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Inosine is one of the most common post-transcriptional modifications. Since its discovery, it has been noted for its ability to contribute to non-Watson-Crick interactions within RNA. Rapidly accumulating evidence points to the widespread generation of inosine through hydrolytic deamination of adenosine to inosine by different classes of adenosine deaminases. Three naturally occurring methyl derivatives of inosine, i.e., 1-methylinosine, 2'-O-methylinosine and 1,2'-O-dimethylinosine are currently reported in RNA modification databases. These modifications are expected to lead to changes in the structure, folding, dynamics, stability and functions of RNA. The importance of the modifications is indicated by the strong conservation of the modifying enzymes across organisms. The structure, binding and catalytic mechanism of the adenosine deaminases have been well-studied, but the underlying mechanism of the catalytic reaction is not very clear yet. Here we extensively review the existing data on the occurrence, biogenesis and functions of inosine and its methyl derivatives in RNA. We also included the structural and thermodynamic aspects of these modifications in our review to provide a detailed and integrated discussion on the consequences of A-to-I editing in RNA and the contribution of different structural and thermodynamic studies in understanding its role in RNA. We also highlight the importance of further studies for a better understanding of the mechanisms of the different classes of deamination reactions. Further investigation of the structural and thermodynamic consequences and functions of these modifications in RNA should provide more useful information about their role in different diseases.

Pseudouridine and N1-methylpseudouridine are the key modifications in the field of mRNA therapeutics and vaccine research. The accuracy of the design and development of therapeutic RNAs containing such modifications requires the accuracy of the secondary structure prediction, that depends on the nearest neighbor (NN) thermodynamic parameters for the standard and modified residues. The development of such NN thermodynamic parameters requires expensive and time-consuming experimental studies. There were some earlier attempts to predict the NN free energies of modified RNA using computational methods but those are either computationally expensive or not accurate enough. Here, we propose a new protocol based on MD simulations, which is able to predict the NN free energy parameters (ΔG◦37) for U-A, Ψ-A and m1Ψ-A pairs in general agreement with the recent experimental reports. We report the NN thermodynamic parameters for different U, Ψ and m1Ψ base pairs, which might be helpful for a deeper understanding of the effect of these modifications in RNA. The presence of m1Ψ resulted in more stable NN pairs compared to those containing U or Ψ. The predicted NN free energy parameters in this study are able to closely reproduce the folding free energies of duplexes containing internal Ψ for which the thermodynamic data were available. Additionally, we report the predicted folding free energies for the duplexes containing internal m1Ψ.

Pseudouridine and N1-methylpseudouridine are the key modifications in the field of mRNA therapeutics and vaccine research. The accuracy of the design and development of therapeutic RNAs containing such modifications requires the accuracy of the secondary structure prediction, that depends on the nearest neighbor (NN) thermodynamic parameters for the standard and modified residues. The development of such NN thermodynamic parameters requires expensive and time-consuming experimental studies. There were some earlier attempts to predict the NN free energies of modified RNA using computational methods but those are either computationally expensive or not accurate enough. Here, we propose a new protocol based on MD simulations, which is able to predict the NN free energy parameters (ΔG◦37) for U-A, Ψ-A and m1Ψ-A pairs in general agreement with the recent experimental reports. We report the NN thermodynamic parameters for different U, Ψ and m1Ψ base pairs, which might be helpful for a deeper understanding of the effect of these modifications in RNA. The presence of m1Ψ resulted in more stable NN pairs compared to those containing U or Ψ. The predicted NN free energy parameters in this study are able to closely reproduce the folding free energies of duplexes containing internal Ψ for which the thermodynamic data were available. Additionally, we report the predicted folding free energies for the duplexes containing internal m1Ψ.

