Transcriptome sequencing to produce SNP-based genetic maps of onion.

We used the Roche-454 platform to sequence from normalized cDNA libraries from each of two inbred lines of onion (OH1 and 5225). From approximately 1.6 million reads from each inbred, 27,065 and 33,254 cDNA contigs were assembled from OH1 and 5225, respectively. In total, 3,364 well supported single nucleotide polymorphisms (SNPs) on 1,716 cDNA contigs were identified between these two inbreds. One SNP on each of 1,256 contigs was randomly selected for genotyping. OH1 and 5225 were crossed and 182 gynogenic haploids extracted from hybrid plants were used for SNP mapping. A total of 597 SNPs segregated in the OH1 × 5225 haploid family and a genetic map of ten linkage groups (LOD ≥8) was constructed. Three hundred and thirty-nine of the newly identified SNPs were also mapped using a previously developed segregating family from BYG15-23 × AC43, and 223 common SNPs were used to join the two maps. Because these new SNPs are in expressed regions of the genome and commonly occur among onion germplasms, they will be useful for genetic mapping, gene tagging, marker-aided selection, quality control of seed lots, and fingerprinting of cultivars.

Monitoring environmental microbiomes: Alignment of microbiology and computational biology competencies within a culturally integrated curriculum and research framework.

We have developed a flexible undergraduate curriculum that leverages the place-based research of environmental microbiomes to increase the number of Indigenous researchers in microbiology, data science and scientific computing. Monitoring Environmental Microbiomes (MEM) provides a curriculum and research framework designed to integrate an Indigenous approach when conducting authentic scientific research and to build interest and confidence at the undergraduate level. MEM has been successfully implemented as a short summer workshop to introduce computing practices in microbiome analysis. Based on self-assessed student knowledge of topics and skills, increased scientific confidence and interest in genomics careers were observed. We propose MEM be incorporated in a scalable course-based research experience for undergraduate institutions, including tribal colleges and universities, community colleges and other minority serving institutions. This coupled curricular and research framework explicitly considers cultural perspectives, access and equity to train a diverse future workforce that is more informed to engage in microbiome research and to translate microbiome science to benefit community and environmental health.

Gene space dynamics during the evolution of Aegilops tauschii, Brachypodium distachyon, Oryza sativa, and Sorghum bicolor genomes.

Nine different regions totaling 9.7 Mb of the 4.02 Gb Aegilops tauschii genome were sequenced using the Sanger sequencing technology and compared with orthologous Brachypodium distachyon, Oryza sativa (rice), and Sorghum bicolor (sorghum) genomic sequences. The ancestral gene content in these regions was inferred and used to estimate gene deletion and gene duplication rates along each branch of the phylogenetic tree relating the four species. The total gene number in the extant Ae. tauschii genome was estimated to be 36,371. The gene deletion and gene duplication rates and total gene numbers in the four genomes were used to estimate the total gene number in each node of the phylogenetic tree. The common ancestor of the Brachypodieae and Triticeae lineages was estimated to have had 28,558 genes, and the common ancestor of the Panicoideae, Ehrhartoideae, and Pooideae subfamilies was estimated to have had 27,152 or 28,350 genes, depending on the ancestral gene scenario. Relative to the Brachypodieae and Triticeae common ancestor, the gene number was reduced in B. distachyon by 3,026 genes and increased in Ae. tauschii by 7,813 genes. The sum of gene deletion and gene duplication rates, which reflects the rate of gene synteny loss, was correlated with the rate of structural chromosome rearrangements and was highest in the Ae. tauschii lineage and lowest in the rice lineage. The high rate of gene space evolution in the Ae. tauschii lineage accounts for the fact that, contrary to the expectations, the level of synteny between the phylogenetically more related Ae. tauschii and B. distachyon genomes is similar to the level of synteny between the Ae. tauschii genome and the genomes of the less related rice and sorghum. The ratio of gene duplication to gene deletion rates in these four grass species closely parallels both the total number of genes in a species and the overall genome size. Because the overall genome size is to a large extent a function of the repeated sequence content in a genome, we suggest that the amount and activity of repeated sequences are important factors determining the number of genes in a genome.

Comparison between the DNA precipitation and alkali unwinding assays for detecting DNA strand breaks and cross-links.

