Association of promoter methylation status of NRF2 and PNPLA3 genes in alcoholic liver disease.

BACKGROUND: Alcoholic liver disease (ALD) is the leading cause of chronic liver disease. In the liver, metabolism of alcohol occurs through multiple mechanisms and it results in the generation of various toxic products. Multiple genetic causes have been identified that are associated with the development and progression of ALD. The present study assessed the promoter site methylation status of nuclear factor erythroid 2-related factor 2 (NRF2) and patatin-like phospholipase domain-containing protein-3 (PNPLA3) genes in different subgroups of ALD. METHODS: The patients recruited were cases of alcohol dependence syndrome with hepatic dysfunction, compensated cirrhosis, decompensated cirrhosis, and acute-on-chronic liver failure due to alcohol as an etiology along with healthy control subjects. Routine biochemical investigations were performed along with methylation-specific polymerase chain reaction (MS-PCR) to qualitatively assess the promoter methylation status of NRF2 and PNPLA3 in all these cases. RESULTS: There was significant difference in methylation status of NRF2 gene in ALD when compared to healthy controls but there was no such difference in PNPLA3. All biochemical and clinical parameters studied were significantly different in subgroups of ALD except the serum aspartate aminotransferase (AST) level. Subgroups of ALD did not show any significant association with NRF2 or PNPLA3 methylation status. Gamma-glutamyl transferase (GGT) and creatinine levels in serum were significantly associated with the methylation status of NRF2 gene while no such association was seen with PNPLA3 gene. Model for end-stage liver disease (MELD) score varied differentially with NRF2 methylation and PNPLA3 methylation but there was no statistical significance. CONCLUSIONS: The present study showed that methylation status of NRF2 and PNPLA3 genes could not differentiate between subgroups of alcoholic liver diseases. However, the unmethylation of NRF2 promoter is associated with higher serum levels of GGT.

First report on embryonic and larval development of 2n/3n mosaic sterlet.

Mosaicism is frequently observed in aquaculture practices, and it adversely affects the production as well as the restoration programme of the sturgeon. The purpose of the present study was the induction of 2n/3n mosaic in sterlet, Acipenser ruthenus L., and compare their embryonic and larval development with diploid control sterlet. Microsatellite DNA loci genotyping was conducted for the identification of the genotypes and parentage analysis. Embryonic development was monitored in experimental groups at every 24 h interval. Identification of individual stages of embryonic development was recorded based on a 36-degree scale of development. Additionally, the BW and body length (LT) of experimental fishes were taken during 110 days of the rearing period. The Fulton's condition coefficient (F), length-weight parameters, and specific growth rate (SGR) coefficient were calculated. The analysis of embryonic development of the 2n/3n mosaic and the diploid control group did not show differences. However, higher mortality (88%) was observed in 2n/3n mosaic groups in comparison to the diploid control groups (55%). BW and body length of 2n/3n mosaic sterlet were slightly lower than the diploid control sterlet, but the differences were not statistically significant. F analysis did not confirm a lower growth performance of the fishes in the 2n/3n mosaic group. Microsatellite DNA loci genotyping confirmed both the incidence of polyspermy and retention of the second polar body. This paper presents the first report on embryonic development and growth performance of 2n/3n mosaic sturgeons.

DNA methylation and gene expression pattern of ACE2 and TMPRSS2 genes in saliva samples of patients with SARS-CoV-2 infection

