RESUMO
BACKGROUND OBJECTIVES: A new indigenously developed technology, coronavirus disease (COVID) Kavach, an IgG immunoglobulin-based enzyme-linked immunosorbent assay (ELISA) kit, was developed in 2020 by the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune, India. The primary objective of this study was to determine the total cost of development of COVID Kavach IgG ELISA and estimate the unit cost (UC) as well. METHODS: The total development cost (TDC) of COVID Kavach and its UC during the early phase of pandemic mitigation were estimated through a micro-costing approach from provider's perspective. An activity-based bottom-up costing approach was used to facilitate data collection from all resources, and analysis was performed using Microsoft Excel version 2016. The micro-costing data were utilized to interpret the breakdown of cost across all inputs and different levels of activity. RESULTS: The TDC of COVID Kavach was estimated to be JOURNAL/ijmer/04.03/02223309-202310000-00007/363FF04/v/2023-11-25T134903Z/r/image-tiff 2,884,032 (US$ 38,265). The UC of providing test results for exposure to severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) was estimated to be JOURNAL/ijmer/04.03/02223309-202310000-00007/363FF04/v/2023-11-25T134903Z/r/image-tiff 300 (US$ 4) during July 2020. The capital and recurrent cost were incurred around 5-10 per cent and 90-95 per cent, respectively, in both the development and UC of COVID Kavach. The major portion of funds (70-80%) was utilized for procurement of laboratory consumables, followed by human resources (8-12%) in the development as well as for UC of COVID Kavach. INTERPRETATION CONCLUSIONS: The estimates from this study can be useful for conducting economic evaluations, which will help in deciding upon the subsidy in government health facilities. The data may be useful to set up laboratory facilities analogous to the National Reference Laboratory located at the ICMR-NIV, Pune and for allotting sufficient budget to develop such assays in government-funded laboratories.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/epidemiologia , Pandemias , Índia/epidemiologia , Imunoglobulina GRESUMO
Rho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibits Escherichia coli Rho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, including Mycobacterium smegmatis, Mycobacterium bovis, Mycobacterium tuberculosis, Xanthomonas campestris, Xanthomonas oryzae, Corynebacterium glutamicum, Vibrio cholerae, Salmonella enterica, and Pseudomonas syringae The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from the E. coli transcription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins. In vivo pulldown assays revealed direct binding of Psu with many of these Rho proteins. In vivo expression of psu induced killing of M. smegmatis, M. bovis, X. campestris, and E. coli expressing S. enterica Rho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the "universal" inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions.IMPORTANCE Bacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of the E. coli transcription terminator Rho. Here we show that apart from antagonizing E. coli Rho, Psu is able to inhibit Rho proteins from various phylogenetically unrelated Gram-negative and Gram-positive pathogens. Upon binding to these Rho proteins, Psu inhibited them by affecting their ATPase and RNA release functions. The expression of Psu in vivo kills various pathogens, such as Mycobacterium and Xanthomonas species. Hence, Psu could be useful for identifying peptide sequences with anti-Rho activities and might constitute part of synergistic antibiotic treatment against pathogens.
Assuntos
Proteínas do Capsídeo/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Fator Rho/antagonistas & inibidores , Terminação da Transcrição Genética/efeitos dos fármacos , Adenosina Trifosfatases/antagonistas & inibidores , Bacteriófagos/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/patogenicidade , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/patogenicidade , Mycobacterium/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Ligação Proteica , Proteínas Recombinantes/metabolismo , Fator Rho/genética , Fator Rho/metabolismo , Homologia de Sequência do Ácido Nucleico , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Xanthomonas/efeitos dos fármacosRESUMO
Real-time reverse transcription polymerase chain reaction (rRT-PCR) is one of the most accurate and extensively used laboratory procedures for diagnosing COVID-19. This molecular test has high diagnostic accuracy (sensitivity and specificity) and is considered as the gold standard for COVID-19 diagnosis. During COVID-19 surge in India, rRT-PCR service was encouraged and supported by the government of India through existing healthcare setup at various levels of healthcare facilities. The primary purpose of this research was to determine the per-unit cost of providing COVID-19 rRT-PCR services at the national reference laboratory at ICMR-National Institute of Virology in Pune during the early phase of COVID-19 pandemic mitigation, from the provider's perspective. The monthly cost for rRT-PCR testing as well as an estimated annual average unit cost for testing that takes account of peaks and troughs in pandemic were investigated. The time frame used to estimate unit cost was one year (July 2020-June 2021). For data collection on all resources spent during the early phase of pandemic, a conventional activity-based bottom-up costing technique was used. Capital costs were discounted and annualized over the estimated life of the item. Apportioning statistics were selected for cost heads like human resources, capital, and equipment based on time allocation, sharing of services, and utilization data. The data was also used to understand the breakdown of costs across inputs and over time and different levels of testing activity. During the initial phase of pandemic mitigation, the per unit cost of providing the COVID-19 rRT-PCR test was estimated to be â¹566 ($7.5) in the month of July 2020, where the total 56318 COVID-19 rRT-PCR tests was performed. The major proportion (87%) of funds was utilized for procuring laboratory consumables, followed by HR (10%), and it was least for stationary & allied items (0.02%). Unit cost was found to be the most sensitive to price variations in lab consumables (21.7%), followed by the number of samples tested (3.9%), salaries paid to HR (2.6%), price of equipment (0.23%), and building rental price (0.14%) in a univariate sensitivity analysis. The unit cost varies over the period of the pandemic in proportion with the prices of consumables and inversely proportional with number of tests performed. Our study would help the Government to understand the value for money they invested for laboratory diagnosis of COVID-19, budget allocation, integration and decentralization of laboratory services so as to help for achieving universal health coverage.
