Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363 bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture. (c) 2007 Elsevier Ltd. All rights reserved.

Genome constitutions of Hystrix patula, H-duthiei ssp duthiei and H-duthiei ssp longearistata (Poaceae : Triticeae) revealed by meiotic pairing behavior and genomic in-situ hybridization

Genomic constitutions of three taxa of Hystrix Moench, H. patula, H. duthiei ssp. duthiei and H. duthiei ssp. longearistata, were examined by meiotic pairing behavior and genomic in-situ hybridization. Meiotic pairing in hybrids of H. patula x Pseudoroegneria spicata (St), H. patula x Elymus wawawaiensis (StH), H. patula x H. duthiei ssp. longearistata, H. patula x Psathyrostachys huashanica (Ns(h)), H. duthiei ssp. duthiei x Psa. huashanica, H. duthiei ssp. longearistata x Psa. huashanica, Leymus multicaulis (NsXm) x H. duthiei ssp. longearistata averaged 6.53, 12.83, 1.32, 0.29, 5.18, 5.11 and 10.47 bivalents per cell, respectively. The results indicate that H. patula has the StH genome and H. duthiei ssp. duthiei and H. duthiei ssp. longearistata have the NsXm genome. Results of genomic in-situ hybridization analysis strongly supported the chromosome pairing data; therefore it is concluded that the type species of Hystrix, H. patula, should be included in Elymus, and that H. duthiei ssp. duthiei and H. duthiei ssp. longearistata should be transferred to Leymus.

Instability of anthocyanin accumulation in Vitis vinifera L. var. Gamay Freaux suspension cultures

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, in Vitis vinifera cell cultures. Therefore, four cell line suspensions of Vitis vinitera L. var. Gamay Freaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of 2.73 +/- 0.15, 1.45 +/- 0.04, 0.77 +/- 0.024 and 0.27 +/- 0.04 CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and 84% for V vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be 9.7%, ranging from 4 to 17%. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities to L-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line, showed greater potential in enhanced the anthocyanin production.

Integration of genomic and phenotypic data

Clare, A. (2005) Integration of genomic and phenotypic data. In Data Analysis and Visualization in Genomics and Proteomics, Eds. Francisco Azuaje and Joaquin Dopazo, Wiley, London. ISBN: 0-470-09439-7

Functional genomic hypothesis generation and experimentation by a robot scientist

King R. D., Whelan, K. E., Jones, F. M., Reiser, P. G. K., Bryant, C. H., Muggleton, S., Kell, D. B. and Oliver, S. G. (2004) Functional genomic hypothesis generation and experimentation by a robot scientist. Nature 427 (6971) p247-252

Allochthonous and autochthonous mire deposits, slope instability and palaeoenvironmental investigations in the Borve Valley, Barra, Outer Hebrides, Scotland

Ashmore, P. Brayshay, B.A Edwards, K.J Gilbertson, D. Grattan, J. Kent, M. Pratt, K. Weaver, R. 'Allochthonous and autochthonous mire deposits, slope instability and palaeoenvironmental investigations in the Borve Valley, Barra, Outer Hebrides, Scotland' The Holocene 2000 10, 1 pp.97-108

Fixational Instability and Natural Image Statistics: Implications for Early Visual Representations

Under natural viewing conditions small movements of the eye, head, and body prevent the maintenance of a steady direction of gaze. It is known that stimuli tend to fade when they a restabilized on the retina for several seconds. However; it is unclear whether the physiological motion of the retinal image serves a visual purpose during the brief periods of natural visual fixation. This study examines the impact of fixational instability on the statistics of the visua1 input to the retina and on the structure of neural activity in the early visual system. We show that fixational instability introduces a component in the retinal input signals that in the presence of natural images, lacks spatial correlations. This component strongly influences neural activity in a model of the LGN. It decorrelates cell responses even if the contrast sensitivity functions of simulated cells arc not perfectly tuned to counterbalance the power-law spectrum of natural images. A decorrelation of neural activity at the early stages of the visual system has been proposed to be beneficial for discarding statistical redundancies in the input signals. The results of this study suggest that fixational instability might contribute to establishing efficient representations of natural stimuli.

