A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research

Regulation of mitochondrial morphogenesis by annexin a6.

Mitochondrial homeostasis is critical in meeting cellular energy demands, shaping calcium signals and determining susceptibility to apoptosis. Here we report a role for anxA6 in the regulation of mitochondrial morphogenesis, and show that in cells lacking anxA6 mitochondria are fragmented, respiration is impaired and mitochondrial membrane potential is reduced. In fibroblasts from AnxA6(-/-) mice, mitochondrial Ca(2+) uptake is reduced and cytosolic Ca(2+) transients are elevated. These observations led us to investigate possible interactions between anxA6 and proteins with roles in mitochondrial fusion and fission. We found that anxA6 associates with Drp1 and that mitochondrial fragmentation in AnxA6(-/-) fibroblasts was prevented by the Drp1 inhibitor mdivi-1. In normal cells elevation of intracellular Ca(2+) disrupted the interaction between anxA6 and Drp1, displacing anxA6 to the plasma membrane and promoting mitochondrial fission. Our results suggest that anxA6 inhibits Drp1 activity, and that Ca(2+)-binding to anxA6 relieves this inhibition to permit Drp1-mediated mitochondrial fission

Intragenic DNA methylation: implications of this epigenetic mechanism for cancer research

Epigenetics is the study of all mechanisms that regulate gene transcription and genome stability that are maintained throughout the cell division, but do not include the DNA sequence itself. The best-studied epigenetic mechanism to date is DNA methylation, where methyl groups are added to the cytosine base within cytosine–guanine dinucleotides (CpG sites). CpGs are frequently clustered in high density (CpG islands (CGIs)) at the promoter of over half of all genes. Current knowledge of transcriptional regulation by DNA methylation centres on its role at the promoter where unmethylated CGIs are present at most actively transcribed genes, whereas hypermethylation of the promoter results in gene repression. Over the last 5 years, research has gradually incorporated a broader understanding that methylation patterns across the gene (so-called intragenic or gene body methylation) may have a role in transcriptional regulation and efficiency. Numerous genome-wide DNA methylation profiling studies now support this notion, although whether DNA methylation patterns are a cause or consequence of other regulatory mechanisms is not yet clear. This review will examine the evidence for the function of intragenic methylation in gene transcription, and discuss the significance of this in carcinogenesis and for the future use of therapies targeted against DNA methylation

Tracing Water Sources of Terrestrial Animal Populations with Stable Isotopes: Laboratory Tests with Crickets and Spiders

Fluxes of carbon, nitrogen, and water between ecosystem components and organisms have great impacts across levels of biological organization. Although much progress has been made in tracing carbon and nitrogen, difficulty remains in tracing water sources from the ecosystem to animals and among animals (the “water web”). Naturally occurring, non-radioactive isotopes of hydrogen and oxygen in water provide a potential method for tracing water sources. However, using this approach for terrestrial animals is complicated by a change in water isotopes within the body due to differences in activity of heavy and light isotopes during cuticular and transpiratory water losses. Here we present a technique to use stable water isotopes to estimate the mean mix of water sources in a population by sampling a group of sympatric animals over time. Strong correlations between H and O isotopes in the body water of animals collected over time provide linear patterns of enrichment that can be used to predict a mean mix of water sources useful in standard mixing models to determine relative source contribution. Multiple temperature and humidity treatment levels do not greatly alter these relationships, thus having little effect on our ability to estimate this population-level mix of water sources. We show evidence for the validity of using multiple samples of animal body water, collected across time, to estimate the isotopic mix of water sources in a population and more accurately trace water sources. The ability to use isotopes to document patterns of animal water use should be a great asset to biologists globally, especially those studying drylands, droughts, streamside areas, irrigated landscapes, and the effects of climate change

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression

Wilder rangelands as a natural climate opportunity: Linking climate action to biodiversity conservation and social transformation

Rangelands face threats from climate and land-use change, including inappropriate climate change mitigation initiatives such as tree planting in grassy ecosystems. The marginalization and impoverishment of rangeland communities and their indigenous knowledge systems, and the loss of biodiversity and ecosystem services, are additional major challenges. To address these issues, we propose the wilder rangelands integrated framework, co-developed by South African and European scientists from diverse disciplines, as an opportunity to address the climate, livelihood, and biodiversity challenges in the world’s rangelands. More specifically, we present a Theory of Change to guide the design, monitoring, and evaluation of wilder rangelands. Through this, we aim to promote rangeland restoration, where local communities collaborate with regional and international actors to co-create new rangeland use models that simultaneously mitigate the impacts of climate change, restore biodiversity, and improve both ecosystem functioning and livelihoods

S100A6 (Calcyclin) is a prostate basal cell marker absent in prostate cancer and its precursors