COVID-19 caused by SARS-CoV-2 became a pandemic affecting the health and economy of the world. Although it was known that this virus uses ACE2 protein along with TMPRSS2 to enter the host cell, the methylation pattern and gene expression of ACE2 and TMPRSS2 genes are not explored in saliva samples of patients infected with COVID-19. The study aimed to quantify promoter methylation of ACE2 and TMPRSS2 along with its mRNA expression in saliva samples of COVID-19 patients in order to understand the regulatory mechanism of these genes in SARS-CoV-2 infection. Saliva samples were collected from thirty male patients with SARS-CoV-2 infection and thirty age-matched healthy control male subjects. Q MS PCR and qRT PCR was performed to quantify the promoter DNA methylation and mRNA expression of ACE2 and TMPRSS2 respectively. Our study didnt find any significant difference between methylation and expression of these two genes in cases compared to control subjects. However there was significant positive correlation between DNA methylation of ACE2 and its gene expression. Among cases, the sample collected [≥]7 days after appearance of symptoms showed higher amount of methylation in both ACE2 and TMPRSS2 genes when compared to sample collected before 7 days. In conclusion, we found that ACE2 and TMPRSS2 methylation plays a role in COVID-19.

Drug-Drug Interaction Studies of Methadone and Antiviral Drugs: Lessons Learned.

Different views appear in the literature on the extent of specific cytochrome P450 (CYP) involvement in methadone metabolism. The aim of this work is to leverage knowledge from drug-drug interaction (DDI) studies in new drug applications between methadone and antiviral medications to better understand methadone disposition and to inform design of future DDI studies with methadone. A database of DDI studies between all FDA-approved human immunodeficiency virus and hepatitis C virus medications and methadone was constructed. The database contains data from 29 DDI studies. Sixteen of the 29 studies had statistically significant changes in methadone area under the concentration-time curve. Methadone exposure was either decreased or unchanged when it was coadministered with weak to strong CYP3A inhibitors or a moderate CYP3A4 inducer. Methadone exposure was reduced when it was coadministered with CYP2B6 inducers. The role of other enzymes (CYP2C9, CYP2C19, and CYP2D6) cannot be fully elucidated from these studies. In conclusion, CYP2B6 plays a prominent role in methadone metabolism, although methadone exposure is not sensitive to CYP3A perturbation. In designing methadone DDI studies, (1) measuring R- and S-methadone is more informative than measuring total methadone, and (2) CYP2B6 genotyping of subjects enrolled in methadone DDI studies should be considered. Finally, there is a need for the development of predictive models to determine the influence of medications on methadone disposition.

Reinforcing mitochondrial functions in aging brain: An insight into Parkinson's disease therapeutics.

Mitochondria, the powerhouse of the neural cells in the brain, are also the seat of certain essential gene signaling pathways that control neuronal functions. Deterioration of mitochondrial functions has been widely reported in normal aging as well as in a spectrum of age-associated neurological diseases, including Parkinson's disease (PD). Evidences accumulated in the recent past provide not only advanced information on the causes of mitochondrial bioenergetics defects and redox imbalance in PD brains, but also much insight into mitochondrial biogenesis, quality control of mitochondrial proteins, and genes, which regulate intra- and extra-mitochondrial signaling that control the general health of neural cells. The mitochondrial quality control machinery is affected in aging and especially in PD, thus affecting intraneuronal protein transport and degradation, which are primarily responsible for accumulation of misfolded proteins and mitochondrial damage in sporadic as well as familial PD. Essentially we considered in the first half of this review, mitochondria-based targets such as mitochondrial oxidative stress and mitochondrial quality control pathways in PD, relevance of mitochondrial DNA mutations, mitophagy, mitochondrial proteases, mitochondrial flux, and finally mitochondria-based therapies possible for PD. Therapeutic aspects are considered in the later half and mitochondria-targeted antioxidant therapy, mitophagy enhancers, mitochondrial biogenesis boasters, mitochondrial dynamics modulators, and gene-based therapeutic approaches are discussed. The present review is a critical assessment of this information to distinguish some exemplary mitochondrial therapeutic targets, and provides a utilitarian perception of some avenues for therapeutic designs on identified mitochondrial targets for PD, a very incapacitating disorder of the geriatric population, world over.

Efficacy of Low-level Laser Therapy, Hyaluronic Acid Gel, and Herbal Gel as Adjunctive Tools in Gingivectomy Wound Healing: A Randomized Comparative Clinical and Histological Study.

Introduction Gingival overgrowth is usually an inflammatory response to plaque present on tooth surfaces. The other causes could be drugs and other systemic conditions. When the local factors are responsible and subgingival scaling does not help, gingivectomy is performed. The gingivectomy wound is raw and heals slowly. Low-level laser therapy (LLLT), hyaluronic acid, and herbal gels aid in healing after a gingivectomy. This study compared the efficacy of LLLT, hyaluronic acid, and herbal gel when used topically after a gingivectomy. This was a single-arm, interventional trial wherein 30 patients aged between 18 and 40 years with moderate gingival overgrowth participated. The study was approved by the institutional ethical committee. (DN/0109-16). The participants signed the consent form and the study was also registered (NCT03569683). Materials and methods The samples were equally divided into three groups. Group A received LLLT immediately postop, and on the first, third, and seventh days after surgery. Group B received hyaluronic acid (Gengigel) while Group C received an herbal gel (Hiora SG) for the same time periods after surgery, respectively. Analysis of variance (ANOVA) followed by a post-hoc Bonferroni test was used to evaluate differences within groups. Intergroup comparison was performed using the independent t-test. A p-value of <0.05 was considered statistically significant. Results The plaque index (PI), gingival index (GI), and gingival enlargement index (GEI) showed good improvement postoperatively within the groups, which was statistically significant. However, on an intergroup comparison, the GEI pertaining only to the laser group showed significant changes. Also, the pain perception analyzed by the visual analog score (VAS) was the least, and histologically, the amount of mature collagen fibers laid down were more in the laser group. Conclusion Patients irradiated with laser after gingivectomy (Group A) showed superior results in the clinical, histological variables as compared to Groups B and C.

Percutaneous intervention for restoration of patency of occluded lower limb arteriovenous dialysis access.

BACKGROUND: Arteriovenous (AV) fistula thrombosis is a serious complication in patients undergoing hemodialysis, often presenting with symptoms of venous hypertension, failure to dialysis and uremic symptoms. Treatment is aimed to provide symptomatic relief and to maintain hemodialysis access site patency. AIM: To describe our initial experience in the endovascular treatment of lower limb AV dialysis access (AV fistula) thrombosis and/or obstruction in patients undergoing hemodialysis. SETTINGS AND DESIGN: This was a retrospective study carried out in a tertiary care center. Study duration was 24 months. Follow-up was variable. MATERIALS AND METHODS: Two patients with chronic kidney disease with stage 5 renal failure undergoing hemodialysis presented with lower limb arteriovenous dialysis access (arteriovenous fistula) failure between July 2014 and September 2016. Both the patients underwent endovascular treatment and were analyzed retrospectively. RESULTS AND CONCLUSION: Both the patient underwent successful endovascular treatment for the failure of the lower limb AV dialysis access thrombosis and/or obstruction. One patient had minimal dye extravasation during manipulation of the guide wire, which ceased spontaneously. On follow-up, both patients maintained patency of the dialysis access and are undergoing successful hemodialysis. One patient had a recurrence of the thrombosis of the fistula at 9th month of the follow-up. Endovascular treatment was tried but we could not succeed. However, we found endovascular treatment safe and effective in treating AV fistula failures.

Nicolau Syndrome (Embolia Cutis Medicamentosa): A Rare and Poorly Recognized Iatrogenic Cause of Cutaneous Thrombotic Vasculopathy.

Nicolau syndrome is a rare form of iatrogenic cutaneous necrosis which affects injection sites. Although classically associated with intramuscular injections, it may develop after subcutaneous or other routes of parenteral drug administration. Clinically, it manifests as necrotic ulcers that often develop in a background of erythematous and livedoid reticular patches. The histopathologic characteristics of Nicolau syndrome are poorly documented in the dermatopathology literature and features only rarely as one of the obscure causes of cutaneous thrombotic vasculopathy. We report a case of Nicolau syndrome developing secondary to subcutaneous injection of cyclizine to familiarize the clinicians and pathologists to this unusual condition. Given that it is potentially avoidable, pathologists should alert the clinicians to the possibility of Nicolau syndrome when a skin biopsy from an injection site shows signs of extensive thrombotic vasculopathy.

