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Despite advances in the management of type 2 diabetes mellitus (T2DM), one-third of patients with diabetes do not achieve the desired glycemic goal. Considering this inadequacy, many agents that activate glucokinase have been investigated over the last two decades but were withdrawn before submission for marketing permission. Dorzagliatin is the first glucokinase activator that has been granted approval for T2DM, only in China. As overstimulation of glucokinase is linked with pathophysiological disturbances such as fatty liver and cardiovascular issues and a loss of therapeutic efficacy with time. This review aims to highlight the benefits of glucokinase activators vis-à-vis the risks associated with chronic enzymatic activation. We discuss the multisystem disturbances expected with chronic activation of the enzyme, the lessons learned with glucokinase activators of the past, the major efficacy and safety findings with dorzagliatin and its pharmacological properties, and the status of other glucokinase activators in the pipeline. The approval of dorzagliatin in China was based on the SEED and the DAWN trials, the major pivotal phase III trials that enrolled patients with T2DM with a mean glycosylated hemoglobin of 8.3-8.4%, and a mean age of 53-54.5 years from multiple sites in China. Patients with uncontrolled diabetes, cardiac diseases, organ dysfunction, and a history of severe hypoglycemia were excluded. Both trials had a randomized double-blind placebo-controlled phase of 24 weeks followed by an open-label phase of 28 weeks with dorzagliatin. Drug-naïve patients with T2DM with a disease duration of 11.7 months were enrolled in the SEED trial while the DAWN trial involved patients with T2DM with a mean duration of 71.5 months and receiving background metformin therapy. Compared with placebo, the decline in glycosylated hemoglobin at 24 weeks was more with dorzagliatin with an estimated treatment difference of - 0.57% in the SEED trial and - 0.66% in the DAWN trial. The desired glycosylated hemoglobin (< 7%) was also attained at more than two times higher rates with dorzagliatin. The glycemic improvement was sustained in the SEED trial but decreased over 52 weeks in the DAWN trial. Hyperlipidemia was observed in 12-14% of patients taking dorzagliatin versus 9-11% of patients receiving a placebo. Additional adverse effects noticed over 52 weeks with dorzagliatin included an elevation in liver enzymes, hyperuricemia, hyperlacticacidemia, renal dysfunction, and cardiovascular disturbances. Considering the statistically significant improvement in glycosylated hemoglobin with dorzagliatin in patients with T2DM, the drug may be given a chance in treatment-naïve patients with a shorter disease history. However, with the waning therapeutic efficacy witnessed in patients with long-standing diabetes, which was also one of the potential concerns with previously tested molecules, extended studies involving patients with chronic and uncontrolled diabetes are needed to comment upon the long-term therapeutic performance of dorzagliatin. Likewise, evidence needs to be generated from other countries, patients with organ dysfunction, a history of severe hypoglycemia, cardiac diseases, and elderly patients before extending the use of dorzagliatin. Apart from monitoring lipid profiles, long-term safety studies of dorzagliatin should involve the assessment of serum uric acid, lactate, renal function, liver function, and cardiovascular parameters., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The current analysis is a part of an ongoing observational study that began in February 2021 in the Sir Sunder Lal Hospital (Varanasi, Uttar Pradesh) in northern India and is expected to continue until June 2022. This analysis aimed to delineate the clinical presentation and risk factors of occurrence and severity of COVID-19 in vaccinated individuals. The study enrolled health-care workers and the elderly receiving the COVID-19 vaccine at one of three centers linked to the study hospital. The participants received the ChAdOx1 nCoV-19 (Oxford-AstraZeneca) vaccine based on the chimpanzee adenovirus platform (manufactured in India by the Serum Institute of India). The adenovirus codes for the spike (S) protein of SARS-CoV-2. Participants were contacted by phone at pre-decided intervals and questioned about the occurrence of COVID-19, clinical presentation, severity, and persistence of symptoms. A logistic regression analysis was performed to predict the risk factors of occurrence and severity of COVID-19. Of the 1,500 participants included in the analysis, 418 developed COVID-19 (27.9%). Fever was the most common symptom (72%), followed by cough (34%) and rhinitis (26%). Cardiovascular involvement was seen in more than 2% of individuals, and 11% had post-COVID-19 complaints. Regression analysis showed 1.6 times greater odds of contracting the disease in females and in those younger than 40 years, 1.4 times greater odds in individuals who were overweight, and 2.9 times greater odds in those receiving only one dose, compared with respective comparators. Individuals receiving two doses at a gap of ≤ 30 days had 6.7 times greater odds of infection than those receiving at a > 60-day interval. There was no association between COVID-19 occurrence in the vaccinees and pre-vaccination history of SARS-CoV-2 infection. Males were at a 3.6 times greater risk, and persons with preexisting lung disease-mainly asthma-had a 5.9 times greater risk of experiencing moderate to severe COVID-19 than comparators. While an extended interval between the two vaccine doses seems to be a better strategy, gender differences and an association of asthma phenotypes with COVID-19 need to be explored.

