In Vivo Gene Knockdown Techniques and the Establishment of Cryopreservation Methods in Culex spp. Mosquitoes

Culex (Cx.) pipiens, known as the common house mosquito (subspecies Cx. pipiens quinquefasciatus and Cx. pipiens pipiens) affects humans and animals through its ability to transmit numerous viruses and parasites such as West Nile Virus (WNV), filarial nematodes, and Plasmodium relictum, the causative agent of avian malaria. RNA interference (RNAi) is an antiviral pathway that is well studied in most arthropods such as Aedes aegypti mosquitoes. In Culex. spp., aspects of the RNAi mechanism are not completely understood, including the role of a potentially antiviral protein, P-element- Induced Wimpy Testis-2 (PIWI2). One of the objectives of this thesis was to knock down PIWI2 gene expression in vivo to investigate its role during virus infection. This was implemented by introducing double-stranded RNA (dsRNA) into mosquitoes to stimulate RNAi-mediated gene silencing of PIWI2. In this study, dsRNA was introduced into adult Cx. quinquefasciatus mosquitoes and pupae via intrathoracic injection or soaking in dsRNA, respectively. However, a significant knockdown of PIWI2 was not achieved using either dsRNA delivery method. Additional experiments testing dsRNA knockdown efficiency in Cx. quinquefasciatus Hsu cells indicated that the dsRNA used for our studies may not have been effective. The next approach would be to conduct further in vivo tests utilizing a different dsRNA production technique and increased quantities of dsRNA. In this study we additionally aimed to contribute to mosquito related research methods. Continuous maintenance of Culex eggs is required for such in vivo studies but is labor-intensive and costly. Thus, the second objective of this thesis was to establish a method for the cryopreservation of Cx. pipiens embryos.Cryopreservation would permit extended storage of Culex mosquito lines without the burden of continual colony rearing. The methodology and effects of dechorionation, permeabilization, and handling on embryo viability were tested. Lab grade sodium hypochlorite and heptane successfully permeabilized 6-9 hr old Culex embryos, but this treatment was lethal. Additional experimentation is needed to determine the optimal conditions for the permeabilization of Culex embryos

AK2 deficiency compromises the mitochondrial energy metabolism required for differentiation of human neutrophil and lymphoid lineages

Reticular dysgenesis is a human severe combined immunodeficiency that is primarily characterized by profound neutropenia and lymphopenia. The condition is caused by mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of mitochondrial AK2 protein expression. AK2 regulates the homeostasis of mitochondrial adenine nucleotides (ADP, ATP and AMP) by catalyzing the transfer of high-energy phosphate. Our present results demonstrate that AK2-knocked-down progenitor cells have poor proliferative and survival capacities and are blocked in their differentiation toward lymphoid and granulocyte lineages. We also observed that AK2 deficiency impaired mitochondrial function in general and oxidative phosphorylation in particular - showing that AK2 is critical in the control of energy metabolism. Loss of AK2 disrupts this regulation and leads to a profound block in lymphoid and myeloid cell differentiation

Melanoma addiction to the long non-coding RNA SAMMSON

Focal amplifications of chromosome 3p13-3p14 occur in about 10% of melanomas and are associated with a poor prognosis. The melanoma-specific oncogene MITF resides at the epicentre of this amplicon. However, whether other loci present in this amplicon also contribute to melanomagenesis is unknown. Here we show that the recently annotated long non-coding RNA (lncRNA) gene SAMMSON is consistently co-gained with MITF. In addition, SAMMSON is a target of the lineage-specific transcription factor SOX10 and its expression is detectable in more than 90% of human melanomas. Whereas exogenous SAMMSON increases the clonogenic potential in trans, SAMMSON knockdown drastically decreases the viability of melanoma cells irrespective of their transcriptional cell state and BRAF, NRAS or TP53 mutational status. Moreover, SAMMSON targeting sensitizes melanoma to MAPK-targeting therapeutics both in vitro and in patient-derived xenograft models. Mechanistically, SAMMSON interacts with p32, a master regulator of mitochondrial homeostasis and metabolism, to increase its mitochondrial targeting and pro-oncogenic function. Our results indicate that silencing of the lineage addiction oncogene SAMMSON disrupts vital mitochondrial functions in a cancer-cell-specific manner; this silencing is therefore expected to deliver highly effective and tissue-restricted anti-melanoma therapeutic responses

Evaluation of OASL and HERC5’s role in the non-lytic clearance of influenza A virus from club cells

Influenza A virus (IAV) is a highly infectious pathogen responsible for causing severe respiratory illness and death in humans and animals worldwide. Due to highly effective strategies to negate host antiviral defenses, IAV leads to the death of nearly all infected cells. Furthermore, IAV induces high levels of genome-damaging oxidative stress within infected cells and suppresses the cellular mismatch repair (MMR) mechanism, thereby inhibiting expression of key antiviral genes, which further contributes to cell death. However, recent studies have demonstrated that a subset of respiratory epithelial cells, called club cells, are able to non-lytically clear IAV and continue to survive following direct infection. These cells are able to maintain genome integrity during IAV infection using MMR activity, thus allowing for sufficient expression of antiviral genes. We hypothesize that several of these antiviral genes are critical to the non-lytic clearance of IAV, particularly HERC5 and OSAL. Through siRNA gene knockdown techniques, we have evaluated the impact of these genes on the non-lytic clearance mechanism of IAV-infected club cells

siRecords: a database of mammalian RNAi experiments and efficacies

RNAi-based gene-silencing techniques offer a fast and cost-effective way of knocking down genes’ functions in an easily regulated manner. Exciting progress has been made in recent years in the application of these techniques in basic biomedical research and therapeutic development. However, it remains a difficult task to design effective siRNA experiments with high efficacy and specificity. We present siRecords, an extensive database of mammalian RNAi experiments with consistent efficacy ratings. This database serves two purposes. First, it provides a large and diverse dataset of siRNA experiments. This dataset faithfully represents the general, diverse RNAi experimental practice, and allows more reliable siRNA design tools to be developed with the overfitting problem well curbed. Second, the database helps experimental RNAi researchers directly by providing them with the efficacy and other information about the siRNAs experiments designed and conducted previously against the genes of their interest. The current release of siRecords contains the records of 17 192 RNAi experiments targeting 5086 genes

A Low Percent Ethanol Method for Immobilizing Planarians

Planarians have recently become a popular model system for the study of adult stem cells, regeneration and polarity. The system is attractive for both undergraduate and graduate research labs, since planarian colonies are low cost and easy to maintain. Also in situ hybridization, immunofluorescence and RNA-interference (RNAi) gene knockdown techniques have been developed for planarian studies. However, imaging of live worms (particularly at high magnifications) is difficult because animals are strongly photophobic; they quickly move away from light sources and out of frame. The current methods available to inhibit movement in planarians include RNAi injection and exposure to cold temperatures. The former is labor and time intensive, while the latter precludes the use of many fluorescent reporter dyes. Here, we report a simple, inexpensive and reversible method to immobilize planarians for live imaging. Our data show that a short 1 hour treatment with 3% ethanol (EtOH) is sufficient to inhibit both the fine and gross movements of Schmidtea mediterranea planarians, of the typical size used (4–6 mm), with full recovery of movement within 3–4 hours. Importantly, EtOH treatment did not interfere with regeneration, even after repeated exposure, nor lyse epithelial cells (as assayed by H&E staining). We demonstrate that a short exposure to a low concentration of EtOH is a quick and effective method of immobilizing planarians, one that is easily adaptable to planarians of all sizes and will increase the accessibility of live imaging assays to planarian researchers

Anti-Angiogenic and Anti-Cancer Effects by Targeting the Protein Kinase G Type-Iα (PKG-Iα) Signaling Pathway and its Downstream Effects on Expression of Inhibitor of Apoptosis Proteins, C-IAP1, Livin and Survivin

Ovarian cancer is often difficult to treat because of the development of resistance to many of the currently-used therapeutic agents (i.e. chemoresistance). The progression and chemoresistance of ovarian cancer can involve tumor angiogenesis, the development of new blood vessels bringing more blood and nutrients to the growing tumor. Tumor angiogenesis also involves the vascular endothelium-induced stimulation of cancer cell growth (1) and the higher expression levels of certain “cell survival proteins”, such as the Inhibitor of Apoptosis Proteins (IAPs, including c-IAP1, Livin and Survivin), which are expressed in both the proliferating cancer cells (2, 3) and the vascular endothelial cells involved in tumor angiogenesis (4)

IRIS: Learning the Underlying Information of Scientific Research Interests Using Heterogeneous Network Representation

Understanding scientific research fields and finding potential relations between seemingly distinct fields can help researchers rapidly grasp their most interested topics with expertises. In this study, we construct a heterogeneous network which contains authors, keywords, papers and institutions, and built an “Integrated Research Interest Space (IRIS)” which can represent both author and keyword nodes. Similar keywords in the sense of research interest and research manner can obvious aggregate together. Authors that are interested in different keywords distributed in different IRIS areas, with strongly associated with research objectives and methodologies of the keywords. The average similarities between authors and their real used keywords is significantly higher than that of randomly chosen author-keyword pairs. Based on these observations, we propose a simple algorithm which attempts to recommend potential interested keywords for researchers, and got meaningful results. Our study may also give useful hints for understanding research interests and discovering potential cross disciplines

Nuclear factor erythroid 2-related factor 2 nuclear translocation induces myofibroblastic dedifferentiation in idiopathic pulmonary fibrosis

AIMS: Oxidants have been implicated in the pathophysiology of idiopathic pulmonary fibrosis (IPF), especially in myofibroblastic differentiation. We aimed at testing the hypothesis that nuclear factor erythroid 2-related factor 2 (Nrf2), the main regulator of endogenous antioxidant enzymes, is involved in fibrogenesis via myofibroblastic differentiation. Fibroblasts were cultured from the lungs of eight controls and eight IPF patients. Oxidants-antioxidants balance, nuclear Nrf2 expression, and fibroblast phenotype (α-smooth muscle actin and collagen I expression, proliferation, migration, and contraction) were studied under basal conditions and after Nrf2 knockdown or activation by Nrf2 or Keap1 siRNA transfection. The effects of sulforaphane (SFN), an Nrf2 activator, on the fibroblast phenotype were tested under basal and pro-fibrosis conditions (transforming growth factor β [TGF-β]). RESULTS: Decreased Nrf2 expression was associated with a myofibroblast phenotype in IPF compared with control fibroblasts. Nrf2 knockdown induced oxidative stress and myofibroblastic differentiation in control fibroblasts. Conversely, Nrf2 activation increased antioxidant defences and myofibroblastic dedifferentation in IPF fibroblasts. SFN treatment decreased oxidants, and induced Nrf2 expression, antioxidants, and myofibroblastic dedifferentiation in IPF fibroblasts. SFN inhibited TGF-β profibrotic deleterious effects in IPF and control fibroblasts and restored antioxidant defences. Nrf2 knockdown abolished SFN antifibrosis effects, suggesting that they were Nrf2 mediated. INNOVATION AND CONCLUSION: Our findings confirm that decreased nuclear Nrf2 plays a role in myofibroblastic differentiation and that SFN induces human pulmonary fibroblast dedifferentiation in vitro via Nrf2 activation. Thus, Nrf2 could be a novel therapeutic target in IPF