S100A6 (Calcyclin) is a calcium-binding protein that has been implicated in a variety of biological functions as well as tumorigenesis. The aim of our study was to investigate the involvement of S100A6 during prostate cancer development and progression. Using immunohistochemistry, the expression of S100A6 was examined in benign (n ¼ 66), premalignant (n ¼ 10), malignant (n ¼ 66) and metastatic prostate (n ¼ 5) tissues arranged in a tissue-microarray or whole sections as well as in prostate cancer cell lines. The S100A6 immunostaining pattern in tissues was compared with that of cytokeratin 5 (a basal cell marker) and 18 (a benign luminal cell marker). In all cases of benign epithelium, intense S100A6 expression was seen in the basal cell layer with absent staining in luminal cells. In all cases of prostatic adenocarcinoma (matched), metastatic lesions and 3/10 high-grade prostatic intraepithelial neoplasia lesions, an absence of S100A6 was seen. Western blotting and RT–PCR analysis of cell lines showed S100A6 expression to be absent in LNCaP, LNCaP-LN3 and LNCaP-Pro5 but present in Du145, PC3, PC-3M and PC-3M-LN4. LNCaP cells treated with 5- Azacytidine, caused re-expression of S100A6 mRNA. Sequencing of bisulphite modified DNA showed CpG methylation within the S100A6 promoter region and exon 1 of LNCaP, LNCaP-LN3 and LNCaP-Pro5 cell lines but not in Du145 cells. Our data suggest that loss of S100A6 protein expression is common in prostate cancer development and may occur at an early stage. The mechanism of loss of expression may involve hypermethylation of CpG sites. The finding of intense S100A6 expression in the basal cells of benign glands but loss of expression in cancer could be useful as a novel diagnostic marker for prostate cancer

A theoretical study of the activity in Rh-catalyzed hydroformylation: the origin of the enhanced activity of the π-acceptor phosphinine ligand

The factors governing the activity in Rh-catalyzed hydroformylation were investigated using a set of computational tools. We performed DFT calculations on the phosphinine- modified Rh catalyst [HRh(CO)3(PC5H2R3)] and compared it to the phosphane-modified HRh(CO)3(PR3) and HRh(CO)2(PR3)2 complexes. The π-acceptor phosphinine ligand coordinates preferentially at the equatorial site of the pentacoordinated Rh complex with the heterocycle perpendicular to the equatorial plane, although the ligand freely rotates around the Rh–P bond. The overall energy barrier can be divided into the following contributions: alkene complex formation, alkene rotation and alkene insertion. In the absence of steric effects (model systems), the overall barrier correlates with the computed barrier for alkene rotation. This proves that π-acceptor ligands reduce back-donation to the alkene, leading to a lower rotational barrier and, consequently, to a higher activity. The Rh–P donor–acceptor interactions were quantified using a modified version of energy decomposition analysis (EDA). In Rh–phosphinine systems, the efficient directionality of the π-back-donation, rather than the overall acceptor ability, is responsible for the high catalytic activity. Introducing steric effects increases the energy required to coordinate the alkene, increasing the overall barrier. The factors governing the activity in Rh–monophosphane catalysts seem to be related to those derived for Rh–diphosphane during the development of a QSAR model (Catal. Sci. Technol. 2012, 2, 1694). To investigate whether the findings for mono- can be extrapolated to diphosphane ligands, we re-examined our previous QSAR model using the Topological Maximum Cross Correlation (TMACC) method based on easy-to-interpret 2D-descriptors. The TMACC descriptors highlight heteroatoms close to phosphorus as activity-increasing atoms, whereas highly substituted carbon atom groups are highlighted as activity-decreasing groups

Clathrin Is Spindle-Associated but Not Essential for Mitosis

Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells

Revealing histological and morphological features of female reproductive system in tree shrew (Tupaia belangeri)

The tree shrew has been used as a primate animal model in neuroscience studies but it has only rarely been employed in the study of reproductive systems. This is mainly because we know very little about the histological features of reproductive organs of the tree shrew. In this study, we have systematically analyzed the histology of reproductive organs of tree shrew, in comparison with human organs. The uterus of female tree shrew is uterus biomes unicolis, which is connected with an enveloped ovary through a thin fallopian tube. Histologically, the fallopian tube consists of folded mucosa, muscularis and serosa. Like other mammalian animals, the different developmental stages (primordial, primary, secondary and Graafian follicles) of ovarian follicles including inner oocyte and outer granulosa cells are embedded in the cortex. The luminal endometrium, middle muscular myometrium and serosa constitute the wall of uterus of tree shrew. The uterine endometrium contains simple columnar ciliated cells and goblet cells, and there are rich uterine glands in underlying stroma. Furthermore, these glands of tree shrew are round and smaller during anestrus, and become much longer when they are in estrus. The uterine endometrium in younger animals was less developed when compared to a mature tree shrew. Compared to human uterine endometrium, the histological features of tree shrew are very similar, indicating that it could potentially be good primate animal model for studying the diseases in reproductive system