Mechanical, tribological, and surface morphological studies on the effects of hybrid ilmenite and silicon dioxide fillers on glass fibre reinforced epoxy composites.

Recently, researchers have been attempting to enhance the mechanical and tribological characteristics of thermosetting epoxy composites by incorporating inorganic nanoparticles and ensuring their uniform distribution throughout the matrix. This study characterises ball-milled ilmenite (FeTiO3-size of 63 nm) and silicon dioxide (SiO2-size of 67.5 nm) fillers added to epoxy in proportions of 0:0, 2.5:2.5, 5:5, and 7.5:7.5% by weight. A liquid ultrasonic technique is used to blend the fillers with the epoxy, and compression moulding is used to fabricate the composite. Mechanical tests were performed based on ASTM standards. Tensile strength, tensile modulus, flexural strength, flexural modulus and elongations at break(tensile and flexural test) of 5:5 wt % are 30.54%, 12.2%, 32.22%, 28.98%,23.78% and 23.53% higher than neat sample respectively. Shore "D" hardness and Izod's impact strength are 4.65% and 98.93% higher at 5:5 wt % than neat sample respectively. Specific wear rate decreased from 2.6 × 10-11 m3/Nm (neat GFRP: 0 wt % glass fibre reinforced polymer composite) to 0.7 × 10-11 m3/Nm at 5:5 wt % filler. Nanoparticles lowered the coefficient of friction by around 16.66%, 60.42%, and 33.33% at sliding distances of 100 m for 2.5:2.5, 5:5, and 7.5:7.5 wt % respectively with the neat sample. A 5:5 wt percent resulted in 76.68% less wear volume loss than pure GFRP. Field emission scanning electron microscopy (FESEM) analysis revealed element distributions, particle size, pullout of fibers, damaged interfaces, filler dispersion, voids, wear debris, interfacial debonding, and cavities. Thus, this approach enhances GFRP composite's mechanical, tribological, and structural properties.

Shorter Treatment for Nonsevere Tuberculosis in African and Indian Children.

BACKGROUND: Two thirds of children with tuberculosis have nonsevere disease, which may be treatable with a shorter regimen than the current 6-month regimen. METHODS: We conducted an open-label, treatment-shortening, noninferiority trial involving children with nonsevere, symptomatic, presumably drug-susceptible, smear-negative tuberculosis in Uganda, Zambia, South Africa, and India. Children younger than 16 years of age were randomly assigned to 4 months (16 weeks) or 6 months (24 weeks) of standard first-line antituberculosis treatment with pediatric fixed-dose combinations as recommended by the World Health Organization. The primary efficacy outcome was unfavorable status (composite of treatment failure [extension, change, or restart of treatment or tuberculosis recurrence], loss to follow-up during treatment, or death) by 72 weeks, with the exclusion of participants who did not complete 4 months of treatment (modified intention-to-treat population). A noninferiority margin of 6 percentage points was used. The primary safety outcome was an adverse event of grade 3 or higher during treatment and up to 30 days after treatment. RESULTS: From July 2016 through July 2018, a total of 1204 children underwent randomization (602 in each group). The median age of the participants was 3.5 years (range, 2 months to 15 years), 52% were male, 11% had human immunodeficiency virus infection, and 14% had bacteriologically confirmed tuberculosis. Retention by 72 weeks was 95%, and adherence to the assigned treatment was 94%. A total of 16 participants (3%) in the 4-month group had a primary-outcome event, as compared with 18 (3%) in the 6-month group (adjusted difference, -0.4 percentage points; 95% confidence interval, -2.2 to 1.5). The noninferiority of 4 months of treatment was consistent across the intention-to-treat, per-protocol, and key secondary analyses, including when the analysis was restricted to the 958 participants (80%) independently adjudicated to have tuberculosis at baseline. A total of 95 participants (8%) had an adverse event of grade 3 or higher, including 15 adverse drug reactions (11 hepatic events, all but 2 of which occurred within the first 8 weeks, when the treatments were the same in the two groups). CONCLUSIONS: Four months of antituberculosis treatment was noninferior to 6 months of treatment in children with drug-susceptible, nonsevere, smear-negative tuberculosis. (Funded by the U.K. Medical Research Council and others; SHINE ISRCTN number, ISRCTN63579542.).

Cost of Management of Diabetes Mellitus: A Pan India Study.

BACKGROUND: Diabetes mellitus is a major noncommunicable disease. While mortality rates are increasing, the costs of managing the disease are also increasing. The all-India average monthly expenditure per person (pppm) is reported to be ₹ 1,098.25, which translates to an annual expenditure of ₹13,179 per person. PURPOSE: While a number of studies have gone into the aspect of the cost of disease management, we do not find any study which has pan-India reach. We also do not find studies that focus on differences (if any) between rural and urban areas, age or on the basis of gender. We planned to report the cost of illness (COI) in diabetes individuals as compared to others from the data of a pan-India trial. METHODS: Government of India commissioned the Indian Yoga Association to study the prevalence of diabetes mellitus in India in 2017. As part of the questionnaire, the cost of treatment was also captured. Data collected from 25 states and union territories were analyzed using the analysis of covriance (ANCOVA) test on SPSS version 21. RESULTS: There was a significant difference (P < .05) between the average expenses per person per month (pppm) of individuals with self-reported known diabetes (₹1,357.65 pppm) and others (unknown and/or nondiabetes individuals-₹ 999.91 pppm). Similarly, there was a significant difference between rural (₹2,893 pppm) and urban (₹4,162 pppm) participants and between those below (₹1,996 pppm) and above 40 years (₹5,059 pppm) of age. CONCLUSION: This preliminary report has shown that the COI because of diabetes is significantly higher than others pointing to an urgent need to promote disease-preventive measures.

Evaluation of metformin in combination with rifampicin containing antituberculosis therapy in patients with new, smear-positive pulmonary tuberculosis (METRIF): study protocol for a randomised clinical trial.

INTRODUCTION: Shorter duration of treatment for the management of drug-susceptible pulmonary tuberculosis (TB) would be a significant improvement in the care of patients suffering from the disease. Besides newer drugs and regimens, other modalities like host-directed therapy are also being suggested to reach this goal. This study's objective is to assess the efficacy and safety of metformin-containing anti-TB treatment (ATT) regimen in comparison to the standard 6-month ATT regimen in the treatment of patients with newly diagnosed sputum smear-positive drug-sensitive pulmonary TB. METHODS AND ANALYSIS: We are conducting a multicentric, randomised open-label controlled clinical trial to achieve the study objective. The intervention group will receive isoniazid (H), rifampicin (R), ethambutol (E) and pyrazinamide (Z) along with 1000 mg of daily metformin (Met) for the first 2 months while the control group will receive only HRZE. After 2 months, both the groups will receive HRE daily for 4 months. The primary endpoint is time to sputum culture conversion. Secondary endpoints will include time to detection of Mycobacterium tuberculosis in sputum, pharmacokinetics and pharmacogenomics of study drugs, drug-drug interactions, safety and tolerability of the various combinations and measurement of autophagy and immune responses in the study participants. ETHICS AND DISSEMINATION: The ethics committee of the participating institutes have approved the study. Results from this trial will contribute to evidence towards constructing a shorter, effective and safe regimen for patients with TB. The results will be shared widely with the National Programme managers, policymakers and stakeholders through open access publications, dissemination meetings, conference abstracts and policy briefs. This is expected to provide a new standard of care for drug-sensitive patients with pulmonary TB who will not only reduce the number of clinic visits and lost to follow-up of patients from treatment but also reduce the burden on the healthcare system. TRIAL REGISTRATION NUMBER: CTRI/2018/01/011176; Pre-results.

Shorter treatment for minimal tuberculosis (TB) in children (SHINE): a study protocol for a randomised controlled trial.

BACKGROUND: Tuberculosis (TB) in children is frequently paucibacillary and non-severe forms of pulmonary TB are common. Evidence for tuberculosis treatment in children is largely extrapolated from adult studies. Trials in adults with smear-negative tuberculosis suggest that treatment can be effectively shortened from 6 to 4 months. New paediatric, fixed-dose combination anti-tuberculosis treatments have recently been introduced in many countries, making the implementation of World Health Organisation (WHO)-revised dosing recommendations feasible. The safety and efficacy of these higher drug doses has not been systematically assessed in large studies in children, and the pharmacokinetics across children representing the range of weights and ages should be confirmed. METHODS/DESIGN: SHINE is a multicentre, open-label, parallel-group, non-inferiority, randomised controlled, two-arm trial comparing a 4-month vs the standard 6-month regimen using revised WHO paediatric anti-tuberculosis drug doses. We aim to recruit 1200 African and Indian children aged below 16 years with non-severe TB, with or without HIV infection. The primary efficacy and safety endpoints are TB disease-free survival 72 weeks post randomisation and grade 3 or 4 adverse events. Nested pharmacokinetic studies will evaluate anti-tuberculosis drug concentrations, providing model-based predictions for optimal dosing, and measure antiretroviral exposures in order to describe the drug-drug interactions in a subset of HIV-infected children. Socioeconomic analyses will evaluate the cost-effectiveness of the intervention and social science studies will further explore the acceptability and palatability of these new paediatric drug formulations. DISCUSSION: Although recent trials of TB treatment-shortening in adults with sputum-positivity have not been successful, the question has never been addressed in children, who have mainly paucibacillary, non-severe smear-negative disease. SHINE should inform whether treatment-shortening of drug-susceptible TB in children, regardless of HIV status, is efficacious and safe. The trial will also fill existing gaps in knowledge on dosing and acceptability of new anti-tuberculosis formulations and commonly used HIV drugs in settings with a high burden of TB. A positive result from this trial could simplify and shorten treatment, improve adherence and be cost-saving for many children with TB. Recruitment to the SHINE trial begun in July 2016; results are expected in 2020. TRIAL REGISTRATION: International Standard Randomised Controlled Trials Number: ISRCTN63579542 , 14 October 2014. Pan African Clinical Trials Registry Number: PACTR201505001141379 , 14 May 2015. Clinical Trial Registry-India, registration number: CTRI/2017/07/009119, 27 July 2017.

High Prevalence of Lipid Abnormalities and Insulin Resistance Among Antiretroviral Naïve HIV-infected Children in India.

BACKGROUND: As large numbers of children are accessing antiretroviral therapy (ART) in India, we evaluated the dietary intake, growth pattern and risk of metabolic complications like dyslipidemia and insulin resistance among ART-naïve HIV-infected children (CLHIV). METHODS: CLHIV 2-12 years of age, at the time of initiating ART in Chennai and Bangalore, were assessed for their dietary intake, anthropometry, blood CD4 cell count, HIV-1 viral load, fasting serum lipids, glucose and insulin. Homeostatic model assessment-insulin resistance was derived. RESULTS: Three hundred and ninety CLHIV (mean age [SD]: 8 [3] yrs; median viral load: 141,000 [25,876-436,000] copies/mL) were started on non-nucleoside reverse transcriptase inhibitor-based ART. Perinatal infection was documented among 97%. Sixty percent of children were in stage 3 or 4 of World Health Organization clinical staging of HIV/AIDS. Food insecurity was seen in 40% of households. A total of 204 children (52.4%) were stunted and 224 (57.6%) were underweight. Stunting seemed to be more prevalent with increasing age (0-4 years: 48%; >9 years: 60%). Mean intakes of calories, iron, folate and calcium were significantly less than recommended dietary allowances across all age groups. Dyslipidemia, in terms of any abnormal triglycerides or total cholesterol or low-density lipoprotein cholesterol (excluding high-density lipoprotein cholesterol), was seen in approximately 40% of children; insulin resistance in 17%; and C-reactive protein in risk range of metabolic syndrome in 24% of children. CONCLUSIONS: In the background of high food insecurity and malnutrition, cardiometabolic abnormalities were seen in 20%-35% of ART-naïve CLHIV in India emphasizing close monitoring of these children for long-term cardiovascular morbidities after initiation of ART.

Plasma and Urinary Type IV Collagen Levels for Early Detection of Nephropathy in Type 2 Diabetes Mellitus Patients.

