Post-transcriptional capping generates coenzyme A-linked RNA.

NAD can be inserted co-transcriptionally via non-canonical initiation to form NAD-RNA. However, that mechanism is unlikely for CoA-linked RNAs due to low intracellular concentration of the required initiator nucleotide, 3'-dephospho-CoA (dpCoA). We report here that phosphopantetheine adenylyltransferase (PPAT), an enzyme of CoA biosynthetic pathway, accepts RNA transcripts as its acceptor substrate and transfers 4'-phosphopantetheine to yield CoA-RNA post-transcriptionally. Synthetic natural (RNAI) and small artificial RNAs were used to identify the features of RNA that are needed for it to serve as PPAT substrate. RNAs with 4-10 unpaired nucleotides at the 5' terminus served as PPAT substrates, but RNAs having <4 unpaired nucleotides did not undergo capping. No capping was observed when the +1A was changed to G or when 5' triphosphate was removed by RNA pyrophosphohydrolase (RppH), suggesting the enzyme recognizes pppA-RNA as an ATP analog. PPAT binding affinities were equivalent for transcripts with +1A, +1 G, or 5'OH (+1A), indicating that productive enzymatic recognition is driven more by local positioning effects than by overall binding affinity. Capping rates were independent of the number of unpaired nucleotides in the range of 4-10 nucleotides. Capping was strongly inhibited by ATP, reducing CoA-RNA production ~70% when equimolar ATP and substrate RNA were present. Dual bacterial expression of candidate RNAs with different 5' structures followed by CoA-RNA CaptureSeq revealed 12-fold enrichment of the better PPAT substrate, consistent with in vivo CoA-capping of RNA transcripts by PPAT. These results suggest post-transcriptional RNA capping as a possible mechanism for the biogenesis of CoA-RNAs in bacteria.

Depressive symptoms and associated factors among persons with physical disabilities in disability care homes of Kathmandu district, Nepal: A mixed method study.

Depression is one of the most common mental disorders, affecting 300 million people worldwide and 75% of these occur in low- and middle-income countries. Persons with physical disabilities are vulnerable groups and are more prone to experience depressive symptoms than the general population. This study investigated the prevalence of depressive symptoms and the associated factors among persons with a physical disability. We conducted a concurrent triangulation mixed methods design using Beck's Depression Inventory scale among 162 persons with physical disabilities in the Kathmandu district. In parallel, eight in-depth interviews were conducted with an interview guideline to collect the participants' perceptions and experiences of disability. Both quantitative and qualitative findings were integrated into the results. We found that about 77% of the participants with a physical disability had experienced depressive symptoms. Unemployment status (adjusted odds ratio (AOR) 2.7, 95% confidence interval (CI) 1.0-7.3) and comorbidity (AOR 2.5, 95% CI 1.0-6.0) had a statistically significant association with depressive symptoms. The majority of people with physical disabilities had negative experiences with societal prejudice and coping with their limitations. They were depressed as well as angry over having to stop their careers, education, and possibilities. Nevertheless, they were significantly happier and less sad than in their earlier years of life because of the possibilities, family environment, improved means of subsistence, therapeutic facilities, and supportive atmosphere at disability care homes. The policymakers should focus on preventing comorbidity and providing technical skills to persons with physical disabilities to improve their employment status and promote a healthy lifestyle.

Accommodation and disability-specific differences in nutritional status of children with disabilities in Kathmandu, Nepal: A cross-sectional study.

BACKGROUND: Worldwide, more than 150 million children < 18 years live with disabilities. These children are more vulnerable to malnutrition regardless of institutional care that they receive, such as daycare or residential care. In Nepal, little is known about the status of malnutrition and factors associated with malnutrition among children with disabilities. This study was conducted to investigate the factors associated with malnutrition based on the types of disability and accommodation. METHODS: This institution-based, cross-sectional study was conducted in 22 institutions in the Kathmandu Valley, Nepal. From these institutions, parents/guardians of all children with disabilities were recruited who were present there on the day of data collection. They were interviewed using a structured questionnaire. The questionnaire included questions on demographic characteristics, disability type and severity, accommodation place, feeding practices, and dietary patterns. The outcome variables, stunting, underweight, and obesity were measured using height-for-age, weight-for-age, and body mass index-for-age, respectively. A generalized linear model was used to investigate the factors associated with stunting and underweight, and multinomial logistic regression was used to identify the factors associated with overweight and obesity. RESULTS: Among the 345 children with disabilities, 45% were stunted, 33% were underweight, 19% were thin, and 12% were overweight. Children with physical disabilities (relative risk ratio = 1.88, 95% confidence interval [CI] = 1.26-2.81) were more likely to be stunted than those with sensory disabilities. Children with autism (adjusted odds ratio [aOR] = 5.56, 95% CI: 1.23-25.23) and intellectual disabilities (aOR = 5.84, 95% CI: 1.59-21.51) were more likely to be overweight and obese than those with sensory disabilities. No evidence was found regarding an association between accommodation type and malnutrition. CONCLUSION: Children with disabilities are vulnerable to malnutrition in several ways. Different types of disabilities are associated with different forms of malnutrition. Considering the types of disabilities, tailor-made approaches should be adopted to improve malnutrition status.

Cotranscriptional Assembly and Native Purification of Large RNA-RNA Complexes for Structural Analyses.

Recent technological developments such as cryogenic electron microscopy (Cryo-EM) and X-ray free electron lasers (XFEL) have significantly expanded the available toolkit to visualize large, complex noncoding RNAs and their complexes. Consequently, the quality of the RNA sample, as measured by its chemical monodispersity and conformational homogeneity, has become the bottleneck that frequently precludes effective structural analyses. Here we describe a general RNA sample preparation protocol that combines cotranscriptional RNA folding and RNA-RNA complex assembly, followed by native purification of stoichiometric complexes. We illustrate and discuss the utility of this versatile method in overcoming RNA misfolding and enabling the structural and mechanistic elucidations of the T-box riboswitch-tRNA complexes.

Factors Associated with Fluid Sequestration in Patients with Acute Pancreatitis: A Prospective Study in Tertiary Centre Hospital in Nepal.

BACKGROUND: Acute pancreatitis (AP) is associated with extensive fluid sequestration. The aim of this study was to determine association of fluid sequestration at 48 hours after hospital admission (FS48) in AP patients with demographics, clinical parameters, and outcomes of AP. METHODS: A prospective observational study was carried out on all adult patients with AP admitted to Tribhuvan University Teaching Hospital, Nepal, from January to September 2017. FS48 was calculated as the difference between fluid input and output in the first 48 hours of admission. The Kruskal-Wallis test with post hoc Dunn's test examined the difference in FS48 between mild AP, moderately severe AP, and severe AP. Linear regression analysis was used to evaluate association between FS48 with patients' characteristics and outcomes of AP. Outcomes of AP assessed included pancreatic necrosis, persistent organ failure, length of stay, and in-hospital mortality. RESULTS: Eighty patients (median age 44 years; 57% male) with a median FS48 of 1610 mL were evaluated. The median FS48 for mild AP, moderately severe AP, and severe AP were 1,180 mL, 2,380 mL, and 3,500 mL, respectively. There was a significant difference in pairwise comparisons between mild AP and moderately severe AP, along with mild AP and severe AP. Younger age, other etiology, and higher creatinine were independently associated with increased FS48. Increased FS48 was significantly associated with pancreatic necrosis, persistent organ failure, and in-hospital mortality. CONCLUSIONS: In our study population, younger age and higher creatinine were predictors of increased FS48. Increased FS48 was associated with poorer outcomes of AP.

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 IFNß-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.

Health research capacity in Nepal: Analysis of the trend and the role of local researchers.

Evidence-based decision-making is less common in low- and middle-income countries where the research capacity remains low. Nepal, a lower-middle-income country in Asia, is not an exception. We conducted a rapid review to identify the trend of health research in Nepal and found more than seven-fold increase in the number of published health-related articles between 2000 and 2018. The proportion of articles with Nepalese researchers as the first authors has also risen over the years, though they are still only in two-thirds of the articles in 2018.

Cloning, expression and purification of the low-complexity region of RanBP9 protein.

Recombinant expression and purification of proteins is key for biochemical and biophysical investigations. Although this has become a routine and standard procedure for many proteins, intrinsically disordered ones and those with low complexity sequences pose difficulties. Proteins containing low complexity regions (LCRs) are increasingly becoming significant for their roles in both normal and pathological processes. Here, we report cloning, expression and purification of N-terminal LCR of RanBP9 protein (Nt-RanBP9). RanBP9 is a scaffolding protein present in both cytoplasm and nucleus that is implicated in many cellular processes. Nt-RanBP9 is a poorly understood region of the protein perhaps due to difficulties posed by the LCR. Indeed, conventional methods presented difficulties in Nt-RanBP9 cloning due to its high GC content resulting in insignificant protein expression. These led us to use a different approach of cloning by expressing the protein as a fusion construct containing mCherry or mEGFP using in vivo DNA recombination methods. Our results indicate that expression of mEGFP-tagged Nt-RanBP9 followed by thrombin cleavage of the tag was the most effective method to obtain the protein with >90% purity and good yields. We report and discuss the challenges in obtaining the N-terminal region of RanBP9, a protein with functional implications in multiple biological processes and neurodegenerative diseases.

Efficient one-pot enzymatic synthesis of dephospho coenzyme A.

Dephospho coenzyme A (depCoA) is the last intermediate for CoA biosynthesis, and it can be used as a transcription initiator to prepare CoA-linked RNA by in vitro transcription. However, commercially available depCoA is expensive. We hereby describe a simple and efficient enzymatic synthesis of depCoA in a single-step from commercially available and inexpensive oxidized pantethine (Ox-Pan) and ATP. A plasmid (pCoaDAa) was constructed to co-express and co-purify two enzymes pantothenate kinase (PanK/coaA) and phosphopantetheine adenylyltransferase (PPAT/coaD). Starting from Ox-Pan and ATP, two different synthetic routes of one-pot reaction catalyzed by PanK and PPAT, followed by a simple column purification step, afforded depCoA and its oxidized dimer (Ox-depCoA) with high yields and purity. The simplicity and low cost of our method should make depCoA easily accessible to a broad scientific community, and promote research on CoA-related areas in biology and biomedicine.

Comparison between external rotation method and milch method for reduction of acute anterior dislocation of shoulder.

BACKGROUND: Anterior dislocation of shoulder account for more than 50% of dislocation that occur in our body. Several methods of reduction are described in literature which are painful or require anaesthesia. AIM: This study was undertaken to compare the External Rotation Method and Milch method for reduction of anterior dislocation of shoulder. MATERIALS AND METHODS: There were total 52 patients with anterior dislocation of shoulder, distributed randomly into 2 groups. Reduction was done by External Rotation Method and Milch Method for each group and their outcome were compared. STATISTICAL ANALYSIS: The data was analysed by using SPSS for Windows (version 16.0) by applying the Chi-Square test. p-values of <0.05 was considered as significant. RESULTS: Among each group of 26 patients, three patients required anaesthesia in External Rotation Method and eight patients required anaesthesia in Milch Method. There was no statistically significant difference in success rate between external rotation (88.46%) and Milch (69.23%) methods of reduction (p=0.09). CONCLUSION: Both methods of reduction can be used for reduction of anterior dislocation of shoulder without anaesthesia but external rotation method was found to be easier and less painful.