Infection prevention and control (IPC) program is obligatory for delivering quality services in any healthcare setup. Lack of administrative support and resource-constraints (under-staffing, inadequate funds) were primary barriers to successful implementation of IPC practices in majority of the hospitals in the developing countries. The Coronavirus Disease 2019 (COVID-19) brought a unique opportunity to improve the IPC program in these hospitals. A PDSA (Plan-Do-Study- Act) model was adopted for this study in a tertiary care hospital which was converted into a dedicated COVID-19 treatment facility in Varanasi, India. The initial focus was to identify the deficiencies in existing IPC practices and perceive the opportunities for improvement. Repeated IPC training (induction and reinforce) was conducted for the healthcare personnel (HCP) and practices were monitored by direct observation and closed-circuit television. Cleaning audits were performed by visual inspection, review of the checklists and qualitative assessment of the viewpoints of the HCP was carried out by the feedbacks received at the end of the training sessions. A total of 2552 HCP and 548 medical students were trained in IPC through multiple offline/onsite sessions over a period of 15 months during the ongoing pandemic. Although the overall compliance to surface disinfection and cleaning increased from 50% to >80% with repeated training, compliance decreased whenever newly recruited HCP were posted. Fear psychosis in the pandemic was the greatest facilitator for adopting the IPC practices. Continuous wearing of personal protective equipment for long duration, dissatisfaction with the duty rosters as well as continuous posting in high-risk areas were the major obstacles to the implementation of IPC norms. Recognising the role of an infection control team, repeated training, monitoring and improvisation of the existing resources are keys for successful implementation of IPC practices in hospitals during the COVID-19 pandemic., Competing Interests: The authors have declared that no competing interests exist.

Membrane-protein interactions play a major role in human physiology as well as in diseases pathology. Interaction of a protein with the membrane was previously thought to be dependent on well-defined three-dimensional structure of the protein. In recent decades, however, it has become evident that a large fraction of the proteome, particularly in eukaryotes, stays disordered in solution and these proteins are termed as intrinsically disordered proteins (IDPs). Also, a vast majority of human proteomes have been reported to contain substantially long disordered regions, called intrinsically disordered regions (IDRs), in addition to the structurally ordered regions. IDPs exist in an ensemble of conformations and the conformational flexibility enables IDPs to achieve functional diversity. IDPs (and IDRs) are found to be important players in cell signaling, where biological membranes act as anchors for signaling cascades. Therefore, IDPs modulate the membrane architectures, at the same time membrane composition also affects the binding of IDPs. Because of intrinsic disorders, misfolding of IDPs often leads to formation of oligomers, protofibrils and mature fibrils through progressive self-association. Accumulation of amyloid-like aggregates of some of the IDPs is a known causative agent for numerous diseases. In this chapter we highlight recent advances in understanding membrane interactions of some of the intrinsically disordered proteins involved in the pathogenesis of human diseases., Competing Interests: Conflict of interest statement The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: We provide the first post-approval safety analysis of COVISHIELD in health care workers (HCWs) in northern India., Methods: This continuing prospective observational study (February 2021 to May 2022) enrolled participants ≥18 years receiving COVISHIELD vaccination. Primary outcome was safety and reactogenicity. Categories (FDA toxicity grading) and outcomes of adverse events following immunization (AEFIs) were recorded, causality assessment performed, and risk factors analysed., Findings: We present the results of an interim analysis of 804 participants. AEFIs following first dose were reported in 321 (40%; systemic involvement in 248). Among 730 participants who completed a 7-day follow-up post second dose, AEFIs occurred in 115 (15.7%; systemic in 99). Majority of AEFIs were mild-moderate and resolved spontaneously. Serious AEFIs, leading to hospitalization was noticed in 1 (0.1%) participant with suspicion of immunization stress related response (ISRR). AEFIs of grade 3 severity (FDA) were recorded in 4 participants (0.5%). No deaths were recorded. Regression analysis showed increased risk of AEFIs in younger individuals, a two times higher odds in females, those with hypertension or with history of allergy; and three times higher odds in individuals with hypothyroidism., Interpretation: COVISHIELD carries an overall favourable safety profile with AEFI rates much less than reported for other adenoviral vaccines. Females, those with hypertension, individuals with history of allergy and hypothyroidism may need watchful vaccine administration. This being an interim analysis and based on healthcare workers who may not reflect the general population demographics, larger inclusive studies are warranted for confirming the findings., Funding: No funding support., Competing Interests: The authors have nothing to declare., (© 2021 The Author(s).)

