BC, we identified 2 targets that are discriminant for CD34+ cells of CML patients without cytogenetic response under Imatinib therapy. These two genes were Dynamin 3 (DNM3) and LIMS1/ PINCH1. Dynamin 3 is a member of the motor proteins implicated in cell motility and cytokinesis and it is implicated in megakaryocyte development whereas LIMS1 / PINCH1 is essentially involved in cell migration and adhesion. In order to validate ETS1 and its target expressions in a novel cohort of CML patients, we have analyzed the expression of DNM3 and PINCH1 in whole blood samples in CML patients ( n= 60 ) as compared to healthy controls (n= 32). We have found that, similar to ETS1, DNM3 and LIMS1 / PINCH1 mRNA were highly upregulated in primary CML and this increase was highly significant. In this new cohort of patients, DNM3 transcript levels were inversely correlated to WBC count (r=-0.32, p-value=0.011) and especially in male gender (r=-0.43, p-value=0.012). The expression of LIMS1 was inversely correlated with the age at diagnosis and the major molecular response at 12 months. Thus, our results show for the first time the major upregulation of ETS1 transcriptional program in CML. ETS1 transcriptional program has been found to correlate with the gene expression pattern involved in the progression of the disease in the transcriptomic analysis of CML CD34+ cells. The novel druggable targets that we identified in the BCR-ABL-activated transcriptional program is currently under investigation to determine their use in patients with resistance to targeted therapies. Disclosures No relevant conflicts of interest to declare.

Background:Systemic lupus erythematosus (SLE) is a chronic, inflammatory, and autoimmune disease that damages vital organs such as the heart. Patients with SLE have a higher risk of developing a cardiovascular (CV) disease than the general population (1).Objectives:The aim of this study was to compare the echocardiographic findings in patients with SLE and controls.Methods:This was a cross-sectional, observational, and comparative study. A total of 38 patients with SLE according to the 2019 EULAR/ACR classification criteria, ≥18 years old and 38 matched controls by age (±5 years) and gender, were recruited for this study. Exclusion criteria were a poor echocardiographic window, patients with a previous CV event, such as myocardial infarction, cerebrovascular event or peripheral arterial disease, and pregnant women. A transthoracic echocardiogram, including speckle tracking technique, was performed, by two certified echocardiographers, in all study subjects. Distribution was evaluated with Kolmogorov-Smirnov test. Comparisons were done with χ2 and Fisher´s Exact test for qualitative variables, and Student’s t test and Mann-Whitney´s U test for quantitative variables. A p-value <0.05 was considered statistically significant.Results:When comparing demographic characteristics there were no significant differences in age, gender, and comorbidities between SLE patients and controls. In the echocardiographic findings a significant difference was found in the left ventricular ejection fraction (LVEF), lower in SLE patients compared to controls (57.02% vs 61.89%, p=0.001), in the global longitudinal strain (GLS), reduced in SLE patients (-19.55% vs -22.00%, p=0.001), in the tricuspid annular plane systolic excursion (TAPSE), lower in SLE patients (22.00mm vs 24.00mm, p=0.011), in the left atrial volume index, higher in SLE patients (28.44ml/m2 vs 22.29ml/m2, p=0.014) and in the presence of mitral regurgitation, more prevalent in SLE patients (31.6% vs 10.5%, p=0.024) (Table 1).Conclusion:Patients with SLE have a worse left ventricular function, evaluated by LVEF and GLS, a worse right ventricular systolic function, evaluated by TAPSE, an increased left atrial volume index and a higher prevalence of mitral regurgitation, which are associated with a higher risk of CV death. It is important to consider including a transthoracic echocardiogram as part of the CV evaluation in patients with SLE, which may result in an early detection of CV abnormalities and an opportune treatment.References:[1]Avina-Zubieta JA, To F, Vostretsova K, et al. Risk of Myocardial Infarction and Stroke in Newly Diagnosed Systemic Lupus Erythematosus: A General Population-Based Study. Arthritis Care Res (Hoboken) 2017;69(6):849-56. doi: 10.1002/acr.23018Table 1.Demographic characteristics and echocardiographic findings.SLE