Pseudouridine (Ψ) is one of the most common post-transcriptional modifications in RNA and has been known to play significant roles in several crucial biological processes. The N1-methyl derivative of pseudouridine i.e N1-methylpseudouridine has also been reported to be important for the stability and function of RNA. Several studies suggest the importance of pseudouridine and N1-methylpseudouridine in mRNA therapeutics. The critical contribution of pseudouridine, especially that of its N1-methyl derivative in the efficiency of the COVID-19 mRNA vaccines, suggests the requirement to better understand the role of these modifications in the structure, stability and function of RNA. In the present study, we have investigated the consequences of the presence of these modifications in the stability of RNA duplex structures by analyzing different structural properties, hydration characteristics and energetics of these duplexes. We have previously studied the structural and thermodynamic properties of RNA duplexes with an internal Ψ-A pair and reported the stabilizing effect of Ψ over U (Deb, I. et al.Sci Rep9, 16278 (2019)). Here, we have extended our work to understand the properties of RNA duplexes with an internal m1Ψ-A pair and also theoretically demonstrate the effect of substituting internal U-G, U-U and U-C mismatches with the Ψ-G, Ψ-U and Ψ-C mismatches and also with the m1Ψ-G, m1Ψ-U and m1Ψ-C mismatches respectively, within dsRNA. Our results indicate the context-dependent stabilization of base stacking interactions by N1-methylpseudouridine compared to uridine and pseudouridine, presumably resulting from the increased molecular polarizability due to the presence of the methyl group.

Inosine is one of the most abundant post-transcriptionally modified ribonucleosides which is known to play a major role in several important biological processes and is of great therapeutic importance. The growing importance of this modified ribonucleoside in therapeutics suggests the requirement of further theoretical studies involving inosine and its derivatives and ensuring the accuracy of their force field parameters is crucial for such theoretical studies to be reliable. The present study reports the validation of the AMBER derived force field parameter sets for inosine as well as examination of the transferability of the available revised sets of glycosidic and gamma torsion parameters corresponding to the respective canonical nucleosides based on detailed comparison of different conformational features from replica exchange molecular dynamics. We also report newly developed sets of partial atomic charges and glycosidic torsion parameters (𝛘KOL0) for inosine and its methyl derivatives. These parameters, in combination with the AMBER FF99 parameters (Cheatham, T. E., III; Cieplak, P.; Kollman, P. A. J. Biomol. Struct. Dyn. 1999, 16, 845 − 862; Aduri, R.; Psciuk, B. T.; Saro, P.; Taniga, H.; Schlegel, H. B.; SantaLucia, J. J. Chem. Theory Comput. 2007, 3, 1464 − 1475), and the recommended bsc0 correction for the gamma torsion (Pérez, A.; Marchán, I.; Svozil, D.; Sponer, J.; Cheatham, T. E.; Laughton, C. A.; Orozco, M. Biophys. J. 2007, 3817 − 3829.), reproduced the conformational properties of inosine and its 1-methyl derivative in agreement with experimental (NMR) data. In this study, we have also predicted the conformational preferences for the other two methyl derivatives of inosine, i.e., 2’-O-methylinosine and 1,2’-O-dimethylinosine using the revised sets of glycosidic torsion parameters.

Pseudouridine is the most abundant post-transcriptional modification in RNA. We have previously shown that the FF99-derived parameters for pseudouridine and some of its naturally occurring derivatives in the AMBER distribution either alone or in combination with the revised 𝛄 torsion parameters (parmbsc0) failed to reproduce their conformational characteristics observed experimentally (Deb I, et al. J. Chem. Inf. Model. 2014, 54 (4):1129–1142; Deb I, et al. J. Comput. Chem., 2016, 37:1576−1588; Dutta N, et al. J. Chem. Inf. Model. 2020, 60 (10):4995–5002). However, the application of the recommended bsc0 correction did lead to an improvement in the description not only of the distribution in the 𝛄 torsional space but also of the sugar pucker distributions. In an earlier study, we examined the transferability of the revised glycosidic torsion parameters (𝛘IDRP) for Ψ to its derivatives. We noticed that although these parameters in combination with the AMBER FF99-derived parameters and the revised 𝛄 torsional parameters resulted in conformational properties of these residues that were in better agreement with experimental observations, the sugar pucker distributions were still not reproduced accurately. Here we report a new set of partial atomic charges for pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine and 2′-O-methylpseudouridine and a new set of glycosidic torsional parameters (𝛘ND) based on chosen glycosidic torsional profiles that most closely corresponded to the NMR data for conformational propensities and studied their effect on the conformational distributions using REMD simulations at the individual nucleoside level. We have also studied the effect of the choice of water model on the conformational characteristics of these modified nucleosides. Our observations suggest that the current revised set of parameters and partial atomic charges describe the sugar pucker distributions for these residues more accurately and that the choice of a suitable water model is important for the accurate description of their conformational properties.

