Development and validation of functional marker targeting an InDel in the major rice blast disease resistance gene Pi54 (Pik h )

Rice blast is one of the most devastating diseases affecting the rice crop throughout the world. In molecular breeding for host plant resistance, functional markers are very useful for enhancing the precision and accuracy in marker-assisted selection (MAS) of target gene(s) with minimum effort, time and cost. Pi54 (which was earlier known as Pik ^h ) is one of the major blast resistance genes and has been observed to show resistance against many isolates of the blast pathogen in India. The gene has been cloned through map-based strategy and encodes a nucleotide-binding site?Cleucine-rich repeat (NBS?CLRR) domain-containing protein. In the present study, we carried out allele mining for this gene and identified a 144-bp insertion/deletion (InDel) polymorphism in the exonic region of the gene. A PCR-based co-dominant molecular marker targeting this InDel, named Pi54 MAS, was developed. Pi54 MAS was observed to perfectly co-segregate with blast resistance in a mapping population with no recombinants. Validation of this marker in 105 genotypes which are either susceptible or resistant to rice blast disease showed that the marker is polymorphic in most of the resistant?Csusceptible genotype combinations and is more accurate than the earlier reported markers for Pi54. Hence this functional, co-dominant marker is suggested for routine deployment in MAS of Pi54 in breeding programs.     

Mammary Gland Reprogramming: Metalloproteinases Couple Form with Function

The adult mammary structure provides for the rapid growth, development, and immunological protection of the live-born young of mammals through its production of milk. The dynamic remodeling of the branched epithelial structure of the mammary gland in response to physiological stimuli that allow its programmed branching morphogenesis at puberty, cyclical turnover during the reproductive cycle, differentiation into a secretory organ at parturition, postlactational involution, and ultimately, regression with age is critical for these processes. Extracellular metalloproteinases are essential for the remodeling programs that operate in the tissue microenvironment at the interface of the epithelium and the stroma, coupling form with function. Deregulated proteolytic activity drives the transition of a physiological mammary microenvironment into a tumor microenvironment, facilitating malignant transformation.Milk production is an evolutionary survival strategy that allows the rapid growth and development of live-born young, as well as being a defining trait of mammals. Milk production at parturition and not at other times conserves valuable energy resources of the mother. It also provides immunological protection to the offspring. Thus, the response of the adult mammary structure to changes in systemic hormones as well as locally derived factors is to expand the ductal network into a milk-producing gland rapidly and to tear it down again when its function is no longer required. The origin of the mammary gland during evolution including its link with the immune system has inspired considerable interest but remains speculative (Oftedal 2002; Vorbach et al. 2006; Widelitz et al. 2007; McClellan et al. 2008). Although the branched epithelial structure of the mammary gland varies in composition and complexity among mammals, the alveolar acinus, its cellular secretory unit, and the tubular ducts, which channel milk for delivery through the teat, have been conserved in mammals.The concept of form and function first arose from the study of mammary epithelial cells and has become a cornerstone in biology, because it applies universally to other parenchymal units (Lee et al. 1984). Here, epithelial cells retain apical-basal polarity and shape by establishing physical contacts with the structural matrix and neighboring cells, and build an adequate form to enable their function of milk production in response to appropriate stimuli (Boudreau and Bissell 1998). The mammary gland encounters constant physiological demands during the female lifespan. To maintain its function, it must repeatedly reacquire its fundamental form with the preservation of cell types, ratios, differentiation state, and matrix integrity. This requires remodeling programs that initially allow development of the mammary gland at puberty, cyclical turnover during the reproductive cycle, differentiation into a secretory organ at parturition, postlactational involution, and ultimately, regression with age. In this article, we describe the essential features of remodeling programs that generally operate at the parenchymal-stromal interface in the mammary tissue microenvironment, and highlight the critical role of extracellular proteolysis in coupling form with function. We also discuss how deregulated protease activity facilitates the transition of a physiological mammary microenvironment into a tumor microenvironment.Our understanding of the mammary gland has been enriched by the use of the mouse as an experimental system, and, thus, observations from murine genetic models, loss- and gain-of-function studies, as well as transplantation assays form the basis of this article. Although the mouse has become an integral part of investigations, there are notable differences between human and murine mammary biology (Cardiff and Wellings 1999). The mammary epithelial ductal system in humans differs considerably with respect to its branching pattern, the stromal, adipocyte, and extracellular matrix (ECM) content, as well as the hormonal triggers that provide the mammotrophic stimuli. Therefore species-specific differences must be considered while making generalizations for the mammary gland.     

E phage gene transfection associated to chemotherapeutic agents increases apoptosis in lung and colon cancer cells

The limited ability of conventional therapies to achieve the long-term survival of metastatic lung and colon cancer patients suggests the need for new treatment options. In this respect, genes encoding cytotoxic proteins have been proposed as a new strategy to enhance the activity of drugs, and combined therapies involving such genes and classical antitumoral drugs have been studied intensively. The E gene from phiX174 encodes a membrane protein with a toxic domain that leads to a decrease in tumour cell growth rates. Therefore, in order to improve the anti-tumour effects of currently used chemotherapeutic drugs on cancer cells, we investigated the association of the E suicide gene with these antineoplastic drugs. The E gene has antitumoral effects in both lung and colon cancer cells. In addition, expression of this gene induces ultrastructural changes in lung cancer transfected cells (A-549), although the significance of these changes remains unknown. The effect of combined therapy (gene and cytotoxic therapy) enhances the inhibition of tumour cell proliferation in comparison to single treatments. Indeed, our in vitro results indicate that an experimental therapeutic strategy based on this combination of E gene therapy and cytotoxic drugs may result in a new treatment strategy for patients with advanced lung and colon cancer.     

Validation of the inactivant binary ethylenimine for inactivating rabies virus for veterinary rabies vaccine production.

The rabies vaccine is produced by inactivation of rabies virus propagated on BHK21 cells. In the rabies inactivation process, BEI is added at a final concentration of 1.6 mM to the viral harvest at 37 degrees C, followed by a second dose of BEI at 24 h post-inactivation. Inactivation was confirmed by the mice innocuity test and tissue culture amplification test as per B.P (Vet) 2004. Validation of test procedure is essential as per cGMP requirement. The dose of BEI was validated by using lower and higher concentrations of BEI in inactivation process. The study indicated that BEI at a lower concentration (0.4 mM) was able to inactivate the rabies virus within 30 h and the routine concentration (1.6 mM) of BEI is effective in inactivating rabies virus within 18 h. The amplification test used for confirming the inactivation of the live virus was validated by spiking the sample with different dilutions of pretitrated live rabies virus. The test revealed that the amplification method is sensitive to detect live rabies virus if present in the inactivated sample. The validation of BEI as an inactivant and the amplification test are discussed.     

Drought Induces Oxidative Stress and Enhances the Activities of Antioxidant Enzymes in Growing Rice Seedlings

When rice seedlings grown for 10 and 20 days were subjected to in vitro drought stress of −0.5 and −2.0 MPa for 24 h, an increase in the concentration of superoxide anion (O₂^.−), increased level of lipid peroxidation and a decrease in the concentration of total soluble protein and thiols was observed in stressed seedlings compared to controls. The concentration of H₂O₂ as well as ascorbic acid declined with imposition of drought stress, however glutathione (GSH) concentration declined only under severe drought stress. The activities of total superoxide dismutases (SODs) as well as ascorbate peroxidase (APX) showed consistent increases with increasing levels of drought stress, however catalase activity declined. Mild drought stressed plants had higher guaiacol peroxidase (GPX) and chloroplastic ascorbate peroxidase (c-APX) activity than control grown plants but the activity declined at the higher level of drought stress. The activities of enzymes involved in regeneration of ascorbate i.e. monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were higher in drought stressed plants compared to controls. Results suggest that drought stress induces oxidative stress in rice plants and that besides SOD, the enzymes of ascorbate-glutathione cycle, which have not been studied in detail earlier under stressful conditions, appear to function as important component of antioxidative defense system under drought stress.     

Male sex drive and the masculinization of the genome

Charles Darwin remarked that "males, with their superior strength, pugnacity, armaments, unwieldly passion and love songs, are almost always the more active and most often, the initiators of sexual interactions". Here, we propose that such male sex drive directly impacts the genome by leading to its progressive masculinization--genes that possess sex-specific effects on male fitness accumulate to a much greater extent and are generally more diverged. The larger proportion of male versus female fitness modifiers in combination with stronger sexual selection may generate evolutionary signatures such as a greater sensitivity to male sterility and a paucity of X-linked male-specific genes. Male sex-drive theory complements the female-choice theory of sexual selection and allows for the genetic variation of costly sexual traits to be continuously replenished.     

The institute for genomic research Osa1 rice genome annotation database

We have developed a rice (Oryza sativa) genome annotation database (Osa1) that provides structural and functional annotation for this emerging model species. Using the sequence of O. sativa subsp. japonica cv Nipponbare from the International Rice Genome Sequencing Project, pseudomolecules, or virtual contigs, of the 12 rice chromosomes were constructed. Our most recent release, version 3, represents our third build of the pseudomolecules and is composed of 98% finished sequence. Genes were identified using a series of computational methods developed for Arabidopsis (Arabidopsis thaliana) that were modified for use with the rice genome. In release 3 of our annotation, we identified 57,915 genes, of which 14,196 are related to transposable elements. Of these 43,719 non-transposable element-related genes, 18,545 (42.4%) were annotated with a putative function, 5,777 (13.2%) were annotated as encoding an expressed protein with no known function, and the remaining 19,397 (44.4%) were annotated as encoding a hypothetical protein. Multiple splice forms (5,873) were detected for 2,538 genes, resulting in a total of 61,250 gene models in the rice genome. We incorporated experimental evidence into 18,252 gene models to improve the quality of the structural annotation. A series of functional data types has been annotated for the rice genome that includes alignment with genetic markers, assignment of gene ontologies, identification of flanking sequence tags, alignment with homologs from related species, and syntenic mapping with other cereal species. All structural and functional annotation data are available through interactive search and display windows as well as through download of flat files. To integrate the data with other genome projects, the annotation data are available through a Distributed Annotation System and a Genome Browser. All data can be obtained through the project Web pages at http://rice.tigr.org.     

Apoptosis of airway epithelial cells in response to meconium

Meconium aspiration syndrome (MAS) is common among newborn children but its mechanism is unclear. The syndrome is known to produce a strong inflammatory reaction in the lungs resulting in massive cell death. In this work we studied lung cell death by apoptosis after meconium aspiration in forty two-week-old rabbit pups. Analyzing lung samples by ISEL-DNA end labeling demonstrated the specific spread of apoptotic bodies throughout the lungs. These bodies were shrunken and smaller in size compared to normal cells and many of them were lacking cell membranes. About 70% of all apoptotic bodies were found among the airway epithelium cell eight hours after meconium instillation. In comparison, among lung alveolar cells, only about 20% cells were apoptotic in the same animals. In meconium-treated lungs and A549 cells, a significant increase of angiotensinogen mRNA and Caspase-3 expression were observed. The pretreatment of cells with Caspase-3 inhibitor ZVAD-fmk significantly inhibited meconium-induced lung cell death by apoptosis. These findings demonstrate the apoptotic process in meconium-instilled lungs or A549 cells in culture. Our results show lung airway epithelial and A549 cell apoptosis after meconium instillation. We suggest that studies of lung airway epithelial cell death are essential to understanding the pathophysiology of MAS and may present a key point in future therapeutic applications.