Double-Stranded RNA-Mediated Suppression of Trypsin-Like Serine Protease (t-SP) Triggers Over-Expression of Another t-SP Isoform in Helicoverpa armigera.

High diversity of digestive proteases is considered to be the key factor in the evolution of polyphagy in Helicoverpa armigera. Serine proteases (SPs) contribute ~85% of the dietary protein digestion in H. armigera. We investigated the dynamics of SP regulation in the polyphagous pest, H. armigera using RNA interference (RNAi). HaTry1, an isoform of SP, expressed irrespective of the composition of the diet, and its expression levels were directly proportional to the larval growth rate. Therefore, HaTry1 was silenced by delivering 10 and 20 µg concentrations of double-stranded RNA through semi-synthetic diet. This led to a drastic reduction in the target gene transcript levels that manifested in a significant reduction in the larval weight initially, but the larvae recovered in later stages despite continuous dsRNA treatment. This was probably due to the compensatory effect by over-expression of HaTry13 (31-folds), another isoform of SP. Phylogenetic analysis of H. armigera SPs revealed that the over-expressed isoform was closely related to the target gene as compared to the other tested isoforms. Further, silencing of both the isoforms (HaTry1 and HaTry13) caused the highest reduction in the larval weight and there was no larval growth recovery. These findings provide a new evidence of the existence of compensatory effect to overcome the effect of silencing individual gene with RNAi. Hence, the study emphasizes the need for simultaneous silencing of multiple isoforms.

Evaluation of fetal echocardiography as a routine antenatal screening tool for detection of congenital heart disease.

BACKGROUND: Fetal echocardiography plays a pivotal role in identifying the congenital heart defects (CHDs) in utero. Though foetal echocardiography is mostly reserved for high risk pregnant women, its role as a routine prenatal screening tool still needs to be defined. Performing foetal echocardiography based on only these indications can lead to a significant numbers of CHD cases going undetected who will be deprived of further management leading to increased early neonatal mortalities. The aim of this study is to assess the incidence of CHDs by fetal echocardiography in an unselected population of pregnant women in comparison with pregnant women with conventional high risk factors for CHD. METHODS: This study enrolled consecutive pregnant women who attended antenatal clinic between 2008 and 2012 in a tertiary care hospital. These pregnant women were categorized into two groups: high risk group included pregnant women with traditional risk factors for CHD as laid down by Pediatric Council of the American Society of Echocardiography and low risk group. Detailed fetal 2 D echocardiography was done. RESULTS: A total of 1,280 pregnant women were included in study. The 118 women were categorized as the high risk group while remaining 1,162 were included in the low risk group. Twenty six cases of CHDs were detected based on abnormal foetal echocardiography (20.3 per 1,000). Two of the 26 cases of CHD occurred in high risk group whereas the remaining 24 occurred in low risk pregnancy. The difference in the incidence of CHDs between the two groups was not significant statistically (P=0.76). CONCLUSIONS: Our study shows no difference in incidence of CHDs between pregnancies associated with high risk factors compared to low risk pregnancies. So we advocate foetal echocardiography should be included as a part of routine antenatal screening and all pregnant women irrespective of risk factors for CHDs.

Diet-delivered RNAi in Helicoverpa armigera--Progresses and challenges.

Helicoverpa armigera (the cotton bollworm) is a significant agricultural pest endemic to Afro-Eurasia and Oceania. Gene suppression via RNA interference (RNAi) presents a potential avenue for management of the pest, which is highly resistant to traditional insecticide sprays. This article reviews current understanding on the fate of ingested double-stranded RNA in H. armigera. Existing in vivo studies on diet-delivered RNAi and their effects are summarized and followed by a discussion on the factors and hurdles affecting the efficacy of diet-delivered RNAi in H. armigera.

Percutaneous left atrial appendage closure in AF using Amplatzer Cardiac Plug: First single center experience from India.

BACKGROUND: Atrial fibrillation (AF) is one of the most common arrhythmias accounting for significant mortality and morbidity, especially in elderly. Though oral anticoagulation (OAC) is an effective mode of prevention of stroke in patients of AF, bleeding complication remains a major concern. Because of these issues, a significant proportion of patients either does not receive or receive suboptimal doses of OAC. METHODS: In such patients, percutaneous left atrial appendage (LAA) closure remains an interesting option. Experience and literature of this procedure from India have been sparse. We report the first single center experience, from India, of percutaneous LAA closure with Amplatzer Cardiac Plug in 10 patients of non-valvular AF. These patients had contraindications for OAC or had high risk of bleeding or labile international normalized ratio (INR) on therapy. RESULTS: We successfully deployed the devices in all of the cases with no major complications perioperatively and on short-term follow-up. We also report a comprehensive review on the technique of percutaneous LAA closure using Amplatzer Cardiac Plug, including some novel modification with our experience of doing percutaneous mitral balloon valvuloplasty.

An analysis of Pseudomonas genomic diversity in take-all infected wheat fields reveals the lasting impact of wheat cultivars on the soil microbiota.

Manipulation of the soil microbiota associated with crop plants has huge promise for the control of crop pathogens. However, to fully realize this potential we need a better understanding of the relationship between the soil environment and the genes and phenotypes that enable microbes to colonize plants and contribute to biocontrol. A recent 2 years of investigation into the effect of wheat variety on second year crop yield in the context of take-all fungal infection presented the opportunity to examine soil microbiomes under closely defined field conditions. Amplicon sequencing of second year soil samples showed that Pseudomonas spp. were particularly affected by the wheat cultivar grown in year one. Consequently, 318 rhizosphere-associated Pseudomonas fluorescens strains were isolated and characterized across a variety of genetic and phenotypic traits. Again, the wheat variety grown in the first year of the study was shown to exert considerable selective pressure on both the extent and nature of Pseudomonas genomic diversity. Furthermore, multiple significant correlations were identified within the phenotypic/genetic structure of the Pseudomonas population, and between individual genotypes and the external wheat field environment. The approach outlined here has considerable future potential for our understanding of plant-microbe interactions, and for the broader analysis of complex microbial communities.

Salvageability of kidney in Grade IV renal trauma by minimally invasive treatment methods.

CONTEXT: Renal trauma is increasingly being managed conservatively. Grade I-III injuries are managed conservatively whereas Grade V injuries may end in surgery. Managing Grade IV renal trauma is individualized and managed accordingly. AIMS: To evaluate retrospectively all Grade IV renal injuries managed in our institute over five years and to review the available literature. SETTINGS AND DESIGN: Reviewing the records of patients who sustained renal trauma and study all Grade IV renal injuries. MATERIALS AND METHODS: We retrospectively analyzed all Grade IV renal injuries (16) managed at our institute between July 2008-August 2013. All patients were treated conservatively initially by hemodynamic stabilization, strict bed rest, if required endoscopic procedures. These patients were followed up with CECT. STATISTICAL ANALYSIS: Descriptive statistics was performed using Microsoft excel spreadsheet 2007. Continuous data were described as mean and range. Categorical data was described as percentages. RESULTS: Sixteen patients with Grade IV renal injury were included in the study. All patients had gross hematuria and 15 had urinary extravasation. D-J Stenting was done in 7 patients; perinephric tube drainage with D-J stentingwas done in 2 patients. One required selective upper pole arterial embolisation. Nephrectomy was not required in any of the patients. In the follow-up period, no patient had delayed complications. CONCLUSIONS: Successful conservative management of Grade IV renal trauma requires constant monitoring both clinically and radiologically, and if properly managed, kidneys can be salvaged in all stable patients as reinforced by our study.

Population genetic structure of malaria vector Anopheles stephensi using mitochondrial cytochrome oxidase II gene in Indian populations.

The genetic differentiation in A. stephensi based on haplotype diversity using Restriction Fragment Length Polymorphism and bysequencing of CO II gene across different localities in India has been analyzed. The presence of only one DraI restriction site in CO II gene conferred to haplotype B indicating that the gene is very much conserved and the gene flow is not affected even by a major geographical distance barrier. The sequencing and analysisof various population parameters revealed seven haplotypes in all populations. The West Bengal population was found to be more genetically diverse than others. The geographic distance between populations was found to be contributing to the genetic differentiation. The sign of demographic expansion were found in three of the five populations. The local geographic barriers were found to be ineffective in prevention of gene flow.

Isolated megalourethra: A rare case report and review of literature.

Megalourethra is a rare congenital disorder of anterior urethra and erectile tissue of penis. It mainly appears in two types-a milder scaphoid type and severe fusiform type. Fusiform type is commonly associated with congenital anomalies of various systems of the body. Isolated megalourethra without other congenital anomalies is extremely rare. We report one such case which was detected postnatally and successfully treated by reduction urethroplasty.

A comparative evaluation of intranasal dexmedetomidine, midazolam and ketamine for their sedative and analgesic properties: a triple blind randomized study.

OBJECTIVES: To evaluate and compare the efficacy and safety of Intranasal (IN) Dexmedetomidine, Midazolam and Ketamine in producing moderate sedation among uncooperative pediatric dental patients. STUDY DESIGN: This randomized triple blind comparative study comprises of eighty four ASA grade I children of both sexes aged 4-14 years, who were uncooperative and could not be managed by conventional behavior management techniques. All the children were randomized to receive one of the four drug groups Dexmedetomidine 1 microg/ kg (D1), 1.5 microg/kg (D2), Midazolam 0.2 mg/kg (M1) and Ketamine 5 mg/kg (K1) through IN route. These drug groups were assessed for efficacy and safety by gauging overall success rate and by monitoring vital signs, respectively. RESULTS: The onset of sedation was significantly rapid with M1 and K1 as compared to D1 and D2 (p = < 0.001). The overall success rate was highest in D2 (85.7%) followed by D1 (81%), K1 (66.7%) and M1 (61.9%), however, the difference among them was not statistically significant (p = > 0.05). Even though all the vital signs were within physiological limits, there was significant reduction in pulse rate (PR) (p = < 0.001) and systolic blood pressure (SBP) (p = < 0.05) among D1 and D2 as compared to M1 and K1. D1, D2 and K1 produced greater intra- and post-operative analgesia as compared to M1. There were no significant adverse effects with any group. CONCLUSION: Dexmedetomidine, Midazolam and Ketamine, all the three drugs evaluated in the present study can be used safely and effectively through IN route in uncooperative pediatric dental patients for producing moderate sedation.

Effect of additional vitamin E and zinc supplementation on immunological changes in peripartum Sahiwal cows.

This study was conducted to exploit ameliorative effect of additional vitamin E and/or zinc supplementation on immune response of peripartum Sahiwal cows. Thirty-two pregnant dry Sahiwal cows were blocked into four treatment groups (n = 8), namely control, zinc (Zn), vitamin E (Vit E) and zinc + vitamin E (Zn + Vit E). Feeding regimen was same in all the groups except that the Sahiwal cows in the zinc-, vitamin E- and zinc + vitamin E-fed groups were additionally supplemented with 60 mg Zn/kg DM, 1000 IU vitamin E and 60 mg/kg + 1000 IU Zn + vitamin E, respectively, from day 60 pre-partum to day 90 post-partum. Blood samples were collected on days -60, -45, -30, -15, -7, -3, 0, 3, 7, 15, 30, 45, 60, 90 and 120 with respect to day of parturition and analysed for total immunoglobulin (TIG), immunoglobulin G (IgG), interleukin-2 (IL-2), vitamin E (Vit E) and zinc (Zn) status. Before calving, cows showed a decrease in plasma TIG, IgG, IL-2, Vit E and Zn levels. However, increased levels of plasma TIG, IgG, IL-2, Vit E and Zn were observed after calving. After calving, Sahiwal cows supplemented with Zn + Vit E had higher plasma TIG, IgG and IL-2 in comparison with cows of control and Zn + Vit E-fed groups. In the present study, plasma vitamin E level was higher in Vit E-fed and Zn + Vit E-fed cows; however, zinc level was higher in Zn- and Zn + Vit E-supplemented cows. In conclusion, a reduced immune response during peripartum period in Sahiwal cows was ameliorated by dietary vitamin E and zinc supplementation.

Efficacy of spinal ropivacaine versus ropivacaine with fentanyl in transurethral resection operations.

BACKGROUND: The low-dose ropivacaine provides differential spinal block to reduce adverse hemodynamic effects in elderly patients. Addition of intrathecal fentanyl with ropivacaine may enhance analgesia and early postoperative mobility. The present study was performed to evaluate the efficacy of intrathecal ropivacaine alone and in combination with fentanyl in transurethral resection operation. METHODS: Sixty male patients aged >50 years of ASA I-III scheduled for elective transurethral resection were included in a prospective, randomized, double-blinded study and they were divided in two groups of 30 each. Group A (n = 30) received intrathecal injection of ropivacaine 2 ml (0.75%) and Group B (n = 30) ropivacaine 1.8 ml (0.75%) with fentanyl 10 µg. The characteristics of onset and regression of sensory and motor blockade, hemodynamic stability, and side effects were observed. Student's t test (for parametric data) and Mann-Whitney U test (for non-parametric data) were used for statistical analyses. RESULTS: There were no significant differences between the two groups for patient demographic data, intraoperative hemodynamic parameters, side effects, and satisfaction to patients and surgeon. The highest level of sensory block was at T10 in group A and T9 in group B (P = 0.001). Duration of motor block was longer in group B being 210.51 ± 61.25 min than in group A being 286.25 ± 55.65 min (P < 0.001). CONCLUSION: The addition of fentanyl to ropivacaine may offer the advantage of shorter duration of complete motor block, hemodynamic stability, and without any increase in the frequency of major side effects.

[Evaluation of reference genes for quantitative real-time PCR normalization in cotton bollworm, Helicoverna armigera].

Reverse-transcription quantitative real-time PCR (RT-qPCR), a sensitive technique is being extensively employed in quantification of gene expression. However this requires normalization with suitable reference gene (RG) which is crucial in minimizing inter sample variations. Information regarding suitable RG is scarce in general and more so in insects, including the cotton bollworm, Helicoverpa armigera, an economically important pest. In management of this pest RNA interference (RNAi), is perceived as a potential tool, which is achieved by double-stranded RNA (dsRNA) delivery. These studies demand accurate quantification of gene silencing. In this study we assessed the suitability of five RGs viz. ß-actin (ACTB), 18S rRNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB) and elongation fator-1-alfa (EF1-α) for gene expression studies in dsRNA treatment and across different developmental stages of H. armigera and ranked using geNorm, NormFinder and BestKeeper software programs. Data analysis revealed that best ranked RGs were varied in dsRNA treatment and in developmental stages. Under dsRNA treatment, 18S and GAPDH were more stable whereas, TUB and GAPDH were more stable across developmental stages. We also demonstrate that inappropriate selection of RG led to erroneous estimation of the target gene, chymotrypsin, expression. These results facilitate accurate quantification of gene expression in H. armigera.