Exploring Potential Drug Target Sites In The Ribosome Using Cisplatin And Its Analogues

Cis-diamminodichloridoplatinum (II), cisplatin, is an antitumor drug that has been used to treat several types of cancers. The reaction of cisplatin with DNA has been studied and discussed extensively in the literature; however, the effects of cisplatin on RNA function are poorly understood. In this thesis, two aspects of cisplatin, its preferred sites of interaction with RNA and its use as a chemical probe to gain accessibility information, were explored. To understand the site-selectivity of cisplatin with RNA, model RNA constructs and full-length 16S rRNA were employed. The binding studies revealed a cisplatin preference for guanosine-rich sequences. Primer extensions in 16S rRNA and MALDI-TOF in model constructs were used to locate the binding sites of cisplatin. HPLC and LC-MS were useful to determine the types and ratios of various adducts formed. Cisplatin and its analogues were employed to probe the accessibility of nucleotides on 16S rRNA, 30S subunits and 70S ribosomes in vitro as well as in vivo. This study revealed that many functionally important sites, such as helix 18, 24, 27, and 34 are accessible to the aquated platinum complex. Thus, these accessible sites can potentially be utilized as a new target sites in the design of structure-based antibiotics. When charge and size of the complex were changed, the binding preference was altered. In addition to the expected consecutive Gs, cisplatin analogues preferentially targeted AG sites on loop or bulge regions. Thus, several new complexes could be synthesized and utilized to gain more information about drug accessibility on the ribosome. The last part of the research focused on the application of siRNA to target non-Hodgkin\u27s lymphoma (NHL). Small interfering RNAs were designed to downregulate the c-Myc expression in NHL cells. Stabilities of designed siRNAs in media and their incorporation into liposomes were studied. Complexes of siRNA, liposomes, and antibody fragments (scFv) could be utilized in future applications to target specifically the c-Myc expression in NHL cells. Overall, this thesis work explored cisplatin binding to RNA and a number of possible new antibiotic target sites on the ribosome were identified. In the long term, further studies with fully functional ribosomes and comparisons with other organisms will have a greater impact on identifying novel drug target sites in pathogenic bacteria

Analysis of Geographical and Seasonal Variation of Dengue Fever in Nepal Based on EWARS Line Listing from 2017 to 2019

Background: Dengue is a mosquito-borne viral disease; rapidly spreading in many countries worldwide in recent years. Dengue has been identified as one of the youngest emerging infectious diseases in Nepal, the first case reported in 2004. In 2006, 32 laboratory-confirmed cases were reported across hospitals in central and western Terai and Kathmandu during the post-monsoon season. The trend for increased magnitude has been continued with the number of outbreaks reported each year in many districts in two to three years intervals. Since 2010, dengue epidemics have continued to affect lowland districts and mid-hill areas. These all reflected need of study on geographical and seasonal variation of dengue fever and many more. This study analyzed the geographical and seasonal variation of Dengue fever in Nepal based on EWARS line listing 2017-2019 and provides recommendations for improvement of the dengue control program in Nepal. Methodology: Quantitative analysis of key variables from the Early Warning and Reporting System (EWARS) line list was applied. EWARS line listing data from 2017to 19 was received from the department of health service, epidemiology and disease control division (EDCD), Nepal and analyzed. Published literature and articles were reviewed to discuss and triangulate the findings. Microsoft excels and SPSS20 software was used for data analysis, and Mendeley software was used for referencing and citations. Based on the line listing data from EDCD, last three years dengue situation, age, gender, seasons, geography and department of registration in the sentinel sites were analyzed and discussed. The Chi-square test in 95 % confidence interval was applied to test hypothesis assessing the relationship of age group and geographical belt on the dengue cases reported in the studied years. Results: Entirely of 2273 individual cases were verified and updated by EDCD; among these 1.32 % of received data were excluded and a total of 2243 samples were analyzed and studied. The male population of the age group 20 to 40 was observed at higher risk of dengue with the infant proportion of 1.43% in Nepal. The notable infant case showed the urgency of actions needed to address this issue. Most of the dengue cases were found registered in from IPD, emergency, OPD and laboratory departments. The geographical distribution of dengue cases reflected the higher case in Terai low land districts with massively escalating in the hill, Kathmandu valley and Mountain belts. The clusters of cases to outbreak situation of dengue suggest diversity and uncertainty of dengue incidence. In the study period, the highest cases were found in Lumbini province followed by Province 01and significance cases were reported from Province 02 and Bagmati province. In contrast, the Sudurpaschim, Gandaki and Karnali provinces reported a minimum number of cases. Mountain district Dhading had reported one of the top five reporting districts in the study period. Similarly, dengue cases fluctuated based on the seasons, predominantly in the monsoon and post-monsoon season in Nepal. The test statistics, Chi-square test suggested a significant association between age group and geographical belts to the year wise dengue cases. Conclusion: Thus Dengue has been tough public health problem with a rising burden and wider geographical and seasonal coverage resulting in a frequent outbreak in Nepal. The changing disease patterns with an uncertainty of prediction increases challenge. It is strongly recommended strategic preparedness and research and developmental works to improve dengue control program for the betterment of the population's health.open석

Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels <i>In Vivo</i>

<div><p>The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in <i>S</i>. <i>pombe</i>. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT <i>in vitro</i> and <i>in vivo</i>, and surprisingly, amounts of transcription <i>in vivo</i>. Because assessing <i>in vivo</i> transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease.</p></div

Library screening of spRpc1.

<p>Library screening of spRpc1.</p

Mapping <i>S</i>. <i>pombe</i> C1 active center LOF mutants onto <i>S</i>. <i>cerevisiae</i> RNAP III structure.

<p><b>A)</b> Active center bridge helix (BH, cyan) and trigger loop (TL, green) as well as the cleft-pore entrance loop (CPEL, brown) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006253#pgen.1006253.ref017" target="_blank">17</a>]; numbers indicate <i>S</i>. <i>cerevisiae</i> positions (with <i>S</i>. <i>pombe</i> positions in parentheses). <b>B)</b> The holoenzyme EC structure with mutations in the funnel and CPEL highlighted as red spheres. α20 and α21 refer to RNAP II motifs; CPEL: cleft-pore entrance loop (see text); other subunits and structural features are indicated. The PDB ID used is 5FJ8 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006253#pgen.1006253.ref017" target="_blank">17</a>]. <b>C)</b> RNAP III EC structure (PDB 5FJ8) into which the <i>S</i>. <i>cerevisiae</i> TFIIS CTD and linker was placed (shown in sticks and ribbon backbone mode). <b>D)</b> A high resolution view of the acidic hairpin CTD of TFIIS with its linker placed into the RNAP III cryo-EM structure shown in C. The inset shows a sequence alignment of the acidic hairpin regions of the CTDs of C11 and TFIIS from <i>S</i>. <i>cerevisiae</i> (sc) and <i>S</i>. <i>pombe</i> (sp), as indicated. The acidic DE residues at the tip of the hairpin are colored cyan to match their stick representation in the structure placement model, which come within 4.9 Å of the RNA 3' end (magenta). Another close contact of 3.8 Å between a C1 residue found mutated in mutant D854N and an invariant Q in TFIIS (the second Q in the sequence alignment) is also indicated. The CPEL, funnel helix loop and α21 are also indicated.</p

Promoter-dependent <i>in vitro</i> transcription reveals oligo(T) length-dependent terminator readthrough by RNAP III C1 mutants.

<p><b>A)</b> Schematic of tRNA gene arrangement used for <i>in vitro</i> transcription by S100 extracts; the plasmids used for transcription differed only in the number of Ts at the test terminator, T1, as listed above the lanes of B & C. <b>B-C)</b> S100 extracts from yKR1-C1 mutants or -C1-Wt control were used as a source of initiation factors and RNAP III as indicated above the lanes. Plasmids containing tRNA genes that differ only in the number of Ts in the oligo(T) terminator as indicated above the lanes were used as templates to program the transcription reactions. RT = readthrough; termination at the failsafe terminator, T = termination at the test terminator, IC = internal control. <b>D)</b> Quantitation of % readthrough as defined on the Y-axis; reactions contained 32P-αGTP.</p

Representative RNAP III C1-E850K mutant exhibits increased elongation.

<p><b>A)</b> Schematic of 3'-tailed templates used for promoter-independent transcription by FLAG-purified RNAPs III (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006253#pgen.1006253.s002" target="_blank">S2 Fig</a> for establishment of assay). The template design on the left was used for panel B and the design on the right was used for panel C. FL = full length 300 nt transcript resulting from termination at 12T, RO = run-off transcript. <b>B)</b> Time course of elongation by purified RNAPs III C1-Wt and C1-E850K as indicated above the lanes. <b>C)</b> RNAP III was stalled at position +22 followed by a synchronized chase time course of transcription elongation (see text). (The blemish between lanes 5 & 6 is not a transcription signal.)</p

DNA oligos.

<p>DNA oligos.</p

Single mutation LOF C1 mutants (from yKR1), phenotypes in yKR1, yJI1.

<p>Single mutation LOF C1 mutants (from yKR1), phenotypes in yKR1, yJI1.</p

C1 mutants cluster map to highly conserved RNAP motifs.

<p><b>A-D)</b> Sequence alignments of homologous regions of RNAP II and III largest subunits of three species, <i>Homo sapiens</i> (hs), <i>Schizosaccharomyces pombe</i> (sp) <i>and Saccharomyces cerevisiae</i> (sc); the bridge helix (BH, A), trigger loop (TL, B), funnel (C) and cleft-pore entrance loop (CPEL, D); black and grey asterisks indicate positions of strong and weaker phenotypes of LOF mutants respectively; # indicates GOF mutations. Upward triangles were placed above residues which when mutated in <i>S</i>. <i>cerevisiae</i> RNAP II caused increased elongation rate, and downward triangles were placed above residues which when mutated in <i>S</i>. <i>cerevisiae</i> RNAP II caused decreased elongation rate as summarized in figure 3 of Kaplan et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006253#pgen.1006253.ref042" target="_blank">42</a>].</p