Assuntos
COVID-19 , Humanos , COVID-19/diagnóstico , COVID-19/epidemiologia , Pandemias , Teste para COVID-19 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índia/epidemiologiaRESUMO
Tyrosinase plays an important role in melanin biosynthesis and protects skin against ultraviolet radiations. Functional deficiency of tyrosinase results in serious dermatological diseases. Tyrosinase also participates in neuromelanin formation in the human brain, which leads to neurodegeneration resulting in Parkinson's disease. In fruits and vegetables, tyrosinase plays a critical role in senescence, causing undesired browning that results in faster deterioration and shorter shelf lines. The only commercially available tyrosinase is mushroom tyrosinase and it shows the highest homology to the mammalian tyrosinase. Although kojic acid is currently used as a tyrosinase inhibitor, they have serious side effects such as dermatitis, carcinogenesis and hepatotoxicity. Therefore, in order to develop a more active and safer tyrosinase inhibitor, 3D QSAR pharmacophore models were generated based on experimentally known inhibitors. The pharmacophore model, Hypo1, was developed with a large cost difference, high correlation coefficient and low RMS deviation. Hypo1 showed a good spatial arrangement; consisting of five-point features including two hydrogen bond acceptor, one hydrogen bond donor and two hydrophobic features. Hypo1 was further validated by cost analysis, test set and Fisher's randomisation method. Hypo1 was used as a 3D query for screening the in-house drug-like databases, and the hits were further selected by applying ADMET, Lipinski's rule of five and fit value criteria. To identify binding conformations, the obtained hits were subjected to molecular docking. Finally, molecular dynamics simulations revealed the appropriate binding modes of hit compounds. To conclude, we propose the final three hit compounds with new structural scaffolds as a virtual candidate as tyrosinase inhibitors.Communicated by Ramaswamy H. Sarma.
Assuntos
Monofenol Mono-Oxigenase , Inibidores de Proteínas Quinases/química , Relação Quantitativa Estrutura-Atividade , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Monofenol Mono-Oxigenase/antagonistas & inibidoresRESUMO
Hypermodified bases present at 3'-adjacent (37th) position in anticodon loop of tRNAPhe are well known for their contribution in modulating codon-anticodon interactions. Peroxywybutosine (o2yW), a wyosine family member, is one of such tricyclic modified bases observed at the 37th position in tRNAPhe. Conformational preferences and three-dimensional structural analysis of peroxywybutosine have not been investigated in detail at atomic level. Hence, in the present study quantum chemical semi-empirical RM1 and multiple molecular dynamics (MD) simulations have been used to study structural significance of peroxywybutosine in tRNAPhe. Full geometry optimizations over the peroxywybutosine base have also been performed using ab-initio HF-SCF (6-31G**), DFT (B3LYP/6-31G**) and semi-empirical PM6 method to compare the salient properties. RM1 predicted most stable structure shows that the amino-carboxy-propyl side chain of o2yW remains 'distal' to the five membered imidazole ring of tricyclic guanosine. MD simulation trajectory of the isolated peroxy base showed restricted periodical fluctuations of peroxywybutosine side chain which might be helpful to maintain proper anticodon loop structure and mRNA reading frame during protein biosynthesis process. Another comparative MD simulation study of the anticodon stem loop with codon UUC showed various properties, which justify the functional implications of peroxywybutosine at 37th position along with other modified bases present in ASL of tRNAPhe. Thus, this study presents an atomic view into the structural properties of peroxywybutosine, which can be useful to determine its role in the anticodon stem loop in context of codon-anticodon interactions and frame shift mutations.
