Variable T-DNA linkage configuration affects inheritance of carotenogenic transgenes and carotenoid accumulation in transgenic indica rice

Transgenics for the expression of β-carotene biosynthetic pathway in the endosperm were developed in indica rice background by introducing phytoene synthase (psy) and phytoene desaturase (crtI) genes through Agrobacterium-mediated transformation, employing non-antibiotic positive selectable marker phosphomannose isomerase (pmi). Twenty-seven transgenic lines were characterized for the structural organization of T-DNA inserts and the expression of transgenes in terms of total carotenoid and β-carotene accumulation in the endosperm. Ten lines were also studied for the inheritance of transgenic loci to the T₁ progenies. Copy number and sites of integration of the transgenes ranged from one to four. Almost 50% of the transgenic lines showed rearrangement of T-DNA inserts. However, most of the rearrangements occurred in the crtI expression cassette which is adjacent to the right T-DNA border. Differences in copy numbers of psy and crtI were also observed indicating partial T-DNA integration. Beyond T-DNA border transfer was also detected in 25% of the lines. Fifty percent of the lines studied showed single Mendelian locus inheritance, while two lines showed bi-locus inheritance in the T₁ progenies. Some of the lines segregating in 3:1 ratio showed two sites of integration on restriction digestion analysis indicating that the T-DNA insertion sites were tightly linked. Three transgenic lines showed nonparental types in the segregating progenies, indicating unstable transgenic locus. Evidences from the HPLC analysis showed that multiple copies of transgenes had a cumulative effect on the accumulation of carotenoid in the endosperm. T₁ progenies, in general, accumulated more carotenoids than their respective parents, the highest being 6.77 μg/g of polished seeds. High variation in the carotenoid accumulation was observed within the T₁ progenies which could be attributed to the variation in the structural organization and expression of transgenes, minor variations in the genetic background within the progeny plants, or differences in the plant microenvironments. The study identified lines worthy of further multiplication and breeding based on transgene structural integrity in the segregating progeny and high expression levels in terms of the β-carotene accumulation.     

Expression in a recombinant murid herpesvirus 4 reveals the in vivo transforming potential of the K1 open reading frame of Kaposi's sarcoma-associated herpesvirus

Murid herpesvirus 4 (commonly called MHV-68) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV) and provides an excellent model system for investigating gammaherpesvirus-associated pathogenesis. MHV-76 is a naturally occurring deletion mutant of MHV-68 that lacks 9,538 bp of the left end of the unique portion of the genome encoding nonessential pathogenesis-related genes. The KSHV K1 protein has been shown to transform rodent fibroblasts in vitro and common marmoset T lymphocytes in vivo. Using homologous recombination techniques, we successfully generated recombinants of MHV-76 that encode green fluorescent protein (MHV76-GFP) and KSHV K1 (MHV76-K1). The replication of MHV76-GFP and MHV76-K1 in cell culture was identical to that of MHV-76. However, infection of BALB/c mice via the intranasal route revealed that MHV76-K1 replicated to a 10-fold higher titer than MHV76-GFP in the lungs at day 5 postinfection (p.i.). We observed type 2 pneumocyte proliferation in areas of consolidation and interstitial inflammation of mice infected with MHV76-K1 at day 10 p.i. MHV76-K1 established a 2- to 3-fold higher latent viral load than MHV76-GFP in the spleens of infected mice on days 10 and 14 p.i., although this was 10-fold lower than that established by wild-type MHV-76. A salivary gland tumor was present in one of four mice infected with MHV76-K1, as well as an increased inflammatory response in the lungs at day 120 p.i. compared with that of mice infected with MHV-76 and MHV76-GFP.     

Spatial Distribution of Heavy Metals in Soil and Flora Associated with the Glass Industry in North Central India: Implications for Phytoremediation

The effect of the glass industry on soil metal characterization was assessed at five test sites at five successive distances in a semi-arid area. A comprehensive profile of Zn, Cd, Pb, Ni, Cu, and As levels in soils was obtained. The spatial distribution patterns of integrated contamination indices for these metals show a similar decreasing trend in distribution as we move further from the industrial cluster. There was significant correlation among individual heavy metal concentrations in the soil samples. Integrated contamination indices indicate that 64% of the sites were in the high contamination range and 28% were in the moderate contamination range. A statistically significant difference (P ≤ 0.001) was obtained for each metal on comparing mean metal content among soil samples. Phytoremedial potential of 12 native plant species was also evaluated. Individual elements displayed remarkably different patterns of accumulation in soils as well as plants. Plants established limited capabilities in mobilizing Zn, Pb, Ni, and Cu in the root zone. While Cd, Cu, As, Zn and Pb were predominantly partitioned in shoots, Ni was equally partitioned between shoots and roots. Interestingly, some plants showed a different partitioning trend at higher concentrations of different metals compared to lower concentrations. Potential species for phytoremediation include Calotropis procera (Milk weed), Chenopodium murale (Goosefoot),Poa annua (Annual bluegrass) and Datura stramonium (Thorn apple). None of the species showed phytoremedial potential for Ni and Cu.     

DNA barcoding,microarrays and next generation sequencing: recent tools for genetic diversity estimation and authentication of medicinal plants

DNA barcoding, microarray technology and next generation sequencing have emerged as promising tools for the elucidation of plant genetic diversity and its conservation. They are proving to be immensely helpful in authenticating the useful medicinal plants for herbal drug preparations. These newer versions of molecular markers utilize short genetic markers in the genome to characterize the organism to a particular species. This has the potential not only to classify the known and yet unknown species but also has a promising future to link the medicinally important plants according to their properties. The newer trends being followed in DNA chips and barcoding pave the way for a future with many different possibilities. Several of these possibilities might be: characterization of unknown species in a considerably less time than usual, identification of newer medicinal properties possessed by the species and also updating the data of the already existing but unnoticed properties. This can assist us to cure many different diseases and will also generate novel opportunities in medicinal drug delivery and targeting.     

Pseudaulacaspis cockerelli (Cooley) hyperparasitizing Dendrophthoe falcata (L.f) Ettingsh

Here, we first report hyperparasitism of Honey Suckeled Mistletoe, Dendrophthoe falcata var. falcata (Loranthaceae) by Pseudaulacaspis cockerelli (Diaspididae). The infected mistletoe was found parasitising branches of the Senna siamea host. Pear-shaped white spots were observed on abaxial and adaxial leaf surfaces, stem and few on the haustorial surfaces of the mistletoe. Close inspection revealed a yellowish-brown spot on individual white spots. These were identified on the basis of morphological features as Cockerell Scales hidden in the white-spot like self-secreted waxy armours. No incidence of such an infection was seen on any part of the mistletoe host, Senna siamea.     

Liposomal budesonide for dry powder inhaler: Preparation and stabilization

The purpose of the study was to prepare stable liposomally entrapped budesonide (BUD) for a dry powder inhaler (DPI) formulation. BUD liposomes composed of egg phosphatidyl choline and cholesterol were prepared by lipid film hydration technique and sonicated to have the desired size (<5 μm). A rapid method was used for separation of free drug by centrifugation at a lower centrifugal force (G value). Liposomal dispersion was subjected to lyophilization after blending BUD with cryoprotectant in varying bulk and mass ratios, and percent drug remaining entrapped after lyophilization was optimized. Comparative drug retention studies on storage of DPI formulations were carried out in accordance with International Conference on Harmonization guidelines. Critical relative humidity of the formulations was determined and reported as one of the manufacturing controls. Sucrose was found to be the most effective cryoprotectant when present on both sides of the lamellae of liposomes in a bulk strength of 500 mM and mass ratio of lipid:sugar; 1∶10. Blending of sorbolac before lyophilization showed better retention of encapsulated drug (95.59%). The respirable fraction of the product (20.69±1.50%) was comparable with that of the control (26.49±1.52%), suggesting that the liposomal BUD can be successfully delivered throughout the broncho-pulmonary tree. The findings demonstrate that liposome of BUD can be prepared with a high entrapment value, stabilized by lyophilization, and delivered as an aerosolized DPI. The stability studies of lyophilized product suggests a shelf-life of one year when stored under refrigeration (2°C–8°C).     

FR-CAPTCHA: CAPTCHA Based on Recognizing Human Faces

A Completely Automated Public Turing test to tell Computers and Humans Apart (CAPTCHA) is designed to distinguish humans from machines. Most of the existing tests require reading distorted text embedded in a background image. However, many existing CAPTCHAs are either too difficult for humans due to excessive distortions or are trivial for automated algorithms to solve. These CAPTCHAs also suffer from inherent language as well as alphabet dependencies and are not equally convenient for people of different demographics. Therefore, there is a need to devise other Turing tests which can mitigate these challenges. One such test is matching two faces to establish if they belong to the same individual or not. Utilizing face recognition as the Turing test, we propose FR-CAPTCHA based on finding matching pairs of human faces in an image. We observe that, compared to existing implementations, FR-CAPTCHA achieves a human accuracy of 94% and is robust against automated attacks.     

In silico characterization,and expression analysis of rice golden 2-like (OsGLK) members in response to low phosphorous

Molecular Biology Reports - The availability of phosphorus (P) affects productivity of rice. Under acidic soil conditions (pH?&lt;?5.5), P is rapidly immobilized in the soil....