The genomic analysis of erythrocyte microRNA expression in sickle cell diseases.

BACKGROUND: Since mature erythrocytes are terminally differentiated cells without nuclei and organelles, it is commonly thought that they do not contain nucleic acids. In this study, we have re-examined this issue by analyzing the transcriptome of a purified population of human mature erythrocytes from individuals with normal hemoglobin (HbAA) and homozygous sickle cell disease (HbSS). METHODS AND FINDINGS: Using a combination of microarray analysis, real-time RT-PCR and Northern blots, we found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs. MicroRNA expression of erythrocytes was different from that of reticulocytes and leukocytes, and contributed the majority of the microRNA expression in whole blood. When we used microRNA microarrays to analyze erythrocytes from HbAA and HbSS individuals, we noted a dramatic difference in their microRNA expression pattern. We found that miR-320 played an important role for the down-regulation of its target gene, CD71 during reticulocyte terminal differentiation. Further investigation revealed that poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation. CONCLUSIONS: In summary, we have discovered significant microRNA expression in human mature erythrocytes, which is dramatically altered in HbSS erythrocytes and their defect in terminal differentiation. Thus, the global analysis of microRNA expression in circulating erythrocytes can provide mechanistic insights into the disease phenotypes of erythrocyte diseases.

Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex.

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MATa alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MATa locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MATa locus appear to have a higher number of changes and substitutions than their MATalpha counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.

Evolution of the sex-related locus and genomic features shared in microsporidia and fungi.

BACKGROUND: Microsporidia are obligate intracellular, eukaryotic pathogens that infect a wide range of animals from nematodes to humans, and in some cases, protists. The preponderance of evidence as to the origin of the microsporidia reveals a close relationship with the fungi, either within the kingdom or as a sister group to it. Recent phylogenetic studies and gene order analysis suggest that microsporidia share a particularly close evolutionary relationship with the zygomycetes. METHODOLOGY/PRINCIPAL FINDINGS: Here we expanded this analysis and also examined a putative sex-locus for variability between microsporidian populations. Whole genome inspection reveals a unique syntenic gene pair (RPS9-RPL21) present in the vast majority of fungi and the microsporidians but not in other eukaryotic lineages. Two other unique gene fusions (glutamyl-prolyl tRNA synthetase and ubiquitin-ribosomal subunit S30) that are present in metazoans, choanoflagellates, and filasterean opisthokonts are unfused in the fungi and microsporidians. One locus previously found to be conserved in many microsporidian genomes is similar to the sex locus of zygomycetes in gene order and architecture. Both sex-related and sex loci harbor TPT, HMG, and RNA helicase genes forming a syntenic gene cluster. We sequenced and analyzed the sex-related locus in 11 different Encephalitozoon cuniculi isolates and the sibling species E. intestinalis (3 isolates) and E. hellem (1 isolate). There was no evidence for an idiomorphic sex-related locus in this Encephalitozoon species sample. According to sequence-based phylogenetic analyses, the TPT and RNA helicase genes flanking the HMG genes are paralogous rather than orthologous between zygomycetes and microsporidians. CONCLUSION/SIGNIFICANCE: The unique genomic hallmarks between microsporidia and fungi are independent of sequence based phylogenetic comparisons and further contribute to define the borders of the fungal kingdom and support the classification of microsporidia as unusual derived fungi. And the sex/sex-related loci appear to have been subject to frequent gene conversion and translocations in microsporidia and zygomycetes.

Bunching-induced optical nonlinearity and instability in cold atoms [Invited].

