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Computational modeling has been applied to simulate the heterogeneity of cancer behavior. The development of Cervical Cancer (CC) is a process in which the cell acquires dynamic behavior from non-deleterious and deleterious mutations, exhibiting chromosomal alterations as a manifestation of this dynamic. To further determine the progression of chromosomal alterations in precursor lesions and CC, we introduce a computational model to study the dynamics of deleterious and non-deleterious mutations as an outcome of tumor progression. The analysis of chromosomal alterations mediated by our model reveals that multiple deleterious mutations are more frequent in precursor lesions than in CC. Cells with lethal deleterious mutations would be eliminated, which would mitigate cancer progression; on the other hand, cells with non-deleterious mutations would become dominant, which could predispose them to cancer progression. The study of somatic alterations through computer simulations of cancer progression provides a feasible pathway for insights into the transformation of cell mechanisms in humans. During cancer progression, tumors may acquire new phenotype traits, such as the ability to invade and metastasize or to become clinically important when they develop drug resistance. Non-deleterious chromosomal alterations contribute to this progression. [ABSTRACT FROM AUTHOR]

Triatoma infestans is the most important Chagas disease vector in South America. Two main evolutionary lineages, named Andean and non-Andean, have been recognized by geographical distribution, phenetic and genetic characteristics. One of the main differences is the genomic size, varying over 30% in their haploid DNA content. Here we realize a genome wide analysis to compare the repetitive genome fraction (repeatome) between both lineages in order to identify the main repetitive DNA changes occurred during T. infestans differentiation process. RepeatExplorer analysis using Illumina reads showed that both lineages exhibit the same amount of non-repeat sequences, and that satellite DNA is by far the major component of repetitive DNA and the main responsible for the genome size differentiation between both lineages. We characterize 42 satellite DNA families, which are virtually all present in both lineages but with different amount in each lineage. Furthermore, chromosomal location of satellite DNA by fluorescence in situ hybridization showed that genomic variations in T. infestans are mainly due to satellite DNA families located on the heterochromatic regions. The results also show that many satDNA families are located on the euchromatic regions of the chromosomes. [ABSTRACT FROM AUTHOR]

Metazoan gene expression is controlled through the action of long stretches of noncoding DNA that contain enhancers—shorter sequences responsible for controlling a single aspect of a gene’s expression pattern. Models built on thermodynamics have shown how enhancers interpret protein concentration in order to determine specific levels of gene expression, but the emergent regulatory logic of a complete regulatory locus shows qualitative and quantitative differences from isolated enhancers. Such differences may arise from steric competition limiting the quantity of DNA that can simultaneously influence the transcription machinery. We incorporated this competition into a mechanistic model of gene regulation, generated efficient algorithms for this computation, and applied it to the regulation of Drosophila even-skipped (eve). This model finds the location of enhancers and identifies which factors control the boundaries of eve expression. This model predicts a new enhancer that, when assayed in vivo, drives expression in a non-eve pattern. Incorporation of chromatin accessibility eliminates this inconsistency. [ABSTRACT FROM AUTHOR]

Reverse transcriptase activity of telomerase adds telomeric repeat sequences at extreme ends of the newly replicated chromosome in actively dividing cells. Telomerase expression is not detected in terminally differentiated cells but is noticeable in 90% of the cancer cells. hTERT (human telomerase reverse transcriptase) expression seems to promote invasiveness of cancer cells. We here present proteomic profiles of cells overexpressing or knocked down for hTERT. This study also attempts to find out the potential interacting partners of hTERT in cancer cell lines. Two-dimensional gel electrophoresis (2-DE) of two different cell lines U2OS (a naturally hTERT negative cell line) and HeLa revealed differential expression of proteins in hTERT over-expressing cells. In U2OS cell line 28 spots were picked among which 23 spots represented upregulated and 5 represented down regulated proteins. In HeLa cells 21 were upregulated and 2 were down regulated out of 23 selected spots under otherwise identical experimental conditions. Some heat shock proteins viz. Hsp60 and Hsp70 and GAPDH, which is a housekeeping gene, were found similarly upregulated in both the cell lines. The upregulation of these proteins were further confirmed at RNA and protein level by real-time PCR and western blotting respectively. [ABSTRACT FROM AUTHOR]

We characterize how genomic variants that alter chromatin accessibility influence regulatory factor binding with a new method called DeltaBind that predicts condition specific factor binding more accurately than other methods based on DNase-seq data. Using DeltaBind and DNase-seq experiments we predicted the differential binding of 18 factors in K562 and GM12878 cells with an average precision of 28% at 10% recall, with the prediction of individual factors ranging from 5% to 65% precision. We further found that genome variants that alter chromatin accessibility are not necessarily predictive of altering proximal factor binding. Taken together these findings suggest that DNase-seq or ATAC-seq Quantitative Trait Loci (dsQTLs), while important, must be considered in a broader context to establish causality for phenotypic changes. [ABSTRACT FROM AUTHOR]

The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT. [ABSTRACT FROM AUTHOR]

Glucocorticoid resistance (GCR), i.e. unresponsiveness to the beneficial anti-inflammatory activities of the glucocorticoid receptor (GR), poses a serious problem in the treatment of inflammatory diseases. One possible solution to try and overcome GCR, is to identify molecules that prevent or revert GCR by hyper-stimulating the biological activity of the GR. To this purpose, we screened for compounds that potentiate the dexamethasone (Dex)-induced transcriptional activity of GR. To monitor GR transcriptional activity, the screen was performed using the lung epithelial cell line A549 in which a glucocorticoid responsive element (GRE) coupled to a luciferase reporter gene construct was stably integrated. Histone deacetylase inhibitors (HDACi) such as Vorinostat and Belinostat are two broad-spectrum HDACi that strongly increased the Dex-induced luciferase expression in our screening system. In sharp contrast herewith, results from a genome-wide transcriptome analysis of Dex-induced transcripts using RNAseq, revealed that Belinostat impairs the ability of GR to transactivate target genes. The stimulatory effect of Belinostat in the luciferase screen further depends on the nature of the reporter construct. In conclusion, a profound discrepancy was observed between HDACi effects on two different synthetic promoter-luciferase reporter systems. The favorable effect of HDACi on gene expression should be evaluated with care, when considering them as potential therapeutic agents. GEO accession number GSE96649. [ABSTRACT FROM AUTHOR]

An increasing number of single cell transcriptome and epigenome technologies, including single cell ATAC-seq (scATAC-seq), have been recently developed as powerful tools to analyze the features of many individual cells simultaneously. However, the methods and software were designed for one certain data type and only for single cell transcriptome data. A systematic approach for epigenome data and multiple types of transcriptome data is needed to control data quality and to perform cell-to-cell heterogeneity analysis on these ultra-high-dimensional transcriptome and epigenome datasets. Here we developed Dr.seq2, a Quality Control (QC) and analysis pipeline for multiple types of single cell transcriptome and epigenome data, including scATAC-seq and Drop-ChIP data. Application of this pipeline provides four groups of QC measurements and different analyses, including cell heterogeneity analysis. Dr.seq2 produced reliable results on published single cell transcriptome and epigenome datasets. Overall, Dr.seq2 is a systematic and comprehensive QC and analysis pipeline designed for parallel single cell transcriptome and epigenome data. Dr.seq2 is freely available at: and . [ABSTRACT FROM AUTHOR]

