Development and Validation of UPLC-MS/MS Method for Rapid Simultaneous Determination of Levothyroxine and Liothyronine in Human Serum

A simple ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method was developed and fully validated to simultaneously determine levothyroxine (LT4) and liothyronine (LT3) in human serum. Sample preparation was done through protein precipitation with acetonitrile. HyPURITY C18 column was selected to achieve rapid separation for LT4 and LT3 within 4 min. Electrospray ionization (ESI) under multiple reaction monitoring (MRM) was used to monitor the ion transitions for LT4 (m/z 777.54→731.52), LT3 (m/z 651.64→ 605.65) and internal standard LT4-D3 (m/z 780.53 →734.19), operating in the positive ion mode. The method was proved to be accurate (82.35% to 113.56%) and precise (0.73% to 8.28%) over concentration range of 50.37 ng/ml – 300.13 ng/ml for LT4 and 0.5 ng/ml – 50.37 ng/ml for LT3. The validated method could be applied for pharmacokinetic study or bioequivalence testing of combination products of LT4 and LT3. Keywords: Levothyroxine; Liothyronine; Ultra Performance Liquid Chromatographic; Mass Spectrometry; Human Seru

Food Storage, Processing and Genetic Stability Studies of Bacillus (Heyndrickxia) coagulans BCP92 (MTCC 25460)

Background and Objective: Bacillus coagulans are spore-forming probiotics that provide health benefits when consumed in adequate amounts. Therefore, they can be added to functional foods to enhance their nutritional values. The aim of the present study was to investigate stability of Bacillus coagulans BCP92 in various functional foods during food processing and storage conditions as well as genetic stability study of the strain using DNA fingerprinting method. Material and Methods: Bacillus coagulans BCP92 was incorporated into a range of functional foods and beverages such as instant coffee, tea, sweet corn soups, oatmeal, upma, gummies, brownies, ice creams, non-alcoholic beverages, chocolates, peanut butter and shrikhand. Viability of the bacteria was assessed using pour plate method under various processing and storage conditions. Genetic stability of B. coagulans was assessed using DNA fingerprinting. Results and Conclusion: The viability was shown in food processing conditions of teas (99.97%), coffees (99.45%), sweet corn soups (99.36%), oatmeal (98.81%), upma (99.57%), gummies (99.67%) and brownies (98.14%). In food-storage conditions, relative viability was as follows: fruit juices (98.91%), lassi (98.72%), energy drinks (98.70%), cold coffees (99.29%), milk chocolates (99.87%), white chocolates (100.13%), dark chocolates (99.20%), shrikhand (99.04%), ice creams (99.45%), and peanut butters (98.32%). Furthermore, DNA fingerprinting showed genetic stability of the probiotic B. coagulans BCP92. In conclusion, B. coagulans BCP92 has shown good viability in various food processing and storage conditions. Moreover, it is genetically stable, thus making it a good candidate for addition to functional foods. Introduction   The potential health benefits of probiotics, which involve improving gut microflora, have been a topic of scientific interests for many years. However, it has only recently begun to receive scientific assessments [1]. Probiotics are living microorganisms that confer health benefits to the host when consumed in sufficient quantities [2]. Several studies have revealed that consumption of probiotics decreases risks of antibiotic-associated diarrhoea [3], symptoms of irritable bowel syndrome (IBS) [4], risks of lactose intolerance [5] and constipation [6] and risks of carcinogens and helps in decreases of obesity and enhancement of immune responses and decreases of cholesterol levels [7]. To confer the specific health benefits of incorporated probiotics in food products, the recommended adequate levels of probiotics (106–107 CFU.ml-1) should be provided in the final products [8]. Preserving viability of the probiotic cultures in foods until the end of shelf life is an important criterion for providing effective probiotic food products [9]. It has been observed that the viability of most probiotic bacteria is lost during processing and storage conditions. Furthermore, only a limited number of bacteria can survive harsh conditions of the gastrointestinal tract (GIT) [10]. An effective way to deliver probiotic bacteria includes incorporation of them into food products, making it easier for the consumers to maintain their gut health, considering that many people choose probiotic food products instead of probiotic capsules and pills [11]. Awareness of the importance of maintaining gut health has led to great increases in demands for probiotic foods. Probiotics are either used as starter cultures in combination with traditional starters or alone and incorporated into dairy products, where many functional characterisations are improved by the addition of probiotics. However, there are several challenges linked to function and stability of the probiotics in dairy products [12]. It is generally accepted that probiotic products should include a minimum concentration of 106–107 CFU.g-1 or CFU.ml-1 and a total concentration should be 108–109 CFU.g-1 consumed daily to exert the probiotic effects [13,14]. Numerous probiotic foods include dairy products such as ice creams, fermented milks, frozen desserts, yoghurts, cheeses, milk powders and cheesecakes, [12,15,16], as well as non-dairy products such as oat drinks, commercial fruit juices, soya milks [17–19]. However, spore-based Bacillus-based probiotics have shown higher survival rates than those others have. In a study by Hashemi et al. [20], it was observed that survival rates of the samples with B. coagulans were higher than those with Lactobacillus acidophilus. Similarly, Soares et al. detected that the Bacillus strains, which included probiotic characteristics, showed a greater viability than that the probiotic strains of Bifidobacterium and Lactobacillus did [21]. Stability of probiotics is always a concern during the storage and processing conditions. The present study focused on the stability assessment of Bacillus coagulans BCP92 under various food processing and storage conditions to assess its potential as a probiotic addictive for enhancing the nutritional values of food products. Furthermore, assessment of genetic stability of the strain was carried out using DNA fingerprinting method. Materials and Methods 2.1 Microbial culture Bacterial spores of Bacillus coagulans BCP92 (MTCC 25460) used in this study were produced at Pellucid Lifesciences, India. Concentration of the prepared B. coagulans spores was 150 billion CFU.g-1 (11.146 log CFU.g-1). Standard pour plate technique was used to assess the total viable bacterial count. The B. coagulans spores were thoroughly mixed in the food product and incubated at 75 °C for 30 min using water bath, followed by rapid cooling down to below 45 °C. Suspension was serially diluted in sterile peptone water. An appropriate quantity of diluents was poured into a sterile petri plate and mixed with molten glucose yeast extract BC agar (Hi media M2102, India) in triplicate. Plates were incubated at 40 °C for 48–72 h. The mean of these viable counts was expressed as log10 CFU.g-1. All the Chemicals and reagents were purchased from Merck, Germany, and the microbiological media were purchased from Hi Media, India. 2.2 Assessment of the stability of Bacillus coagulans BCP92 under food processing conditions The B. coagulans BCP92 was added to a variety of food products and beverages such as instant coffees, teas, sweet corn soup powders, oatmeal, upma, cornflakes, gummies and brownies to assess its stability under certain processing conditions. The B. coagulans BCP92 stability under food processing conditions was studied by comparing the initial count with that after the processing conditions. 2.2.1 Instant coffees and teas Instant coffee (7.5 g) and tea (1.9 g) powders were mixed with 14 mg each of B. coagulans powder. The prepared mixtures were dissolved in 100 ml of hot water (80–85 °C). Samples from tea and coffee were collected at 0 and 30 min and viable counts of B. coagulans BCP92 were analysed using standard pour plate method. 2.2.2 Sweet-corn soup powders and oatmeal Briefly, B. coagulans BCP92 (14 mg) was mixed uniformly with sweet-corn soup powder (10 g.serving-1) and oatmeal (100 g.serving-1) separately. Mixture was cooked in 150 ml hot water (80–85 °C).  Cooked samples were collected at 0 and 30 min to analyse the viable cell count of B. coagulans BCP92 using pour plate method. 