We report a new nonlinear optical process that occurs in a cloud of cold atoms at low-light-levels when the incident optical fields simultaneously polarize, cool, and spatially-organize the atoms. We observe an extremely large effective fifth-order nonlinear susceptibility of χ(⁵) = 7.6 × 10⁻¹⁵ (m/V)⁴, which results in efficient Bragg scattering via six-wave mixing, slow group velocities (∼ c/10⁵), and enhanced atomic coherence times (> 100 μs). In addition, this process is particularly sensitive to the atomic temperatures, and provides a new tool for in-situ monitoring of the atomic momentum distribution in an optical lattice. For sufficiently large light-matter couplings, we observe an optical instability for intensities as low as ∼ 1 mW/cm² in which new, intense beams of light are generated and result in the formation of controllable transverse optical patterns.

Rapid behavioral and genomic responses to social opportunity.

From primates to bees, social status regulates reproduction. In the cichlid fish Astatotilapia (Haplochromis) burtoni, subordinate males have reduced fertility and must become dominant to reproduce. This increase in sexual capacity is orchestrated by neurons in the preoptic area, which enlarge in response to dominance and increase expression of gonadotropin-releasing hormone 1 (GnRH1), a peptide critical for reproduction. Using a novel behavioral paradigm, we show for the first time that subordinate males can become dominant within minutes of an opportunity to do so, displaying dramatic changes in body coloration and behavior. We also found that social opportunity induced expression of the immediate-early gene egr-1 in the anterior preoptic area, peaking in regions with high densities of GnRH1 neurons, and not in brain regions that express the related peptides GnRH2 and GnRH3. This genomic response did not occur in stable subordinate or stable dominant males even though stable dominants, like ascending males, displayed dominance behaviors. Moreover, egr-1 in the optic tectum and the cerebellum was similarly induced in all experimental groups, showing that egr-1 induction in the anterior preoptic area of ascending males was specific to this brain region. Because egr-1 codes for a transcription factor important in neural plasticity, induction of egr-1 in the anterior preoptic area by social opportunity could be an early trigger in the molecular cascade that culminates in enhanced fertility and other long-term physiological changes associated with dominance.

Comparative genomic data of the Avian Phylogenomics Project.

BACKGROUND: The evolutionary relationships of modern birds are among the most challenging to understand in systematic biology and have been debated for centuries. To address this challenge, we assembled or collected the genomes of 48 avian species spanning most orders of birds, including all Neognathae and two of the five Palaeognathae orders, and used the genomes to construct a genome-scale avian phylogenetic tree and perform comparative genomics analyses (Jarvis et al. in press; Zhang et al. in press). Here we release assemblies and datasets associated with the comparative genome analyses, which include 38 newly sequenced avian genomes plus previously released or simultaneously released genomes of Chicken, Zebra finch, Turkey, Pigeon, Peregrine falcon, Duck, Budgerigar, Adelie penguin, Emperor penguin and the Medium Ground Finch. We hope that this resource will serve future efforts in phylogenomics and comparative genomics. FINDINGS: The 38 bird genomes were sequenced using the Illumina HiSeq 2000 platform and assembled using a whole genome shotgun strategy. The 48 genomes were categorized into two groups according to the N50 scaffold size of the assemblies: a high depth group comprising 23 species sequenced at high coverage (>50X) with multiple insert size libraries resulting in N50 scaffold sizes greater than 1 Mb (except the White-throated Tinamou and Bald Eagle); and a low depth group comprising 25 species sequenced at a low coverage (~30X) with two insert size libraries resulting in an average N50 scaffold size of about 50 kb. Repetitive elements comprised 4%-22% of the bird genomes. The assembled scaffolds allowed the homology-based annotation of 13,000 ~ 17000 protein coding genes in each avian genome relative to chicken, zebra finch and human, as well as comparative and sequence conservation analyses. CONCLUSIONS: Here we release full genome assemblies of 38 newly sequenced avian species, link genome assembly downloads for the 7 of the remaining 10 species, and provide a guideline of genomic data that has been generated and used in our Avian Phylogenomics Project. To the best of our knowledge, the Avian Phylogenomics Project is the biggest vertebrate comparative genomics project to date. The genomic data presented here is expected to accelerate further analyses in many fields, including phylogenetics, comparative genomics, evolution, neurobiology, development biology, and other related areas.

