Green fluorescent protein gene as a tool to examine the efficacy of Agrobacterium-delivered CRISPR/Cas9 reagents to generate targeted mutations in the potato genome

CRISPR/Cas9 has emerged as a simple, yet efficient gene editing tool to generate targeted mutations in desired genes in crops plants. Agrobacterium tumefaciens, a reliable and inexpensive DNA-delivery mechanism into plant cells, has been used for the generation of CRISPR/Cas9-mediated mutations in crop plants, including potato. However, little information is available as to the progression of gene knockout during various stages of culture following the introduction of CRISPR components in this species. In the current study, the green fluorescent protein (gfp) transgene was first introduced in the genome of a potato variety, Yukon Gold. Two GFP-expressing lines, one with a single gfp copy integrated and another with four gfp copies integrated, were subjected to CRISPR/Cas9-mediated mutations in the transgene(s) using three different gRNAs. Disappearance of GFP fluorescence was monitored during the entire culture/regeneration process. Although all three gRNAs successfully knocked out the transgene(s), their efficiencies differed greatly and did not completely match the predicted scores by some guide RNA prediction tools. The nature of mutations in various knockout events was analyzed. Several lines containing four gfp-copies showed four different types of mutations. These findings suggest that it is possible to target all four alleles of a desired native gene in the tetraploid potato.

     

Network-based approaches for linking metabolism with environment

Progress in the reconstruction of genome-wide metabolic maps has led to the development of network-based computational approaches for linking an organism with its biochemical habitat.     

Global Functional Atlas of Escherichia coli Encompassing Previously Uncharacterized Proteins

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a “systems-wide” functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.     

Photostability Issues in Pharmaceutical Dosage Forms and Photostabilization

Photodegradation is one of the major pathways of the degradation of drugs. Some therapeutic agents and excipients are highly sensitive to light and undergo significant degradation, challenging the quality and the stability of the final product. The adequate knowledge of photodegradation mechanisms and kinetics of photosensitive therapeutic entities or excipients is a pivotal aspect in the product development phase. Hence, various pharmaceutical regulatory agencies, across the world, mandated the industries to assess the photodegradation of pharmaceutical products from manufacturing stage till storage, as per the guidelines given in the International Conference on Harmonization (ICH). Recently, numerous formulation and/or manufacturing strategies has been investigated for preventing the photodegradation and enhancing the photostability of photolabile components in the pharmaceutical dosage forms. The primary focus of this review is to discuss various photodegradation mechanisms, rate kinetics, and the factors that influence the rate of photodegradation. We also discuss light-induced degradation of photosensitive lipids and polymers. We conclude with a brief note on different approaches to improve the photostability of photosensitive products.     

Prediction and validation of the unexplored RNA‐binding protein atlas of the human proteome

Detecting protein‐RNA interactions is challenging both experimentally and computationally because RNAs are large in number, diverse in cellular location and function, and flexible in structure. As a result, many RNA‐binding proteins (RBPs) remain to be identified. Here, a template‐based, function‐prediction technique SPOT‐Seq for RBPs is applied to human proteome and its result is validated by a recent proteomic experimental discovery of 860 mRNA‐binding proteins (mRBPs). The coverage (or sensitivity) is 42.6% for 1217 known RBPs annotated in the Gene Ontology and 43.6% for 860 newly discovered human mRBPs. Consistent sensitivity indicates the robust performance of SPOT‐Seq for predicting RBPs. More importantly, SPOT‐Seq detects 2418 novel RBPs in human proteome, 291 of which were validated by the newly discovered mRBP set. Among 291 validated novel RBPs, 61 are not homologous to any known RBPs. Successful validation of predicted novel RBPs permits us to further analysis of their phenotypic roles in disease pathways. The dataset of 2418 predicted novel RBPs along with confidence levels and complex structures is available at http://sparks-lab.org (in publications) for experimental confirmations and hypothesis generation. Proteins 2014; 82:640–647. © 2013 Wiley Periodicals, Inc.