The induction efficiency of maize embryonic callus is highly dependent on the genotype, and only a few lines possess a high capacity for callus formation. Although certain genes and pathways have been reported to contribute to the regulation of callus induction, to the best of our knowledge, the functions of the small interfering RNAs (siRNAs) involved in this process remain unknown. In this study, we identified 861 differentially expressed siRNAs and 576 target genes in the callus induction process. These target genes were classified into 3 clusters, and their functions involve controlling metalloexopeptidase activity, catalase activity, transcription regulation, and O-methyltransferase activity. In addition, certain genes related to auxin transport and stem cell or meristem development (e.g., PLT5-like, ARF15, SAUR-like, FAS1-like, Fea3, SCL5, and Zmwox2A) were regulated by the differentially expressed siRNAs. Moreover, zma-siR004119-2 directly cleaves the 5’ UTR of Homeobox-transcription factor 25, which further leads to the down-regulation of its expression. Twelve 24-nt-siRNAs led to the hyper-methylation of GRMZM2G013465, which further decreases its expression. These results suggest that differentially expressed siRNAs regulate callus formation by controlling the expression of their target genes. [ABSTRACT FROM AUTHOR]

Differential DNA methylation with hyperglycemia is significantly associated with Type 2 Diabetes (T2D). Longtime extended exposure to high blood glucose levels can affect the epigenetic signatures in all organs. However, the relevance of the differential DNA methylation changes with hyperglycemia in blood with pancreatic islets remains unclear. We investigated differential DNA methylation in relation to glucose homeostasis based on the Oral Glucose Tolerance Test (OGTT) in a population-based cohort. We found a total of 382 differential methylation sites from blood DNA in hyperglycemia and type 2 diabetes subgroups using a longitudinal and cross-sectional approach. Among them, three CpG sites were overlapped; they were mapped to the MSI2 and CXXC4 genes. In a DNA methylation replication study done by pyrosequencing (n = 440), the CpG site of MSI2 were shown to have strong associations with the T2D group (p value = 2.20E-16). The differential methylation of MSI2 at chr17:55484635 was associated with diabetes-related traits, in particular with insulin sensitivity (QUICKI, p value = 2.20E-16) and resistance (HOMA-IR, p value = 1.177E-07). In human pancreatic islets, at the single-base resolution (using whole-genome bisulfite sequencing), the 292 CpG sites in the ±5kb at chr17:55484635 were found to be significantly hypo-methylated in donors with T2D (average decrease = 13.91%, 95% confidence interval (CI) = 4.18~ 17.06) as compared to controls, and methylation patterns differed by sex (-9.57%, CI = -16.76~ -6.89) and age (0.12%, CI = -11.17~ 3.77). Differential methylation of the MSI2 gene (chr17:55484635) in blood and islet cells is strongly related to hyperglycemia. Our findings suggest that epigenetic perturbation on the target site of MSI2 gene in circulating blood and pancreatic islets should represent or affect hyperglycemia. [ABSTRACT FROM AUTHOR]

Age estimation remains one of the most challenging tasks in forensic practice when establishing a biological profile of unknown skeletonised remains. Morphological methods based on developmental markers of bones can provide accurate age estimates at a young age, but become highly unreliable for ages over 35 when all developmental markers disappear. This study explores the changes in the biomechanical properties of bone tissue and matrix, which continue to change with age even after skeletal maturity, and their potential value for age estimation. As a proof of concept we investigated the relationship of 28 variables at the macroscopic and microscopic level in rib autopsy samples from 24 individuals. Stepwise regression analysis produced a number of equations one of which with seven variables showed an R2 = 0.949; a mean residual error of 2.13 yrs ±0.4 (SD) and a maximum residual error value of 2.88 yrs. For forensic purposes, by using only bench top machines in tests which can be carried out within 36 hrs, a set of just 3 variables produced an equation with an R2 = 0.902 a mean residual error of 3.38 yrs ±2.6 (SD) and a maximum observed residual error 9.26yrs. This method outstrips all existing age-at-death methods based on ribs, thus providing a novel lab based accurate tool in the forensic investigation of human remains. The present application is optimised for fresh (uncompromised by taphonomic conditions) remains, but the potential of the principle and method is vast once the trends of the biomechanical variables are established for other environmental conditions and circumstances. [ABSTRACT FROM AUTHOR]

Polydactyly, a common heritable limb malformation in vertebrates, is characterized by supernumerary digits. In chickens, basic characteristics and rough dominant genes have been explored in past decades; however, the elaborate pattern of inheritance and the determinant gene remain obscure. In this study, different types of polydactylism were classified by the numbers and the shapes of toes, including the newly defined subtypes of B’ and G, for the Beijing fatty chicken, a native breed of chicken from China. Through experiments on hybridization, we demonstrated a complete dominant inheritance of polydactyly instead of an incomplete penetrance or genetic modification of the previous conjecture. In particular, by using the F2 population of the five-digit purebred line of Beijing fatty chicken backcrossed to Shiqiza chicken and by using restriction-site associated DNA based markers, we performed a genome-wide association study on the trait of polydactyly. Furthermore, whole genome resequencing strategy was applied to sweep SNPs across the whole genome. An outlier-based Fst approach was employed to search for signatures of selection, and results indicated that the determinant mutation was found in the region ranging from 8.3 Mb to 8.7 Mb, where the polydactyly candidate gene LMBR1 was located. The G/T mutation of rs80659072 was identified to be highly associated with polydactyly in our resequencing and was validated in random samples from an expanded population. Thus, we confirmed that LMBR1 was the causative gene of polydactyly in the Beijing fatty chicken by using GWAS with restriction-site associated DNA based markers and resequencing. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphisms (SNPs) in the human angiotensinogen (hAGT) gene may modulate its transcription and affect the regulation of blood pressure via activation of the renin-angiotensin aldosterone system (RAAS). In this regard, we have identified polymorphisms in the 2.5 Kb promoter of the hAGT gene that form two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C & -1178G/A). hAGT gene with Hap -6A/-217A (Hap I) is associated with increased blood pressure whereas, Hap -6G/-217G (Hap II) is associated with normal blood pressure in human subjects. Since RAAS over activity contributes to hypertension in obesity, we have made transgenic mice (TG) containing either Hap I or Hap II of the hAGT gene to understand the role of obesity on its transcriptional regulation. Although, a high-fat diet (60% Kcal from fat, 12 weeks) elevates hAGT and mAGT regardless of haplotype, this effect is significantly (p<0.05) accentuated in Hap I mice, in both adipose and liver tissues. Chromatin Immuno- precipitation (ChIP) assay shows an increased binding of transcription factors including, GR, CEBPβ and STAT3 to the chromatin of the Hap I TG mice after high-fat diet as compared to Hap II TG mice (p<0.05). Differential plasma levels of hAGT in Hap II and I mice, after high-fat diet, further corroborate the variable transcriptional regulation of the hAGT, governed by gene-haplotypes. Taken together, our results show that SNPs in the Hap-I of the hAGT gene promote high-fat diet-induced binding of transcription factors GR, CEBP-β and STAT3, which lead to elevated expression of the hAGT gene in hepatic and adipose tissues. [ABSTRACT FROM AUTHOR]

Integrative analyses of high-throughput ‘omic data, such as DNA methylation, DNA copy number alteration, mRNA and protein expression levels, have created unprecedented opportunities to understand the molecular basis of human disease. In particular, integrative analyses have been the cornerstone in the study of cancer to determine molecular subtypes within a given cancer. As malignant tumors with similar morphological characteristics have been shown to exhibit entirely different molecular profiles, there has been significant interest in using multiple ‘omic data for the identification of novel molecular subtypes of disease, which could impact treatment decisions. Therefore, we have developed intNMF, an integrative approach for disease subtype classification based on non-negative matrix factorization. The proposed approach carries out integrative clustering of multiple high dimensional molecular data in a single comprehensive analysis utilizing the information across multiple biological levels assessed on the same individual. As intNMF does not assume any distributional form for the data, it has obvious advantages over other model based clustering methods which require specific distributional assumptions. Application of intNMF is illustrated using both simulated and real data from The Cancer Genome Atlas (TCGA). [ABSTRACT FROM AUTHOR]