Computational Inference of Genome-Wide Protein-DNA Interactions Using High-Throughput Genomic Data

Transcriptional regulation has been studied intensively in recent decades. One important aspect of this regulation is the interaction between regulatory proteins, such as transcription factors (TF) and nucleosomes, and the genome. Different high-throughput techniques have been invented to map these interactions genome-wide, including ChIP-based methods (ChIP-chip, ChIP-seq, etc.), nuclease digestion methods (DNase-seq, MNase-seq, etc.), and others. However, a single experimental technique often only provides partial and noisy information about the whole picture of protein-DNA interactions. Therefore, the overarching goal of this dissertation is to provide computational developments for jointly modeling different experimental datasets to achieve a holistic inference on the protein-DNA interaction landscape.

We first present a computational framework that can incorporate the protein binding information in MNase-seq data into a thermodynamic model of protein-DNA interaction. We use a correlation-based objective function to model the MNase-seq data and a Markov chain Monte Carlo method to maximize the function. Our results show that the inferred protein-DNA interaction landscape is concordant with the MNase-seq data and provides a mechanistic explanation for the experimentally collected MNase-seq fragments. Our framework is flexible and can easily incorporate other data sources. To demonstrate this flexibility, we use prior distributions to integrate experimentally measured protein concentrations.

We also study the ability of DNase-seq data to position nucleosomes. Traditionally, DNase-seq has only been widely used to identify DNase hypersensitive sites, which tend to be open chromatin regulatory regions devoid of nucleosomes. We reveal for the first time that DNase-seq datasets also contain substantial information about nucleosome translational positioning, and that existing DNase-seq data can be used to infer nucleosome positions with high accuracy. We develop a Bayes-factor-based nucleosome scoring method to position nucleosomes using DNase-seq data. Our approach utilizes several effective strategies to extract nucleosome positioning signals from the noisy DNase-seq data, including jointly modeling data points across the nucleosome body and explicitly modeling the quadratic and oscillatory DNase I digestion pattern on nucleosomes. We show that our DNase-seq-based nucleosome map is highly consistent with previous high-resolution maps. We also show that the oscillatory DNase I digestion pattern is useful in revealing the nucleosome rotational context around TF binding sites.

Finally, we present a state-space model (SSM) for jointly modeling different kinds of genomic data to provide an accurate view of the protein-DNA interaction landscape. We also provide an efficient expectation-maximization algorithm to learn model parameters from data. We first show in simulation studies that the SSM can effectively recover underlying true protein binding configurations. We then apply the SSM to model real genomic data (both DNase-seq and MNase-seq data). Through incrementally increasing the types of genomic data in the SSM, we show that different data types can contribute complementary information for the inference of protein binding landscape and that the most accurate inference comes from modeling all available datasets.

This dissertation provides a foundation for future research by taking a step toward the genome-wide inference of protein-DNA interaction landscape through data integration.

Patient beliefs and behaviors about genomic risk for type 2 diabetes: Implications for prevention

Copyright © Taylor & Francis Group, LLC 2015.Type 2 diabetes is a major health burden in the United States, and population trends suggest this burden will increase. High interest in, and increased availability of, testing for genetic risk of type 2 diabetes presents a new opportunity for reducing type 2 diabetes risk for many patients; however, to date, there is little evidence that genetic testing positively affects type 2 diabetes prevention. Genetic information may not fit patients illness representations, which may reduce the chances of risk-reducing behavior changes. The present study aimed to examine illness representations in a clinical sample who are at risk for type 2 diabetes and interested in genetic testing. The authors used the Common Sense Model to analyze survey responses of 409 patients with type 2 diabetes risk factors. Patients were interested in genetic testing for type 2 diabetes risk and believed in its importance. Most patients believed that genetic factors are important to developing type 2 diabetes (67%), that diet and exercise are effective in preventing type 2 diabetes (95%), and that lifestyle changes are more effective than drugs (86%). Belief in genetic causality was not related to poorer self-reported health behaviors. These results suggest that patients interest in genetic testing for type 2 diabetes might produce a teachable moment that clinicians can use to counsel behavior change.