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

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

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

High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding.

Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called "MultiCLEM," allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.This work was financially supported by grants from the Deutsche Forschungsgemeinschaft (SFB1129 Z2 to J.A.G. Briggs), EMBL (to J.A.G. Briggs), the Medical Research Council (MC_UP_1201/16 to J.A.G. Briggs), and the German Ministry of Education and Research (031A605 to K.R. Patil). The Schuldiner laboratory is supported by the European Research Council CoG 646604 Peroxisystem, the Deutsche Forschungsgemeinschaft (grant SFB1190 and a Deutsch-Israelische Projektkooperation [DIP] collaborative grant). N. Gabrielli was supported by the EMBL interdisciplinary postdoctoral program. M. Schuldiner is an incumbent of the Dr. Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics

Annotated chemical patent corpus: A gold standard for text mining

Exploring the chemical and biological space covered by patent applications is crucial in early-stage medicinal chemistry activities. Patent analysis can provide understanding of compound prior art, novelty checking, validation of biological assays, and identification of new starting points for chemical exploration. Extracting chemical and biological entities from patents through manual extraction by expert curators can take substantial amount of time and resources. Text mining methods can help to ease this process. To validate the performance of such methods, a manually annotated patent corpus is essential. In this study we have produced a large gold standard chemical patent corpus. We developed annotation guidelines and selected 200 full patents from the World Intellectual Property Organization, United States Patent and Trademark Office, and European Patent Office. The patents were pre-annotated automatically and made available to four independent annotator groups each consisting of two to ten annotators. The annotators marked chemicals in different subclasses, diseases, t

RANTES/CCL5 and risk for coronary events: Results from the MONICA/KORA Augsburg case-cohort, Athero-express and CARDIoGRAM studies

Background: The chemokine RANTES (regulated on activation, normal T-cell expressed and secreted)/CCL5 is involved in the pathogenesis of cardiovascular disease in mice, whereas less is known in humans. We hypothesised that its relevance for atherosclerosis should be reflected by associations between CCL5 gene variants, RANTES serum concentrations and protein levels in atherosclerotic plaques and risk for coronary events. Methods and Findings: We conducted a case-cohort study within the population-based MONICA/KORA Augsburg studies. Baseline RANTES serum levels were measured in 363 individuals with incident coronary events and 1,908 non-cases (mean follow-up: 10.2±

The Cholecystectomy As A Day Case (CAAD) Score: A Validated Score of Preoperative Predictors of Successful Day-Case Cholecystectomy Using the CholeS Data Set

Background Day-case surgery is associated with significant patient and cost benefits. However, only 43% of cholecystectomy patients are discharged home the same day. One hypothesis is day-case cholecystectomy rates, defined as patients discharged the same day as their operation, may be improved by better assessment of patients using standard preoperative variables. Methods Data were extracted from a prospectively collected data set of cholecystectomy patients from 166 UK and Irish hospitals (CholeS). Cholecystectomies performed as elective procedures were divided into main (75%) and validation (25%) data sets. Preoperative predictors were identified, and a risk score of failed day case was devised using multivariate logistic regression. Receiver operating curve analysis was used to validate the score in the validation data set. Results Of the 7426 elective cholecystectomies performed, 49% of these were discharged home the same day. Same-day discharge following cholecystectomy was less likely with older patients (OR 0.18, 95% CI 0.15–0.23), higher ASA scores (OR 0.19, 95% CI 0.15–0.23), complicated cholelithiasis (OR 0.38, 95% CI 0.31 to 0.48), male gender (OR 0.66, 95% CI 0.58–0.74), previous acute gallstone-related admissions (OR 0.54, 95% CI 0.48–0.60) and preoperative endoscopic intervention (OR 0.40, 95% CI 0.34–0.47). The CAAD score was developed using these variables. When applied to the validation subgroup, a CAAD score of ≤5 was associated with 80.8% successful day-case cholecystectomy compared with 19.2% associated with a CAAD score >5 (p < 0.001). Conclusions The CAAD score which utilises data readily available from clinic letters and electronic sources can predict same-day discharges following cholecystectomy

Improved transformation of <i>Agrobacterium</i> assisted by silver nanoparticle

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, nanoparticlemediated 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