Fluorescent proteins and epitope tags are often used as protein fusion tags to study target proteins. One prevailing technique in the budding yeast Saccharomyces cerevisiae is to fuse these tags to a target gene at the precise chromosomal location via homologous recombination. However, several limitations hamper the application of this technique, such as the selectable markers not being reusable, tagging of only the C-terminal being possible, and a “scar” sequence being left in the genome. Here, we describe a strategy to solve these problems by tagging target genes based on a pop-in/pop-out and counter-selection system. Three fluorescent protein tag (mCherry, sfGFP, and mKikGR) and two epitope tag (HA and 3×FLAG) constructs were developed and utilized to tag HHT1, UBC13 or RAD5 at the chromosomal locus as proof-of-concept. [ABSTRACT FROM AUTHOR]

Barley net blotch caused by the necrotrophic fungus Pyrenophora teres is a major barley disease in Norway. It can cause grain shriveling and yield losses, and resistance in currently grown cultivars is insufficient. In this study, a set of 589 polymorphic SNP markers was used to map resistance loci in a population of 109 doubled haploid lines from a cross between the closely related Norwegian cultivars Arve (moderately susceptible) and Lavrans (moderately resistant). Resistance to three net form net blotch (P. teres f. teres) single spore isolates was evaluated at the seedling stage in the greenhouse and at the adult plant stage under field conditions during three years. Days to heading and plant height were scored to assess their influence on disease severity. At the seedling stage, three to four quantitative trait loci (QTL) associated with resistance were found per isolate used. A major, putatively novel QTL was identified on chromosome 5H, accounting for 23–48% of the genetic variation. Additional QTL explaining between 12 and 16.5% were found on chromosomes 4H, 5H, 6H and 7H, with the one on 6H being race-specific. The major QTL on 5H was also found in adult plants under field conditions in three years (explaining up to 55%) and the 7H QTL was found in field trials in one year. Additional adult plant resistance QTL on 3H, 6H and 7H were significant in single years. The resistance on chromosomes 3H, 5H, 6H and 7H originates from the more resistant parent Lavrans, while the resistance on 4H is conferred by Arve. The genetic markers associated with the QTL found in this study will benefit marker-assisted selection for resistance against net blotch. [ABSTRACT FROM AUTHOR]

We examined age-related biochemical and histological changes in the fat bodies and hemolymph of Osmia rufa males and females. We analysed solitary bees during diapause, in October and in April; as well as the flying insects following diapause, in May and June. The trophocyte sizes, as well as the numbers of lipid droplets were the greatest at the beginning of diapause. Subsequently, they decreased along with age. Triglyceride and glucose concentrations systematically decreased in fat body cells but increased in the hemolymph from October to June. Concentrations/activities of (enzymatic and non-enzymatic) antioxidant and proteolytic systems, as well as phenoloxidase, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels were constant during the diapause, usually lower in the males than the females. Prior to the diapause/overwintering, the concentrations/activities of all the compounds were higher in the fat bodies than in the hemolymph. Later in the spring and in the summer, they increased in the hemolymph and on the body surfaces, while decreasing in the fat bodies. The global DNA methylation levels increased with age. Higher levels were always observed in the males than in the females. The study will promote better understanding of bee evolution and will be useful for the protection and management of solitary bees, with benefits to the environment and agriculture. [ABSTRACT FROM AUTHOR]

Toxic effects of cadmium (Cd), a common soil pollutant, are still not very well understood, particularly in regard to its epigenetic impact. Therefore, the aim of this study was to assess DNA methylation changes and their persistence in the earthworm Lumbricus terrestris upon chronic low dose Cd exposure using methylation sensitive amplification polymorphism (MSAP). Moreover, the biomarker response and fitness of the earthworms, as well as the expression of detoxification-related genes (metallothionein (MT) and phytochelatin synthase (PCS)) was evaluated. Low levels of Cd caused an increase in genome-wide DNA methylation, which remained partly modified, even after several months of recovery in unpolluted soil. Increased cellular stress seemed to decrease after two weeks of exposure whereas fitness parameters remained unaffected by Cd, probably as a result from the activation of detoxification mechanisms like the expression of MTs. Interestingly, even though the level of Cd exposure was very low, MT expression levels indicate the development of acclimation mechanisms. Taken together, this study demonstrates that acclimation, as well as epigenetic modifications can occur already in moderately polluted environments. In addition, these effects can have long-lasting impacts on key species of soil invertebrates and might persist long after the actual heavy metal challenge has passed. [ABSTRACT FROM AUTHOR]

The genus Deschampsia P. Beauv (Poaceae) involves a group of widespread polymorphic species. Some of them are highly tolerant to stressful and variable environmental conditions, and D. antarctica is one of the only two vascular plants growing in Antarctic. This species is a source of useful for selection traits and a valuable model for studying an environmental stress tolerance in plants. Genome diversity and comparative chromosomal phylogeny within the genus have not been studied yet as karyotypes of most Deschampsia species are poorly investigated. We firstly conducted a comparative molecular cytogenetic analysis of D. antarctica (Antarctic Peninsula) and related species from various localities (D. cespitosa, D. danthonioides, D. elongata, D. flexuosa (= Avenella flexuosa), D. parvula and D. sukatschewii by fluorescence in situ hybridization with 45S and 5S rDNA, DAPI-banding and sequential rapid in situ hybridization with genomic DNA of D. antarctica, D. cespitosa, and D. flexuosa. Based on patterns of distribution of the examined markers, chromosomes of the studied species were identified. Within these species, common features as well as species peculiarities in their karyotypic structure and chromosomal distribution of molecular cytogenetic markers were characterized. Different chromosomal rearrangements were detected in D. antarctica, D. flexuosa, D. elongata and D. sukatschewii. In karyotypes of D. antarctica, D. cespitosa, D. elongata and D. sukatschewii, 0–3 B chromosomes possessed distinct DAPI-bands were observed. Our findings suggest that the genome evolution of the genus Deschampsia involved polyploidy and also different chromosomal rearrangements. The obtained results will help clarify the relationships within the genus Deschampsia, and can be a basis for the further genetic and biotechnological studies as well as for selection of plants tolerant to extreme habitats. [ABSTRACT FROM AUTHOR]

To compensate for the sex difference in the number of X chromosomes, human females, like human males have only one active X. The other X chromosomes in cells of both sexes are silenced in utero by XIST, the Inactive X Specific Transcript gene, that is present on all X chromosomes. To investigate the means by which the human active X is protected from silencing by XIST, we updated the search for a key dosage sensitive XIST repressor using new cytogenetic data with more precise resolution. Here, based on a previously unknown sex bias in copy number variations, we identify a unique region in our genome, and propose candidate genes that lie within, as they could inactivate XIST. Unlike males, the females who duplicate this region of chromosome 19 (partial 19 trisomy) do not survive embryogenesis; this preimplantation loss of females may be one reason that more human males are born than females. [ABSTRACT FROM AUTHOR]

Background and aims: Physical activity has preventive as well as therapeutic benefits for overweight subjects. In this study we aimed to examine effects of in vivo exercise on in vitro metabolic adaptations by studying energy metabolism in cultured myotubes isolated from biopsies taken before and after 12 weeks of extensive endurance and strength training, from healthy sedentary normal weight and overweight men. Methods: Healthy sedentary men, aged 40–62 years, with normal weight (body mass index (BMI) < 25 kg/m2) or overweight (BMI ≥ 25 kg/m2) were included. Fatty acid and glucose metabolism were studied in myotubes using [14C]oleic acid and [14C]glucose, respectively. Gene and protein expressions, as well as DNA methylation were measured for selected genes. Results: The 12-week training intervention improved endurance, strength and insulin sensitivity in vivo, and reduced the participants’ body weight. Biopsy-derived cultured human myotubes after exercise showed increased total cellular oleic acid uptake (30%), oxidation (46%) and lipid accumulation (34%), as well as increased fractional glucose oxidation (14%) compared to cultures established prior to exercise. Most of these exercise-induced increases were significant in the overweight group, whereas the normal weight group showed no change in oleic acid or glucose metabolism. Conclusions: 12 weeks of combined endurance and strength training promoted increased lipid and glucose metabolism in biopsy-derived cultured human myotubes, showing that training in vivo are able to induce changes in human myotubes that are discernible in vitro. [ABSTRACT FROM AUTHOR]

Background: We use our earlier experimental studies of the catalytic mechanism of DNA methyltransferases to prepare in silico a family of novel mechanism-based inhibitors of human Dnmt1. Highly specific inhibitors of DNA methylation can be used for analysis of human epigenome and for the creation of iPS cells. Results: We describe a set of adenosyl-1-methyl-pyrimidin-2-one derivatives as novel mechanism-based inhibitors of mammalian DNA methyltransferase Dnmt1. The inhibitors have been designed to bind simultaneously in the active site and the cofactor site and thus act as transition-state analogues. Molecular dynamics studies showed that the lead compound can form between 6 to 9 binding interactions with Dnmt1. QM/MM analysis showed that the upon binding to Dnmt1 the inhibitor can form a covalent adduct with active site Cys1226 and thus act as a mechanism-based suicide-inhibitor. The inhibitor can target DNA-bond and DNA-free form of Dnmt1, however the suicide-inhibition step is more likely to happen when DNA is bound to Dnmt1. The validity of presented analysis is described in detail using 69 modifications in the lead compound structure. In total 18 of the presented 69 modifications can be used to prepare a family of highly specific inhibitors that can differentiate even between closely related enzymes such as Dnmt1 and Dnmt3a DNA methyltransferases. Conclusions: Presented results can be used for preparation of some highly specific and potent inhibitors of mammalian DNA methylation with specific pharmacological properties. [ABSTRACT FROM AUTHOR]

etal olerance roteins (MTPs) are a gene family of cation efflux transporters that occur widely in plants and might serve an essential role in metal homeostasis and tolerance. Our research describes the identification, characterization, and localization of OsMTP11, a member of the MTP family from rice. OsMTP11 was expressed constitutively and universally in different tissues in rice plant. Heterologous expression in yeast showed that OsMTP11 complemented the hypersensitivity of mutant strains to Mn, and also complemented yeast mutants to other metals, including Co and Ni. Real time RT-PCR analysis demonstrated OsMTP11 expression was substantially enhanced following 4 h under Cd, Zn, Ni, and Mn treatments, suggesting possible roles of OsMTP11 involvement in heavy metal stress responses. Promoter analysis by transgenic assays with GUS as a reporter gene and mRNA in situ hybridization experiments showed that OsMTP11 was expressed specifically in conducting tissues in rice. DNA methylation assays of genomic DNA in rice treated with Cd, Zn, Ni, and Mn revealed that decreased DNA methylation levels were present in the OsMTP11 promoter region, which was consistent with OsMTP11 induced-expression patterns resulting from heavy metal stress. This result suggested that DNA methylation is one of major factors regulating expression of OsMTP11 through epigenetic mechanisms. OsMTP11 fused to reen luorescent rotein (GFP) localized to the entire onion epidermal cell cytoplasm, while vacuolar membrane exhibited increased GFP signals, consistent with an OsMTP11 function in cation sequestration. Our results indicated that OsMTP11 might play vital roles in Mn and other heavy metal transportation in rice. [ABSTRACT FROM AUTHOR]

Introduction: Obesity is the principal component in the Metabolic Syndrome (MetS) that determines the progression of metabolic complications. Metabolically healthy obese (MHO) individuals seem to be protected against those complications. Telomere length (TL) as a novel marker of cellular aging had a complex relationship to the MetS. The principal aim of this study was to investigate the TL in MHO, and to study the association between TL and the worsening of the metabolic condition. Material and methods: We have determined the absolute TL (aTL) in 400 women (mean age of 46.76 ± 15.47 years; range: 18–86 years), grouped according to the metabolic condition in three groups: metabolically healthy non-obese women (MHNO), MHO and obese women with MetS (MSO); and grouped according to the number of components of MetS. Results: We found that MHO displays significantly higher aTL than MSO (p = 0.033; r = -4.63; 95% CI r = -8.89 / -0.37), but did not differ from MHNO. A decrease in aTL with the progressive increase in the number of MetS components was also observed (p < 0.001; r = -2.06; 95% CI r = -3.13 / -0.99). In this way, our results indicate that aTL is influenced by the presence of MetS, but it is not affected by the presence of obesity. Discussion: We found that shorter aTL is not associated with MHO, but is related to MetS and with the increased number of metabolic abnormalities. [ABSTRACT FROM AUTHOR]

Background: Cancers of the oral cavity are primarily oral squamous cell carcinomas (OSCCs). Many of the OSCCs present at late stages with an exceptionally poor prognosis. A probable limitation in management of patients with OSCC lies in the insufficient knowledge pertaining to the linkage between copy number alterations in OSCC and oral tumourigenesis thereby resulting in an inability to deliver targeted therapy. Objectives: The current study aimed to identify copy number alterations (CNAs) in OSCC using array comparative genomic hybridization (array CGH) and to correlate the CNAs with clinico-pathologic parameters and clinical outcomes. Materials and methods: Using array CGH, genome-wide profiling was performed on 75 OSCCs. Selected genes that were harboured in the frequently amplified and deleted regions were validated using quantitative polymerase chain reaction (qPCR). Thereafter, pathway and network functional analysis were carried out using Ingenuity Pathway Analysis (IPA) software. Results: Multiple chromosomal regions including 3q, 5p, 7p, 8q, 9p, 10p, 11q were frequently amplified, while 3p and 8p chromosomal regions were frequently deleted. These findings were in confirmation with our previous study using ultra-dense array CGH. In addition, amplification of 8q, 11q, 7p and 9p and deletion of 8p chromosomal regions showed a significant correlation with clinico-pathologic parameters such as the size of the tumour, metastatic lymph nodes and pathological staging. Co-amplification of 7p, 8q, 9p and 11q regions that harbored amplified genes namely CCND1, EGFR, TPM2 and LRP12 respectively, when combined, continues to be an independent prognostic factor in OSCC. Conclusion: Amplification of 3q, 5p, 7p, 8q, 9p, 10p, 11q and deletion of 3p and 8p chromosomal regions were recurrent among OSCC patients. Co-alteration of 7p, 8q, 9p and 11q was found to be associated with clinico-pathologic parameters and poor survival. These regions contain genes that play critical roles in tumourigenesis pathways. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma, which accounts for the majority of pancreatic cancers, is a lethal disease with few therapeutic options. Genomic profiling of pancreatic ductal adenocarcinoma has identified a complex and heterogeneous landscape. Understanding the molecular characteristics of pancreatic ductal adenocarcinoma will facilitate the identification of potential therapeutic strategies. We analyzed the gene expression profiles of primary tumors from patients compared to normal pancreas and identified high co-overexpression of core components of the spindle assembly checkpoint, including the protein kinase TTK (also known as MPS-1). We found overexpression of TTK protein in a subset of pancreatic ductal adenocarcinoma primary tumors and cell lines. siRNA-mediated depletion or catalytic inhibition of TTK resulted in an aberrant cell cycle profile, multi- and micro-nucleation, induction of apoptosis, and decreased cell proliferation and transformed growth. Selective catalytic inhibition of TTK caused override of the spindle assembly checkpoint-induced cell cycle arrest. Interestingly, we identified ubiquitin specific peptidase 16 (Usp16), an ubiquitin hydrolase, as a phosphorylation substrate of TTK. Usp16 regulates chromosomal condensation and G2/M progression by deubiquitinating histone H2A and polo-like kinase 1. Phosphomimetic mutants of Usp16 show enhanced proteosomal degradation and may prolong the G2/M transition allowing for correction of replication errors. Taken together, our results suggest a critical role for TTK in preventing aneuploidy-induced cell death in pancreatic cancer. [ABSTRACT FROM AUTHOR]

Austronesian diffusion is considered one of the greatest dispersals in human history; it led to the peopling of an extremely vast region, ranging from Madagascar in the Indian Ocean to Easter Island in Remote Oceania. The Y-chromosome haplogroup O3a2b*-P164(xM134), a predominant paternal lineage of Austronesian populations, is found at high frequencies in Polynesian populations. However, the internal phylogeny of this haplogroup remains poorly investigated. In this study, we analyzed -seventeen Y-chromosome sequences of haplogroup O3a2b*-P164(xM134) and generated a revised phylogenetic tree of this lineage based on 310 non-private Y-chromosome polymorphisms. We discovered that all available O3a2b*-P164(xM134) samples belong to the newly defined haplogroup O3a2b2-N6 and samples from Austronesian populations belong to the sublineage O3a2b2a2-F706. Additionally, we genotyped a series of Y-chromosome polymorphisms in a large collection of samples from China. We confirmed that the sublineage O3a2b2a2b-B451 is unique to Austronesian populations. We found that O3a2b2-N6 samples are widely distributed on the eastern coastal regions of Asia, from Korea to Vietnam. Furthermore, we propose- that the O3a2b2a2b-B451 lineage represents a genetic connection between ancestors of Austronesian populations and ancient populations in North China, where foxtail millet was domesticated about 11,000 years ago. The large number of newly defined Y-chromosome polymorphisms and the revised phylogenetic tree of O3a2b2-N6 will be helpful to explore the origin of proto-Austronesians and the early diffusion process of Austronesian populations. [ABSTRACT FROM AUTHOR]

The goal of our study was to investigate the contribution of ABCB1 expression to the risk of clopidogrel resistance (CR). Platelets functions were measured using the Verify-Now P2Y12 assay. Applying Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP), the single-nucleotide polymorphisms (SNPs) was tested. Using bisulphite pyrosequencing assay, we investigated the association of the ABCB1 DNA methylation levels and CR. It was shown that female, hypertension, and lower albumin levels increased the risk of CR (P<0.05). If patients did not have hypoproteinaemia or had hypertension, the SNP in rs1045642 was associated with CR (CC vs. TT: albumin ≥35, P = 0.042; hypertension, P = 0.045; C vs. T: albumin ≥35, P = 0.033; hypertension, P = 0.040). Additionally, the platelet inhibition of the CT+TT genotype in rs1128503 was larger than that of the CC genotype (P = 0.021). Multivariate logistic regression analysis showed that male, higher albumin and hsCRP decreased the risk of CR, and the stent size maybe positively correlated with CR. The SNP in rs1045642 was related to all-cause mortality (P = 0.024). We did not find any relationship between the methylation levels of the ABCB1 promoter and CR. In conclusions, our study indicated that ABCB1 polymorphisms might be useful in further evaluating the pathogenesis of CR. [ABSTRACT FROM AUTHOR]

Objective: Vitamin D deficiency has been linked to increased risk of multiple sclerosis (MS) and poor outcome. However, the specific role that vitamin D plays in MS still remains unknown. In order to identify potential mechanisms underlying vitamin D effects in MS, we profiled epigenetic changes in vitamin D receptor (VDR) gene to identify genomic regulatory elements relevant to MS pathogenesis. Methods: Human T cells derived from whole blood by negative selection were isolated in a set of 23 relapsing-remitting MS (RRMS) patients and 12 controls matched by age and gender. DNA methylation levels were assessed by bisulfite cloning sequencing in two regulatory elements of VDR. mRNA levels were measured by RT-qPCR to assess changes in VDR expression between patients and controls. Results: An alternative VDR promoter placed at exon 1c showed increased DNA methylation levels in RRMS patients (median 30.08%, interquartile range 19.2%) compared to controls (18.75%, 9.5%), p-value<0.05. Moreover, a 6.5-fold increase in VDR mRNA levels was found in RRMS patients compared to controls (p-value<0.001). Conclusions: An alternative promoter of the VDR gene shows altered DNA methylation levels in patients with multiple sclerosis, and it is associated with VDR mRNA upregulation. This locus may represent a candidate regulatory element in the genome relevant to MS pathogenesis. [ABSTRACT FROM AUTHOR]

Compaction of DNA in chromatin is a hallmark of the eukaryotic cell and unravelling its structure is required for an understanding of DNA involving processes. Despite strong experimental efforts, many questions concerning the DNA packing are open. In particular, it is heavily debated whether an ordered structure referred to as the “30 nm fibre” exist in vivo. Scanning probe microscopy has become a cutting edge technology for the high-resolution imaging of DNA- protein complexes. Here, we perform high-resolution atomic force microscopy of non-cross-linked chromatin arrays in liquid, under different salt conditions. A statistical analysis of the data reveals that array compaction is salt dependent in a non-monotonic fashion. A simple physical model can qualitatively explain the observed findings due to the opposing effects of salt dependent stiffening of DNA, nucleosome stability and histone-histone interactions. While for different salt concentrations different compaction states are observed, our data do not provide support for the existence of regular chromatin fibres. Our studies add new insights into chromatin structure, and with that contribute to a further understanding of the DNA condensation. [ABSTRACT FROM AUTHOR]

It is now well established that eukaryote genomes have a common architectural organization into topologically associated domains (TADs) and evidence is accumulating that this organization plays an important role in gene regulation. However, the mechanisms that partition the genome into TADs and the nature of domain boundaries are still poorly understood. We have investigated boundary regions in the Drosophila genome and find that they can be identified as domains of very low H3K27me3. The genome-wide H3K27me3 profile partitions into two states; very low H3K27me3 identifies Depleted (D) domains that contain housekeeping genes and their regulators such as the histone acetyltransferase-containing NSL complex, whereas domains containing moderate-to-high levels of H3K27me3 (Enriched or E domains) are associated with regulated genes, irrespective of whether they are active or inactive. The D domains correlate with the boundaries of TADs and are enriched in a subset of architectural proteins, particularly Chromator, BEAF-32, and Z4/Putzig. However, rather than being clustered at the borders of these domains, these proteins bind throughout the H3K27me3-depleted regions and are much more strongly associated with the transcription start sites of housekeeping genes than with the H3K27me3 domain boundaries. While we have not demonstrated causality, we suggest that the D domain chromatin state, characterised by very low or absent H3K27me3 and established by housekeeping gene regulators, acts to separate topological domains thereby setting up the domain architecture of the genome. [ABSTRACT FROM AUTHOR]

Aim of the study: To assess the feasibility of partial HPV genotyping and methylation analysis of CADM1, MAL, and miR124-2 genes as triage tests in assaying self-collected cervical samples positive for high-risk HPV on primary screening, and to review the literature regarding host cellular gene methylation analysis of self-collected cervical samples. Material and methods: Women residing in North-East Italy who had failed to respond to the invitation to participate in an organized population-based program were invited to provide a self-sample. Their stored baseline (self-collected) and follow-up (clinician-collected) cervical samples were included in the study. DNA was extracted from HPV-positive (Qiagen’s Hybrid Capture 2, HC2) samples. Partial genotyping with separate detection of HPV types 16 and 18 was performed with a hybrid capture-based method and a quantitative PCR assay. Methylation was assayed with a quantitative methylation-specific PCR. Results: High-risk HPV infection was detected in 48% of baseline and 71% of follow-up HC2-positive samples. Methylation was demonstrated respectively in 15% and 23.5% of baseline and follow-up samples and chiefly involved a single gene (miR124-2). Invalid quantitative PCR results were recorded in 5% of self-collected samples. The specificity of miR124-1, MAL, and CADM1 methylation was 84%, 94%, and 98%, respectively, and the specificity of the three markers combined was 84%. Sensitivity was not estimated due to the lack of CIN2+ samples. The systematic review showed that different methylation assays yield different accuracy values. Conclusion: Self-collected samples are suitable for methylation assays included in reflex triage testing. The reproducibility and accuracy of the methylation tests described in the literature should be improved. [ABSTRACT FROM AUTHOR]

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a ‘phenotype of interest’ (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with ‘Fuzzy-Spreader’-like morphologies were also identified through a visual screen. The second, a ‘mutant of interest’ (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid. [ABSTRACT FROM AUTHOR]

Background: In the past decade many Genome-wide Association Studies (GWAS) were performed that discovered new associations between single-nucleotide polymorphisms (SNPs) and various phenotypes. Imputation methods are widely used in GWAS. They facilitate the phenotype association with variants that are not directly genotyped. Imputation methods can also be used to combine and analyse data genotyped on different genotyping arrays. In this study we investigated the imputation quality and efficiency of two different approaches of combining GWAS data from different genotyping platforms. We investigated whether combining data from different platforms before the actual imputation performs better than combining the data from different platforms after imputation. Methods: In total 979 unique individuals from the AMC-PAS cohort were genotyped on 3 different platforms. A total of 706 individuals were genotyped on the MetaboChip, a total of 757 individuals were genotyped on the 50K gene-centric Human CVD BeadChip, and a total of 955 individuals were genotyped on the HumanExome chip. A total of 397 individuals were genotyped on all 3 individual platforms. After pre-imputation quality control (QC), Minimac in combination with MaCH was used for the imputation of all samples with the 1,000 genomes reference panel. All imputed markers with an r2 value of <0.3 were excluded in our post-imputation QC. Results: A total of 397 individuals were genotyped on all three platforms. All three datasets were carefully matched on strand, SNP ID and genomic coordinates. This resulted in a dataset of 979 unique individuals and a total of 258,925 unique markers. A total of 4,117,036 SNPs were available when imputation was performed before merging the three datasets. A total of 3,933,494 SNPs were available when imputation was done on the combined set. Our results suggest that imputation of individual datasets before merging performs slightly better than after combining the different datasets. Conclusions: Imputation of datasets genotyped by different platforms before merging generates more SNPs than imputation after putting the datasets together. [ABSTRACT FROM AUTHOR]

Introduction: Colorectal cancer belongs to the most frequent occurring malignancies. A prediction of the clinical outcome and appropriate choice of neoadjuvant chemotherapy needs personalized insight to the main driving pathways. Because most CRCs have polyps as progenitor lesions, studying the pathways driving to adenomagenesis is no less important. Goals: Our purpose was the evaluation of microsatellite stability status within conventional colon adenomas and also β-catenin, BRAFV600E and p53 contribution. Material and methods: The cohort included 101 cases of typical colon adenomas with high grade epithelial dysplasia according to WHO. An immunohistochemistry method was used for the depiction of the expression of targeted proteins, as also their heterogeneity. Results: Generally, we noted a 10% frequency of MSI events where MSI-H reached a 5% share occurred within the left colon and rectal polyps. β-catenin nuclear overexpression was noted with a 70% frequency and p53 with close to a 24% frequency. In addition, we found a presence of micro-serration foci more often within tubular adenomas, where focal MSI took place more often. Our results indicate that MSI events occur more often than had been theorized earlier. It results in tumour heterogeneity, more complex underlying pathways and finally ontogenetic molecular-diversity of tumours besides similar occurring histopathological features. [ABSTRACT FROM AUTHOR]

The first step of the benzoxazinoid (BX) synthesis pathway is catalyzed by an enzyme with indole-3-glycerol phosphate lyase activity encoded by 3 genes, Bx1, TSA and Igl. A gene highly homologous to maize and wheat Bx1 has been identified in rye. The goal of the study was to analyze the gene and to experimentally verify its role in the rye BX biosynthesis pathway as a rye ortholog of the Bx1 gene. Expression of the gene showed peak values 3 days after imbibition (dai) and at 21 dai it was undetectable. Changes of the BX content in leaves were highly correlated with the expression pattern until 21 dai. In plants older than 21 dai despite the undetectable expression of the analyzed gene there was still low accumulation of BXs. Function of the gene was verified by correlating its native expression and virus-induced silencing with BX accumulation. Barley stripe mosaic virus (BSMV)-based vectors were used to induce transcriptional (TGS) and posttranscriptional (PTGS) silencing of the analyzed gene. Both strategies (PTGS and TGS) significantly reduced the transcript level of the analyzed gene, and this was highly correlated with lowered BX content. Inoculation with virus-based vectors specifically induced expression of the analyzed gene, indicating up-regulation by biotic stressors. This is the first report of using the BSMV-based system for functional analysis of rye gene. The findings prove that the analyzed gene is a rye ortholog of the Bx1 gene. Its expression is developmentally regulated and is strongly induced by biotic stress. Stable accumulation of BXs in plants older than 21 dai associated with undetectable expression of ScBx1 indicates that the function of the ScBx1 in the BX biosynthesis is redundant with another gene. We anticipate that the unknown gene is a putative ortholog of the Igl, which still remains to be identified in rye. [ABSTRACT FROM AUTHOR]

Background and aim: The biological functions of non-coding RNAs (ncRNAs) have been widely identified in many human diseases. In the present study, the relationship between long non-coding RNA HOTAIR and microRNA-17-5p (miR-17-5p) and their roles in osteogenic differentiation and proliferation in non-traumatic osteonecrosis of femoral head (ONFH) were investigated. Methods: The expression levels of HOTAIR and miR-17-5p in the mesenchymal stem cells (MSCs) derived from patients with non-traumatic ONFH and osteoarthritis (OA) were examined by real-time PCR. BMP-2 induced human MSCs from bone marrow (hMSC-BM) were used for osteogenic differentiation. Results: It was observed that the expression level of miR-17-5p was lower and the level of HOTAIR was higher in samples of non-traumatic ONFH compared with OA. HOTAIR downregulation induced by si-HOTAIR led to the increase of miR-17-5p expression and the decrease of miR-17-5p target gene SMAD7 expression. The values of osteogenic differentiation markers, including RUNX2 and COL1A1 mRNA expression and ALP activity, were also elevated by si-HOTAIR. However, the increase of these values was canceled by miR-17-5p inhibitor or SMAD7 upregulation. Conclusion: HOTAIR played a role in regulating osteogenic differentiation and proliferation through modulating miR-17-5p and its target gene SMAD7 in non-traumatic ONFH. [ABSTRACT FROM AUTHOR]

The application of circulating tumor DNA(ctDNA) represents a non-invasive method for tumor detection. Its prognostic significance in patients with colorectal cancer is controversial. We performed a systematic review of data from published studies to assess the prognostic values of ctDNA in patients with colorectal cancer. We searched Medline, Embase, Web of Science, the Cochrane Library, and Scopus databases to identify eligible studies reporting disease-free survival (DFS) and overall survival (OS) stratified by ctDNA prior to December 6, 2016. We evaluated the quality and design of these studies. A total of 22 studies were eligible for systematic review. Among them, 11 studies investigated the prognostic value of ctDNA on disease-free survival (DFS). Seven of 11 studies showed that ctDNA was an independent variable to estimate the probability of DFS by multivariate analyses. Thirteen studies assessed the relationship between ctDNA and overall survival (OS). Eight of 13 studies showed that ctDNA was an independent predictor of worse OS through the use of multivariate analyses. This analysis provides evidence that ctDNA may be a prognostic biomarker, negatively correlated with the survival of patients with colorectal cancer. [ABSTRACT FROM AUTHOR]

