Selection of stable housekeeping genes for gene expression studies in different varieties of black pepper (Piper nigrum L.)

Real Time quantitative PCR (RT-qPCR) is a widely used technique to study the transcript level modulation of genes during developmental stages of crop plants as well as in stress responses. Suitable reference genes have not been validated in many plants including black pepper. In this study, expression stability of six commonly used housekeeping genes viz., actin, ?-tubulin, elongation factor, initiation factor, ubiquitin and glyceraldehyde 3- phosphate dehydrogenase were evaluated by RT-qPCR during the growth of the black pepper inflorescence of varieties viz., Panniyur 1, Karimunda and Thekken. The results were analyzed using geNorm and Normfinder statistical algorithms. Stable reference gene is critical for the accurate normalization of target gene data in RT-qPCR. In this study actin, elongation factor and initiation factor were identified as the most stable housekeeping gene in different black pepper varieties viz., Thekken, Panniyur 1 and Karimunda respectively. Actin in combination with GAPDH and elongation factor were obtained as optimal reference genes for Thekken. It is the first report on identification of stable housekeeping gene in different varieties of black pepper and can aid in expression studies in black pepper for yield improvement. The study will aid in normalization of gene expression studies in different varieties of black pepper

A Rapid Protocol for Somatic Embryogenesis Mediated Regeneration in Banana (Musa Spp.) Cv. Nendran

A simple and rapid protocol for somatic embryogenesis in banana cv. Nendran (AAB) using immature male flowers (IMF) has been developed. The IMF produced palewhite to yellow, globular embryogenic callus on MS medium supplemented with BA (0.05 - 0.50mgL-1) and picloram (0.50 - 2.00mgL-1) with explant response of to 30 per cent. Addition of ascorbic acid (20mgL-1) and Gelrite© (0.45 per cent) to callus induction medium reduced interference from phenolic exudation. Embryogenesis was induced (33.3 to 60 per cent) on semisolid (0.30 per cent Gelrite©) MS medium supplemented with BA 2mgL-1 + IAA 0.5mgL-1. The somatic embryos showed 60-80 per cent germination on half- strength semisolid MS medium with BA 2mgL-1 + IAA 0.5mgL-1. Transfer of germinated embryos to semisolid MS medium supplemented with BA 2mgL-1 + NAA 1mgL-1under 14 h light /8h dark photoperiod resulted in hundred percent conversion to plantlets. This protocol takes merely 6 months for producing plantlets from immature flower buds through somatic embryogenesis, without any intermediate liquid cultures

Improved transformation of Agrobacterium assisted by silver nanoparticles

In transgenic plant development, the low transformation efficiency of Agrobacterium with exogenous DNA is the major constraint, and hence, methods to improve its transformation efficiency are needed. Recently, nanoparticle-mediated gene transfer has evolved as a key transformational tool in genetic transformation. Since silver nanoparticles (AgNPs) can induce pores on the cell membrane, their efficacy in the improvement of conventional calcium chloride freeze-thaw technique of transformation of Agrobacterium was explored in this study. Agrobacterium cells in the exponential growth phase were exposed to different concentrations of AgNPs (0.01, 1, 5, 10, and 20 mg/l), and the half-maximal effective concentration (EC50) was determined via Probit analysis using the SPSS software. Transformation efficiency of AgNPs alone and in combination with calcium chloride was compared with that of the conventional calcium chloride freeze-thaw technique. AgNPs at a concentration of 0.01 mg/l in combination with calcium chloride (20 mM) showed a ten fold increase in the transformation efficiency (3.33 log CFU (colony-forming unit/microgram) of DNA) of Agrobacterium tumefaciens strain EHA 105 with plasmid vector pART27 compared with the conventional technique (2.31 log CFU/μg of DNA). This study indicates that AgNPs of size 100 nm can eliminate the freeze-thaw stage in the conventional (Agrobacterium transformation technique, with a 44% improvement in efficiency. The use of AgNPs (0.01 mg/l) along with 20 mM calcium chloride was found to be an economically viable method to improve the transformation of Agrobacterium with exogenous plasmid DNA

Algicidal Effects of Green Synthesized Silver Nanoparticles using Tinospora cordifolia on Chlamydomonas reinhardtii

Eco-friendly anti-algal agents are in demand for preventing the growth of unwanted algae. Green synthesized nanoparticles exhibit antimicrobial properties and have been used as a better alternative against chemical and physical processes. In the present study, treatment of silver nitrate with leaf extracts (5% w/v) of Tinospora cordifolia, a plant with proven antimicrobial effects, exhibited UV-visible absorption maxima between 440-460 nm after 1h indicating bioreduction of silver to nanoparticles. The green synthesised silver nanoparticles (5 mgl-1) exhibited inhibition zones against Chlamydomonas reinhardtii in in vitro agar assays. Treatment with green synthesised silver nanoparticles during exponential phase of algal growth resulted in significant reduction in algal population, carbohydrate, protein and chlorophyll contents confirming the anti-algal potential. This is the first report on the growth inhibitory potential of green synthesised silver nanoparticles against green algae

Productivity and nitrogen use efficiency of rice under conventional and organic nutrition

The current study demonstrates the influence of conventional and organic nutrient management practices on nitrogen use efficiency, growth, yield, and physiological and biochemical parameters in four rice varieties, namely, Jaiva, Ezhome 2, Jyothi and Uma. Growth parameters, grain yield per hill, and physiological and biochemical parameters were higher under conventional management for all rice varieties. Although the nitrogen use efficiency of each variety varied significantly with nutrient management practices, the variation was least in Jaiva (23.8%), which is the organic rice variety released by Kerala Agricultural University. The rice varieties Jaiva and Ezhome 2 showed consistency in the grain weight per panicle under both conventional (Jaiva- 4.57 g, Ezhome 2- 5.86 g) and organic (Jaiva, 4.24 g, Ezhome 2, 4.54 g) management. The soil nitrogen content at the tillering stage (0.66**) showed a significantly higher positive correlation with nitrogen use efficiency under organic management. The results of the study provide a better understanding of factors that can lead to a sustained yield in organic rice production in terms of nitrogen use efficiency

Antimicrobial resistance and virulence characteristics of Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio harveyi from natural disease outbreaks of marine/estuarine fishes

Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio harveyi are the causative agents of the most severe diseases of marine and brackish aquaculture systems. These are also associated with serious ailments in humans. The present paper unravels the virulence features/genes and antimicrobial resistance (AMR) phenotypes/genes of these Vibrio spp. isolated from natural disease outbreaks of marine/estuarine fishes of India for the first time. Results on virulence features showed that V. vulnificus infected fish can pose public health risk. While, it has been found clinically that V. parahaemolyticus without tdh and trh genes are pathogenic to fish, even though they are reported to be, not associated with human diseases. It was significant to note that virulence features of V. harveyi were induced at increased salinity. Analysis based on the percentage prevalence of susceptible isolates and variation coefficient of zone diameters categorized 17 antibiotics in terms of their efficiency against each fish pathogenic species. Multiple antibiotic resistance index (MARi) of the isolates ranged between 0.058 and 0.47. Results on MARi and percentage of multidrug resistance strains indicated that >50% of the isolates were from low antibiotic usage area. The study generated tetH sequence from V. parahaemolyticus for the first time, and the sequence revealed high identity to that of clinical strains. Presence of tetB/ tetH gene was identified as the predictor for the resistance against the first generation tetracycline, the most commonly used antibiotic against Vibrio spp. in aquaculture practices. The data on associations between AMR features predicted certain cross-resistance between antimicrobials within Vibrio spp. Altogether, the paper serves as the baseline for epizootic tracking of public health significant vibrios from diseased fishes, to devise practical guidelines for antibiotic use and to formulate efficient control measures against three Vibrio spp. in aquaculture, targeting final applications in the implementation of national green and healthy aquaculture practices

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

Society of Dermatology Hospitalists supportive care guidelines for the management of Stevens-Johnson syndrome/toxic epidermal necrolysis in adults

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening conditions with high morbidity and mortality. Supportive care management of SJS/TEN is highly variable. A systematic review of the literature was performed by dermatologists, ophthalmologists, intensivists, and gynecologists with expertise in SJS/TEN to generate statements for supportive care guideline development. Members of the Society of Dermatology Hospitalists with expertise in SJS/TEN were invited to participate in a modified, online Delphi-consensus. Participants were administered 9-point Likert scale questionnaires regarding 135 statements. The RAND/UCLA Appropriateness Method was used to evaluate and select proposed statements for guideline inclusion; statements with median ratings of 6.5 to 9 and a disagreement index of ≤1 were included in the guideline. For the final round, the guidelines were appraised by all of the participants. Included are an evidence-based discussion and recommendations for hospital setting and care team, wound care, ocular care, oral care, urogenital care, pain management, infection surveillance, fluid and electrolyte management, nutrition and stress ulcer prophylaxis, airway management, and anticoagulation in adult patients with SJS/TEN

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention