A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Genomic-Assisted Enhancement in Stress Tolerance for Productivity Improvement in Sorghum

Sorghum [Sorghum bicolor (L.) Moench], the fifth most important cereal crop in the world after wheat, rice, maize, and barley, is a multipurpose crop widely grown for food, feed, fodder, forage, and fuel, vital to the food security of many of the world’s poorest people living in fragile agroecological zones. Globally, sorghum is grown on ~42 million hectares area in ~100 countries of Africa, Asia, Oceania, and the Americas. Sorghum grain is used mostly as food (~55%), in the form of flat breads and porridges in Asia and Africa, and as feed (~33%) in the Americas. Stover of sorghum is an increasingly important source of dry season fodder for livestock, especially in South Asia. In India, area under sorghum cultivation has been drastically come down to less than one third in the last six decades but with a limited reduction in total production suggesting the high-yield potential of this crop. Sorghum productivity is far lower compared to its genetic potential owing to a limited exploitation of genetic and genomic resources developed in the recent past. Sorghum production is challenged by various abiotic and biotic stresses leading to a significant reduction in yield. Advances in modern genetics and genomics resources and tools could potentially help to further strengthen sorghum production by accelerating the rate of genetic gains and expediting the breeding cycle to develop cultivars with enhanced yield stability under stress. This chapter reviews the advances made in generating the genetic and genomics resources in sorghum and their interventions in improving the yield stability under abiotic and biotic stresses to improve the productivity of this climate-smart cereal

Functional imaging using fluorine ((19)F) MR methods: basic concepts

Kidney-associated pathologies would greatly benefit from noninvasive and robust methods that can objectively quantify changes in renal function. In the past years there has been a growing incentive to develop new applications for fluorine ((19)F) MRI in biomedical research to study functional changes during disease states. (19)F MRI represents an instrumental tool for the quantification of exogenous (19)F substances in vivo. One of the major benefits of (19)F MRI is that fluorine in its organic form is absent in eukaryotic cells. Therefore, the introduction of exogenous (19)F signals in vivo will yield background-free images, thus providing highly selective detection with absolute specificity in vivo. Here we introduce the concept of (19)F MRI, describe existing challenges, especially those pertaining to signal sensitivity, and give an overview of preclinical applications to illustrate the utility and applicability of this technique for measuring renal function in animal models. This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This introduction chapter is complemented by two separate chapters describing the experimental procedure and data analysis

Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein

Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively ‘regulating’ connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and other tissues, and this connexin’s role in therapeutic and adverse effects of statins in a range of disease states

Transgenic mouse models support a protective role of type I IFN response in SARS-CoV-2 infection-related lung immunopathology and neuroinvasion

Summary: Type I interferon (IFN-I) response is the first line of host defense against invading viruses. In the absence of definite mouse models, the role of IFN-I in SARS-CoV-2 infection remains perplexing. Here, we develop two mouse models, one with constitutively high IFN-I response (hACE2; Irgm1−/−) and the other with dampened IFN-I response (hACE2; Ifnar1−/−), to comprehend the role of IFN-I response. We report that hACE2; Irgm1−/− mice are resistant to lethal SARS-CoV-2 infection. In contrast, a severe SARS-CoV-2 infection along with immune cell infiltration, cytokine storm, and enhanced pathology is observed in the lungs and brain of hACE2; Ifnar1−/− mice. The hACE2; Irgm1−/−Ifnar1−/− double-knockout mice display loss of the protective phenotype observed in hACE2; Irgm1−/− mice, suggesting that heightened IFN-I response accounts for the observed immunity. Taking the results together, we demonstrate that IFN-I protects from lethal SARS-CoV-2 infection, and Irgm1 (IRGM) could be an excellent therapeutic target against SARS-CoV-2