Chemoenzymatic Study of CoA-Linked RNA in Bacteria

The ability of RNA to store genetic information and to catalyze biochemical transformations led to the speculation of the existence of RNA world before the evolution of contemporary ribonucleoprotein (RNP) world. Recent discovery of RNA molecules containing metabolic cofactors including coenzyme A and its various thioesters at their 5’ end further supported the RNA world hypothesis as these CoA-linked RNA molecules could be the molecular fossils with very ancient origin. As both RNA and Coenzyme A are believed to have co-existed since last universal common ancestor (LUCA) or even before, the CoA-RNA conjugates in current biology may reveal fundamental molecular secrets involved in evolution. Furthermore, these CoA-RNA conjugates may not be just remnants of evolution, rather may have an important functional significance in contemporary metabolism and gene regulation. The successful characterization of these conjugates will expand our knowledge in RNA function and CoA function. However, the sequence, metabolism and biological role of these RNA species are still unknown. The aim of this study is to capture CoA-RNA sequences form E. coli total RNA to uncover their sequences and to investigate their biogenesis. The successful characterization of CoA-RNA requires a specific protocol to capture them. The development of such protocol requires an easy access to synthetic CoA-RNA. While our lab previously developed a method to incorporate CoA into RNA co-transcriptionally by using dephospho CoA as a transcription initiator, the limited availability of dephospoCoA restricts an easy access to synthetic CoA-RNA. In the first section, a simple and easy method for the synthesis of dephospho CoA and its oxidized dimer was developed. Two enzymes of CoA biosynthetic pathway, PanK and PPAT were cloned in a single plasmid, and purified in a single enzyme preparation. The synthesis of dephsphoCoA was achieved by the enzyme cocktail and pure product was obtained by a simple reverse phase column chromatography. The method was extended further to synthesize various dephosphoCoA analogs including amino dephosphoCoA and biotin dephosphoCoA. In the second section, two strategies were investigated for their application in CoA-RNA capture. In first strategy, various acyl-CoA ligases including acetyl CoA synthetase (ACS), Malonyl CoA synthetase (MatB), Succinyl CoA synthetase (SucCD), medium chain fatty acyl CoA synthetase (FadK) and Long chain fatty acyl CoA synthetase were cloned, expressed and tested their ability to accept biotinylated fatty acid and CoA-RNA as substrates. In the second strategy, various pantetheine and phosphopantetheine analogs with biotin as a purification handle, [14-C] acetate as a reporter tag, and variable number of positive charges to mediate cellular uptake were synthesized. During the synthesis of such analogs, a novel method for the selective protection of secondary alcohols in presence of their primary counterparts was developed. In the third section of this work, a mechanism of CoA-RNA biogenesis was explored. Synthesized phosphopantetheine analog was used to investigate whether CoA-RNA can be generated post-transcriptionally. We found that a CoA biosynthetic enzyme, PPAT (coaD), can accept ATP-RNA as its substrate and yields CoA-RNA. our results showed that the suitability of ATP initiated RNA to serve as a PPAT substrate is determined by its 5’ structure. An RNA having at least four unstructured nucleotides at the 5’ end can participate in PPAT catalyzed phosphopantetheine transfer reaction. Furthermore, the rate of the reaction was independent to the number of 5’ unstructured nucleotides at least for the range of 4-10. These findings established the post-transcriptional transfer of 4’-phosphopantetheine to ATP-RNA as another mode of CoA-RNA biogenesis besides previously characterized RNAP mediated co-transcriptional incorporation. Collectively, we have developed an easy and simple method to prepare dephospho CoA and its analogs, investigated chemo-enzymatic strategies for CoA-RNA capture and, discovered a post-transcriptional mechanism of CoA-RNA biogenesis. Our results suggest that the existence of CoA-RNA in extant biology may be evolutionary. Future studies may lead to development of specific CoA-RNA capture and will expand our understandings on how ribozyme catalysis of the RNA world was transformed into protein enzyme-based catalysis of contemporary world

Pesticides handling practices among potato growers in Kavrepalanchok, Nepal

The rate of pesticide application in the agricultural field is surging. Farmers are getting exposed to pesticide hazards from the misuse and unsafe handling of pesticides. The study was conducted among 101 potato growers in Kaverpalanchok district of Nepal in 2018. The objective of the study was to assess the knowledge, status, and pesticide handling practices among potato growers. Around 94% farmers applied pesticides against early and late blight. Only 5% growers treated seed before potato sowing. About 93 and 73% farmers sprayed pesticides 2-10 times/season into the field and spent 2-6 hours/pesticide spray respectively. More than 2/3rd growers did not read the pesticide labels, and nearly 95% growers received information on pesticide applications from agrovet rather than authorized government bodies. Only 13% farmers had received Integrated Pest Management (IPM) training. However, 1/4th of them had practiced IPM techniques. The majority of the growers used masks, rubber boots, and long-sleeved clothes during pesticide handling. Nearly 2/3rd growers threw pesticide containers anywhere in the environment. Concerned authorities should provide IPM training, skill-building programs on pesticide handling and awareness on waiting period and environmental hazards to avoid pesticide risk

Exploring the emerging impacts of open aid data and budget data in Nepal

Nepal underwent a second Constituent Assembly (CA) election in November 2013, an important precursor for transparent and accountable governance. This case study explores whether and how open data can make a relevant contribution to governance and inclusive citizen empowerment. The project sought to understand interactions of key stakeholders and to develop recommendations for intelligent action in the future. In countries like Nepal, where internet access is low, mainstream media are vital open data intermediaries. The study revealed that there is a gap between open data efforts and the information needs and practices of civil society and journalists in Nepal

Intra-articular autologous conditioned plasma reduces pain in early osteoarthritis and improves stiffness in advanced osteoarthritis knee: a prospective observation

Background: In this study, we aimed to investigate the effects of single-dose intra-articular autologous conditioned plasma injection as the treatment for early and advanced osteoarthritis knee. Methods: A single centre-based prospective observational analysis was conducted among patients who opt for conservative management by intra-articular autologous conditioned plasma Injection between July 2022 to June 2023. Total 46 patients were included and analysed in study on 1, 3, and 6 months’ follow-up after ACP injection. The WOMAC score and its sub scores were analysed and compared pre-procedure and on subsequent follow ups. Results: A total of 46 patients were analysed in the study, with male predominance of 56.5% with mean BMI of 27.37±5.35. On KL grading, 34.8% patients had KL grade II Osteoarthritis, 52.2% had grade III and 13.0% had grade IV. On comparison of WOMAC score and its sub-scales, all the values were found to be statically significant while comparing before injection (p value <0.0001) and one month follow up and before injection and six months’ follow-up (p value <0.0001). Conclusions: Six months following intra-articular autologous conditioned plasma injection, there was a noteworthy decrease in pain in early stages of osteoarthritis and improvement in knee stiffness in advanced stages of osteoarthritis as compared to the pre-treatment state

Germplasm rescue and rebuilding local seed systems in red zone areas

Native agrobiodiversity become endangered and even lost due to natural disasters in red zone area such as earthquake that hit Nepal on April and May 2015. Endangered agricultural genetic resources should be rescued and revival of disrupted local seed system was essential for sustainable and productive agriculture in earthquake affected areas. The objectives of this paper are to document methods employed to rescue germplasm and rebuild local seed systems in earthquake affected areas to restore lost crop diversity and strengthen local seed systems. Among 14 severely hit districts by April 2015 earthquake in Nepal, 10 districts were selected for germplasm rescue and rebuilding local seed system. We surveyed households and sensitized relevant stakeholders. Earthquake affected areas were declared as red zones and status of crop landraces were assessed through survey, five cell analysis and focus group discussion. Three germplasm rescue techniques, namely direct rescue, diversity fair and indirect rescue were applied. Local seed systems were rebuilt through diversity fair, diversity block, seed exchange, repatriation and diversity kits. Collected accessions were characterized, multiplied and conserved in National Genebank and community seed banks. About 5-10% of total local crop diversity (based on the landraces) were lost due to earthquake in these districts. A total of 921 accessions of 61 crops along with 284 rare and endangered crop landraces were collected and rescued from 35 VDCs of 10 severely earthquake affected districts. Climate analogue sites and climate smart germplasm were identified for some of earthquake affected areas, and five landraces of four crops were repatriated. Participatory seed exchange, diversity fairs and 200 diversity kits (containing 3 to 5 varieties) were employed to revive the local seed systems. Farmers’ preferred landraces were conserved in Community Seed Bank in Lamjung and Dolakha and all collections were conserved in Naional Genebank, Khumaltar, Nepal. The study concludes that multiple approaches and tools are necessary for germplasm rescue and rebuilding local seed systems from red zone area &nbsp

Exploring the emerging impacts of open aid data and budget data in Nepal

This study provides a baseline of information regarding the aid and budget data landscape in Nepal. It presents an understanding of interactions of key stakeholders working in the open data ecosystem, and documents Nepal’s governance context. The study focuses on three groups of stakeholders within the open data ecosystem: data providers, data intermediaries and data users, and reveals that there is a gap between open data efforts, and the information needs and practices of civil society and journalists. It is part of the larger project “From Data to Development: Exploring the Emerging Impact of Open Government Data in Developing Countries”

Exploring International Educators\u27 Learning About Local and Global Social Justice in a Virtual Community of Practice

In this chapter, the authors report themes that emerged when a cross-cultural team of researchers involved in a virtual international community of practice (Global Social Justice in Education-GSJE) investigated reflections on activities focused on social justice in local and global contexts. The findings suggested that the activities elicited GSJE community members\u27 understandings of the complexities of social justice associated with naming practices, privilege, and the arts within their own and across contexts. The authors discuss implications of the activities to advance diverse educators\u27 understanding of social justice in global and local contexts. They also unpack the opportunities and challenges that resulted from the collaborative research process and the writing of this chapter completed by the international author team comprised of 11 education scholars from seven countries

Dicer Represses the Interferon Response and the Double-Stranded RNA-Activated Protein Kinase Pathway In Mouse Embryonic Stem Cells

Recent studies have demonstrated that embryonic stem cells (ESCs) are deficient in expressing type I interferons (IFN), the cytokines that play key roles in antiviral responses. However, the underlying molecular mechanisms and biological implications of this finding are poorly understood. In this study, we developed a synthetic RNA-based assay that can simultaneously assess multiple forms of antiviral responses. Dicer is an enzyme essential for RNA interference (RNAi), which is used as a major antiviral mechanism in invertebrates. RNAi activity is detected in wild-type ESCs but is abolished in Dicer knockout ESCs (D-/-ESCs) as expected. Surprisingly, D-/-ESCs have gained the ability to express IFN, which is otherwise deficient in wild-type ESCs. Furthermore, D-/-ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), an enzyme that is also involved in antiviral response. D-/-ESCs show increased sensitivity to the cytotoxicity resulting from RNA transfection. The effects of dsRNA can be partly replicated with a synthetic B2RNA corresponding to the retrotransposon B2 short interspersed nuclear element. B2RNA has secondary structure features of dsRNA and accumulates in D-/-ESCs, suggesting that B2RNA could be a cellular RNA that activates PKR and contributes to the decreased cell proliferation and viability of D-/-ESCs. Treatment of D-/-ESCs with a PKR inhibitor and IFNβ-neutralizing antibodies increased cell proliferation rate and cell viability. Based on these findings, we propose that, in ESCs, Dicer acts as a repressor of antiviral responses and plays a key role in the maintenance of proliferation, viability, and pluripotency of ESCs