Assuntos
Anticódon/genética , Códon/genética , Guanosina/análogos & derivados , Nucleosídeos/genética , RNA de Transferência de Fenilalanina/genética , Anticódon/química , Códon/química , Mutação da Fase de Leitura/genética , Guanosina/química , Guanosina/genética , Conformação Molecular , Simulação de Dinâmica Molecular , Nucleosídeos/química , Biossíntese de Proteínas/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA de Transferência de Fenilalanina/químicaRESUMO
Lack of naturally occurring modified nucleoside 5-taurinomethyluridine (τm5U) at the 'wobble' 34th position in tRNALeu causes mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). The τm5U34 specifically recognizes UUG and UUA codons. Structural consequences of τm5U34 to read cognate codons have not been studied so far in detail at the atomic level. Hence, 50ns multiple molecular dynamics (MD) simulations of various anticodon stem loop (ASL) models of tRNALeu in presence and absence of τm5U34 along with UUG and UUA codons were performed to explore the dynamic behaviour of τm5U34 during codon recognition process. The MD simulation results revealed that τm5U34 recognizes G/A ending codons by 'wobble' as well as a novel 'single' hydrogen bonding interactions. RMSD and RMSF values indicate the comparative stability of the ASL models containing τm5U34 modification over the other models, lacking τm5U34. Another MD simulation study of 55S mammalian mitochondrial rRNA with tRNALeu showed crucial interactions between the A-site residues, A918, A919, G256 and codon-anticodon bases. Thus, these results could improve our understanding about the decoding efficiency of human mt tRNALeu with τm5U34 to recognize UUG and UUA codons.
Assuntos
Códon/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Leucina/metabolismo , Uridina/análogos & derivados , Animais , Anticódon/metabolismo , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , RNA/metabolismo , RNA Mitocondrial , RNA Ribossômico/metabolismo , RNA de Transferência de Leucina/química , Uridina/metabolismoRESUMO
Transfer RNAs (tRNAs) contain various uniquely modified nucleosides thought to be useful for maintaining the structural stability of tRNAs. However, their significance for upholding the tRNA structure has not been investigated in detail at the atomic level. In this study, molecular dynamic simulations have been performed to assess the effects of methylated nucleic acid bases, N (2)-methylguanosine (m(2)G) and N (2)-N (2)-dimethylguanosine (m 2 (2) G) at position 26, i.e., the hinge region of E. coli tRNA(Phe) on its structure and dynamics. The results revealed that tRNA(Phe) having unmodified guanosine in the hinge region (G26) shows structural rearrangement in the core of the molecule, resulting in lack of base stacking interactions, U-turn feature of the anticodon loop, and TΨC loop. We show that in the presence of the unmodified guanosine, the overall fold of tRNA(Phe) is essentially not the same as that of m(2)G26 and m 2 (2) G26 containing tRNA(Phe). This structural rearrangement arises due to intrinsic factors associated with the weak hydrogen-bonding patterns observed in the base triples of the tRNA(Phe) molecule. The m(2)G26 and m 2 (2) G26 containing tRNA(Phe) retain proper three-dimensional fold through tertiary interactions. Single-point energy and molecular electrostatics potential calculation studies confirmed the structural significance of tRNAs containing m(2)G26 and m 2 (2) G26 compared to tRNA with normal G26, showing that the mono-methylated (m(2)G26) and dimethylated (m 2 (2) G26) modifications are required to provide structural stability not only in the hinge region but also in the other parts of tRNA(Phe). Thus, the present study allows us to better understand the effects of modified nucleosides and ionic environment on tRNA folding.
Assuntos
Guanosina/metabolismo , Modelos Moleculares , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/metabolismo , Pareamento de Bases , Ligação de Hidrogênio , Magnésio/metabolismo , Metilação , RNA Fúngico/química , RNA Fúngico/metabolismo , TermodinâmicaRESUMO
Hypermodified nucleosides lysidine (L) and N(6)-threonylcarbamoyladenosine (t(6)A) influence codon-anticodon interactions during the protein biosynthesis process. Lysidine prevents the misrecognition of the AUG codon as isoleucine and that of AUA as methionine. The structural significance of these modified bases has not been studied in detail at the atomic level. Hence, in the present study we performed multiple molecular dynamics (MD) simulations of anticodon stem loop (ASL) of tRNA(Ile) in the presence and absence of modified bases 'L' and 't(6)A' at the 34th and 37th positions respectively along with trinucleotide 'AUA' and 'AUG' codons. Hydrogen bonding interactions formed by the tautomeric form of lysidine may assist in reading the third base adenine of the 'AUA' codon, unlike the guanine of the 'AUG' codon. Such interactions might be useful to restrict codon specificity to recognize isoleucine tRNA instead of methionine tRNA. The t(6)A side chain interacts with the purine ring of the first codon nucleotide adenine, which might provide base stacking interactions and could be responsible for restricting extended codon-anticodon recognition. We found that ASL tRNA(Ile) in the absence of modifications at the 34th and 37th positions cannot establish proper hydrogen bonding interactions to recognize the isoleucine codon 'AUA' and subsequently disturbs the anticodon loop structure. The binding free energy calculations revealed that tRNA(Ile) ASL with modified nucleosides prefers the codon AUA over AUG. Thus, these findings might be useful to understand the role of modified bases L and t(6)A to recognize the AUA codon instead of AUG.
Assuntos
Códon , Lisina/análogos & derivados , Nucleosídeos/química , Nucleosídeos de Pirimidina/química , Adenosina/análogos & derivados , Adenosina/química , Anticódon/química , Sequência de Bases , Simulação por Computador , Ligação de Hidrogênio , Lisina/química , Metionina/química , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ribossomos/química , Eletricidade EstáticaRESUMO
Conformational preferences of hypermodified nucleoside 5-taurinomethyluridine 5'-monophoshate 'p-τm(5)U' (-CH2-NH2(+)-CH2-CH2-SO3(-)) have been investigated using semi-empirical RM1 method. Automated geometry optimization using ab initio molecular orbital HF-SCF (6-31G**) and DFT (B3LYP/6-31G**) calculations have also been made to compare the salient features. The RM1 preferred most stable conformation of 'p-τm(5)U' has been stabilized by hydrogen bonding interactions between O(11a) HN(8), O1P(34) HN(8), and O1P(34) HC(10). Another conformational study of 5-taurinomethyluridine side chain has also been performed in context of anticodon loop bases of E. coli tRNA(Leu). The atom O(11a) of τm(5)U(34) side chain interacts with adenosine (A35) as well as ribose-phosphate backbone which might provide structural stability to the anticodon loop. The glycosyl torsion angle of τm(5)U retains 'anti'-conformation. The solvent accessible surface area calculations revealed the role of τm(5)U in tRNA(Leu) anticodon loop. MD simulation results are found in agreement with RM1 preferred stable structure. The MEPs calculations of τm(5)U(34):G3 model show unique potential tunnels between the hydrogen bond donor and acceptor atoms as compared to τm(5)U(34):A3 model. Thus, these results could pave the way to understand the role of τm(5)U(34) to recognize UUG/UUA codons at atomic level in the mitochondrial disease, MELAS.
Assuntos
Anticódon/metabolismo , Conformação Molecular , Uridina/análogos & derivados , Escherichia coli/genética , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Eletricidade Estática , Uridina/química , Uridina/metabolismoRESUMO
Modified nucleic acid bases are most commonly found in tRNA. These may contain modifications from simple methylation to addition of bulky groups. Methylation of the four canonical nucleotide bases at a wide variety of positions is particularly prominent among the known modification. Methylation of N2 group of guanine is a relatively common modification in tRNA and rRNA. N2-methylguanosine (m(2)G) is the second most often encountered nucleoside in E. coli tRNAs. N2, N2- dimethylguanosine (m(2) 2G) is found in the majority of eukaryotic tRNAs and involved in forming base pair interactions with adjacent bases. Hence, in order to understand the structural significance of these methylated nucleic acid bases we have carried out molecular dynamics simulation to see the salvation effect. The results obtained shows iso-energetic conformational behaviors for m(2)G and m(2) 2G. The simulation trajectory of m(2)G shows regular periodical fluctuations suggesting that m(2)G is equally stable as either s-cis or s-trans rotamers. The two rotamers of m(2)G may interact canonically or non-canonically with opposite base as s-trans m(2)G26:C/A/U44 and s-cis m(2)G26:A/U44. The free rotations around the C-N bond could be the possible reason for these iso-energetic conformations. Dimethylation of G has almost no influence on base pairing with either A or U. Thus, these results reveal that modified nucleosides m(2)G and m(2) 2G may play an important role to prevent tRNA from adopting the unusual mitochondrial like conformation.