BACKGROUND: Diabetic nephropathy is a major complication with high morbidity and mortality, and leads to end stage renal disease (ESRD). Type IV collagen is the main component of the glomerular basement membrane (GBM) and the extracellular matrix. The thickening of the GBM is due to accumulation of type IV collagen and alterations in its structure and composition. AIM: The aim of this study was to evaluate the association of plasma and urine type IV collagen with albuminuria status and to determine the clinical implications of type IV collagen as a marker in the early stage of diabetic nephropathy. MATERIALS AND METHODS: A total of 150 type 2 diabetes mellitus patients with more than 5 year diabetic duration in the age group of 35 to 60 years were selected for this study and 50 age and sex matched healthy individuals were selected as control group. Type IV collagen (Plasma and urine), Insulin were analyzed by ELISA method and micro albumin was analyzed by turbilatex method. Routine investigations fasting plasma glucose, post prandial glucose, lipid profile parameters, serum urea and creatinine were analyzed by using Auto analyzer. RESULTS: The plasma and urinary type IV collagen levels were significantly higher in the normoalbuminuric group with diabetes than in the control group, and increased with increasing severity of albuminuria among diabetics. Both plasma and urine type IV collagen levels showed positive correlation with albumin creatinine ratio (ACR) and regression analysis showed significant influence with ACR and also positive significant correlation of ACR with FPG, PPG, HbA1C, HOMA-IR, negative correlation with HDL cholesterol was observed. CONCLUSION: Plasma and urinary type IV collagen can be helpful in the prediction of the subsequent development of albuminuria in type 2 diabetic patients.

Anisotropic interface induced formation of Sb nanowires on GaSb(111)A substrates.

The growth of Sb nanowires on GaSb(111)A substrates is studied by in situ azimuthal scan reflection high-energy electron diffraction (ARHEED). Bulk and layer contributions can be distinguished in the ARHEED transmission pattern through the Sb nanowires. The three-dimensional structure of the growing Sb nanowires is identified by post-growth atomic force microscopy (AFM) and x-ray diffraction (XRD). The lattice match of the Sb crystal along the [Formula: see text] and the GaSb crystal along [Formula: see text] directions lead to a preferential orientation of the Sb nanowires. The Sb adsorption and desorption kinetics is studied by thermal desorption spectroscopy.

SU-E-T-569: A Dosimetric Comparison of Helical Tomotherapy versus Intensity Modulated Proton for Lung Cancer.

PURPOSE: To evaluate the dosimetric difference between helical tomotherapy (HT) and intensity modulated proton therapy (IMPT) treatment for lung cancer patients. METHODS: Five patients treated by HT at University of Wisconsin Carbone Cancer Center were selected. HT plans were generated on TomoTherapy treatment planning station (TomoTherapy Inc., USA). The field widths were set to 2.5 cm for all patients in this study. The IMPT plans were generated using the same planning CT and contours with our in-house treatment planning system. Three to five field spot scanning IMPT were used to deliver uniform doses to the targets while minimizing the irradiated lung volume. The proton spots used has a Gaussian sigma of 6mm and are placed on a rectangular grid. The dose distribution of each proton spot is calculated using a pencil beam algorithm with tissue heterogeneity corrections. All the dosimetric analyses are performed using normalized total dose. Alpha/beta ratios were set to 3 for normal tissues and 10 for tumors. RESULTS: IMPT plans showed improvement of critical structure avoidance and target dose uniformity for all patients. Reductions in mean lung doses of between 81% to 27% were observed in the IMPT plans relative to the HT. The equivalent uniform dose of the target improved from 49.2 Gy in HT plan to 60.04 Gy in IMPT for patient #2, and equivalent for other cases. The maximum doses to cord were reduced by 20.5 Gy on average using IMPT. In two patient cases, the normal tissue complication probabilities were reduced by 53% and 14% with IMPT. CONCLUSION: IMPT provides improved dose homogeneity on the target and normal structure sparing compared with HT in the treatment of non-small cell carcinoma in lung. Significant reduction of mean lung dose was demonstrated, as well as toxicity to organs at risk adjacent to the target.

Efficacy and safety of once-daily nevirapine- or efavirenz-based antiretroviral therapy in HIV-associated tuberculosis: a randomized clinical trial.

BACKGROUND: Nevirapine (NVP) can be safely and effectively administered once-daily but has not been assessed in human immunodeficiency virus (HIV)-infected patients with tuberculosis (TB). We studied the safety and efficacy of once-daily NVP, compared with efavirenz (EFV; standard therapy); both drugs were administered in combination with 2 nucleoside reverse-transcriptase inhibitors. METHODS: An open-label, noninferiority, randomized controlled clinical trial was conducted at 3 sites in southern India. HIV-infected patients with TB were treated with a standard short-course anti-TB regimen (2EHRZ(3)/4RH(3); [2 months of Ethambutol, Isoniazid, Rifampicin, Pyrazinamide / 4 months of Isoniazid and Rifampicin] thrice weekly) and randomized to receive once-daily EFV at a dose of 600 mg or NVP at a dose of 400 mg (after 14 days of 200 mg administered once daily) with didanosine 250/400 mg and lamivudine 300 mg after 2 months. Sputum smears and mycobacterial cultures were performed every month. CD4+ cell count, viral load, and liver function test results were monitored periodically. Primary outcome was a composite of death, virological failure, default, or serious adverse event (SAE) at 24 weeks. Both intent-to-treat and per protocol analyses were done, and planned interim analyses were performed. RESULTS: A total of 116 patients (75% [87 patients] of whom had pulmonary TB), with a mean age of 36 years, a median CD4+ cell count of 84 cells/mm(3), and a median viral load of 310 000 copies/mL, were randomized. At 24 weeks, 50 of 59 patients in the EFV group and 37 of 57 patients in the NVP group had virological suppression (P = .024). There were no deaths, 1 SAE, and 5 treatment failures in the EFV arm, compared with 5 deaths, 2 SAEs, and 10 treatment failures in the NVP arm. The trial was halted by the data and safety monitoring board at the second interim analysis. Favorable TB treatment outcomes were observed in 93% of the patients in the EFV arm and 84% of the patients in the NVP arm (P = .058). CONCLUSIONS: Compared with a regimen of didanosine, lamivudine, and EFV, a regimen of once-daily didanosine, lamivudine, and NVP was inferior and was associated with more frequent virologic failure and death. Clinical Trials Registration. NCT00332306.

Bioaccessibility of Carotenoids and Tocopherols in Marine Microalgae, Nannochloropsis sp. and Chaetoceros sp.

Microalgae can produce various natural products such as pigments, enzymes, unique fatty acids and vitamin that benefit humans. The objective of the study is to study the bioaccessibility of carotenoids (ß-carotene and lycopene) and vitamin E (α- and ß-tocopherol) of Nannochloropsis oculata and Chaetoceros calcitrans. Analyses were carried out for both the powdered forms of N. oculata and C. calcitrans, and the dried extract forms of N. oculata and C. calcitrans. In vitro digestion method together with RP-HPLC was used to determine the bioaccessibility of carotenoids and vitamin E for both forms of microalgae. Powdered form of N. oculata had the highest bioaccessibility of ß-carotene (28.0 ± 0.6 g kg-1), followed by dried extract N. oculata (21.5 ± 1.1 g kg-1), dried extract C. calcitrans (16.9 ± 0.1 g kg-1), and powdered C. calcitrans (15.6 ± 0.1 g kg-1). For lycopene, dried extract of N. oculata had the highest bioaccessibility of lycopene (42.6 ± 1.1 g kg-1), followed by dried extract C. calcitrans (41.9 ± 0.6 g kg-1), powdered C. calcitrans (39.7 ± 0.1 g kg-1) and powdered N. oculata (32.6 ± 0.7 g kg-1). Dried extract C. calcitrans had the highest bioaccessibility of α-tocopherol (72.1 ± 1.2 g kg-1). However, ß-tocopherol was not detected in both dried extract and powdered form of C. calcitrans. In conclusion, all samples in their dried extract forms were found to have significantly higher bioaccessibilities than their powdered forms. This may be due to the disruption of the food matrix contributing to a higher bioaccessibility of nutrients shown by the dried extract forms.

Bioaccessibility of Carotenoids and Tocopherols in Marine Microalgae, Nannochloropsis sp. and Chaetoceros sp

Microalgae can produce various natural products such as pigments, enzymes, unique fatty acids and vitamin that benefit humans. The objective of the study is to study the bioaccessibility of carotenoids (β-carotene and lycopene) and vitamin E (α- and β- tocopherol) of Nannochloropsis oculata and Chaetoceros calcitrans. Analyses were carried out for both the powdered forms of N. oculata and C. calcitrans, and the dried extract forms of N. oculata and C. calcitrans. In vitro digestion method together with RP-HPLC was used to determine the bioaccessibility of carotenoids and vitamin E for both forms of microalgae. Powdered form of N. oculata had the highest bioaccessibility of β-carotene (28.0 ± 0.6 g kg-1), followed by dried extract N. oculata (21.5 ± 1.1 g kg-1), dried extract C. calcitrans (16.9 ± 0.1 g kg-1), and powdered C. calcitrans (15.6 ± 0.1 g kg-1). For lycopene, dried extract of N. oculata had the highest bioaccessibility of lycopene (42.6 ± 1.1 g kg- 1), followed by dried extract C. calcitrans (41.9 ± 0.6 g kg-1), powdered C. calcitrans (39.7 ± 0.1 g kg-1) and powdered N. oculata (32.6 ± 0.7 g kg-1). Dried extract C. calcitrans had the highest bioaccessibility of α-tocopherol (72.1 ± 1.2 g kg-1). However, β-tocopherol was not detected in both dried extract and powdered form of C. calcitrans. In conclusion, all samples in their dried extract forms were found to have significantly higher bioaccessibilities than their powdered forms. This may be due to the disruption of the food matrix contributing to a higher bioaccessibility of nutrients shown by the dried extract forms

Novel substituted methylenedioxy lignan suppresses proliferation of cancer cells by inhibiting telomerase and activation of c-myc and caspases leading to apoptosis.

Conventional solvent fractionation and bioactivity based target assays were used to identify a new anti-cancer molecule from Phyllanthus urinaria, a herbal medicinal plant used in South India. At each step of the purification process the different fractions that were isolated were tested for specific anti-proliferative activity by assays measuring the inhibition of [(3)H]thymidine incorporation, and trypan blue drug exclusion. The ethyl acetate fraction that contained the bioactivity was further purified and resolved by HPLC on a preparative column. The purity of each of the fractions and their bioactivity were checked. Fraction 3 demonstrated a single spot on TLC and showed maximum anti-proliferative activity. This fraction was further purified and the structure was defined as 7'-hydroxy-3',4',5,9,9'-pentamethoxy-3,4-methylene dioxy lignan using NMR and mass spectrometry analysis. The pure compound and the crude ethyl acetate fraction which showed anti-proliferative activities were examined for ability to target specific markers of apoptosis like bcl2, c-myc and caspases and for effects on telomerase. Four specific cancer cell lines HEp2, EL-1 monocytes, HeLa and MCP7 were used in this study. The results indicate that 7'-hydroxy-3',4',5,9,9'-pentamethoxy-3,4-methylene dioxy lignan was capable of inhibiting telomerase activity and also could inhibit bcl2 and activate caspase 3 and caspase 8 whose significance in the induction of apoptosis is well known. We believe that this compound could serve as a valuable chemotherapeutic drug after further evaluations.

Purification, characterization, and cloning of the cDNA of human signal recognition particle RNA 3'-adenylating enzyme.

The 3'-terminal adenylic acid residue in several human small RNAs including signal recognition particle (SRP) RNA, nuclear 7SK RNA, U2 small nuclear RNA, and ribosomal 5S RNA is caused by a post-transcriptional adenylation event (Sinha, K., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853-6859). Using the Alu portion of the SRP RNA as a substrate in an in vitro adenylation assay, we purified an adenylating enzyme that adds adenylic acid residues to SRP/Alu RNA from the HeLa cell nuclear extract. All the peptide sequences obtained by microsequencing of the purified enzyme matched a unique human cDNA corresponding to a new adenylating enzyme having homologies to the well characterized mRNA poly(A) polymerase. The amino terminus region of the human SRP RNA adenylating enzyme showed approximately 75% homology to the amino terminus of the human mRNA poly(A) polymerase that includes the catalytic domain. The carboxyl terminus of the human SRP RNA adenylating enzyme showed less than 25% homology to the carboxyl terminus of poly(A) polymerase, which interacts with other factors and provides specificity. The SRP RNA adenylating enzyme is coded for by a gene located on chromosome 2 in contrast to the poly(A) polymerase gene, which is located on chromosome 14. A recombinant protein for the SRP RNA adenylating enzyme was prepared, and its activity was compared with the purified enzyme from HeLa cells. The data indicate that in addition to the SRP RNA adenylating enzyme, other factors may be required to carry out accurate 3'-end adenylation of SRP RNA.

Effect of 3' terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes.

It is known that several small RNAs including human and Xenopus signal recognition particle (SRP) RNA, U2 small nuclear RNA (snRNA) and 7SK RNAs are posttranscriptionally adenylated, whereas U6 snRNA and ribosomal 5S RNA are posttranscriptionally uridylated on their 3' ends. In this study, we provide evidence that a small fraction of U6 snRNA and 5S ribosomal RNA molecules from human as well as Xenopus oocytes contain a single posttranscriptionally added adenylic acid residue on their 3' ends. These data show that U6 snRNA and 5S rRNAs are posttranscriptionally modified on their 3' ends by both uridylation and adenylation. Although the SRP RNA, 7SK RNA, 5S RNA, and U6 snRNA with the uridylic acid residue on their 3' ends were readily uridylated, all these RNAs with posttranscriptionally added adenylic acid residue on their 3' ends were not uridylated in vitro, or when U6 snRNA with 3' A(OH) was injected into Xenopus oocytes. These results show that the presence of a single posttranscriptionally added adenylic acid residue on the 3' end of SRP RNA, U6 snRNA, 5S rRNA, or 7SK RNA prevents 3' uridylation. These data also show that adenylation and uridylation are two competing processes that add nucleotides on the 3' end of some small RNAs and suggest that one of the functions of the 3' adenylation may be to negatively affect the 3' uridylation of small RNAs.

Evolutionary conservation of post-transcriptional 3' end adenylation of small RNAs: S. cerevisiae signal recognition particle RNA and U2 small nuclear RNA are post-transcriptionally adenylated.

The 3' terminal nucleotide of several human small RNAs, including Signal Recognition Particle (SRP) RNA, 7SK RNA, U2 small nuclear RNA and ribosomal 5S RNA was previously characterized and a fraction of these RNAs was found to contain a single post-transcriptionally added adenylic acid residue on their 3' ends. Here we report the development of a reverse transcription-polymerase chain reaction (RT-PCR) assay for determining and quantifying the extent of post-transcriptional adenylation of RNAs from different species. Using this assay, we found that a fraction of S. cerevisiae U2 small nuclear RNA and S. cerevisiae SRP RNA contain a post-transcriptionally added adenylic acid residue on their 3' ends. Sequencing analysis confirmed this adenylation to be post-transcriptional. Corresponding small RNAs in Xenopus oocytes also contained this post-transcriptional adenylation on their 3' ends. These data show that post-transcriptional adenylation on the 3' end of several small RNA molecules is conserved through evolution. Xenopus SRP RNA from both cytoplasmic and nuclear compartments contained post-transcriptionally added adenylic acid residue on its 3' end. In addition, the Alu portion of SRP RNA was adenylated, when injected into the cytoplasm of frog oocytes. These data show that this novel adenylating machinery, capable of specifically adding a single adenylic acid to the 3' end of some RNA molecules, is present and functional in both nucleus and cytoplasm.

Inhibition of translation of mRNAs containing gamma-monomethylphosphate cap structure in frog oocytes and in mammalian cells.

The gamma-monomethylphosphate cap structure is found in several eukaryotic small RNAs including nuclear U6, U6atac, 7SK, plant nucleolar U3, and rodent cytoplasmic B2 RNAs. In the case of human U6 snRNA, the 5' end sequence corresponding to nucleotides 1-25 serves as the capping signal and directs the formation of methylphosphate cap structure. In this study, we show that the U6 RNA capping signal, when introduced at the 5' end of RNAs, can efficiently direct the methylphosphate cap formation in RNAs of up to 2.7 kb long, as well as in different mRNAs. These data show that the methylphosphate capping signal functions in mRNAs having different primary sequences and different lengths. Presence of the methylphosphate cap structure on the 5' end of a luciferase mRNA with EMCV 5' noncoding region, which is translated in an IRES-dependent pathway, resulted in a 6- to 100-fold inhibition of translation compared to the same mRNA with a 5' triphosphate when microinjected into frog oocytes or expressed in mouse cells in tissue culture. Thus, conversion of the pppG structure to a methyl-pppG structure on the 5' end of an mRNA, which is translated in an IRES-dependent pathway, results in severe inhibition of translation. These data show that the 5' end motif of mRNAs plays an important role even in the IRES-mediated mRNA translation.

Influence of culture parameters on paper mill effluent decolourization by a white rot fungus Ganoderma lucidum.

Efficacy of a white rot fungus G. lucidum for reduction of colour of paper mill effluent under various growth conditions was evaluated. G. lucidum cultured in IBME medium supported maximum colour reduction on 18th day of fungal growth. The optimization of growth parameters further improved colour reduction. The 18 day old culture at 4 g/l inoculum concentration resulted in maximum decolourization (89%) of the effluent with pH adjusted to 6.5 at 35 degrees C along with maximum reduction in biological oxygen demand and chemical oxygen demand. Relative contribution of lignin peroxidase and laccase to the decolourization of paper mill effluent by G. lucidum was also observed.

Post-transcriptional adenylation of signal recognition particle RNA is carried out by an enzyme different from mRNA Poly(A) polymerase.

A fraction of the signal recognition particle (SRP) RNA from human, rat, Xenopus, and Saccharomyces cerevisiae cells contains a single post-transcriptionally added adenylic acid residue on its 3'-end; in the case of human SRP RNA, over 60% of the SRP RNA molecules contain a nontemplated adenylic acid residue on their 3'-ends (Sinha, K. M., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853-6859). In this study, we investigated the enzyme that is involved in this 3'-end adenylation of SRP RNA. A U1A protein peptide conjugated to albumin completely inhibited the polyadenylation of a SV40 mRNA by HeLa cell nuclear extract in vitro; however, the 3'-end adenylation of human SRP RNA or Alu RNA, which corresponds to 5' and 3'-ends of SRP RNA, was not affected by this U1A peptide conjugate. SRP RNA from mutant strains of S. cerevisiae with a temperature-sensitive mRNA poly(A) polymerase grown at a restrictive temperature of 37 degrees C also contained a post-transcriptionally added adenylic acid residue just like SRP RNA from wild-type cells and mutant cells grown at permissive temperature of 23 degrees C. In addition, binding of SRP 9/14-kDa protein heterodimer was required for adenylation of Alu RNA in vitro. These lines of evidence, along with other data, show that post-transcriptional adenylation of SRP and Alu RNAs is carried out by a novel enzyme that is distinct from the mRNA poly(A) polymerase, CCA-adding enzyme, and nonspecific terminal transferase.

Accurate 3' end processing and adenylation of human signal recognition particle RNA and alu RNA in vitro.

Human signal recognition particle (SRP) RNA is transcribed by RNA polymerase III and terminates with -GUCUCUUUUOH on its 3' end. Our previous studies showed that the three terminal uridylic acid residues of human SRP RNA are post-transcriptionally removed and a single adenylic acid residue is added, resulting in a 3' end sequence of -GUCUCUAOH (Sinha, K. M., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853-6859). In this study we show that the Alu RNA, corresponding to the 5' and 3' ends of SRP RNA, is also accurately processed and adenylated in vitro. Alu RNAs containing 7 or 11 additional nucleotides on the 3' end were accurately processed and then adenylated. Deletion analysis showed that an 87-nucleotide-long motif comprising of the 5' and 3' ends, including stem IV of the Alu RNA, is sufficient and necessary for the 3' end processing and adenylation. A 73-nucleotide-long construct with deletion of stem IV, required for the binding of SRP 9/14-kDa proteins, was neither processed nor adenylated. The adenylated Alu RNA as well as adenylated SRP RNA were bound to the SRP 9/14-kDa heterodimer and were immunoprecipitated by specific antibodies. A significant fraction of SRP RNA in the nucleoli was found to be processed and adenylated. These data are consistent with nascent SRP and/or Alu RNAs first binding to SRP 9/14-kDa protein heterodimer, followed by the removal of extra sequence on the 3' end and then the addition of one adenylic acid residue in the nucleus, before transport into the cytoplasm.