2.2.3 Upma and cornflakes Ready-to-eat breakfast upma and cornflakes were purchased from a local market. Upma (50 g·serving-1) was added into warm water (80–85 °C) and cooked for 2 min. Then, 14 mg of B. coagulans BCP92 were added to the mixture, stirred well and cooked for 2 min. Pour plate method was used to assess viable count of B. coagulans BCP92 in samples of 0 and 30 min. One serve of 100-g cornflakes was mixed with 150 ml of hot milk and cooked for 5 min. Then, 14 mg B. coagulans BCP92 were added to the mixture and set for 5 min. Samples were collected at 0 min and 30 min to assess the viable count of B. coagulans BCP92 using pour plate method. 2.2.4 Gummies All the requirements for gummies such as sugar, flavour, sodium citrate, citric acid, corn syrup, water and pectin were mixed for a batch of gummies for 1 kg material to investigate the bacterial survival under processing conditions. All the ingredients of gummies were mixed and heated to dissolve all the contents. Once all the ingredients dissolved, they were mixed with B. coagulans BCP92 (14 mg.3 g-1) at not greater than 85–90 °C in the final mixture before it solidifies. Viable count of B. coagulans BCP92 was measured in gummies and the molten sample once mixed using pour plate method. 2.2.5 Brownies All the necessary ingredients for preparing brownies such as flour, salt, cocoa powder, eggs, brown sugar, vanilla essence, vegetable oil and butter were mixed thoroughly to prepare the batter, then B. coagulans BCP92 (14 mg.30 g·serving-1) was added to the batter and baked at 175 °C for 22–25 min. Viable count of B. coagulans BCP92 was calculated in the brownies before and after baking using pour plate method. 2.3 Storage stability of Bacillus coagulans BCP92 in various food matrices Stability of B. coagulans BCP92 was assessed under standard food storage conditions for ice creams (-20 °C), peanut butters (22–25 °C), non-alcoholic beverages, chocolates and shrikhand (4 °C) based on the ICH Guideline Q1A(R2) [22]. 2.3.1 Ice creams A batch of ice cream was homogenously mixed with B. coagulans BCP92 (14 mg.100 ml serving-1) and 100 ml of ice cream were dispensed in a sterile ice cream cup. The ice cream was stored at -20 °C for 6 m. The primary bacterial count of the B. coagulans BCP92 was carried out immediately after mixing of B. coagulans BCP92. Samples were collected monthly for enumeration up to 6 m of storage. Pour-plate technique in triplicates was used to carry out the bacterial count. 2.3.2 Non-alcoholic beverages Four various types of commercial beverages of fruit juices, energy drinks, lassi and cold coffees were purchased from a local market. The B. coagulans BCP92 (14 mg.serving-1) was inoculated into four sterile flasks containing fruit juices (100 ml), energy drinks (250 ml), cold coffees (200 ml) and lassi (180 ml). All flasks were sealed and stored at 4 °C. Viability of B. coagulans BCP92 in all beverages was assessed using pour plate method. Stored samples were collected for analysis on Days 0, 30, 60, 90, 120 and 180. All analyses were carried out with three replicates. 2.3.3 Chocolates Three types of chocolates were purchased from a local market, including white, dark and milk chocolates. Chocolate bars were melted by heating at 45–50 °C. Then, B. coagulans BCP92 (14 mg.100 ml serving-1) powder was thoroughly mixed with the molten chocolates. The mixture of chocolate and probiotics was stored at 4 °C. Viability of B. coagulans BCP92 in chocolates was assessed using pour plate method. Stored samples were collected for analysis on Days 0, 30, 60, 90, 120 and 180. All analyses were carried out with three replicates.  2.3.4 Peanut butter Peanut butter was purchased from the local market. Samples (32 g·serving-1) were inoculated with B. coagulans BCP92 (14 mg·serving-1). Peanut butter and probiotic powder were mixed uniformly and stored at room temperature (RT). Then, B. coagulans BCP92 viability was assessed after 0, 30, 60, 90, 120 and 180 days of storage. 2.3.5 Shrikhand Shrikhand was purchased from a local market, added into probiotic B. coagulans BCP92 (14 mg.100 g.serving-1) and stored at 4 °C. Stored samples were collected for analysis on Days 0, 30, 60, 90, 120 and 180. All analyses were carried out with three replicates.  2.4 Genetic stability of Bacillus coagulans BCP92 For the comparisons from two various stages of the production process, primary cultures in the form of VIAL and final product batch samples were used. Generally, DNA was isolated from each sample. Quality of the DNA was assessed on 1.0% agarose gel and a single band of high-molecular weight DNA was observed. Moreover, DNA fingerprinting of the cultures was carried out using rep-PCR method and MSP-PCR fingerprinting. Two types of rep-PCR fingerprinting were applied [23] using BOX (50-CTACGGCAAGGCGACGCTGACG-30) and (GTG)5 primers (5-GTGGTGGTGGTGGTG-3) [24]. Briefly, 20 ul of PCR amplicons were separated on 2% agarose gel and banding patterns were analyzed using Gel-analyzer software. The dendrogram was plotted using unweighted pair-group method and arithmetic averages with correlation levels expressed as proportions of the Pearson correlation coefficient. 2.5 Statistical analysis Viable count of B. coagulans BCP92 was expressed as log10 CFU.serving-1 in food processing conditions and log10 CFU g-1.ml-1 in food storage conditions. All analyses were carried out with three replicates. Results included averages of the three independent determinations. Differences between the two values were calculated using student’s t-test. Level of the significance for all statistical tests was p < 0.05. Results and Discussion 3.1 Stability of Bacillus coagulans BCP92 in food processing conditions  Stability of B. coagulans BCP92 was studied by measuring viability of the bacteria in various food matrices under certain processing conditions (Figure 1). The primary viable count of B. coagulans BCP92 in tea was 9.31 ±0.02 log10 CFU serving-1 and in coffee was 9.38 ±0.02 log10 CFU serving-1. After 30 min, these values were 9.30 ±0.05 and 9.33 ±0.02 log10 CFU serving-1, respectively. The B. coagulans preserved 99.97% of its viability in tea. In instant coffee, viability of B. coagulans after processing was preserved up to 99.45%. Viability of B. coagulans BCP92 was assessed in sweet corn soup and oatmeal during processing. The B. coagulans was incorporated into corn soup powder and oatmeal by adding hot water. In soup preparation, the primary B. coagulans of 9.38 ±0.03 log10 CFU serving-1 preserved a 99.36% viable count after 30 min. The oatmeal primary concentration was 9.25 ±0.03 log10 CFU serving-1 and after 30 min, it preserved 98.81% viability (Figure 1). Upma primary concentration was 9.20 ±0.05 log10 CFU serving-1 and after 30 min, it preserved 99.57% viability. The viability studies on cornflakes showed a primary count of 9.22 ±0.02 log10 CFU.serving-1, and after 30 min, it preserved 99.78% of viability (Figure 1). Viability was studied in gummies as well. For gummies before and after processing, they showed 99.67% of relative viability per gummy. The viable count of B. coagulans BCP92 spores in gummy processing conditions showed slight non-significant  decreases in count (Figure 1). Viability studies in brownies showed that the primary concentration of probiotics in each brownie was 9.22 log10 CFU serving-1. After heating, this showed a 98.14% relative survival rate (Figure 1). Studies have shown use of probiotics in functional foods. Polo et al. [25] reported use of B. coagulans in herbal teas. Majeed et al. [26] reported viability of B. coagulans up to 2 y of shelf life when stored with tea and coffee powders. Kahraman et al. [27] and Miranda et al. [28] studied B. coagulans stability in gummies and showed B. coagulans survivals during production and processing. Majeed et al. [29] reported stability of B. coagulans in various food matrices such as hot fudge toppings, chocolate fudges (97.23%) and peanut butters (99.6%) with viability over 95% as well as its viability in apple juices (99.3%) of baked products. Almada et al. [30] showed that eight strains of Bacillus in various baking, cooking and drying processes affected γ of the Bacillus strains; of which, B. coagulans reported higher resistance. Foods containing spore probiotics are becoming popular due to their resistance to heat processes, low water activity, acidic pH and heat stability [31]. In this study, B. coagulans BCP92 showed high viability in various foods during food processing conditions, with viabilities ranging 98.14–99.97% in food products such as teas, coffees, sweet corn soups, oatmeal, upma, gummies and brownies. 3.2 Storage stability of Bacillus coagulans BCP92 in various food storage conditions Incorporation of B. coagulans BCP92 into a food product was studied to assess viability and stability of B. coagulans BCP92 during storage and its possible use as a food ingredient (Table 1). The relative viability of B. coagulans in ice creams was 99.41%, The primary viable count of B. coagulans in ice creams was 7.32 ±0.04 log10 CFU ml-1 and the final count was 7.28 ±0.06 log10 CFU ml-1 over 6 m of storage (Table 1). Studies show use of ice creams as vehicles for probiotics. Due to exposure of the cells to various stress factors associated with formulation, overrun, melting and storage, losses in viability occur [9]. Fruit juices, energy drinks and cold coffees showed primary counts of 7.34 ±0.01, 6.92 ±0.01 and 7.00 ±0.03 log10 CFU ml-1, respectively. After storage up to 6 m, the preserved viability rates were up to 98.87, 98.74 and 99.23%, respectively (Table 1). Viability of B. coagulans was assessed in various types of chocolates. Results revealed that B. coagulans BCP92 preserved its high viability throughout the entire 180-d storage time. Viability of B. coagulans in milk, white and dark chocolates were 99.99, 100 and 99.16%, respectively (Table 1) Stability studies in shrikhand and lassi showed consistency of B. coagulans BCP92 as the primary concentration of B. coagulans was 7.32 ±0.08 and 7.03 ±0.02 log10 CFU ml-1 and after the study, it preserved its 99.08 and 98.80% relative viabilities, respectively (Table 1). Stability studies in peanut butters demonstrated a good viability of 98.40% from primary 7.76 ±0.02 log10 CFU ml-1 per serving after 6 m of storage (Table 1). Various studies report stability of non-spore-forming probiotics, showing survival of probiotics during storage [32,33]. However, probiotic Bacillus strain showed a higher survival rate [20, 21] than Lactobacillus and Bifidobacterium during storage under GIT conditions when studied in cheeses, pasteurized orange juices and breads [21]. A study with L. acidophilus and B. coagulans in ice creams stored at -18 °C for 90 d showed a higher survival rate in  B. coagulans than L. acidophilus [20]. Marcial-Coba et al. [33] microencapsulated Akkermansia muciniphila and L. casei in dark chocolates. In another study, Cielecka-Piontek et al. [34] demonstrated stability of B. animalis subsp. Lactis, Saccharomyces boulardii and B. coagulans GBI-30, 6086 in chocolates. Similar results were reported by Silva et al. [35] in L. acidophilus LA3 and B. animalis subsp. lactis BLC1, showing the highest viabilities of approximately 7.7 and 7.3 log CFU.g-1 in semisweet chocolates, respectively. Lavrentev et al. [36] showed use of B. coagulans as a starter culture and its viability for 60 d and reported satisfactory results for stability and quality characteristics of the product. Maity et al. reported the B. coagulans stability in various food matrices under processing conditions, including lemon iced teas (99.46%), green teas (98.48%), masala teas (98.96%), lemon teas (99.59%), instant coffees (99.79%), upma (99.89%), corn soups (99.79%) and noodles (99.68%) [37]. The B. coagulans BCP92 preserved its stability over 6 m in foods such as fruit juices, lassi, energy drinks, cold coffees, chocolates, shrikhand, ice creams and peanut butters with relative viabilities ranging 98.32–100.13%. In the present study, non-significant decreases were observed in all the food matrices under food processing and storage conditions, showing the versatile nature of B. coagulans BCP92. 3.3 Genetic stability Samples of “VIAL” (primary sample) and “final product” were provided for the study. Generally, DNA was extracted and DNA fingerprinting was carried out using BOX primers sets, followed by PCR analysis and dendrogram plotting. Genotype of each strain could be differentiated by the distribution of PCR bands and the two samples were closely linked to each other based on DNA fingerprinting patterns in the experiments (Figures 2 and 3). Genomic safety and probiotics attributes showed that B. coagulans BCP92 was safe [38]. Genomic fingerprinting also showed genetic stability of B. coagulans BCP92 in the production cycle; in which, it showed the genetic stability in primary and final samples of production. Majeed et al. reported genetic stability of three various sample batches [24]. Genetic stability studies using DNA fingerprinting verified stability of B. coagulans BCP92. Conclusion The present study reported stability of B. coagulans BCP92 in various food matrices under processing and storage conditions. The B. coagulans BCP92 tolerated the low pH of juices, low-temperature storage and heating during food processing conditions. These findings focused on the potential of these food products as carrier vehicles for the delivery and stability of spore-forming probiotics. The B. coagulans BCP92 was also genetically stable in the production process, which was a positive indication of genetic stability of culture. Hence, this finding suggests use of spore-forming probiotic B. coagulans BCP92 in functional foods for gut health improvement and gastrointestinal disorders. Further studies can be carried out on incorporating B. coagulans BCP92 in food products and assessing them on human subjects to gauge their effects on human health. Additional studies on food supplemented with B. coagulans BCP92 can help deeper understanding of its potential benefits for human health and sensory profiling, ultimately leading to advancements in the field of nutrition and wellness.   Conflict of Interest The authors report no conflict of interest. References Behnsen J, Deriu E, Sassone-Corsi M, Raffatellu M. Probiotics: Properties, examples and specific applications. Cold Spring Harb perspect Med. 2013; 3: a01007

Effect of gamma ray irradiation doses on pollen viability and in-vitro germination in Citrus

Induction of haploid plants in fruit crops through gamma irradiated pollen technique is of paramount importance in modern fruit breeding to reduce the breeding cycle. But the major problem of this technique is the sensitivity of pollen grains to higher irradiation doses. Present work analyzed the viability, in vitro germinability and pollen tube growth of gamma ray irradiated pollens of 2 different Citrus species: C. limetta, and C. sinensis. Both viability and in vitro germination capacity of irradiated pollen decreased gradually with increasing concentrations of irradiation in both the pollen parent; however, at highest irradiation dose (400 Gy), reduction of pollen viability and in vitro germination capacity, as compared to respective controls was recorded minimum in C. limetta (11.07 and 29.78%, respectively). In vitro pollen tube length as measured at 24, 48 and 72 hr after incubation, was found maximum in C. limetta (267.83, 303.24 and 325.57 μm, respectively). Our experiment revealed that pollen of C. limetta has higher resistance to gamma ray irradiation as compared to C. sinensis, hence C. limetta can be employed as more reliable pollen parent in haploidy programme of Citrus

Effect of farmyard manure, organic manure and balanced fertilizers application on the productivity and soil fertility in pearl millet (Pennisetum glaucum)- mustard (Brassica juncea) cropping sequence in sandy loam soil of semi-arid regions

The experiment was conducted at the research farm of Department of Genetics and Plant Breeding, CCS Haryana Agricultural University, Hisar from 2007-08 to 2009-10 to study the effect of farmyard manure and balanced fertilizers on production potential, economic viability and soil properties in the pearl millet [Pennisetum glaucum (L) R. Br.]- mustard [Brassica juncea (L.) Czernj. and Coss.] cropping sequence. The study consisted of 12 treatment combinations of farmyard manure and inorganic fertilizers with micronutrients were laid out in Factorial Randomized Block Design with three replications. The mean data revealed that use of balanced fertilizers (Potash, gypsum, ZnSO4 and FeSO4) along with application of 5.0 tonnes FYM/ha in pearl millet-mustard crop sequence produced 7.5 per cent higher pearl millet grain yield (3.59 tonnes/ha) and 8.2 per cent more mustard seed yield (1.96 tonnes/ha) than no FYM application (3.34 tonnes/ha by pearl millet and 1.81 tonnes/ha by mustard). Among different balanced nutrient treatments; recommended dose of nitrogen and phosphorous for both the crops, i.e. 120 kg N + 60 kg P/ha for pearl millet and 80 kg N + 30 kg P/ha for mustard along with 5.0 tonnes FYM/ha + 20 kg K2O/ha + 200 kg gypsum +10 kg ZnSO4 /ha + 10 kg FeSO4/ha (F5) produced maximum pearl millet grain yield (3.91 tonnes/ha) and mustard seed yield (2.31 t/ha), pearl millet equivalent yield (11.91 t/ha) and gross returns (Rupees93 051/ha) whereas, maximum net returns (RUPEES 44 529 /ha) and B:C ratio (1:93) were observed in the treatment 5.0 t FYM /ha + RD of N and P +20 kg K2O/ha + 200 kg gypsum/ha + 10 kg ZnS04 kg/ha (F4). The F5 treatment decreased pH (7.8) and EC (0.33 ds/m), improved the organic carbon (0.37%), available N (205 kg/ha), P2O5 (17.7 kg/ha), K2O (331 kg/ha), Fe (3.48 ppm), Zn (2.30 ppm) and S (4.30 ppm) status in comparison to values of 8.0 and 0.36. ds/m 0.34%, 196 kg/ha, 17.3 kg/ha, 323.7 kg/ha, 3.36 ppm, 2.15 ppm and 4.45 ppm, respectively in RD of N and P + 20 kg K2O/ha + 200 kg gypsum/ha + 10 kg ZnSO4/ha + 10 kg FeSO4/ha treatment when no FYM was used. The quality traits; protein content in pearl millet, oil content and oil yield were also found superior in the F5 treament than all other combinations of balanced fertilizers with and without 5.0 tonnes FYM/ha

Fission and cluster decay of 76^{76}Sr nucleus in the ground-state and formed in heavy-ion reactions

Calculations for fission and cluster decay of $^{76}Sr$ are presented for this nucleus to be in its ground-state or formed as an excited compound system in heavy-ion reactions. The predicted mass distribution, for the dynamical collective mass transfer process assumed for fission of $^{76}Sr$, is clearly asymmetric, favouring $\alpha$-nuclei. Cluster decay is studied within a preformed cluster model, both for ground-state to ground-state decays and from excited compound system to the ground-state(s) or excited states(s) of the fragments.Comment: 14 pages LaTeX, 5 Figures available upon request Submitted to Phys. Rev.

Avian Influenza (H5N1) Virus of Clade 2.3.2 in Domestic Poultry in India

South Asia has experienced regular outbreaks of H5N1 avian influenza virus since its first detection in India and Pakistan in February, 2006. Till 2009, the outbreaks in this region were due to clade 2.2 H5N1 virus. In 2010, Nepal reported the first outbreak of clade 2.3.2 virus in South Asia. In February 2011, two outbreaks of H5N1 virus were reported in the State of Tripura in India. The antigenic and genetic analyses of seven H5N1 viruses isolated during these outbreaks were carried out. Antigenic analysis confirmed 64 to 256-fold reduction in cross reactivity compared with clade 2.2 viruses. The intravenous pathogenicity index of the isolates ranged from 2.80–2.95 indicating high pathogenicity to chickens. Sequencing of all the eight gene-segments of seven H5N1 viruses isolated in these outbreaks was carried out. The predicted amino acid sequence analysis revealed high pathogenicity to chickens and susceptibility to the antivirals, amantadine and oseltamivir. Phylogenetic analyses indicated that these viruses belong to clade 2.3.2.1 and were distinct to the clade 2.3.2.1 viruses isolated in Nepal. Identification of new clade 2.3.2 H5N1 viruses in South Asia is reminiscent of the introduction of clade 2.2 viruses in this region in 2006/7. It is now important to monitor whether the clade 2.3.2.1 is replacing clade 2.2 in this region or co-circulating with it. Continued co-circulation of various subclades of the H5N1 virus which are more adapted to land based poultry in a highly populated region such as South Asia increases the risk of evolution of pandemic H5N1 strains

International Review and Advisory Committee

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Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy