Yeast-based High-throughput Screens To Identify Novel Compounds Active Against Brugia Malayi

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Lymphatic filariasis is caused by the parasitic worms Wuchereria bancrofti, Brugia malayi or B. timori, which are transmitted via the bites from infected mosquitoes. Once in the human body, the parasites develop into adult worms in the lymphatic vessels, causing severe damage and swelling of the affected tissues. According to the World Health Organization, over 1.2 billion people in 58 countries are at risk of contracting lymphatic filariasis. Very few drugs are available to treat patients infected with these parasites, and these have low efficacy against the adult stages of the worms, which can live for 7-15 years in the human body. The requirement for annual treatment increases the risk of drug-resistant worms emerging, making it imperative to develop new drugs against these devastating diseases. Methodology/Principal Findings We have developed a yeast-based, high-throughput screening system whereby essential yeast genes are replaced with their filarial or human counterparts. These strains are labeled with different fluorescent proteins to allow the simultaneous monitoring of strains with parasite or human genes in competition, and hence the identification of compounds that inhibit the parasite target without affecting its human ortholog. We constructed yeast strains expressing eight different Brugia malayi drug targets (as well as seven of their human counterparts), and performed medium-throughput drug screens for compounds that specifically inhibit the parasite enzymes. Using the Malaria Box collection (400 compounds), we identified nine filarial specific inhibitors and confirmed the antifilarial activity of five of these using in vitro assays against Brugia pahangi. Conclusions/Significance We were able to functionally complement yeast deletions with eight different Brugia malayi enzymes that represent potential drug targets. We demonstrated that our yeast-basedscreening platform is efficient in identifying compounds that can discriminate between human and filarial enzymes. Hence, we are confident that we can extend our efforts to the construction of strains with further filarial targets (in particular for those species that cannot be cultivated in the laboratory), and perform high-throughput drug screens to identify specific inhibitors of the parasite enzymes. By establishing synergistic collaborations with researchers working directly on different parasitic worms, we aim to aid antihelmintic drug development for both human and veterinary infections.101Bill and Melinda Gates FoundationGrand Challenges Explorations [OP1087646, OPP1098441]Sao Paulo Research Foundation [2015/19103-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Telomerase Directed Gene Therapy

Stabilisation of telomere length is considered to be an essential step in cellular immortalisation in vitro and in human cancers. The telomerase ribonucleoprotein reverse transcriptase catalyses the addition of new telomeric repeat sequence to the ends of linear eukaryotic chromosomes and counteracts the cell division associated telomeric attrition that leads to cellular senescence. Its expression has been detected in approximately 85% of all human malignancies but is not detectable in the majority of normal somatic tissues and, therefore, telomerase represents an attractive target for the development of novel molecular therapeutics. Although telomerase activity is modulated on a number of levels, a primary level of regulation is the transcription of the telomerase sub-unit genes. In the present study, I describe the development of a transcriptionally directed cytotoxic gene therapy approach targeted against telomerase positive cancer cells. Transfection experiments using fragments of the human telomerase RNA component (hTERC) and the human telomerase reverse transcriptase (hTERT) promoters revealed large differences in promoter activity between mortal cells and cancer cells. The promoter fragments were sub-cloned into plasmids containing the coding sequence of nitroreductase (NTR), a bacterial enzyme that catalyses the chemical reduction of the non-toxic pro-drug CB1954 resulting in the formation of a powerful bi-functional alkylating agent that kills both dividing and nondividing cells. Stable cell lines harbouring hTERC-NTR and hTERT-NTR expression vectors were sensitised to CB1954 to an extent that was dependent on hTERC and hTERT promoter activity, with cell lines that had high promoter activities showing significant sensitisation, while those with low promoter activities were not significantly sensitised. The hTERC-NTR and hTERT-NTR expression constructs were cloned into adenovirus (Ad) delivery vehicles and the efficiencies of infection and expression of NTR were characterised in infected cell lines. The major RNA species that was expressed in infected cells was a splice variant that encoded a truncated NTR protein, but the function of NTR was not significantly impaired. Infection with the Ad-hTERC-NTR and Ad-hTERT-NTR gene therapy vectors resulted in a sensitisation to CB1954 that was dually dependent on promoter activity and infection efficiency. Two cancer cell lines that had high hTERC and hTERT promoter activities were significantly sensitised to CB1954, while a mortal foetal lung fibroblast cell strain and a normal adult human mammary epithelial strain, in addition to a bladder cancer cell line with low promoter activity, were not sensitised despite efficient infection with adenovirus. Therefore, the data presented herein support the further development of telomerase-nitroreductase expression vectors for anti-cancer gene therapy

Strategies to prevent motoneuron degeneration in models of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder, characterised by progressive motoneuron degeneration in the spinal cord, motor cortex and brainstem. The pathogenic mechanisms underlying selective motoneuron degeneration are unclear and currently there is no effective treatment. In this Thesis, strategies designed to prevent motoneuron degeneration are investigated in both in vivo and in vitro models of ALS. Furthermore, interaction between motoneurons and astrocytes is studied to examine the influence of astrocytes on disease progression. In the SODlG93A mouse model of ALS, the glial cell genotype influences the susceptibility of motoneurons to degeneration (Gong et al., 2000 Clement et al., 2003). In this Thesis, the effect of mutant SOD1 expression in astrocytes on motoneuron properties is examined in an in vitro co-culture system using confocal microscopy. Cannabinoids exert anti-excitotoxic, anti-inflammatory and anti-oxidant actions, all of which may contribute to ALS pathogenesis. In these experiments, the potential neuroprotective effect of manipulating the endocannabinoid system is investigated in vivo in SODl093* mice. Augmentation of the endocannabinoid system, by pharmacological and genetic manipulation, ameliorates disease symptoms in SOD1 mice. Furthermore, genetic ablation of the CBi receptor significantly extends lifespan in SOD 1093A mice. Finally, the effect of ablating the expression of mutant SOD1 protein in vitro in primary motoneurons using viral delivery of targeted small interfering RNA (siRNA) is assessed. The successful transfection and ablation of the mutant protein in vitro, as shown in this Thesis, has since been tested successfully in vivo. The results of this Thesis show that manipulation of the endocannabinoid system and siRNA technology may be successful therapeutic approaches in ALS. The results also indicate that mutant SOD1 expression in astrocytes has a deleterious influence on mitochondrial function in motoneurons even under resting conditions. Therefore, specific targeting of astrocytes may also be an appropriate strategy to prevent motoneuron degeneration in ALS

MDM2 negatively regulates the human telomerase RNA gene promoter

BACKGROUND: We have previously demonstrated that NF-Y and Sp1 interact with the human telomerase RNA (hTR) promoter and play a central role in its regulation. We have also shown that pRB activates the hTR promoter, but the mechanism of pRb directed activation is unknown. It has recently been reported that pRB induces Sp1 activity by relieving inhibition mediated by mdm2. The aim was to investigate possible roles for mdm2 in hTR promoter regulation. METHODS: Chromatin immunoprecipitation was used to determine binding of mdm2 to the hTR promoter. Transfection and luciferase assays were used to investigate mdm2 repression of the promoter activity and interaction with known transcriptional modulators. RESULTS: Here we show using chromatin immunoprecipitation that mdm2 specifically binds the hTR promoter in vivo. Transient co-transfection experiments using an hTR promoter luciferase reporter construct show that hTR promoter activity is inhibited by over-expression of mdm2 in 5637 bladder carcinoma cells (p53 and pRB negative, low mdm2). Titration of mdm2 was able to antagonise activation of hTR promoter activity mediated by pRB or Sp1 over-expression, although in the presence of pRB, mdm2 could not repress promoter activity below basal levels. Using an Sp1 binding site mutation construct we showed that mdm2 repression did not absolutely require Sp1 binding sites in the hTR promoter, suggesting the possibility of pRB/Sp1 independent mechanisms of repression. Finally, we show that NF-Y mediated transactivation of the hTR promoter was also suppressed by mdm2 in a dose-dependent manner. CONCLUSIONS: These studies suggest that mdm2 may inhibit the hTR promoter by multiple mechanisms. Mdm2 may directly repress activation by both pRB and Sp1, or activation by NF-Y. Furthermore, the ability of mdm2 to interact and interfere with components of the general transcription machinery might partly explain the general repressive effect seen here. Elucidation of new regulators affecting hTR basal promoter activity in cancer cells provides a basis for future studies aimed at improving our understanding of the differential hTR expression between normal and cancer cells

The president, the state and the Cold War: comparing the foreign policies of Presidents Truman and Reagan

US foreign policy during the Cold War has been analysed from a number of perspectives, generating large bodies of literature attempting to explain its origins, its development and its conclusion. Within the discipline of International Relations these debates have tended to be led by scholars focusing on events at the system level. However, there are still many questions left only partially explained. In large part this is because these accounts restrict themselves to a single level of analysis, either the international system, or the structure of the state and society. The first level of analysis, focusing on the role of individuals, has largely been excluded from International Relations. It is often left to historians to incorporate the role of individual decision makers into their studies. The problem for international relations students, however, is that their arguments run the risk of determinism. They come close to advocating that the course of history is shaped by these external forces and there is little if no room for alternate courses to be steered. They have, intentionally or otherwise, removed human agency and choice from the equation. This thesis argues that structural theories, and any approach that limits itself to one level of analysis, are inadequate to explain the development of US foreign policy. Instead, it is necessary to incorporate the first level of analysis in order to bring human agency back into International Relations and provide a more detailed explanation of US foreign policy. The present study proposes an analytical framework which incorporates presidential agency into a multi-level analysis of US foreign policy during the Cold War. Drawing on Foreign Policy Analysis, International Relations theory, presidential studies and the historiography of US foreign policy, this thesis constructs a multi-level case study comparison of the foreign policies of Presidents Truman and Reagan. It argues that the worldview of the president is central to agenda setting in US foreign policy making and that the management style of the president influences both decision-making and the implementation of US foreign policy. Evidence to support this is drawn from detailed empirical analysis of Truman’s foreign policy of containment in Korea and Reagan’s foreign policy of rollback in Nicaragua

Identification of putative regulatory upstream ORFs in the yeast genome using heuristics and evolutionary conservation.

BACKGROUND: The translational efficiency of an mRNA can be modulated by upstream open reading frames (uORFs) present in certain genes. A uORF can attenuate translation of the main ORF by interfering with translational reinitiation at the main start codon. uORFs also occur by chance in the genome, in which case they do not have a regulatory role. Since the sequence determinants for functional uORFs are not understood, it is difficult to discriminate functional from spurious uORFs by sequence analysis. RESULTS: We have used comparative genomics to identify novel uORFs in yeast with a high likelihood of having a translational regulatory role. We examined uORFs, previously shown to play a role in regulation of translation in Saccharomyces cerevisiae, for evolutionary conservation within seven Saccharomyces species. Inspection of the set of conserved uORFs yielded the following three characteristics useful for discrimination of functional from spurious uORFs: a length between 4 and 6 codons, a distance from the start of the main ORF between 50 and 150 nucleotides, and finally a lack of overlap with, and clear separation from, neighbouring uORFs. These derived rules are inherently associated with uORFs with properties similar to the GCN4 locus, and may not detect most uORFs of other types. uORFs with high scores based on these rules showed a much higher evolutionary conservation than randomly selected uORFs. In a genome-wide scan in S. cerevisiae, we found 34 conserved uORFs from 32 genes that we predict to be functional; subsequent analysis showed the majority of these to be located within transcripts. A total of 252 genes were found containing conserved uORFs with properties indicative of a functional role; all but 7 are novel. Functional content analysis of this set identified an overrepresentation of genes involved in transcriptional control and development. CONCLUSION: Evolutionary conservation of uORFs in yeasts can be traced up to 100 million years of separation. The conserved uORFs have certain characteristics with respect to length, distance from each other and from the main start codon, and folding energy of the sequence. These newly found characteristics can be used to facilitate detection of other conserved uORFs.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Dynamic telomerase gene suppression via network effects of GSK3 inhibition

<b>Background</b>: Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression. <b>Methodology/Principal Findings</b>: In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3′-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFκB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc. <b>Conclusions/Significance</b>: Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting