MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

We demonstrate the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNAmanipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaster toolkit.     

In vitro nuclear egress of herpes simplex virus type 1 capsids

During their life cycles, viruses typically undergo many transport events throughout the cell. These events depend on a variety of both viral and host proteins and are often not fully understood. Such studies are often complicated by asynchronous infections and the concurrent presence of various viral intermediates in the cells, making it difficult to molecularly define each step. In the case of the herpes simplex virus type 1, the etiological agent of cold sores and many other illnesses, the viral particles undergo an intricate series of transport steps during its life cycle. Upon entry by fusion with a cellular membrane, they travel to the host cell nucleus where the virus replicates and assembles new viral particles. These particles then travel across the two nuclear envelopes and transit through the trans-Golgi network before finally being transported to and released at the cell surface. Though viral components and some host proteins modulating these numerous transport events have been identified, the details of these processes remain to be elucidated. To specifically address how the virus escapes the nucleus, we set up an in vitro model that reproduces the unconventional route used by herpes simplex type 1 virus to leave nuclei. This has not only allowed us to clarify the route of capsid egress of the virus but is now useful to define it at the molecular level.     

p38 MAPK-mediated regulation of Xbp1s is crucial for glucose homeostasis

Here we show that p38 mitogen-activated protein kinase (p38 MAPK) phosphorylates the spliced form of X-box binding protein 1 (Xbp1s) on its Thr48 and Ser61 residues and greatly enhances its nuclear migration in mice, whereas mutation of either residue to alanine substantially reduces its nuclear translocation and activity. We also show that p38 MAPK activity is markedly reduced in the livers of obese mice compared with lean mice. Further, we show that activation of p38 MAPK by expression of constitutively active MAP kinase kinase 6 (MKK6Glu) greatly enhances nuclear translocation of Xbp1s, reduces endoplasmic reticulum stress and establishes euglycemia in severely obese and diabetic mice. Hence, our results define a crucial role for phosphorylation on Thr48 and Ser61 of Xbp1s in the maintenance of glucose homeostasis in obesity, and they suggest that p38 MAPK activation in the livers of obese mice could lead to a new therapeutic approach to the treatment of type 2 diabetes.     

UBE4B promotes Hdm2-mediated degradation of the tumor suppressor p53

The TP53 gene (encoding the p53 tumor suppressor) is rarely mutated, although frequently inactivated, in medulloblastoma and ependymoma. Recent work in mouse models showed that the loss of p53 accelerated the development of medulloblastoma. The mechanism underlying p53 inactivation in human brain tumors is not completely understood. We show that ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase, physically interacts with p53 and Hdm2 (also known as Mdm2 in mice). UBE4B promotes p53 polyubiquitination and degradation and inhibits p53-dependent transactivation and apoptosis. Notably, silencing UBE4B expression impairs xenotransplanted tumor growth in a p53-dependent manner and overexpression of UBE4B correlates with decreased expression of p53 in these tumors. We also show that UBE4B overexpression is often associated with amplification of its gene in human brain tumors. Our data indicate that amplification and overexpression of UBE4B represent previously undescribed molecular mechanisms of inactivation of p53 in brain tumors.     

A high-throughput assay for modulators of NNT activity in permeabilized yeast cells

Nicotinamide nucleotide transhydrogenase (NNT) mutant mice show glucose intolerance with impaired insulin secretion during glucose tolerance tests. Uncoupling of the β cell mitochondrial metabolism due to such mutations makes NNT a novel target for therapeutics in the treatment of pathologies such as type 2 diabetes. The authors propose that increasing NNT activity would help reduce deleterious buildup of reactive oxygen species in the inner mitochondrial matrix. They have expressed human Nnt cDNA for the first time in Saccharomyces cerevisiae, and transhydrogenase activity in mitochondria isolated from these cells is six times greater than is seen in wild-type mitochondria. The same mitochondria have partially uncoupled respiration, and the cells have slower growth rates compared to cells that do not express NNT. The authors have used NNT's role as a redox-driven proton pump to develop a robust fluorimetric assay in permeabilized yeast. Screening in parallel a library of known pharmacologically active compounds (National Institute of Neurological Disorders and Stroke collection) against NNT ± cells, they demonstrate a robust and reproducible assay suitable for expansion into larger and more diverse compound sets. The identification of NNT activators may help in the elucidation of the role of NNT in mammalian cells and assessing its potential as a therapeutic target for insulin secretion disorders.     

Phylogenetic constraints on digesta separation: Variation in fluid throughput in the digestive tract in mammalian herbivores

The relevance of the mean retention time (MRT) of particles through the gastrointestinal tract (GIT) is well understood and MRT(particle)GIT is an important parameter in digestion models. Solute markers have been used to estimate MRT(solute)GIT (or 'fluid passage') in animals, but the relevance of this measure is less evident and is usually sought in its relation to MRT(particle)GIT. The ratio between the two measures indicates the degree of 'digesta washing', with little washing occurring at ratios of 1, aborad washing at ratios >1 (where the solute marker travels faster than the particle marker), and orad (retrograde) washing at ratios <1 (where the solute marker travels slower than the particle marker). We analysed digesta washing in a dataset of 98 mammalian species including man of different digestion types (caecum, colon and nonruminant foregut fermenters, and ruminants), controlling for phylogeny; a subset of 72 species allowed testing for the influence of food intake level. The results indicate that MRT(solute)GIT and the degree of digesta washing are related to digestion type, whereas variation in MRT(particle)GIT is influenced mainly by effects of body mass and food intake. Thus, fluid throughput and digesta washing emerge as important correlates of digestive anatomy. Most importantly, primates appear constrained to little digesta washing compared to non-primate mammalian herbivores, regardless of their digestion type. These results may help explain the absence of primates from certain herbivore niches and represent a drastic example of a physiologic limitation in a phylogenetic group. More experimental research is required to illuminate relative benefits and costs of digesta washing.     

Molecular and structural basis of ESCRT-III recruitment to membranes during archaeal cell division

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Highlights? A conserved protein, CdvA, recruits ESCRT-III to membranes during cell division ? Recruitment is mediated by a peptide-winged helix domain interaction ? We have determined the structure of this complex ? CdvA and a single ESCRT-III protein can drive membrane deformation in vitro