Southern right whales (SRWs, Eubalena australis) are polymorphic for an X-linked pigmentation pattern known as grey morphism. Most SRWs have completely black skin with white patches on their bellies and occasionally on their backs; these patches remain white as the whale ages. Grey morphs (previously referred to as partial albinos) appear mostly white at birth, with a splattering of rounded black marks; but as the whales age, the white skin gradually changes to a brownish grey color. The cellular and developmental bases of grey morphism are not understood. Here we describe cellular and ultrastructural features of grey-morph skin in relation to that of normal, wild-type skin. Melanocytes were identified histologically and counted, and melanosomes were measured using transmission electron microscopy. Grey-morph skin had fewer melanocytes when compared to wild-type skin, suggesting reduced melanocyte survival, migration, or proliferation in these whales. Grey-morph melanocytes had smaller melanosomes relative to wild-type skin, normal transport of melanosomes to surrounding keratinocytes, and normal localization of melanin granules above the keratinocyte nuclei. These findings indicate that SRW grey-morph pigmentation patterns are caused by reduced numbers of melanocytes in the skin, as well as by reduced amounts of melanin production and/or reduced sizes of mature melanosomes. Grey morphism is distinct from piebaldism and albinism found in other species, which are genetic pigmentation conditions resulting from the local absence of melanocytes, or the inability to synthesize melanin, respectively. [ABSTRACT FROM AUTHOR]

Studies have demonstrated that nucleotide-binding site–leucine-rich repeat (NBS–LRR) genes respond to pathogen attack in plants. Characterization of NBS–LRR genes in peanut is not well documented. The newly released whole genome sequences of Arachis duranensis and Arachis ipaënsis have allowed a global analysis of this important gene family in peanut to be conducted. In this study, we identified 393 (AdNBS) and 437 (AiNBS) NBS–LRR genes from A. duranensis and A. ipaënsis, respectively, using bioinformatics approaches. Full-length sequences of 278 AdNBS and 303 AiNBS were identified. Fifty-one orthologous, four AdNBS paralogous, and six AiNBS paralogous gene pairs were predicted. All paralogous gene pairs were located in the same chromosomes, indicating that tandem duplication was the most likely mechanism forming these paralogs. The paralogs mainly underwent purifying selection, but most LRR 8 domains underwent positive selection. More gene clusters were found in A. ipaënsis than in A. duranensis, possibly owing to tandem duplication events occurring more frequently in A. ipaënsis. The expression profile of NBS–LRR genes was different between A. duranensis and A. hypogaea after Aspergillus flavus infection. The up-regulated expression of NBS–LRR in A. duranensis was continuous, while these genes responded to the pathogen temporally in A. hypogaea. [ABSTRACT FROM AUTHOR]

Brachypodium distachyon (Brachypodium) is now intensively utilized as a model grass species in various biological studies. Its favorable cytological features create a unique foundation for a convenient system in mutagenesis, thereby potentially enabling the ‘hot spots’ and ‘cold spots’ of DNA damage in its genome to be analyzed. The aim of this study was to analyze the involvement of 5S rDNA, 25S rDNA, the Arabidopsis-type ()n telomeric sequence and the Brachypodium-originated centromeric BAC clone CB33J12 in the micronuclei formation in Brachypodium root tip cells that were subjected to the chemical clastogenic agent maleic hydrazide (MH). To the best of our knowledge, this is the first use of a multicolor fluorescence in situ hybridization (mFISH) with four different DNA probes being used simultaneously to study plant mutagenesis. A quantitative analysis allowed ten types of micronuclei, which were characterized by the presence or absence of specific FISH signal(s), to be distinguished, thus enabling some specific rules governing the composition of the MH-induced micronuclei with the majority of them originating from the terminal regions of chromosomes, to be identified. The application of rDNA sequences as probes showed that 5S rDNA-bearing chromosomes are involved in micronuclei formation more frequently than the 25S rDNA-bearing chromosomes. These findings demonstrate the promising potential of Brachypodium to be a useful model organism to analyze the effects of various genotoxic agents on the plant nuclear genome stability, especially when the complex FISH-based and chromosome-specific approaches such as chromosome barcoding and chromosome painting will be applied in future studies. [ABSTRACT FROM AUTHOR]

It has been proposed that copy number variations (CNVs) are associated with increased risk of autism spectrum disorder (ASD) and, in conjunction with other genetic changes, contribute to the heterogeneity of ASD phenotypes. Array comparative genomic hybridization (aCGH) and exome sequencing, together with systems genetics and network analyses, are being used as tools for the study of complex disorders of unknown etiology, especially those characterized by significant genetic and phenotypic heterogeneity. Therefore, to characterize the complex genotype-phenotype relationship, we performed aCGH and sequenced the exomes of two affected siblings with ASD symptoms, dysmorphic features, and intellectual disability, searching for de novo CNVs, as well as for de novo and rare inherited point variations—single nucleotide variants (SNVs) or small insertions and deletions (indels)—with probable functional impacts. With aCGH, we identified, in both siblings, a duplication in the 4p16.3 region and a deletion at 8p23.3, inherited by a paternal balanced translocation, t(4, 8) (p16; p23). Exome variant analysis found a total of 316 variants, of which 102 were shared by both siblings, 128 were in the male sibling exome data, and 86 were in the female exome data. Our integrative network analysis showed that the siblings’ shared translocation could explain their similar syndromic phenotype, including overgrowth, macrocephaly, and intellectual disability. However, exome data aggregate genes to those already connected from their translocation, which are important to the robustness of the network and contribute to the understanding of the broader spectrum of psychiatric symptoms. This study shows the importance of using an integrative approach to explore genotype-phenotype variability. [ABSTRACT FROM AUTHOR]

Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future. [ABSTRACT FROM AUTHOR]

Birds are characterized by a low proportion of repetitive DNA in their genome when compared to other vertebrates. Among birds, species belonging to Piciformes order, such as woodpeckers, show a relatively higher amount of these sequences. The aim of this study was to analyze the distribution of different classes of repetitive DNA—including microsatellites, telomere sequences and 18S rDNA—in the karyotype of three Picidae species (Aves, Piciformes)—Colaptes melanochloros (2n = 84), Colaptes campestris (2n = 84) and Melanerpes candidus (2n = 64)–by means of fluorescence in situ hybridization. Clusters of 18S rDNA were found in one microchromosome pair in each of the three species, coinciding to a region of (CGG)10 sequence accumulation. Interstitial telomeric sequences were found in some macrochromosomes pairs, indicating possible regions of fusions, which can be related to variation of diploid number in the family. Only one, from the 11 different microsatellite sequences used, did not produce any signals. Both species of genus Colaptes showed a similar distribution of microsatellite sequences, with some difference when compared to M. candidus. Microsatellites were found preferentially in the centromeric and telomeric regions of micro and macrochromosomes. However, some sequences produced patterns of interstitial bands in the Z chromosome, which corresponds to the largest element of the karyotype in all three species. This was not observed in the W chromosome of Colaptes melanochloros, which is heterochromatic in most of its length, but was not hybridized by any of the sequences used. These results highlight the importance of microsatellite sequences in differentiation of sex chromosomes, and the accumulation of these sequences is probably responsible for the enlargement of the Z chromosome. [ABSTRACT FROM AUTHOR]

