Role of preoperative zinc, magnesium and budesonide gargles in Postoperative Sore Throat (POST) - a randomised control trial.

BACKGROUND: Post-operative sore throat (POST) has an incidence ranging from 21 to 80%. To prevent the development of POST, several pharmacological measures have been tried. Aim of this study was to compare the efficacy of preoperative zinc, magnesium and budesonide gargles in reducing the incidence and severity of POST in patients who underwent endotracheal intubation for elective surgeries. METHODS: We conducted a prospective, randomized, double-blind, controlled equivalence trial in 180 patients admitted for elective surgical procedures under general anaesthesia. Patients were randomised into three groups; group Z received 40 mg Zinc, group M received 250 mg Magnesium Sulphate and group B received 200 µg Budesonide in the form of 30 ml tasteless and colourless gargle solutions. Sore throat assessment and haemodynamic recording was done postoperatively at immediate recovery (0 h) and 2, 4, 6, 8, 12 and 24 h post-operatively. POST was graded on a four-point scale (0-3). RESULTS: POST score was comparable at all recorded time points i.e. 0,2,4,6,8,12 and 24 h. Maximum incidence was seen at 8 h in group B (33.3%) and the minimum incidence was at 24 h in group Z (10%) (p > 0.05). It was found that the incidence of POST was more in the surgeries lasting longer than 2 h in all groups. This difference was found to be statistically significant in Groups M and B. The incidence of POST was found to be comparable between laparoscopic and open procedures. CONCLUSION: Magnesium, zinc and budesonide have an equivocal effect in the prevention of POST at different time points. The incidence of sore throat increases significantly in surgeries lasting more than two hours if magnesium or budesonide have been used as premedicant. Duration of surgery is an independent predictor for POST. TRIAL REGISTRATION: CTRI/2021/05/033741 Date-24/05/2021(Clinical Trial Registry of India).

Comparison of the effect of two recruitment manoeuvres to conventional ventilation on lung atelectasis in paediatric laparoscopic surgery- a prospective randomised controlled trial.

BACKGROUND: There is a high incidence of pulmonary atelectasis during paediatric laparoscopic surgeries. The authors hypothesised that utilising a recruitment manoeuvre or using continuous positive airway pressure may prevent atelectasis compared to conventional ventilation. OBJECTIVE: The primary objective was to compare the degree of lung atelectasis diagnosed by lung ultrasound (LUS) using three different ventilation techniques in children undergoing laparoscopic surgeries. DESIGN: Randomised, prospective three-arm trial. SETTING: Single institute, tertiary care, teaching hospital. PATIENTS: Children of ASA PS 1 and 2 up to the age of 10 years undergoing laparoscopic surgery with pneumoperitoneum lasting for more than 30 min. INTERVENTION: Random allocation to one of the three study groups: CG group: Inspiratory pressure adjusted to achieve a TV of 5-8 ml/kg, PEEP of 5 cm H2O, respiratory rate adjusted to maintain end-tidal carbon dioxide (ETCO2) between 30-40 mm Hg with manual ventilation and no PEEP at induction. RM group: A recruitment manoeuvre of providing a constant pressure of 30 cm H2O for ten seconds following intubation was applied. A PEEP of 10 cm H2O was maintained intraoperatively. CPAP group: Intraoperative maintenance with PEEP 10 cm H2O with CPAP of 10 cm H2O at induction using mechanical ventilation was done. OUTCOME MEASURES: Lung atelectasis score at closure assessed by LUS. RESULTS: Post induction, LUS was comparable in all three groups. At the time of closure, the LUS for the RM group (8.6 ± 4.9) and the CPAP group (8.8 ± 6.8) were significantly lower (p < 0.05) than the CG group (13.3 ± 3.8). In CG and CPAP groups, the score at closure was significantly higher than post-induction. The PaO2/FiO2 ratio was significantly higher (p < 0.05) for the RM group (437.1 ± 44.9) and CPAP group (421.6 ± 57.5) than the CG group (361.3 ± 59.4) at the time of pneumoperitoneum. CONCLUSION: Application of a recruitment manoeuvre post-intubation or CPAP during induction and maintenance with a high PEEP leads to less atelectasis than conventional ventilation during laparoscopic surgery in paediatric patients. TRIAL REGISTRY: CTRI/2019/08/02058.

MIR159 regulates multiple aspects of stamen and carpel development and requires dissection and delimitation of differential downstream regulatory network for manipulating fertility traits.

Unravelling genetic networks regulating developmental programs are key to devising and implementing genomics assisted trait modification strategies. It is crucial to understand the role of small RNAs, and the basis of their ability to modify traits. MIR159 has been previously reported to cause defects in anther development in Arabidopsis; however, the complete spectrum and basis of the defects remained unclear. The present study was therefore undertaken to comprehensively investigate the role of miR159 from Brassica juncea in modulating vegetative and reproductive traits. Owing to the polyploid nature of Brassica, paralogous and homeologous copies of MIR159A, MIR159B, and, MIR159C were identified and analysis of the precursor uncovered extensive structural and sequence variation. The MIR159 locus with mature miR159 with perfect target complimentarily with MYB65, was cloned from Brassica juncea var. Varuna for functional characterization by generating constitutively over-expressing lines in Arabidopsis thaliana Col-0. Apart from statistically significant difference in multiple vegetative traits, drastic differences were observed in stamen and pistil. Over-expression of miR159a led to shortening of filament length and loss of tetradynamous condition. Anthers were apiculate, with improper lobe formation, and unsynchronized cellular growth between connective tissue and another lobe development. Analysis revealed arrested meiosis/cytokinesis in microspores, and altered lignin deposition pattern in endothecial walls thus affecting anther dehiscence. In the gynoecium, flaccid, dry stigmatic papillae, and large embryo sac in the female gametophyte was observed. Over-expression of miR159a thus severely affected pollination and seed-set. Analysis of the transcriptome data revealed components of regulatory networks of anther and carpel developmental pathway, and lignin metabolism that are affected. Expression analysis allowed us to position the miR159a-MYB65 module in the genetic network of stamen development, involved in pollen-grain maturation; in GA-mediated regulation of stamen development, and in lignin metabolism. The study, on one hand indicates role of miR159a-MYB65 in regulating multiple aspects of reproductive organ development that can be manipulated for trait modification, but also raises several unaddressed questions such as relationship between miR159a and male-meiosis, miR159a and filament elongation for future investigations. Accession numbers: KC204951-KC204960. Project number PRJNA1035268. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01377-7.

Evaluation of an advanced critical care echocardiography program: a mixed methods study.

PURPOSE: Limited data exist on advanced critical care echocardiography (CCE) training programs for intensivists. We sought to describe a longitudinal echocardiography program and investigate the effect of distributed conditional supervision vs predefined en-bloc supervision, as well as the effect of an optional echocardiography laboratory rotation, on learners' engagement. METHODS: In this mixed methods study, we enrolled critical care fellows and faculty from five University of Toronto-affiliated intensive care units (ICU) between July 2015 and July 2018 in an advanced training program, comprising theoretical lectures and practical sessions. After the first year, the program was modified with changes to supervision model and inclusion of a rotation in the echo laboratory. We conducted semistructured interviews and investigated the effects of curricular changes on progress toward portfolio completion (150 transthoracic echocardiograms) using a Bayesian framework. RESULTS: Sixty-five learners were enrolled and 18 were interviewed. Four (9%) learners completed the portfolio. Learners reported lack of time and supervision, and skill complexity as the main barriers to practicing independently. Conditional supervision was associated with a higher rate of submitting unsupervised echocardiograms than unconditional supervision (rate ratio, 1.11, 95% credible interval, 1.08 to 1.14). After rotation in the echocardiography laboratory, submission of unsupervised echocardiograms decreased. CONCLUSION: Trainees perceived lack of time and limited access to supervision as major barriers to course completion. Nevertheless, successful portfolio completion was related to factors other than protected time in the echocardiography laboratory or unconditional direct supervision in ICU. Further research is needed to better understand the factors promoting success of CCE training programs.

RéSUMé: OBJECTIF: Il n'existe que peu de données sur les programmes de formation avancés en échocardiographie pour les soins intensifs (écho-USI) destinés aux intensivistes. Nous avons cherché à décrire un programme longitudinal d'échocardiographie et à étudier l'effet d'une supervision conditionnelle distribuée vs une supervision prédéfinie en bloc, ainsi que l'effet d'une rotation facultative en laboratoire d'échocardiographie, sur le niveau d'implication des apprenants. MéTHODE: Dans cette étude à méthodes mixtes, nous avons recruté des fellows en soins intensifs et des professeurs de cinq unités de soins intensifs (USI) affiliées à l'Université de Toronto entre juillet 2015 et juillet 2018 pour participer à un programme de formation avancée comprenant des conférences théoriques et des séances pratiques. Après la première année, le programme a été modifié en apportant des changements au modèle de supervision et en incluant une rotation dans le laboratoire d'écho. Nous avons mené des entretiens semi-structurés et étudié les effets des changements du programme d'études sur les progrès vers la réussite de la formation (150 échocardiogrammes transthoraciques) en utilisant un cadre bayésien. RéSULTATS: Soixante-cinq apprenants étaient inscrits et 18 ont été interviewés. Quatre (9 %) apprenants ont complété la formation. Les apprenants ont signalé que le manque de temps et de supervision ainsi que la complexité des compétences constituaient les principaux obstacles à une pratique autonome. La supervision conditionnelle était associée à un taux plus élevé de soumission d'échocardiogrammes non supervisés que la supervision inconditionnelle (ratio de taux, 1,11, intervalle crédible à 95 %, 1,08 à 1,14). Après la rotation dans le laboratoire d'échocardiographie, la soumission d'échocardiogrammes non supervisés a diminué. CONCLUSION: Les stagiaires ont perçu le manque de temps et l'accès limité à la supervision comme des obstacles majeurs à la réussite de la formation. Néanmoins, l'achèvement du cours était lié à des facteurs autres que le temps protégé au laboratoire d'échocardiographie ou la supervision directe inconditionnelle aux soins intensifs. D'autres recherches sont nécessaires pour mieux comprendre les facteurs favorisant le succès des programmes de formation en écho-USI.

Training in radiographic caries detection and staging using an interactive tool.

INTRODUCTION: The objective of the study was to compare a dental student's practical ability to detect and stage radiographic caries per International Caries Detection and Assessment System (ICDAS), following a traditional lecture and a lecture containing an interactive session using an audience response system (ARS). Associations between the order of instructions and student performance were also evaluated. MATERIALS AND METHODS: Eighty-three dental students were randomly assigned to groups A and B. On the first day, group A received a traditional lecture and group B received content using the ARS. All students then took an electronic quiz (T1) identifying and staging caries on radiographs per ICDAS. For the second day, group A received the content using the ARS system and group B received a traditional lecture. All students subsequently took a second electronic quiz (T2). Two survey questions about the learning experience were also included. RESULTS: Wilcoxon rank-sum analysis of scores from consenting students (81) showed no difference between the quiz 1 scores of two groups (p=.61). Whilst not statistically significant (p = .07), the group that had the ARS initially scored marginally higher on quiz 2. Survey results showed that most participants preferred either the ARS alone (49.38%) or a combination of the ARS and a traditional lecture (40.74%). A majority of them (80%) found the ARS helpful. CONCLUSION: When training students in practical skills of detection and staging radiographic presence of dental caries per ICDAS, hands-on learning tools, such as an ARS, complement traditional lectures.

Sirtuin 6 deficiency transcriptionally up-regulates TGF-ß signaling and induces fibrosis in mice.

Caloric restriction has been associated with increased life span and reduced aging-related disorders and reduces fibrosis in several diseases. Fibrosis is characterized by deposition of excess fibrous material in tissues and organs and is caused by aging, chronic stress, injury, or disease. Myofibroblasts are fibroblast-like cells that secrete high levels of extracellular matrix proteins, resulting in fibrosis. Histological studies have identified many-fold increases of myofibroblasts in aged organs where myofibroblasts are constantly generated from resident tissue fibroblasts and other cell types. However, it remains unclear how aging increases the generation of myofibroblasts. Here, using mouse models and biochemical assays, we show that sirtuin 6 (SIRT6) deficiency plays a major role in aging-associated transformation of fibroblasts to myofibroblasts, resulting in tissue fibrosis. Our findings suggest that SIRT6-deficient fibroblasts transform spontaneously to myofibroblasts through hyperactivation of transforming growth factor ß (TGF-ß) signaling in a cell-autonomous manner. Importantly, we noted that SIRT6 haploinsufficiency is sufficient for enhancing myofibroblast generation, leading to multiorgan fibrosis and cardiac dysfunction in mice during aging. Mechanistically, SIRT6 bound to and repressed the expression of key TGF-ß signaling genes by deacetylating SMAD family member 3 (SMAD3) and Lys-9 and Lys-56 in histone 3. SIRT6 binding to the promoters of genes in the TGF-ß signaling pathway decreased significantly with age and was accompanied by increased binding of SMAD3 to these promoters. Our findings reveal that SIRT6 may be a potential candidate for modulating TGF-ß signaling to reduce multiorgan fibrosis during aging and fibrosis-associated diseases.

Effect of age, comorbidity and remission status on outcome of COVID-19 in patients with hematological malignancies.

BACKGROUND: There is scarcity of data on outcome of COVID-19 in patients with hematological malignancies. Primary objective of study was to analyse the 14-day and 28-day mortality. Secondary objectives were to correlate age, comorbidities and remission status with outcome. METHODS: Retrospective multicentre observational study conducted in 11 centres across India. Total 130 patients with hematological malignancies and COVID-19 were enrolled. RESULTS: Fever and cough were commonest presentation. Eleven percent patients were incidentally detected. Median age of our cohort was 49.5 years. Most of our patients had a lymphoid malignancy (n = 91). One-half patients (52%) had mild infection, while moderate and severe infections contributed to one-fourth each. Sixty seven patients (52%) needed oxygen For treatment of COVID-19 infection, half(n = 66) received antivirals. Median time to RT-PCR COVID-19 negativity was 17 days (7-49 days). Nearly three-fourth (n = 95) of our patients were on anticancer treatment at time of infection, of which nearly two-third (n = 59;64%) had a delay in chemotherapy. Overall, 20% (n = 26) patients succumbed. 14-day survival and 28-day survival for whole cohort was 85.4% and 80%, respectively. One patient succumbed outside the study period on day 39. Importantly, death rate at 1 month was 50% and 60% in relapse/refractory and severe disease cohorts, respectively. Elderly patients(age ≥ 60) (p = 0.009), and severe COVID-19 infection (p = 0.000) had a poor 14-day survival. The 28-day survival was significantly better for patients in remission (p = 0.04), non-severe infection (p = 0.00), and age < 60 years (p = 0.05). CONCLUSIONS: Elderly patients with hematological malignancy and severe covid-19 have worst outcomes specially when disease is not in remission.

SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity.

Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.

Recapitulating pathophysiology of skeletal muscle diseases in vitro using primary mouse myoblasts on a nanofibrous platform.

Tissue engineering approaches are used to mimic the microenvironment of the skeletal muscle in vitro. However, the validation of a bioengineered muscle as a model to study diseases is inadequate. Here, we present polycaprolactone nanofibers as a robust platform that mimics cellular organization and recapitulates critical functions of the myotubes observed in vivo. We isolated myoblasts from mice following a simplified protocol and cultured them on aligned nanofibers. Myotubes grown on aligned nanofibers maintained alignment for 14 days and exhibited a time-dependent increase in levels of p-AKT upon insulin stimulation. Treatment with matrix-assisted integrin inhibitor led to reduction in p-AKT levels, underscoring the critical role of environment on the biological processes. We demonstrate the suitability of myotubes grown on nanofibrous platform to study corticosteroid-induced muscle degeneration. This study, thus, demonstrates that aligned nanofibers retain myotubes in culture for longer duration and recapitulate the functions of skeletal muscle under pathophysiological conditions.

Oral melatonin as part of multimodal anxiolysis decreases emergence delirium in children whereas midazolam does not: A randomised, double-blind, placebo-controlled study.

BACKGROUND: Pre-operative anxiety is a risk factor for emergence delirium in children and a multimodal approach including sedatives and nonpharmacological measures is the current strategy to tackle this anxiety. The efficacy of oral melatonin as a component of multimodal anxiolytic strategy to decrease emergence delirium is not well studied. OBJECTIVE: The aim of this study was to evaluate the efficacy of a multimodal anxiolytic strategy including oral melatonin or midazolam to decrease emergence delirium after sevoflurane anaesthesia. DESIGN: A randomised, double-blind, parallel arm, placebo-controlled trial. SETTING: Tertiary care teaching hospital from July 2019 till January 2020. PARTICIPANTS: Children in the age group of 3 to 8 years who received sevoflurane anaesthesia for elective ambulatory procedures. INTERVENTIONS: Children were randomised to receive oral premedication with either melatonin 0.3 mg kg-1, midazolam 0.3 mg kg-1 or honey as placebo. All the children received standardised nonpharmacological measures involving multiple techniques to allay anxiety. The anaesthetic plan was also standardised. MAIN OUTCOME MEASURES: The primary outcome was the incidence of emergence delirium as assessed by the Watcha scale in the postanaesthesia care unit. The secondary outcomes were pre-operative anxiety assessed using a modified Yale Preoperative Anxiety scale, patient compliance with mask induction using the Induction Compliance Checklist and postoperative sedation. RESULTS: Data from 132 children were analysed. Melatonin significantly reduced the incidence of emergence delirium compared to placebo: 27 vs. 50%, respectively, an absolute risk reduction of 23.3 [95% confidence interval 3.7 to 42.9), P = 0.03]. Melatonin also significantly reduced the risk of emergence delirium compared with midazolam: 27 vs. 56%, respectively, an absolute risk reduction of 29.2 (95% CI 9.5 to 48.8). The midazolam group had a similar incidence of emergence delirium as placebo. Sedation scores were similar in the three groups postoperatively. The incidence and score of pre-operative anxiety as well as the compliance with mask induction were similar in the three groups. CONCLUSIONS: A multimodal anxiolytic approach including oral melatonin, as opposed to oral midazolam, significantly reduced emergence delirium after sevoflurane anaesthesia. TRIAL REGISTRATION: CTRI/2019/06/019850 in Clinical Trial Registry of India (www.ctri.nic.in).

Self assembly and hydrogelation of N-terminal modified tetrapeptide for sustained release and synergistic action of antibacterial drugs against methicillin resistant S. aureus.

Self assembly is a ubiquitous process of complex bio-molecules to perform various biological functions. This bottom-up approach applies in engineering of various nanostructures in different technological and biomedical applications. Here we report design and synthesis of phenolic acid conjugated tetra peptides which self assembled in uniform nanofibrils upon dissolution in aqueous solutions at physiological pH and formed stiff and transparent hydrogel. Gel inversion assay, HR-TEM, FT-IR, CD spectroscopy and rheometric analysis characterized the developed hydrogel (HG-2). This gel exhibits characteristics of thixotropy and injectability. Structure-gelation relationship studies of peptide revealed the importance of π-π interactions in self assembly and hydrogelation. Further, this hydrogel used for entrapment and sustained release of antibiotics, rifampicin and ciprofloxacin at physiological pH and temperature for 5 days. The hydrogelator peptide has shown moderate antibacterial activity alone, whereas in combination with rifampicin and ciprofloxacin showed a remarkable synergistic antibacterial activity against clinically relevant multidrug resistant methicillin resistant S. aureus (MRSA). Interestingly, this hydrogel neither cause significant damage to hRBCsnor to human keratinocyte up to hydrogelation concentrations tested by haemolytic and MTT assay. These characteristics of present peptide hold future promising soft materials for treatment of infections and drug delivery applications.

Evidence Supporting Clinical Use of Proportional Assist Ventilation: A Systematic Review and Meta-Analysis of Clinical Trials.

BACKGROUND: While proportional assist ventilation (PAV), generates pressure in proportion to effort without a preselected target, proportional assist ventilation plus (PAV+) measures compliance and resistance, calculates work of breathing, and adjusts support to a preset assistance level. OBJECTIVE: To summarize randomized controlled trials (RCTs) comparing invasive or noninvasive PAV or PAV+ in critically ill patients. Data Sources: We searched multiple databases to April 2017 without language restrictions and conference proceedings from 5 meetings to identify randomized parallel-group and crossover RCTs that compared invasive or noninvasive PAV or PAV+ to another mode in critically ill adults or children and reported at least 1 clinically important outcome. RESULTS: We identified 14 RCTs (11 parallel group and 3 crossover) assessing PAV (n = 7) and PAV+ (n = 7) involving 931 adult patients. We found no effect of noninvasive PAV (vs noninvasive pressure support [PS]) on intubation (risk ratio 0.92 [0.59 to 1.43], I2 = 0%) or invasive PAV (vs invasive PS) on percentage rapid eye movement sleep (mean difference [MD] -2.93% [-14.20 to ±8.34], I2 = 43%). Compared to invasive PS, invasive PAV+ showed a nonsignificant increase in weaning time (MD +0.54 [-0.67 to +1.75] hours, I2 = 0%), but no effect on hospital mortality, reintubation, or tracheostomy. CONCLUSIONS: Current evidence does not support the use of invasive or noninvasive PAV or invasive PAV+ in critically ill adults. Amid low to moderate heterogeneity, we identified 3 promising areas for future research including assessing the role of noninvasive PAV as an initial support strategy in patients with acute respiratory failure, invasive PAV on sleep quality during invasive ventilation, and possibly invasive PAV+ for weaning.

What a pediatric anesthesiologist should know about COVID-19.

The COVID-19 pandemic has posed unprecedented challenges and has unique implications for pediatric anesthesiologists. While children have a less severe clinical course compared to adults, they might be an important component in the transmission link by being asymptomatic carriers. Thus, it is essential to have practice guidelines for pediatric health care providers to limit transmission while providing safe and optimum care to our patients. Here we provide a brief review of the unique epidemiology and clinical characteristics of COVID-19 inflicted children. We have also reviewed various pediatric anesthesia guidelines and summarized the same to provide insight into the goals of management. We share the protocols that have been formulated and adopted in the pediatric anesthesia wing of our tertiary care hospital. This article lays special emphasis on the preparation of specialized protocols, designated areas, and training of personnel expected to be involved in patient care. The operating room should be well equipped with weight and age-appropriate equipment and drugs. Special attention should be paid to minimize aerosol generation via premedication and physical barriers. Induction and airway handling should be performed rapidly and securely with minimum personnel present. Disconnections should be avoided during maintenance. Extubation and transfer of children should be smooth. These protocols and guidelines are being constantly reviewed and updated as new evidence emerges. Our goal as pediatric anesthesiologists is to provide anesthesia that is safe for the child while preventing and minimizing the risk of infection to health care workers.

SIRT2 deacetylase represses NFAT transcription factor to maintain cardiac homeostasis.

Heart failure is an aging-associated disease that is the leading cause of death worldwide. Sirtuin family members have been largely studied in the context of aging and aging-associated diseases. Sirtuin 2 (SIRT2) is a cytoplasmic protein in the family of sirtuins that are NAD+-dependent class III histone deacetylases. In this work, we studied the role of SIRT2 in regulating nuclear factor of activated T-cells (NFAT) transcription factor and the development of cardiac hypertrophy. Confocal microscopy analysis indicated that SIRT2 is localized in the cytoplasm of cardiomyocytes and SIRT2 levels are reduced during pathological hypertrophy of the heart. SIRT2-deficient mice develop spontaneous pathological cardiac hypertrophy, remodeling, fibrosis, and dysfunction in an age-dependent manner. Moreover, young SIRT2-deficient mice develop exacerbated agonist-induced hypertrophy. In contrast, SIRT2 overexpression attenuated agonist-induced cardiac hypertrophy in cardiomyocytes in a cell-autonomous manner. Mechanistically, SIRT2 binds to and deacetylates NFATc2 transcription factor. SIRT2 deficiency stabilizes NFATc2 and enhances nuclear localization of NFATc2, resulting in increased transcription activity. Our results suggest that inhibition of NFAT rescues the cardiac dysfunction in SIRT2-deficient mice. Thus, our study establishes SIRT2 as a novel endogenous negative regulator of NFAT transcription factor.

Toll-like receptor 2 deficiency hyperactivates the FoxO1 transcription factor and induces aging-associated cardiac dysfunction in mice.

Toll-like receptors (TLRs) are a family of pattern-recognition receptors involved in innate immunity. Previous studies have shown that TLR2 inhibition protects the heart from acute stress, including myocardial infarction and doxorubicin-induced cardiotoxicity in animal models. However, the role of TLR2 in the development of aging-associated heart failure is not known. In this work, we studied aging-associated changes in structure and function of TLR2-deficient mice hearts. Whereas young TLR2-KO mice did not develop marked cardiac dysfunction, 8- and 12-month-old TLR2-KO mice exhibited spontaneous adverse cardiac remodeling and cardiac dysfunction in an age-dependent manner. The hearts of the 8-month-old TLR2-KO mice had increased fibrosis, cell death, and reactivation of fetal genes. Moreover, TLR2-KO hearts displayed reduced infiltration by macrophages, increased numbers of myofibroblasts and atrophic cardiomyocytes, and higher levels of the atrophy-related ubiquitin ligases MuRF-1 and atrogin-1. Mechanistically, TLR2 deficiency impaired the PI3K/Akt signaling pathway, leading to hyperactivation of the transcription factor Forkhead box protein O1 (FoxO1) and, in turn, to elevated expression of FoxO target genes involved in the regulation of muscle wasting and cell death. AS1842856-mediated chemical inhibition of FoxO1 reduced the expression of the atrophy-related ubiquitin ligases and significantly reversed the adverse cardiac remodeling while improving the contractile functions in the TLR2-KO mice. Interestingly, TLR2 levels decreased in hearts of older mice, and the activation of TLR1/2 signaling improved cardiac functions in these mice. These findings suggest that TLR2 signaling is essential for protecting the heart against aging-associated adverse remodeling and contractile dysfunction in mice.

Sirtuin 6 mediated stem cell cardiomyogenesis on protein coated nanofibrous scaffolds.

The cellular niche provides combination of biomolecular and biophysical cues to control stem cell fate. Three-dimensional (3D) aligned nanofibrous scaffolds can effectively augment stem cell cardiomyogenesis. This work aims to understand the role of biomolecular signals from extracellular matrix (ECM) proteins and leverage them to further promote cardiomyogenesis on nanofibrous scaffolds. Human mesenchymal stem cells (hMSCs) were cultured on 3D aligned polycaprolactone scaffolds coated with different ECM proteins. Among multiple coatings tested, collagen coated fibers were most effective in promoting cardiomyogenesis as determined from increased expression of cardiac biomarkers and intracellular calcium flux. At molecular level, enhanced differentiation on collagen coated fibers was associated with an increased level of sirtuin 6 (SIRT6). Depletion of SIRT6 using siRNA attenuated the differentiation process through activation of Wnt signaling pathway. This study, thus, demonstrates that protein coated scaffolds can augment cardiomyogenic differentiation of stem cells through a combination of topographical and biomolecular signals.

Sequence and functional characterization of MIRNA164 promoters from Brassica shows copy number dependent regulatory diversification among homeologs.

The impact of polyploidy on functional diversification of cis-regulatory elements is poorly understood. This is primarily on account of lack of well-defined structure of cis-elements and a universal regulatory code. To the best of our knowledge, this is the first report on characterization of sequence and functional diversification of paralogous and homeologous promoter elements associated with MIR164 from Brassica. The availability of whole genome sequence allowed us to identify and isolate a total of 42 homologous copies of MIR164 from diploid species-Brassica rapa (A-genome), Brassica nigra (B-genome), Brassica oleracea (C-genome), and allopolyploids-Brassica juncea (AB-genome), Brassica carinata (BC-genome) and Brassica napus (AC-genome). Additionally, we retrieved homologous sequences based on comparative genomics from Arabidopsis lyrata, Capsella rubella, and Thellungiella halophila, spanning ca. 45 million years of evolutionary history of Brassicaceae. Sequence comparison across Brassicaceae revealed lineage-, karyotype, species-, and sub-genome specific changes providing a snapshot of evolutionary dynamics of miRNA promoters in polyploids. Tree topology of cis-elements associated with MIR164 was found to re-capitulate the species and family evolutionary history. Phylogenetic shadowing identified transcription factor binding sites (TFBS) conserved across Brassicaceae, of which, some are already known as regulators of MIR164 expression. Some of the TFBS were found to be distributed in a sub-genome specific (e.g., SOX specific to promoter of MIR164c from MF2 sub-genome), lineage-specific (YABBY binding motif, specific to C. rubella in MIR164b), or species-specific (e.g., VOZ in A. thaliana MIR164a) manner which might contribute towards genetic and adaptive variation. Reporter activity driven by promoters associated with MIR164 paralogs and homeologs was majorly in agreement with known role of miR164 in leaf shaping, regulation of lateral root development and senescence, and one previously un-described novel role in trichome. The impact of polyploidy was most profound when reporter activity across three MIR164c homeologs were compared that revealed negligible overlap, whereas reporter activity among two homeologs of MIR164a displays significant overlap. A copy number dependent cis-regulatory divergence thus exists in MIR164 genes in Brassica juncea. The full extent of regulatory diversification towards adaptive strategies will only be known when future endeavors analyze the promoter function under duress of stress and hormonal regimes.

Highly preserved roles of Brassica MIR172 in polyploid Brassicas: ectopic expression of variants of Brassica MIR172 accelerates floral transition.

Functional characterization of regulatory genes governing flowering time is a research priority for breeding earliness in crop Brassicas. Highly polyploid genomes of Brassicas pose challenges in unraveling homeolog gene function. In Arabidopsis, five MIR172 paralogs control flowering time and floral organ identity by down-regulating AP2 and AP2-like genes. The impact of homeolog diversification on MIR172 loci, however, needs to be examined in morphologically diverse Brassicas. Herein, we analyze fractionation status and phylogeny of MIR172 and target AP2 from Brassicas and compare functionality of MIR172 variants representing distinct sub-genomes and progenitor genomes. Copy number analysis revealed higher retention of MIR172 loci relative to AP2 in diploid and amphi-diploid Brassica species. Dendrogram of 87 MIR172 sequences from Brassicaceae showed five major clusters corresponding to MIR172a-MIR172e which further separated into sub-genome and progenitor genome specific clades. Similar groupings were observed in the phylogeny of 11 Brassica AP2 and AP2-like genes. Over-expression of a pair of natural variants for each of MIR172b, MIR172d and MIR172e representing sub-genomes, progenitor genomes and species of Brassicas displayed floral acceleration in all transgenic lines indicating a strong selection pressure on MIR172. All gain-of-function lines, except 35S::MIR172e and 35S::MIR172e' displayed floral organ defects implying altered target spectrum of MIR172e relative to MIR172b and MIR172d. Expression of MIR172e caused marginal earliness in flowering time in B. juncea. In conclusion, this study demonstrates tightly preserved role of homeologs and natural variants of MIR172 family in mediating flowering in Brassicas and suggests their deployment for introgression of early flowering trait.

Mode of treatment governs curcumin response on doxorubicin-induced toxicity in cardiomyoblasts.

Doxorubicin (Dox) is an effective anti-cancer drug with severe reported cardiotoxicity. Cardiovascular risks associated with present cancer therapeutics demand urgent attention. There has been a growing interest in naturally occurring compounds to improve the therapeutic index as well as prevent non-tumour tissues from sustaining chemotherapy-induced damages. In the present study, the effects of curcumin, a polyphenol isolated from Curcuma longa and well known for its anti-oxidative, anti-cancerous and anti-inflammatory properties, was studied in relation to the Dox-induced cardiotoxicity. As literature suggests conflicting role of curcumin in Dox-induced cardiotoxicity, concentration- and time-dependent studies were conducted to study the different curcumin effects. H9C2 cardiomyoblasts were used in the study and cell viability assays were done to study Dox-induced cellular death. Drug uptake assay for Dox was performed followed by cellular growth inhibition analysis by FACS Calibur. Morphological alterations, intracellular ROS levels and mitochondrial integrity were observed by fluorescent-based microscopic studies. Catalases and superoxide dismutase-inbuilt anti-oxidant enzyme activities were studied, and it was observed that Dox-dependent cardiotoxicity occurs through ROS overproduction by exaggerating the inbuilt anti-oxidant mechanism. Expression analysis for cell death and ROS markers-BCl2, Bax, SOD, catalase-was investigated by semi-quantitative RT-PCR, and the Dox-induced stress on cardiac cells was confirmed. Initiator and effector caspases activity analysis also confirmed these findings. Our study proposes that curcumin exerts time-dependent responses on Dox-induced cardiotoxicity, where parallel treatment potentiates and pre-treatment suppresses the Dox-induced toxicity in H9C2 cardiomyoblasts. In conclusion, pre-treatment of curcumin suppresses the Dox-induced cardiotoxicity and holds a great potential as future cardio-oncological therapeutics.