Objectives: Antiretroviral therapy (ART) has immense survival benefit on human immunodeficiency virus (HIV)-infected people. However, every year, a proportion of patients were failing to the first-line drugs. The aim of this study is to characterize the patients developing first-line failure within 5 years of ART. Materials and Methods: A retrospective observational study was carried out at the Centre of Excellence in HIV care, School of Tropical Medicine, Kolkata. A total of 190 referred patients' data of suspected first-line treatment failure who failed first-line ART within 5 years of initiation were collected and analyzed using R software. Results: Among 190 patients, 100 (52.4%) patients had virologic failure. Male patients 78 (41.05%) outnumbered females 22 (11.57%) and needed to switch to the second-line drugs. The median age was 37 years (range 8–65 years), and the median duration of first-line ART taken was 2.85 years. Among the first-line failed patients, zidovudine, lamivudine, and nevirapine (23.6%) was the most common antiretroviral regimen and 77 (40.5%) referred in the WHO stage I of illness. Seventy-three (38.42%) patients were referred for immunological failure, 26 (13.7%) for both immunological and clinical failure, and only 1 (0.52%) had only clinical failure at the time of referral. We found a significant association of suboptimal adherence (P < 0.05) and high viral load in this study. Conclusion: This study enables that poor adherence was the most important factor responsible for the first-line treatment failure. As adherence is a dynamic process, interventions in every visit following ART initiation should be optimized, and a multidisciplinary approach toward adherence is needed to get the highest treatment outcome benefit.

FESEM and X-ray diffraction studies were carried out to assess the microstructure and phases present in a Dispersalloy admix type Ag-Sn-Cu dental amalgam. Electrochemical behaviour of the amalgam was assessed by electrochemical impedance spectroscopy (EIS) measurements in natural saliva, artificial saliva and 0.90 wt.% NaCl solution. EIS was also measured of the amalgam exposed for varying time in 0.90 wt.% NaCl solutions. Results of the impedance measurements showed that the 0.90 wt.% NaCl solution is more corrosive than saliva. The rough and porous surfaces of dental amalgam are energetically inhomogeneous which contribute frequency dispersion of impedance response.

Differential scanning calorimetry (DSC) technique has been used to examine the reaction sequence in dental amalgam alloy powders of (1) Ag–Cu–Sn lathe-cut particles (single composition type in dentistry terminology), (2) mixture of lathe-cut Ag–Sn–low Cu and spherical particles of Ag–Cu eutectic composition (admix type) and (3) their amalgams, i.e. alloys of liquid Hg and these dental amalgam alloy powders. All the peaks in the DSC curves have been identified and discussed. DSC curves of single composition-type powders showed as many as six distinct peaks, whereas admix-type powders showed only four peaks. Quite interestingly, amalgam of single composition powders exhibited only three peaks, whereas amalgam of admix type displayed four distinct peaks. The noticeable differences of peak positions in the DSC curves of the amalgam powders and their amalgams and the associated chemical reactions have been explained. DSC curves of the alloy powders and their amalgams studied at four different heating rates have showed peak shifting and the variation of change of enthalpy (ΔH) as well with the heating rates (v h). The peak temperatures and the heat evolved and absorbed of all the peaks have been determined with the help of built-in software available in the DSC unit.

Introduction and Objectives: A common adverse effect of Efavirenz (EFV) – a first line drug used in treatment of patients with HIV infections is skin rash. In case of EFV-induced skin rash, the usual practice is to switch to a Protease Inhibitor (PI), as another non-nucleoside reverse transcriptase inhibitor (NNRTI) might have cross-­reactivity with a higher incidence of skin rash which is often severe. The aim of the study was therefore to determine whether with careful evaluation and stratification, using pre­defined criteria of patients who developed rash with EFV, it might be possible to find a subset of patients among whom Nevirapine (NVP) may be safely used, sparing PIs for second-­line treatment. Methods: Of 7000 ART naive patients initiated on EFV in an 18 month period from November 2014 to April 2016, 97(1.9%) developed a rash. Patients developing rash with EFV were carefully selected using pre­defined criteria: grade of rash (only Grade I/II rash were selected), hepatic function, age, gender and CD4 levels (males > 400 cells/cmm and females >250 cells/cmm were excluded if hepatic function was deranged), co­-infection status (Hepatitis B/C were excluded), opportunistic infections, concomitant use of other drugs. Based on these stratification criteria, 23 of the 97 patients were selected for challenge with NVP. Results: Of the 23 selected patients, only 3 (13%) patients developed recurrence of rash, all being mild in nature. Conclusions: In carefully selected patients, challenge with NVP can be done in case of rash with EFV as the theory of cross­-reactivity does not always hold true. This might help spare PIs in the initial phase, which could then be used as second line therapy - a particularly helpful strategy in developing countries, where newer drugs are still not available under public health programmes.