patients(n=38)Controls(n=38)pAge years, median (p25-p75)37.5 (24.5-43.2)45.0 (24.0-50.0)NSWomen, n (%)34 (89.5)37 (97.4)NST2DM, n (%)1 (2.6)3 (7.9)NSHTN, n (%)9 (23.7)3 (7.9)NSDyslipidemia, n (%)3 (7.9)3 (7.9)NSObesity, n (%)1 (2.6)5 (13.2)NSActive smoking, n (%)7 (18.4)3 (7.9)NSEchocardiographic findingsLeft ventricle indexed mass g/m2, median (p25-p75)64.00 (50.66-87.09)60.30 (52.97-76.74)0.009RWT, median (p25-p75)0.37 (0.30-0.46)0.38 (0.33-0.45)NSLeft ventricular ejection fraction %, mean ± SD57.02 ± 7.3761.89 ± 5.230.001Global longitudinal strain %, median (p25-p75)-19.55 (-21.02 –-15.95)-22.00 (-21.00 –-20.00)0.001Left atrium indexed volume ml/m2, median (p25-p75)28.44 (20.34-33.56)22.29 (17.97-27.13)0.014TAPSE mm, median (p25-p75)22.00 (20.00-24.00)24.00 (21.00-25.00)0.011Valvular abnormalitiesAortic regurgitation, n (%)2 (5.3)1 (2.6)NSMitral regurgitation, n (%)12 (31.6)4 (10.5)0.024Tricuspid regurgitation, n (%)16 (42.1)17 (44.7)NSSLE, systemic lupus erythematosus; NS, not significant; T2DM, type 2 diabetes mellitus; HTN, hypertension; RWT, relative wall thickness; TAPSE, tricuspid annular plane systolic excursion.Acknowledgements:We have no acknowledgements to declare.Disclosure of Interests:None declared

Background:In rheumatoid arthritis, circulating autoantibodies like anticyclic citrullinated peptides and rheumatoid factor may be present for years prior to symptom onset. In RA, there are high levels of circulating tumor necrosis factors, interleukins, matrix metalloproteinases, growth factors, and adhesion molecules that cause tissue damage1,2. This can affect different organs in different ways. It is possible that in the heart, they cause inflammation of cardiac tissue 3,4.Objectives:This study was conducted to determine if patients with RA, an inflammatory arthritis, and osteoarthritis (OA), a degenerative arthritis, differ in the prevalence of cardiac arrhythmias.Methods:This was a retrospective study. We enrolled 300 consecutive patients, 150 with RA and 150 with OA. Patients were assessed for age, race, gender, body mass index (BMI), smoking status, hypertension (HTN), hyperlipidemia (HLD), diabetes mellitus (DM), cardiovascular disease (CVD), coronary artery disease (CAD), cardiac arrhythmias including conduction abnormalities, cerebrovascular accident (CVA), congestive heart failure (CHF), abdominal aortic aneurysm (AAA), pulmonary embolism (PE), deep vein thrombosis (DVT), and peripheral vascular disease (PVD). RA and OA groups were compared with the chi square test and the Mann-Whitney test.Results:The RA and OA groups did not differ on the demographic characteristics of age or race. The presence of DM was similar in the two groups. The RA group had more women than the OA group (12.7% vs. 5.3%, p=0.026). The OA group had a higher mean BMI (33.1 vs 30.0, p<0.001) and higher prevalence of HLD (78.9% vs 64.6%, p=0.007) and HTN (79.3% vs. 66.7%, p=0.013). RA patients had a higher rate of smoking (70.0% vs 56.7%, p=0.017) and longer duration of smoking (34.3 years vs. 28.2 years, p=0.003). The RA group had a higher prevalence of CVD (58.7% vs. 42.7%, p= 0.006). Further data analysis among CVD showed there was no difference between the two groups for the prevalence of CAD, CHF, CVA, AAA, PVD or DVT/PE. RA patients had a higher prevalence of cardiac arrhythmias (31.3% vs. 12.0%, p<0.001). Among all types of cardiac arrhythmias, no significant difference was found between two groups for the prevalence of supraventricular tachycardia, ventricular arrhythmias, or conduction abnormalities. Patients with RA had a significantly higher prevalence of atrial fibrillation (15.3% vs. 6.0%, p = 0.015).Conclusion:Cardiac arrhythmias occur more frequently in RA patients than in OA patients. Among all the arrhythmias, atrial fibrillation has a significantly higher prevalence in RA patients.References:[1]Maradit-Kremers H, et al. Increased unrecognized coronary heart disease and sudden deaths in rheumatoid arthritis: A population-based cohort study. Arthritis Rheum. 2005; 52: 402-411[2]Seferovic PM, et al. Cardiac arrhythmias and conduction disturbances in autoimmune rheumatic diseases. Rheumatology. 2006; 45: 39-42[3]Avina-Zubiets JA, et al. Risk of incident cardiovascular events in patients with rheumatoid arthritis: A met-analysis of observational studies. Ann Rheum Dis 2012; 71: 1524-1529[4]Lazzerini PE, Capechi L, et al. Systemic Inflammation and arrhythmic risk: Lessons from rheumatoid arthritis. European Heart Journal. 2017; 38: 1717-1727Disclosure of Interests:None declared

Unambiguous gender identification (ID) is needed to assess parameters in studies of population dynamics, behavior, and evolutionary biology of Caribbean Flamingo (Phoenicopterus ruber ruber) and other birds. Due to its importance for management and conservation, molecular (DNA-based) avian gender ID assays targeting intron-size differences of the Chromosome Helicase ATPase DNA Binding (CHD) gene of males (CHD-Z) and females (CHD-W) have been developed. Male (ZZ) and female (WZ) genotypes are usually scored as size polymorphisms through agarose or acrylamide gels. For certain species, W-specific restriction sites or multiplex polymerase chain-reaction (PCR) involving CHD-W specific primers are needed. These approaches involve a minimum of three steps following DNA isolation: PCR, gel electrophoresis, and photo-documentation, which limit high throughput scoring and automation potential. In here, a short amplicon (SA) High-resolution Melting Analysis (HRMA) assay for avian gender ID is developed. SA-HRMA of an 81-Base Pair (bp) segment differentiates heteroduplex female (WZ) from homoduplex male (ZZ) genotypes by targeting Single-nucleotide Polymorphisms (SNPs) instead of intron-size differences between CHD-Z and CHD-W genes. To demonstrate the utility of the approach, the gender of Caribbean Flamingo (P. ruber ruber) (17 captive from the Dallas Zoo and 359 wild from Ria Lagartos, Yucatan, Mexico) was determined. The assay was also tested on specimens of Lesser Flamingo (P. minor), Chilean Flamingo (P. chilensis), Saddle-billed Stork (Ephippiorhynchus senegalensis), Scarlet Ibis (Eudocimus ruber), White-bellied Stork (Ciconia abdimii), Roseate Spoonbill (Platalea ajaja), Marabou Stork (Leptoptilos crumeniferus), Greater Roadrunner (Geococcyx californianus), and Attwater's Prairie Chicken (Tympanuchus cupido attwateri). Although the orthologous 81 bp segments of Z and W are highly conserved, sequence alignments with 50 avian species across 15 families revealed mismatches affecting one or more nucleotides within the SA-HRMA forward or reverse primers. Most mismatches were located along the CHD-Z gene that may generate heteroduplex curves and thus gender ID errors. For such cases, taxon and species-specific primer sets were designed. The SA-HRMA gender ID assay can be used in studies of avian ecology and behavior, to assess sex-associated demographics and migratory patterns, and as a proxy to determine the health of the flock and the degree by which conservation and captive breeding programs are functioning.

Comparative endocrinology is the study of the endocrine glands and their hormones in different species of animals. It is undergoing a renaissance because of the new tools and techniques provided by genome sequencing and molecular biology. Until relatively recently, characterization and detection of hormones in lower vertebrates relied on biological assays and protein chemistry approaches, whereas now gene sequences can be readily revealed from whole genome sequencing. Gene expression and synthesis can be used to develop antibodies and other reagents for sensitive assays and revealing physiological experiments can be carried out. Endocrinology traditionally used a range of animal species, including many lower vertebrates. Comparative endocrinology became a separate specialty only in the last 50 years when endocrinologists concentrated on rodents as their model animals. In 1933, Riddle demonstrated that an avian pituitary factor that promoted growth of the pigeon crop-sac was identical to a mammalian pituitary factor that earlier had been found to initiate and maintain milk secretion in mammals. Riddle called this avian factor prolactin and the response of the crop-sac provided a sensitive assay for the detection of human prolactin in pituitary extracts. Pigeon prolactin was the first pituitary hormone to be crystallized and purified in 1937 and led to the purification of mammalian prolactin. Prolactin has a number of roles in lower vertebrates, including a vital role as a hypercalcaemic factor in fish. The first part of this chapter focuses on the calcium-regulating factors including parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and stanniocalcin (STC), and the second part will discuss comparative endocrinology of thyroid hormones and transthyretin (a thyroid hormone distributor in blood the cerebrospinal fluid).