The motive of this article is to review the pharmacological and clinical aspects of esketamine (ESK), an NMDA-receptor antagonist approved recently by the FDA for treatment-resistant depression (TRD). PubMed/Medline database was searched using keywords 'esketamine' and 'depression', 'S-ketamine' and 'depression', and 'NMDA antagonist' and 'depression'. Individual trials were searched from ClinicalTrials.gov. We included English-language articles evaluating pharmacokinetics and pharmacodynamics of intranasal (IN) esketamine, along with clinical trial data related to its efficacy and safety in patients diagnosed with TRD. Compared to placebo, IN esketamine causes significant and rapid improvement in depression. Dizziness, vertigo, headache, increase in blood pressure are some of its common adverse effects. With the growing number of patients of TRD, additional effective and safe treatment is the need of the hour. Esketamine appears to be an effective therapy when combined with oral antidepressants in patients with TRD. It is of special value due to the rapid onset of its action. Long-term clinical studies are, however, needed to ascertain its safety profile.

Context  Due to the wide spectrum of clinical illness in coronavirus disease 2019 (COVID-19) patients, it is important to stratify patients into severe and nonsevere categories. Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been evaluated rapidly by a few studies worldwide for its association with severe disease, but practically none have been conducted in the Indian population. This study was undertaken to examine the role of NLR and PLR in predicting severe disease in Indian patients. Objectives  The objective was to study the association of NLR and PLR observed at the time of admission with maximum disease severity during hospitalization and to study their role in predicting disease severity. Material and Methods  A total of 229 COVID-19 patients were admitted at the center during the study period. After applying inclusion and exclusion criteria, 191 patients were included in the study. The demographic, clinical, and laboratory (complete blood count, NLR, and PLR) data of all patients were obtained at the time of admission. Maximum disease severity of all patients was assessed during hospitalization. Statistical Analysis  Chi-square and Mann-Whitney U tests were used to assess statistical significance. Receiver operating characteristic curve (ROC) was plotted for NLR and PLR to estimate the cutoff values and sensitivity and specificity using Youden's index for predicting severe disease. Logistic regression analysis was used to estimate the odds ratios (OR) and 95% confidence intervals. Results  Mean NLR and PLR were significantly higher in severe patients (NLR = 7.41; PLR = 204) compared with nonsevere patients (NLR = 3.30; PLR = 121). ROC analysis showed that NLR, in comparison to PLR, had a higher area under the curve (AUC) of 0.779, with a larger OR of 1.237 and cutoff of 4.1, and showed 69% sensitivity and 78% specificity in predicting severe disease. Cut off for PLR was 115.3, which showed 79% sensitivity and 62% specificity in predicting severe disease. Conclusion  NLR and PLR, both showing acceptable AUCs, can be used as screening tools to predict disease severity. However, NLR was a better predictor of disease severity., Competing Interests: Author ContributionsPermissionConflict of Interest R. J. and A. G. designed the study, collected the data, and prepared the manuscript. R. J., A. G., A. S., and B. P. contributed in data and statistical analyses. S. M. and T. T. were involved in manuscript editing and review. All authors had full access to the data and vouch for the accuracy of the analyses. All authors approved the final manuscript and supported the decision to submit for publication. Permission from the Institutional Ethical Committee was taken prior to the study. None., (The Indian Association of Laboratory Physicians. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Ribosome hibernation is a prominent cellular strategy to modulate protein synthesis during starvation and the stationary phase of bacterial cell growth. Translational suppression involves the formation of either factor-bound inactive 70S monomers or dimeric 100S hibernating ribosomal complexes, the biological significance of which is poorly understood. Here, we demonstrate that the Escherichia coli 70S ribosome associated with stationary phase factors hibernation promoting factor or protein Y or ribosome-associated inhibitor A and the 100S ribosome isolated from both Gram-negative and Gram-positive bacteria are resistant to unfolded protein-mediated subunit dissociation and subsequent degradation by cellular ribonucleases. Considering that the increase in cellular stress is accompanied by accumulation of unfolded proteins, such resistance of hibernating ribosomes towards dissociation might contribute to their maintenance during the stationary phase. Analysis of existing structures provided clues on the mechanism of inhibition of the unfolded protein-mediated disassembly in case of hibernating factor-bound ribosome. Further, the factor-bound 70S and 100S ribosomes can suppress protein aggregation and assist in protein folding. The chaperoning activity of these ribosomes is the first evidence of a potential biological activity of the hibernating ribosome that might be crucial for cell survival under stress conditions., (© 2020 Federation of European Biochemical Societies.)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has spread worldwide and caused widespread devastation. In the absence of definitive therapy, symptomatic management remains the standard of care. Repurposing of many existing drugs, including several anti-viral drugs, is being attempted to tackle the COVID-19 pandemic. However, most of them have failed to show significant benefit in clinical trials. An attractive approach may be to target host proteases involved in SARS-CoV-2 pathogenesis. The priming of the spike (S) protein of the virus by proteolytic cleavage by the transmembrane serine protease-2 (TMPRSS2) is necessary for the fusion of the virus to the host cell after it binds to its receptor angiotensin converting enzyme-2 (ACE2). There are other proteases with varying spatiotemporal locations that may be important for viral entry and subsequent replication inside the cells, and these include trypsin, furin and cathepsins. In this report, we have discussed the tentative therapeutic role of inhibitors of TMPRSS2, cathepsin, trypsin, furin, plasmin, factor X and elastase in infection caused by SARS-CoV-2. Both available evidence, as well as hypotheses, are discussed, with emphasis on drugs which are approved for other indications such as bromhexine, ammonium chloride, nafamostat, camostat, tranexamic acid, epsilon amino-caproic acid, chloroquine, ulinastatin, aprotinin and anticoagulant drugs. Simultaneously, novel compounds being tested and problems with using these agents are also discussed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

A 55 year old patient of COVID-19, with no known comorbidities presented with fever, myalgia and headache and at presentation had leukopenia and thrombocytopenia, however did not have any bleeding manifestations. The patient's inflammatory markers including ferritin and C-reactive protein were elevated at admission. Later in the course of illness went on to develop severe thrombocytosis and leukocytosis. We discuss the course and outcome of illness in an unusual case of COVID-19 with severe and diametrically opposite haematological abnormalities., Competing Interests: None to declare., (Copyright © 2014, Medical University Publishing House Craiova.)