A new DNA precipitation assay used together with the alkali unwinding assay may provide a rapid means of detecting DNA damage in addition to strand breaks based on the relative amount of damage measured by the two assays. X-rays, Adriamycin, 4-nitroquinoline-N-oxide, N-methyl-N'-nitrosoguanidine, bleomycin, RSU 1172, and five other drugs produced the same relative amount of strand breakage by using the DNA precipitation and alkali unwinding assays. However, strand breaks produced by the bifunctional alkylating agents bis(2-chloroethyl)nitrosourea, RSU 1069, and RSU 1131 were detected with greater efficiency by the DNA precipitation assay, while the unwinding assay measured more strand breaks than the precipitation assay after damage by the topoisomerase inhibitors VP-16 and VM-26 and the DNA-condensing agents acridine orange and pyronin Y. Based on the reported mechanisms of action of these drugs, and studies with known DNA cross-linking agents, it appears that in addition to DNA strand breaks, the alkali unwinding assay is more sensitive to interstrand than to DNA-protein cross-links, while the DNA precipitation assay can be used to detect both types of cross-links. While quantification of specific lesions is not possible with this approach, the concomitant use of these two assays may provide a rapid and simple method for screening genotoxic drugs for DNA damage, and may also help to differentiate between DNA lesions which include strand breaks, interstrand and protein cross-links, DNA-phosphate adducts, and DNA-drug precipitates.

Cloning and sequencing of the grass carp (Ctenopharyngodon idellus) growth hormone gene.

An enriched lambda gt11 library for screening the grass carp (Ctenopharyngodon idellus) growth hormone gene was constructed using HindIII digested genomic DNA extracted from the spleen of the fish. Probing this library with a homologous cDNA, a clone carrying the growth hormone gene was obtained. The gene is 2501 bp long and consists of five exons and four introns. The sequence of nucleotide in the exons is almost identical to that of the cDNA except for ten positions. Analysis of the 5' sequence up to 1220 bp from the +1 position reveals the presence of a TATA box as well as a number of consensus regulatory sequences. Comparison of the grass carp growth hormone gene with those of other fish indicates that there is a high degree of homology with the gene from the common carp but not with those from the rainbow trout and Atlantic salmon.

RNA localization and germ cell determination in Xenopus.

In many organisms the proper development of the embryo depends on the asymmetrical distribution of maternal RNAs and proteins in the egg. Although the Xenopus oocyte is radially symmetrical it contains distinct populations of maternal RNAs that are localized either in the animal or vegetal pole. The process of localization of RNAs in Xenopus oocytes occurs during the long period of oocyte differentiation and growth that is accompanied by the elaboration of oocyte polarity. Some of the vegetally localized RNAs, such as Vg1, VegT, and Xwnt11, are involved in axial patterning and germ layer specification. Others, such as Xdazl and Xcat2, which are located in the germ plasm, are likely to play a role in the specification of germ cell fate. We will discuss the different aspects of RNA localization in Xenopus in the context of the differentiation of the germ cells and the development of the oocyte polarity.

Contribution of METRO pathway localized molecules to the organization of the germ cell lineage.

To elucidate the potential role of localized components in the specification of the germ cell lineage we analyzed the composition of the germ plasm in Xenopus laevis oocytes and early embryos with respect to the vegetally-localized RNAs. We focused on Xlsirts, Xcat2, and Xwnt11 transcripts that are localized to the vegetal cortex through a region of the mitochondrial cloud called the messenger transport organizer (METRO) that also contains the nuage or germ plasm. At the ultrastructural level Xcat2 mRNA was detected on germinal granules while Xlsirts and Xwnt11 were associated with a fibrillar network of the germ plasm in stage-1 and stage-4 oocytes. In embryos, we found that all three RNAs remained associated with the germ plasm. Vg1 mRNA, a transcript localized through the late pathway, was excluded from the germ plasm in oocytes and embryos. Addtionally, we detected the protein spectrin within 16 cell nests of germ cells, in a structure reminiscent of the Drosophila spectrosome. Spectrin was detected in the mitochondrial cloud and was found in the germ plasm during embryogenesis. These data indicate that the various RNAs found within METRO and the protein spectrin are integral components of the Xenopus germ plasm with the RNAs being associated with different subcellular structures. They also suggest that the pathway through which RNAs are localized during oogenesis may be an important factor in biasing their distribution into specific cell lineages. The presence of Xwnt11 in the germ cell lineage suggests that a wnt-directed signaling pathway may be involved in germ cell specification. differentiation or migration.

The vegetally localized mRNA fatvg is associated with the germ plasm in the early embryo and is later expressed in the fat body.

Vegetally localized RNAs in Xenopus oocytes have been implicated in the establishment of the primary germ layers and the formation and development of the primordial germ cells. fatvg mRNA is localized through the late pathway to the vegetal cortex. Like Vg1 mRNA fatvg is distributed throughout the entire cortex; however, unlike Vg1 there is a small fraction of the fatvg mRNA that is associated with the mitochondrial cloud. In early cleavage stage embryos, fatvg mRNA is associated with the germ plasm located at the tips of the vegetal blastomeres of the embryo. While several localized RNAs that follow the Message Transport Organizer (METRO) pathway have been found in the germ plasm in embryos, fatvg is a late pathway RNA that is associated with the germ plasm. In tadpoles, fatvg mRNA shows a novel pattern of expression which is distinct from the germ cell lineage and is detected at the dorso-anterior margin of the endodermal mass along the midline in two clusters of cells. fatvg mRNA expression is also detected later in the developing fat bodies, the major adipose tissues of the frog.

Nuclear transplantation from stably transfected cultured cells of Xenopus.

By nuclear transplantation we have generated embryos from enucleated Xenopus eggs and nuclei of stably transfected Xenopus cell lines. We have devised a novel method of transplantation in which cell permeabilization is controlled by a temperature effect on streptolysin O-treated cells. This method is easier and quicker to operate than the conventional cell rupture technique. Single nuclei from cell lines transfected with the lacZ reporter gene were transplanted to Xenopus eggs in which the egg nuclei were destroyed by UV irradiation. We show that the lacZ transgene is transmitted from donor cells to nuclear transplant embryos. Expression of the lacZ transgene has been controlled by the elongation factor 1-alpha promoter (Krieg et al., Dev. Biol. 133: 93-100, 1989). In the nuclear transplant embryos, beta-galactosidase transcripts are expressed at the expected time of development, that is after the mid-blastula transition. In addition, we show that early embryo-specific genes, not expressed in cultured cells, are normally activated in nuclear transplant embryos. Therefore, expression of these genes can be used to monitor the effects of transfected test genes. Although most of the nuclear transplant embryos do not develop beyond the gastrula stage, explants of equatorial tissue from these embryos can undergo differentiation characterized by the expression of muscle and notochord markers. The use of nuclear transplantation, as described here, provides a means of avoiding the mosaic expression of DNA or mRNA injected into Xenopus eggs.

A rapid algorithm for solution of the equations of multiple equilibrium systems-RAMESES.

The solution of systems of equilibrium is an important though commonplace operation, rendered difficult by non-linearity. Previous methods of numerical solution have suffered from slow convergence, unreliability, and inefficient structure. An algorithm, RAMESES, is presented in matrix algebra terms, which is both simple in structure and efficient, involving only one matrix inversion per system and giving an exact solution of the set of dependent equilibrium equations at each iteration.

Impairment of endothelial function--a possible mechanism for atherosclerosis of a high-fat meal intake.

INTRODUCTION: Endothelial dysfunction is known to occur in patients with coronary artery disease. Flow-mediated dilation of the brachial artery using Doppler ultrasound is a non-invasive technique for the assessment of endothelial function. The objective of the study was to use the above method to evaluate the pathophysiology of high-fat (HF) intake on endothelial function in a local population. A popular local dish "nasi-lemak", a source of high saturated fat content from coconut milk, was chosen to represent a local high-fat meal (LHF). In addition, the effects of a Western high-fat (WHF) ("McDonald's") meal and a low-fat (LF) meal control on endothelial function were studied. MATERIALS AND METHODS: The study population consisted of 10 healthy male non-smoker (mean age 22 +/- 2 years) with normal body mass index, normal fasting sugar and lipid profiles. Nitric oxide dependent flow-mediated dilation and nitric oxide independent (GTN) dilation was assessed by Doppler flow in the brachial artery before and 4 hours after each meal on separate occasions by 2 experienced sonographers blinded to the type of meals. RESULTS: The baseline brachial artery size, baseline vessel flow and increase in flow after cuff deflation were similar for each of the six arterial studies. In response to reactive hyperaemia after cuff deflation, the endothelium-dependent dilation was significantly different between the meals. There was a marked decrease in endothelium-dependent dilation after the WHF meal compared to the LF meal (8.6 +/- 2.2% vs. -0.8 +/- 1.1%, P < 0.006). There was also a marked decrease in endothelium-dependent dilation after the LHF meal compared to the LF meal (7.7 +/- 2.1% vs. -0.8 +/- 1.1%, P < 0.001). When comparing between the two HF meals, the change in endothelium-dependent dilation was not significant (7.7 vs. 8.6%, P = 0.678). GTN-induced dilation was not significantly different before and after the LF, WHF or LHF (0.1 +/- 0.5% vs. 0.2 +/- 0.9% vs. 1.3 +/- 0.5%, P = 0.094). CONCLUSION: The results suggest that in a local population, impairment of endothelial function is a possible mechanism in the pathophysiology of atherosclerosis from HF intake, beyond just affecting lipid levels. This effect is observed after both a LHF and a WHF meal intake. This technique to study endothelial function may be a useful non-invasive screening tool in the study of other HF diet choices and provides further information for the education of the influence of dietary choices on atherosclerosis.

Rehabilitation outcomes following traumatic spinal cord injury in a tertiary spinal cord injury centre: a comparison with an international standard.

STUDY DESIGN: Retrospective descriptive analysis of data of patients with traumatic spinal cord injury (SCI) in a tertiary SCI centre. OBJECTIVES: To identify the characteristics of the rehabilitation outcomes of patients with different levels of traumatic SCI and to compare the results with data reported in the American Consortium for Spinal Cord Medicine. SETTING: A newly established tertiary SCI centre in Tai Po Hospital, Tai Po, Hong Kong. METHODS: A total of 33 patients with traumatic SCI admitted in 2002 were included in the study. They were classified into different ASIA subgroups based on their levels and completeness of injury. The functional status changes measured by the Functional Independence Measure (FIM) (on admission, placement and upon discharge, and at 1 and 3 months post discharge) and discharge placement were recorded as rehabilitation outcomes. RESULTS: A total, 24 patients were tetraplegic while nine were paraplegic. Seven and two from tetraplegic and paraplegic groups were readmitted with late complications due to urinary tract infection, spasticity and/or occurrence of pressure sores. The mean age was found to be 48.36 (SD=15.64) years. In all, 16 (48.48%) sustained the injury from falling from height. The trend of FIM motor scores at discharge across different ASIA subgroups appeared to be comparable to those reported in the American Consortium for Spinal Cord Medicine with scores generally lower. Significant functional improvements during the hospital phase were found in the two tetraplegic and paraplegic ASIA D subgroups (t3=3.430, P<0.05; t2=4.083, P=0.55, respectively). Significant differences were also revealed among subgroups (F(7,32)=6.625, P<0.0005) with lower level tetraplegic groups appearing to stay much longer in the rehabilitation centre. In all, 64.5% of newly diagnosed patients returned to live in the community. CONCLUSIONS: This report gives a preliminary overview on the characteristics of rehabilitation outcomes in one of the SCI centres in Hong Kong in relation to the international standard. Longitudinal study with larger population and community integration outcomes might be included in the future to reveal a better picture in the SCI rehabilitation in Hong Kong.

Patterning and lineage specification in the amphibian embryo.

Xenopus has been widely used to study early embryogenesis because the embryos allow for efficient functional assays of gene products by the overexpression of RNA. The first asymmetry of the embryo is initiated during oogenesis and is manifested by the darkly pigmented animal hemisphere and lightly pigmented vegetal hemisphere. Upon fertilization a second asymmetry, the dorsal-ventral asymmetry, is established, with the sperm entry site defining the prospective ventral region. During the cleavage stage, a vegetal cortical cytoplasm (VCC)/beta-catenin signaling pathway is differentially activated on the prospective dorsal side of the embryo. The overlapping of the VCC/beta-catenin and transforming growth factor beta (TGF-beta) pathways in the dorsal vegetal quadrant specifies dorsal-vental axis formation by regulating formation of the Spemann organizer, including the anterior endomesoderm. The organizer initiates gastrulation to form a triploblastic embryo in which the mesoderm layer is located between the ectoderm layer and the endoderm layer. The interplay between maternal and zygotic TGF-beta s and the T-box transcription factors in the vegetal hemisphere initiates the specification of germ-layer lineages. TGF-beta signaling originating from the vegetal region induces mesoderm in the equatorial region, and initiates endoderm differentiation directly in the vegetal region. The ectoderm develops from the animal region, which does not come into contact with the vegetal TGF-beta signals. A large number of the downstream components and transcriptional targets of early developmental pathways have been identified and characterized. This review gives an overview of recent advances in the understanding of the functional roles and interactions of the molecular players important for axis determination and germ-layer specification during early Xenopus embryogenesis.

fatvg encodes a new localized RNA that uses a 25-nucleotide element (FVLE1) to localize to the vegetal cortex of Xenopus oocytes.

Vegetally localized transcripts have been implicated in a number of important biological functions, including cell fate determination and embryonic patterning. We have isolated a cDNA, fatvg, which encodes a localized maternal transcript that exhibits a localization pattern reminiscent of Vg1 mRNA. fatvg is the homologue of a mammalian gene expressed in adipose tissues. The fatvg transcript, unlike Vg1 which localizes strictly through the Late pathway, also associates with the mitochondrial cloud that is characteristic of the METRO or Early pathway. This suggests that fatvg mRNA may utilize both the METRO and Late pathways to localize to the vegetal cortex during oogenesis. We have dissected the cis-acting localization elements of fatvg mRNA and compared these elements with Vg1 mRNA. Our results indicate that, like most localized RNAs, in a variety of systems, transcripts of fatvg contain localization elements in the 3'UTR. The 3'UTR of fatvg mRNA contains multiple elements that are able to function independently; however, it functions most efficiently when all of the elements are present. We have defined a short 25-nucleotide element that can direct vegetal localization as a single copy. This element differs in sequence from previously described Vg1 localization elements, suggesting that different localization elements are involved in the localization of RNAs through the Late pathway.

Mitochondrial ribosomal RNA in the germinal granules in Xenopus embryos revisited.

Germ cells of various animals contain a determinant that is called the germ plasm. In amphibians such as Xenopus laevis, the germ plasm is composed of mitochondria and electron dense germinal granules that are embedded in a fibrillar matrix. Previous reports indicated that one of the components of germinal granules was mitochondrial large and small ribosomal RNA (mtlrRNA and mtsrRNA). Utilizing a modified procedure for electron microscopy in situ hybridization, we investigated the distribution of these RNAs along with other components of the germ plasm in Xenopus laevis embryos. We found, that contrary to previous reports, the mtlrRNA and mtsrRNA were located in close vicinity to the germinal granules but were not major constituents of granules. The majority of the mtlrRNA and mtlsrRNAs was present inside the mitochondria and in the germ plasm matrix.

Hermes is a localized factor regulating cleavage of vegetal blastomeres in Xenopus laevis.

We have identified the RNA-binding protein Hermes in a screen for vegetally localized RNAs in Xenopus oocytes. The RNA localizes to the vegetal cortex through both the message transport organizer (METRO) and late pathways. Hermes mRNA and protein are both detected at the vegetal cortex of the oocyte; however, the protein is degraded within a several hour period during oocyte maturation. Injection of antisense morpholino oligonucleotides (HE-MO) against Hermes caused a precocious reduction in Hermes protein present during maturation and resulted in a phenotype characterized by cleavage defects in vegetal blastomeres. The phenotype can be partially rescued by injecting Hermes mRNA. These results demonstrate that the localized RNA-binding protein Hermes functions during oocyte maturation to regulate the cleavage of specific vegetally derived cell lineages. Hermes most likely performs its function by regulating the translation or processing of one or more target RNAs. This is an important mechanism by which the embryo can generate unique cell lineages. The regulation of region-specific cell division is a novel function for a localized mRNA.

Hemoptysis: comparative study of the role of CT and fiberoptic bronchoscopy.

PURPOSE: A prospective study was performed to compare results of computed tomography (CT) and fiberoptic bronchoscopy in diagnosis of cancer in patients with hemoptysis. MATERIALS AND METHODS: Ninety-one patients (64 men, 27 women) with hemoptysis underwent thin-section CT and fiberoptic bronchoscopy. RESULTS: CT scans demonstrated all 27 tumors seen at bronchoscopy and an additional seven, five of which were beyond bronchoscopic range. In patients with normal chest radiographs, bronchial carcinoma was detected in 5% at both bronchoscopy and CT. In patients with abnormal findings on radiographs, bronchoscopy allowed both location and histologic diagnosis in 78% of carcinomas but was unreliable in locating peripheral tumors demonstrated at CT. CT was insensitive in demonstrating early mucosal abnormalities, bronchitis, squamous metaplasia, and a benign papilloma, all detected at bronchoscopy. CONCLUSION: The initial examination should be bronchoscopy when there is high clinical suspicion of carcinoma and relevant radiographic abnormality, and CT when strong clinical suspicion of carcinoma is not substantiated at bronchoscopy in patients with normal findings on chest radiographs.

Tumorhead, a Xenopus gene product that inhibits neural differentiation through regulation of proliferation.

Tumorhead (TH) is a novel maternal gene product from Xenopus laevis containing several basic domains and a weak coiled-coil. Overexpression of wild-type TH resulted in increased proliferation of neural plate cells, causing expansion of the neural field followed by neural tube and craniofacial abnormalities. Overexpressed TH protein repressed neural differentiation and neural crest markers, but did not inhibit the neural inducers, pan-neural markers or mesodermal markers. Loss of function by injection of anti-TH antibody inhibited cell proliferation. Our data are consistent with a model in which tumorhead functions in regulating differentiation of the neural tissues but not neural induction or determination through its effect on cell proliferation.