Transformations of grapevine pathogens Eutypa lata and Phaeomoniella chlamydospora : a one year project thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand

A transformation system has been developed for the grapevine pathogenic fungi Eutypa lata and Phaeomoniella chlamydospora using a positive selection system based on the Escherichia coli hygromycin B phosphotransferase gene (hph). The system developed could give large, stable transformants at frequencies between 0.7 and 6.5 transformants per µg of DNA. A second type of colony also grew on the selective media. These were believed to be abortive transformants. The first type of transformants were characterized using classical molecular biological technologies such as PCR and Southern hybridization, and the transformation was shown to be successful. Plasmids (pBCH-gfp and pCT74) containing a gfp reporter gene were also transformed into these two fungal species. Expression of the gfp gene was checked using a fluorescence microscope and gfp-expressing E. lata transformants were inoculated onto the host plants blackcurrant and grapevine. Confocal observation of the movement of fungal mycelia in wood tissues was performed but its interaction with host plant was not established in the time available. Purified gfp-expressing P. chlamydospora transformants were also obtained. A vector containing a fragment of the P. chlamydospora putative toxin gene moxY was constructed and transformed into P. chlamydospora. Putative moxY gene disruption transformants were screened with PCR followed by Southern hybridization. The putative moxY gene disruption transformants were spore purified and further confirmed with Southern hybridization. Whilst both PCR and Southern hybridization confirmed disruption of the moxY gene, clear evidence for the presence of an additional wild type moxY was also seen in the same transformants. This led to the suggestions that either P. chlamydospora is a natural diploid, or that moxY is essential for growth and that selective pressures led to the formation of a wild type: moxY-hph diploid

Ectopic expression of RNA-dependent RNA polymerase in Drosophila melanogaster and microRNAs identification in Helicoverpa armigera

Drosophila melanogaster, along with all insects and the vertebrates, lacks an RNA-dependent RNA polymerase (RdRp) gene. We therefore asked whether the C. elegans RdRp genes could be introduced into Drosophila in order to study the effects on RNAi. This system also allowed us to ask whether there were any effects on development, possibly due to interference with microRNA (miRNA) regulation of genes. To address these questions, we introduced the two C. elegans RdRp genes, rrf-1 and ego-1, into D. melanogaster through microinjection. Activation of the gene was performed by crossing these lines to flies carrying the GAL4 transgene under the control of various Drosophila enhancers. Activation of the RdRp genes did not elicit any observable phenotypic changes in the eyes, wing discs, or whole body of adult flies. RT-PCR confirmed the successful RdRp gene expression. We also asked whether these RdRps were capable of enhancing RNAi of a specific, known target gene, triggered by a dsRNA corresponding to that gene. In order to test this, we selected pebble as the candidate endogenous and eGFP as an exogenous gene. Expression of RdRps in RNAi results in differential silencing activities: rrf-1 enhances transitive RNAi and ego-1 silences transgenes by a non-RNAi pathway, the silencing by ego-1 is independent of dsRNA-dependent RNAi, appearing to be transcriptional and restricted to transgenes. We therefore postulate that ego-1 transcriptionally silences transgenes through mitotic unpaired gene silencing mechanism in Drosophila. For identification of miRNAs in Helicoverpa armigera, initially, next generation Illumina sequencings were performed with three Helicoverpa total RNA samples from different tissues and different stages, i.e, neonate, gut, and pupae. about 1.26M unique sequences from 19-24 nts long were harvested. To identify candidate miRNAs, sequence alignment with miRNAs retrieved from miRBase from similar species and the pipeline miRDeep analysis were used. Sequence comparison matched 76 distinct miRNAs while miRDeep prediction gave 164 predicted miRNAs, this gave final 204 miRNAs in total, among which 76 miRNAs are conserved and 128 are novel. 15 miRNAs from above are uniquely expressed in the gut; three of them were confirmed with Northern blot and Hybridization

Genomic innovations, transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa pest species

BACKGROUND: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests. RESULTS: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes. CONCLUSIONS: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant

Different Preparations of Intravenous Immunoglobulin Vary in Their Efficacy to Neutralize Streptococcal Superantigens: Implications for Treatment of Streptococcal Toxic Shock Syndrome

Eight different batches of intravenous immunoglobulin from 3 different manufacturers were tested for neutralizing activities against all currently known streptococcal superantigens. Statistically significant variation among different intravenous immunoglobulin preparations (P < .0001) and between individual streptococcal superantigens (P < .0001) was observed. These results might be helpful for optimizing the type and dose of intravenous immunoglobulin used in adjunctive therapy for severe invasive streptococcal disease

Horizontal well workover technology present situation and development trend

In the downhole operation of horizontal Wells, the pipe string is subjected to the combined action of many kinds of loads. The pipe string may bend sinusoidal or spiral under the action of axial compressive stress, which will also increase the friction between the pipe string and the wellbore wall. Because of the existence of friction, to the milling, salvage, unstuck, sand and other construction to bring adverse effects. This paper describes the characteristics of horizontal well in fishing, sand washing, unlocking and casing milling. The paper introduces the development of horizontal well application, such as drilling technology matching for complex structure wells, high-level completion of lateral Wells, radial lateral wells in low permeability reservoirs and supporting technology for completion and production of horizontal wells

Protein kinase C is involved in arsenic trioxide-induced apoptosis and inhibition of proliferation in human bladder cancer cells

Objective: Arsenic trioxide (ATO) is a potent antitumor agent used to treat acute promyelocytic leukemia, and recently solid tumors including bladder cancers. However, a mechanism to explain its antitumor activity in bladder cancers is unclear. Here, we investigated the role of protein kinase C (PKC) in ATO-induced apoptosis and inhibition of proliferation in bladder cancer cells. Methods: T24 human bladder carcinoma cells were incubated with different concentrations of ATO in the presence or absence of PMA (PKC activator) or H7 (PKC inhibitor). Cell proliferation was assessed by MTT assay, and apoptosis by TUNEL and electron microscopy. Flow cytometry was used to analyze cell cycle distribution, radioimmunoassay to measure PKC activity, and Western blot analysis to detect caspase-3. Results: ATO inhibited proliferation and induced apoptosis of T24 cells in a dose-dependent manner, caused an increase of percentage of cells in the G1 phase and a decrease in the S and G2 phases, and upregulated the expression of activated caspase-3 and reduced PKC activity. These effects were abrogated by PMA, but enhanced by H7. Conclusions: PKC is involved in the anticancer activity of ATO for T24 bladder cancer cells, suggesting that targeting the PKC pathway may represent a potential approach to enhance the efficacy of ATO to treat bladder cancers

Image_4_Identification of B cell marker genes based on single-cell sequencing to establish a prognostic model and identify immune infiltration in osteosarcoma.tif

BackgroundTumor-infiltrating B cells play a crucial role in the promotion or inhibition of tumor development. However, the role of B cells in osteosarcoma remains largely unknown. The aim of this study was to investigate the effect of B cells on the prognosis and immunity infiltration of osteosarcoma.MethodsMarker genes of B cells were identified based on the single-cell sequencing results of osteosarcoma in the GEO database. The prognostic model was established by the TCGA database and verified by the GEO data. The divergence in immune infiltration between the low-risk and high-risk groups was then compared according to the established prognostic model. Finally, the differential genes in the low-risk and high-risk groups were enriched and analyzed.ResultsA total of 261 B cell marker genes was obtained by single-cell sequencing and a prognostic model of 4 B cell marker genes was established based on TCGA data. The model was found to have a good prediction performance in the TCGA and GEO data. A remarkable difference in immune infiltration between the low-risk and high-risk groups was also observed. The obtained results were verified by enrichment analysis.ConclusionIn summary, a prognostic model with good predictive performance was established that revealed the indispensable role of B cells in the development of osteosarcoma. This model also provides a predictive index and a novel therapeutic target for immunotherapy for clinical patients.</p

Image_1_Identification of B cell marker genes based on single-cell sequencing to establish a prognostic model and identify immune infiltration in osteosarcoma.tif

BackgroundTumor-infiltrating B cells play a crucial role in the promotion or inhibition of tumor development. However, the role of B cells in osteosarcoma remains largely unknown. The aim of this study was to investigate the effect of B cells on the prognosis and immunity infiltration of osteosarcoma.MethodsMarker genes of B cells were identified based on the single-cell sequencing results of osteosarcoma in the GEO database. The prognostic model was established by the TCGA database and verified by the GEO data. The divergence in immune infiltration between the low-risk and high-risk groups was then compared according to the established prognostic model. Finally, the differential genes in the low-risk and high-risk groups were enriched and analyzed.ResultsA total of 261 B cell marker genes was obtained by single-cell sequencing and a prognostic model of 4 B cell marker genes was established based on TCGA data. The model was found to have a good prediction performance in the TCGA and GEO data. A remarkable difference in immune infiltration between the low-risk and high-risk groups was also observed. The obtained results were verified by enrichment analysis.ConclusionIn summary, a prognostic model with good predictive performance was established that revealed the indispensable role of B cells in the development of osteosarcoma. This model also provides a predictive index and a novel therapeutic target for immunotherapy for clinical patients.</p