Background: Polypharmacy is a significant problem in the elderly. The veteran population is unique in terms of better access to health-care services and higher educational qualifications. However, the studies to assess the burden and effects of polypharmacy in this cohort are rare. Hence, the research was done to find the prevalence of polypharmacy in the veteran population., Methods: In this cross-sectional observational study, we included veterans with more than 35 years visiting the medical outpatient department. All participants were interviewed about polypharmacy after taking informed consent., Results: Out of 394 patients included in the study, 110 were prescribed five or more medicines for their illnesses (27.91%: 95% confidence interval [CI] 21.1%-30%). More than 95% (377/394) of the study participants were unaware of the concept of polypharmacy. There was high compliance to treatment in veterans (97.46%, 95% CI 95.1%-98.6%)., Conclusion: This study shows that the prevalence of polypharmacy is significant in veterans, including patients in their fourth and fifth decades of life, despite fewer morbidities., Competing Interests: There are no conflicts of interest., (Copyright: © 2021 Indian Journal of Community Medicine.)

Background: Poor dissolution of Etodolac is one of the major challenges in achieving the desired therapeutic effect in oral therapy., Objective: This study aimed to assess the potential of the liquisolid compact technique in increasing the rate of dissolution of Etodolac and thus its bioavailability., Methods: Liquisolid compacts were prepared using PEG 400, Avicel PH-200 and Aerosil 200 as non-volatile liquid, carrier and coating material, respectively. The optimisation was carried out by applying a 32 full factorial design using Design expert software 11.0.3.0 to examine the effects of independent variables (load factor and carrier: coating ratio) on dependent variables (angle of repose and % cumulative drug release at 30 min [Q _{30 min]). Assessment of bioavailability was based on a pharmacokinetic study on rabbits and pharmacodynamics evaluation on rats, respectively., Results: The formulation M3 was identified as the optimised formulation based on the better flow (lower angle of repose) and a higher rate of dissolution (Q 30 min >95%). The higher dissolution rate could be due to conversion of Etodolac into an amorphous molecularly dispersed state, availability of larger surface area, enhancement of aqueous solubility and enhanced wetting of drug particles. Studies with DSC, XRD, and SEM verified the transformation of Etodolac from crystalline to amorphous state, a key factor responsible for improving the dissolution rate. The pharmacokinetic profile of M3 was prominent, demonstrating higher absorption of Etodolac in comparison to oral suspension and immediate-release conventional tablets in rabbits. Liquisolid formulation exhibited a 27% increment in paw thickness as compared to 57% and 46% increments for oral suspension and immediate-release conventional tablets, respectively, after 7 hrs in the carrageenan-induced paw model in rats., Conclusion: The results indicated the liquisolid compact technique to be a promising strategy to enhance the bioavailability of Etodolac., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)}

Background and Aims: Diabetes is a frequent comorbidity in patients with Severe COVID-19 infection associated with a worse prognosis. Hypercoagulability with elevation in D-dimer levels has been demonstrated in patients with COVID-19. This study aims to study D-dimer levels in people with diabetes compared to those without diabetes among patients with COVID-19 infection., Methods: In this observational study 98 moderate and severely ill patients with COVID-19 infection were included at a dedicated COVID hospital. The study group was divided into patients with diabetes and without diabetes. Peak D-dimer was measured in both the groups and compared using appropriate statistical tests., Results: In our study peak D-dimer levels were 1509 ± 2420 ng/mL (Mean ± SD) in people with diabetes and 515 ± 624 ng/mL (Mean ± SD) in patients without diabetes. Patients with diabetes had higher D-dimer levels which were statistically significant., Conclusions: This study shows COVID-19 patients with diabetes had significantly higher D-dimer levels. Therefore, it is possible that COVID-19 infection with diabetes is more likely to cause hypercoagulable state with a worse prognosis. However clinical implications of these findings will need to be seen in further studies., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2020 Diabetes India. Published by Elsevier Ltd. All rights reserved.)

Insulin aggregation is the leading cause of considerable reduction in the amount of active drug molecules in liquid formulations manufactured for diabetes management. Phenolic compounds, such as phenol and m-cresol, are routinely used to stabilize insulin in a hexameric form during its commercial preparation. However, long term usage of commercial insulin results in various adverse secondary responses, for which toxicity of the phenolic excipients is primarily responsible. In this study we aimed to find out a nontoxic insulin stabilizer. To that end, we have selected resveratrol, a natural polyphenol, as a prospective nontoxic insulin stabilizer because of its structural similarity with commercially used phenolic compounds. Atomic force microscopy visualization of resveratrol-treated human insulin revealed that resveratrol has a unique ability to arrest hINS in a soluble oligomeric form having discrete spherical morphology. Most importantly, resveratrol-treated insulin is nontoxic for HepG2 cells and it effectively maintains low blood glucose in a mouse model. Cryo-electron microscopy revealed 3D morphology of resveratrol-stabilized insulin that strikingly resembles crystal structures of insulin hexamer formulated with m-cresol. Significantly, we found that, in a condition inductive to amyloid fibrillation at physiological pH, resveratrol is capable of stabilizing insulin more efficiently than m-cresol. Thus, this study describes resveratrol as an effective nontoxic natural molecule that can be used for stabilizing insulin in a bioactive oligomeric form during its commercial formulation.

Pathogenesis of Alzheimer's disease (AD), the most common type of dementia, involves misfolding and aggregation of the extracellular amyloid-β (Aβ) protein where the intermediate oligomers, formed during the aggregation progression cascade, are considered the prime toxic species. Here, we identify an active peptide fragment from a medicinal plant-derived ( Aristolochia indica ) fibrinolytic enzyme having anti-amyloidogenic effects against Aβ fibrillation and toxicity. Liquid chromatography with tandem mass spectrometry (LC-MS/MS), followed by computational analysis of the peptide pool generated by proteolytic digestion of the enzyme, identifies two peptide sequences with predictive high-propensity binding to Aβ 42 . Microscopic visualizations in conjunction with biochemical and biophysical assessments suggest that the synthetic version of one of the peptides (termed here P active , GFLLHQK) arrests Aβ molecules in off-pathway oligomers that can no longer participate in the cytotoxic fibrillation pathway. In contrast, the other peptide (termed P 1 ) aggravates the fibrillation process. Further investigations confirm the strong binding affinity of P active with both Aβ 42 monomers and toxic oligomers by biolayer interferometric assays. We have also shown that, mechanistically, P active binding induces conformational alterations in the Aβ molecule along with modification of Aβ hydrophobicity, one of the key players in aggregation. Importantly, the biostability of P active in human blood serum and its nontoxic nature make it a promising therapeutic candidate against Alzheimer's, for which no disease-modifying treatments are available to date., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

The ability of the ribosome to assist in the folding of proteins both in vitro and in vivo is well documented. The interaction of an unfolded protein with the peptidyltransferase center of the bacterial large ribosomal subunit is followed by release of the protein in a folding-competent state and rapid dissociation of ribosome into its subunits. Our studies demonstrate that the 50S subunit-associated antiassociation ability of an unfolded protein might contribute significantly to its ability to mediate energy-independent and stable dissociation of the ribosome into its subunits. The stoichiometry of the protein present with respect to the ribosome is an important factor in determining whether the ribosome has a chaperoning effect on protein folding or if the protein acts as a 50S subunit antiassociation factor. Sustained interaction of the protein with the ribosome at higher protein concentrations and the hindrance in the formation of the central intersubunit bridge B2a could underlie the antiassociation activity of unfolded proteins. The ribosome dissociation and antiassociation activity of unfolded proteins could make the ribosome susceptible to cellular ribonucleases, thereby initiating ribosome degradation, which is a well-documented phenomenon under nutrient deprivation conditions., (© 2017 Federation of European Biochemical Societies.)

An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our in vitro studies unexpectedly reveal that the partial unfolding or aggregation of lysozyme under reducing conditions in presence of the ribosome can induce aggregation of ribosomal components. Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading to RNA-protein aggregate formation. Protein aggregation during the refolding of non-disulfide containing protein BCAII at high concentrations also induces ribosome aggregation. BCAII aggregation was also stimulated in presence of the large subunit rRNA. Our observations imply that the specific sequestration of the translation machine by aggregating proteins might contribute to their cytotoxicity., Competing Interests: The authors declare no competing financial interests.

The ability of the ribosome to assist in folding of proteins both in vitro and in vivo is well documented and is a nontranslational function of the ribosome. The interaction of the unfolded protein with the peptidyl transferase centre (PTC) of the bacterial large ribosomal subunit is followed by release of the protein in the folding competent state and rapid dissociation of ribosomal subunits. Our study demonstrates that the PTC-specific antibiotics, chloramphenicol and blasticidin S inhibit unfolded protein-mediated subunit dissociation. During post-termination stage of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. Ribosome dissociation mediated by RRF and induced at low magnesium concentration was also inhibited by the antibiotics indicating that the PTC antibiotics exert an associative effect on ribosomal subunits. In vivo, the antibiotics can also reduce the ribosomal degradation during carbon starvation, a process requiring ribosome subunit dissociation. This study reveals a new mode of action of the broad-spectrum antibiotics chloramphenicol and blasticidin., Significance and Impact of the Study: Ribosome synthesizes protein in all organisms and is the target for multiple antimicrobial agents. Our study demonstrates that chloramphenicol and blasticidin S that target the peptidyl transferase centre of the bacterial ribosome can then inhibit dissociation of 70S ribosome mediated by (i) unfolded protein, (ii) translation factors or (iii) low Mg +2 concentrations in vitro and thereby suppresses ribosomal degradation during carbon starvation in vivo. The demonstration of this new mode of action furthers the understanding of these broad-spectrum antibiotics that differentially inhibit protein synthesis in prokaryotic and eukaryotic cells., (© 2016 The Society for Applied Microbiology.)

Japanese encephalitis (JE) is a major public health problem in Asia and worldwide and it is responsible mainly for viral acute encephalitis syndrome (AES). The sole etiologic agent of JE is Japanese encephalitis virus (JEV). Although JE/AES cases have been regarded traditionally as a disease of children, a growing number of patients with JE/AES cases are also seen in the adult age group every year in the state of West Bengal, India in spite of vaccination. Therefore, a systematic study was performed to differentiate and characterize the clinico-pathological parameters and viral diversity among the patients of different age groups. Viral diversity was also evaluated from the JE/AES cases, depending on their disease severity. A total of 441 JE/AES cases were included in this study. By MAC-ELISA, 111 samples were found JEV IgM positive and among the IgM negative cases, 26 samples were found RT-PCR positive against JEV infection. Neck rigidity, abnormal behavior, convulsion, protein in CSF, WBC in CSF, and aspartate transaminase in blood differed significantly among the patients of pediatric-adolescent and adult group in both IgM positive and RT-PCR positive cases. Viral diversity was increased significantly in the pediatric-adolescent group compared to adult patients. Interestingly, with the rise in disease severity the viral diversity was found to be increased among the patients, irrespective of their age distribution. Based on clinico-pathological parameters and analysis of viral diversity, it can be concluded that viral diversity which occurs naturally is likely to affect disease severity, especially in the patients of pediatric-adolescent group., (© 2015 Wiley Periodicals, Inc.)

Background: The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3' -CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity., Results: The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3'-CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability., Conclusion: Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.

Background: Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone 'foldases' that are distinct from chaperone' holdases' that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC) located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA) is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains., Results: We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII) and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein., Conclusion: The ribosome can behave like a 'holdase' chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.

Background: Increasing virulence of Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen is of grave concern because it causes a neurotrophic killer disease Japanese Encephalitis (JE) which, in turn, is responsible globally for viral acute encephalitis syndrome (AES). Despite the availability of vaccine, JE/AES cases and deaths have become regular features in the different rural districts of West Bengal (WB) state, India, indicating either the partial coverage of vaccine or the emergence of new strain of JEV. Therefore, a study was undertaken to characterize and compare the complete envelope (E) protein gene based molecular changes/patterns of JEVs circulating in WB., Methods: Total of 98 AES case-patients' samples were tested to detect the presence of JEV specific immunoglobulin M (IgM) antibody by Mac-ELISA method. Only JEV IgM negative samples with a history of ≤3 days' illness were screened for virus isolation and RT-PCR. E gene sequences of JEV isolates were subjected to molecular phylogeny and immunoinformatics analysis., Results: Present study confirmed JEV etiology in 39.7% and 29.1% of patients presenting ≤15 days' febrile illness, as determined by Mac-ELISA and RT-PCR respectively. Phylogenetic analysis based on complete E gene sequences of JEV isolates showed the co-circulation of JEV genotype I (GI) with genotype III (GIII). This study also demonstrated that isolate-specific crucial amino acid substitutions were closely related to neurovirulence/neuroinvasiveness of JE. On the basis of immunoinformatics analysis, some substitutions were predicted to disrupt T-cell epitope immunogenicity/antigenicity that might largely influence the outcome of vaccine derived from JEV GIII SA14-14-2 strain and this has been observed in a previously vaccinated boy with mild JE/AES due to JEV GI infection., Conclusions: Based on molecular evolutionary and bioinformatic approaches, we report evolution of JEV at a local level. Such naturally occurring evolution is likely to affect the disease profile and the vaccine efficacy to protect against JEV GI may demand careful evaluation.