The left-handed polyproline II (PPII) type helical structures are thought to play a very important role in many essential biological processes, particularly in recognition mechanisms. However, reliable characterisation of PPII conformation in solution can be experimentally challenging. Computational simulation of these structures offers an attractive alternative, but the accuracy of the results is dependent on the accuracy of the force field employed. In this report, we present the results of simulation of the structural and dynamical properties of a proline-rich viral fusion peptide for which a solution NMR study reported a substantial stretch of PPII conformation in the central region. The suggested mode of action of the p15 fusion peptide depended on the exposure of the flanking N-terminal hydrophobic residues to solvent thereby facilitating their interaction with the target membrane. Our simulations with a set of four force field and water model combinations consisting of (AMBER ff99SB*-ILDNP + TIP3P), (OPLS-AA + SPC/E), (AMBER ff03ws + TIP4P/2005 water with scaled protein-water interactions) and (CHARMM36m + TIP3P) showed a general agreement with the NMR results for all the four force field and water model combinations. The central region encompassing positions 9-15 showed a large PPII propensity, reduced flexibility and lower conformational entropy. The PPII conformations were stable and satisfied the Burgi-Dunitz criteria without the participation of any significant water bridging interaction. However, comparison of the experimentally observed chemical shifts with the distribution of shifts predicted from the simulated ensembles showed a much better agreement for the CHARMM36m + TIP3P and AMBER ff03ws + TIP4P/2005 combinations. The models based on these two force fields also generated conformations which were in much better agreement with the NMR model than the much more compact structures predicted by the AMBER ff99SB*-ILDNP and OPLS-AA force fields and predicted a substantially larger solvent accessible surface area in accordance with the suggested mechanism of action of the peptide.

Ag–Sn–Cu lathe cut particles of dental amalgam powders are mixed with mercury with an approximate 1:1 ratio. Mercury and amalgam powder mixtures are triturated at nominal pressure and at higher pressure applied intermittently, and then condensed into an acrylic mould with reasonably higher condensation pressure as well, followed by setting at room temperature for different time period. The microstructural features of the amalgam alloy powders and its amalgams of various states are studied by FESEM, X-ray elemental mapping, X-ray diffraction, differential scanning calorimetry and by hardness measurement as well. Results showed that the amalgams triturated with higher pressure have yielded finer matrix γ1 (Ag2Hg3), presence of Ag-rich β1 and finer unreacted γ (Ag3Sn) phases with reduced porosity and higher hardness.

Introduction Disseminated histoplasmosis is a treatable common opportunistic infection in HIV infected people and not uncommon in others in a tropical country like India. The objective of our study was to evaluate clinical-pathological correlation and treatment outcome of disseminated histoplasmosis treated with conventional Amphotericin B in an endemic area.Material and methodsThis was a retrospective observational study of twenty-two cases of disseminated histoplasmosis admitted to a tertiary care hospital from January 2009 to December 2012 and treated with Amphotericin B followed by oral Itraconazole therapy for one year. Results of treatment outcome including relapse and mortality were analyzed in January 2014.ResultsHistoplasmosis was diagnosed in patients with advanced HIV (72%) illness with a mean CD4 count of 63.43/l. Tuberculosis and diabetes were other co-morbid illnesses and it was less common among immunocompetent patients (9%). Fifty percent of the patients presented with cutaneous lesions along with systemic manifestations while 27% had only mucocutaneous lesions. Adrenal histoplasmosis (18%) was common in HIV negative subjects. HIV positive patients showed excellent response to Amphotericin B followed by Itraconazole therapy. In 27% HIV positive patients, the disease manifested as IRIS (immune reconstitution inflammatory syndrome). Relapse was seen in 2(9%) patients. After one year of completion of therapy 16 patients were cured, 3 patients (13.6%) died in the early part of treatment and one was lost to follow up. Treatment response in HIV infected patients showed excellent results but long term maintenance itraconazole therapy was inevitable (12-32 months).ConclusionsIt is concluded that early diagnosis and treatment can prevent a fatal disease like disseminated histoplasmosis with conventional Amphotericin B followed by Itraconazole. Adrenal histoplasmosis was common among the HIV negative population. Extensive follow up is required to identify early relapse which may need further prolongation of therapy for cure.

Although cases of Selective Serotonin Reuptake Inhibitor (SSRI) induced akathisia have often been reported in literature, this adverse effect has not adequately been mentioned in major pharmacology textbooks. As a result, SSRIinduced akathisia is very frequently under-recognized. A review of literature showed that almost all frequently used SSRIs such as Fluvoxamine, Fluoxetine, Sertraline, Citalopram have been reported to be causing akathisia. SSRI-induced restless legs syndrome and movement disorders have also been reported. However, Escitalopram-induced akathisia is rare. In our review of literature, we could find only one single case of Escitalopram-induced severe akathisia. And this specific SSRI drug has rarely been implicated with occurrence of restless legs syndrome and extra-pyramidal side-effects like dytonia etc. Here, we present a case of Escitalopram-induced severe akathisia - a 53year old female, who had developed severe akathisia after taking Escitalopram for a few days. According to the Barnes Akathisia Rating Scale (BARS), her Global Clinical Assessment of Akathisia Score was 5 i.e. severe akathisia. As per Naronjo Adverse Drug Reaction Scale the probability of association of this adverse reaction with Escitalopram was 7 (i.e. probable). Her symptoms continued in spite of prompt discontinuation of the drug. But, she improved rapidly with the use of Propranolol and Clonazepam. On the last follow-up, she was free from any symptoms. As new generation antidepressants are rarely associated with extra-pyramidal symptoms, the recognition of such adverse effects requires a high index of suspicion. Early recognition of the symptoms and discontinuation of the offending agent along with supportive therapy like a short course of benzodiazepines, beta-adrenergic antagonists or anticholinergics may rapidly relieve the patient from this distressing symptom.

Aim Hypersensitivity to the anti-retroviral agents is a common problem encountered in the treatment of HIV/AIDS patients. However, in resource-limited settings, where only fixed-dose combinations of the drugs are the only available option and expensive facilities of pharmacological analysis are not available, isolating the offending agent can be a challenging task. Background We, at our centre, follow a protocol to isolate such offending drug – a protocol of gradual de-challenge and re-challenge – by a method of exclusion. Materials and Methods Here, we have described a series of four cases, providing examples of usage of our own protocol to isolate the offending drug by a method of exclusion. Results and Conclusion In a resource-limited setting, where we have to use fixed-dose combination of anti-retroviral agents, management of a patient with drug hypersensitivity is a problem. Our protocol of systematic de-challenge and re-challenge, as exemplified in these cases, can be of help where expensive facilities are not available.

There is a large concentration of chemical firms around Mumbai, and the Maharashtra Pollution Control Board has been actively monitoring them. Such monitoring is likely to have an impact on the productive efficiency of these firms. The firms argue that additional costs to minimise environmental damage have reduced their efficiency. We try to find out whether these water-polluting firms in the chemical industry around Mumbai are actually unable to cope with the additional cost of pollution abatement, or whether by using cleaner practices, are able to improve their efficiency, and support the ‘win-win’ opportunities as claimed by the Porter Hypothesis. The study is carried out by estimating an Output Distance Function using a stochastic production function. The panel data of fifty water-polluting small-to medium-scale firms for three-year period of 2004–06 was collected in a primary survey of chemical industries around Mumbai. We find that the polluting firms are technically more efficient than those firms which adhere to pollution norms. Thus, the Porter Hypothesis does not hold for the sample.

Methods Data of patients referred to School of Tropical Medicine, Kolkata for suspected treatment failure (TF) was analyzed for the period of December2008 to November2013 to ascertain the positive predictive value (PPV) of immunologic and/or clinical parameter for confirming virologic failure (VF) and to observe the short term outcome of secondline treatment. Paired ttest was used for data analysis.