Wheat breeding programs generate a large amount of variation which cannot be completely explored because of limited phenotyping throughput. Genomic prediction (GP) has been proposed as a new tool which provides breeding values estimations without the need of phenotyping all the material produced but only a subset of it named training population (TP). However, genotyping of all the accessions under analysis is needed and, therefore, optimizing TP dimension and genotyping strategy is pivotal to implement GP in commercial breeding schemes. Here, we explored the optimum TP size and we integrated pedigree records and genome wide association studies (GWAS) results to optimize the genotyping strategy. A total of 988 advanced wheat breeding lines were genotyped with the Illumina 15K SNPs wheat chip and phenotyped across several years and locations for yield, lodging, and starch content. Cross-validation using the largest possible TP size and all the SNPs available after editing (~11k), yielded predictive abilities (rGP) ranging between 0.5–0.6. In order to explore the Training population size, rGP were computed using progressively smaller TP. These exercises showed that TP of around 700 lines were enough to yield the highest observed rGP. Moreover, rGP were calculated by randomly reducing the SNPs number. This showed that around 1K markers were enough to reach the highest observed rGP. GWAS was used to identify markers associated with the traits analyzed. A GWAS-based selection of SNPs resulted in increased rGP when compared with random selection and few hundreds SNPs were sufficient to obtain the highest observed rGP. For each of these scenarios, advantages of adding the pedigree information were shown. Our results indicate that moderate TP sizes were enough to yield high rGP and that pedigree information and GWAS results can be used to greatly optimize the genotyping strategy. [ABSTRACT FROM AUTHOR]

Accurate maps of promoters and enhancers are required for understanding transcriptional regulation. Promoters and enhancers are usually mapped by integration of chromatin assays charting histone modifications, DNA accessibility, and transcription factor binding. However, current algorithms are limited by unrealistic data distribution assumptions. Here we propose GenoSTAN (Genomic STate ANnotation), a hidden Markov model overcoming these limitations. We map promoters and enhancers for 127 cell types and tissues from the ENCODE and Roadmap Epigenomics projects, today’s largest compendium of chromatin assays. Extensive benchmarks demonstrate that GenoSTAN generally identifies promoters and enhancers with significantly higher accuracy than previous methods. Moreover, GenoSTAN-derived promoters and enhancers showed significantly higher enrichment of complex trait-associated genetic variants than current annotations. Altogether, GenoSTAN provides an easy-to-use tool to define promoters and enhancers in any system, and our annotation of human transcriptional cis-regulatory elements constitutes a rich resource for future research in biology and medicine. [ABSTRACT FROM AUTHOR]

The promoter region of the imprinted gene MEST contains several motifs capable of forming G-quadruplex (G4) structures, which appear to contribute to consistent allelic dropout during polymerase chain reaction (PCR) analysis of this region. Here, we extend our previous analysis of MEST G4 structures by applying fluorescent footprinting techniques to assess non B-DNA structure and topology in dsDNA from the full MEST promoter region, under conditions that mimic PCR. We demonstrate that the buffer used for PCR provides an extremely favourable milieu for G4 formation, and that cytosine methylation helps maintain G4 structures during PCR. Additionally, we demonstrate G4 formation at motifs not previously identified through bioinformatic analysis of the MEST promoter, and provide nucleotide level resolution for topological reconstruction of these structures. These observations increase our understanding of the mechanisms through which methylation and G4 contribute towards allelic drop-out during PCR. [ABSTRACT FROM AUTHOR]

Rice blast is a destructive disease caused by Magnaporthe oryzae, and it has a large impact on rice production worldwide. Compared with leaf blast resistance, our understanding of panicle blast resistance is limited. The japonica landrace Jiangnanwan from Taihu Lake region in China shows highly resistance to panicle and leaf blast. In this study, three generations (F2:5, F2:6, F2:7) consisting of 221 RILs (recombination inbreeding lines), developed from the cross of Jiangnanwan and Suyunuo, a susceptible-blast japonica variety, were evaluated for panicle blast resistance in the fields and leaf blast resistance in greenhouse in Nanjing in 2013, 2014 and 2015. A blast resistance gene Pi-jnw1 referring to panicle blast resistance and leaf blast resistance was identified in the three generations and located in the region of RM27273 and RM27381 in chromosome 11. The RIL18 line harboring Pi-jnw1 was selected to be backcrossed with Suyunuo to develop BC2F2 populations. According to the genotyping of 1,150 BC2F2 individuals and panicle blast and leaf blast resistance evaluation of 47 recombinants between RM27150 and RM27381, Pi-jnw1 was finally mapped to the 282 kb region between markers W28 and BS39. This study revealed that Jiangnanwan harboring a panicle blast and leaf blast resistance gene Pi-jnw1 could be a genetic source for breeding new rice cultivars with panicle blast resistance. [ABSTRACT FROM AUTHOR]

For over a century, the live bearing guppy, Poecilia reticulata, has been used to study sexual selection as well as local adaptation. Natural guppy populations differ in many traits that are of intuitively adaptive significance such as ornamentation, age at maturity, brood size and body shape. Water depth, light supply, food resources and predation regime shape these traits, and barrier waterfalls often separate contrasting environments in the same river. We have assembled and annotated the genome of an inbred single female from a high-predation site in the Guanapo drainage. The final assembly comprises 731.6 Mb with a scaffold N50 of 5.3 MB. Scaffolds were mapped to linkage groups, placing 95% of the genome assembly on the 22 autosomes and the X-chromosome. To investigate genetic variation in the population used for the genome assembly, we sequenced 10 wild caught male individuals. The identified 5 million SNPs correspond to an average nucleotide diversity (π) of 0.0025. The genome assembly and SNP map provide a rich resource for investigating adaptation to different predation regimes. In addition, comparisons with the genomes of other Poeciliid species, which differ greatly in mechanisms of sex determination and maternal resource allocation, as well as comparisons to other teleost genera can begin to reveal how live bearing evolved in teleost fish. [ABSTRACT FROM AUTHOR]

Objective: To study the feasibility and the diagnostic and prognostic interest of automated analysis of 1p, 19q, 9p and 10q status by FISH technique in oligodendroglial tumors. Methods: We analyzed a retrospective series of 33 consecutive gliomas with oligodendroglial histology (originally diagnosed as 24 oligodendrogliomas and 9 oligoastrocytomas). For all cases, automated FISH analysis of 1p, 19q, 9p and 10q status were performed and compared to clinical and histological data, ATRX, IDH1R132H and alpha-internexin status (studied by immunohistochemistry) and overall survival (OS). Manual analysis of 9p and 10q status were also performed and compared to automated analysis to verify the concordance of the two methods. Results: The 33 gliomas were reclassified into 13 low-grade oligodendrogliomas (OII), 10 anaplastic oligodendrogliomas (OIII), 3 diffuse astrocytomas (AII), 3 anaplastic astrocytomas (AIII) and 4 glioblastomas (GBM) according to the WHO 2016 histological criteria. The 1p and/or 19q imbalanced status were restricted to astrocytomas with no correlation to their grade or their OS. Chromosome 9p deletion was restricted to OIII (70%) and GBM (100%) and was correlated with a shorter OS in the total cohort (p = 0.0007), the oligodendroglioma cohort (p = 0.03) and the astrocytoma cohort (p = 0.001). Concordance between 9p manual and automated analysis was satisfactory (81%, κ = 0.69). Chromosome 10q deletion was restricted to GBMs (50%) and was correlated with a poor OS in both the total cohort (p = 0.003) and the astrocytoma (AS) cohort (p = 0.04). Concordance between manual and automated analysis was satisfactory (79%, κ = 0.62). Conclusion: Automated analysis of 1p, 19q, 9p and 10q status by FISH is a reliable technique which allows for refined classification of oligodendroglial tumors. 1p and/or 19q imbalanced status is evidence of astrocytic differentiation. 9p deletion is found in high grade oligodendrogliomas and astrocytomas with a poor OS. 10q is related to GBM status and a poor OS. [ABSTRACT FROM AUTHOR]