Biological N-removal treatment of piggery wastewater in the upflow anaerobic-anoxic-aerobic floating filter (UA(3)FF) bioreactor based on the concept of nitritation-denitritation was studied along with the changes in internal recycle ratio and dissolved oxygen concentration (DO). Consecutive changes in the recirculation ratio between the anoxic and aerobic reactors has resulted in abundance and composition shifts of N-cycling bacteria as well as other bacterial groups, reflecting different survival strategies across (bio/physico)chemical milieu. The DO concentration was optimized to achieve nitritation in the aerobic reactor and denitritation in the anoxic reactor. Optimal nitritation-denitritation (270 and 130 g NO(2) (-)-N produced or reduced/m(3) filter media/day) was obtained at DO of 1.0-1.5 mg/l, inter-reactor recirculation ratio of 1:1-2:1, HRT of 24 h, pH of 7.6 +/- 0.3, and temperature of 28 +/- 4 degrees C. Since only well known nitrifying and denitrifying taxa were found, nitritation-denitritation was likely carried out by these bacteria rather than the yet unidentified novel taxa. Archaeal nitrifiers recently discovered to be important in the global N-cycle were not detected.

Anaerobic ammonium oxidizing (anammox) bacteria present in microbial communities in two laboratory-scale upflow anoxic reactors supplied with small amounts of ammonium (<3 mg/l) at low temperature were detected and quantified. The reactors, operated at 20 degrees C, were seeded with an immobilized microbial consortium (IMC) and anaerobic granules (AG) from an upflow anaerobic sludge blanket (UASB) treating brewery wastewater. Our results showed that complete ammonium and nitrite removal with greater than 92% total nitrogen removal efficiency was achieved in the reactor inoculated with both the IMC and AG, while that of the reactor inoculated with only the IMC was lower than 40%; enrichment was successful after the addition of AG. Quantitative fluorescence in situ hybridization (FISH) analysis confirmed that anammox bacteria were present only in the reactor inoculated with an IMC and AG. The copy number of the 16S-rRNA gene of the anammox bacteria calculated by most probable number-polymerase chain reaction (MPN-PCR) from the total DNA extracted from both reactors (2.5 x 10(4) copies/mug of DNA) was two orders lower than that of the domain bacteria (2.5 x 10(6) copies/mug of DNA). The results revealed that immobilized multiple seed sludges were optimal for anammox enrichment at low temperature and ammonium concentrations.

A combination of anammox and denitrification process was studied for 300 days in low ammonium-fed bioreactors under the support of organic carbon. Nutrient profiles, (15)N-labelling techniques and qualitative fluorescence in situ hybridization (FISH) probes were used to confirm the nitrogen removal pathways and intercompetition among different bacteria populations. About 80% of nitrogen removal was achieved throughout the study period. The results confirmed that anammox bacteria were absent in the bioreactor inoculated with anaerobic granules only but they were present and active in the central anoxic parts of biopellets in the bioreactor inoculated with mixed microbial consortium from activated sludge and anaerobic granules. It also showed that the anammox bacteria were successfully enriched in the low ammonium-fed bioreactors. Results of this study clearly demonstrated that anammox and denitrification processes could coexist in same environment and anammox bacteria were less competitive than denitrifying bacteria.