Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions

© 2020, Prigge et al. The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply-mutant lines. We find that loss of all six TIR1/AFB proteins results in early embryo defects and eventually seed abortion, and yet a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout development. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1. Surprisingly, AFB1 has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family

Supplemental Ascorbate Diminishes DNA Damage Yet Depletes Glutathione and Increases Acute Liver Failure in a Mouse Model of Hepatic Antioxidant System Disruption

Cellular oxidants are primarily managed by the thioredoxin reductase-1 (TrxR1)- and glutathione reductase (Gsr)-driven antioxidant systems. In mice having hepatocyte-specific codisruption of TrxR1 and Gsr (TrxR1/Gsr-null livers), methionine catabolism sustains hepatic levels of reduced glutathione (GSH). Although most mice with TrxR1/Gsr-null livers exhibit long-term survival, ~25% die from spontaneous liver failure between 4- and 7-weeks of age. Here we tested whether liver failure was ameliorated by ascorbate supplementation. Following ascorbate, dehydroascorbate, or mock treatment, we assessed survival, liver histology, or hepatic redox markers including GSH and GSSG, redox enzyme activities, and oxidative damage markers. Unexpectedly, rather than providing protection, ascorbate (5 mg/mL, drinking water) increased the death-rate to 43%. In adults, ascorbate (4 mg/g × 3 days i.p.) caused hepatocyte necrosis and loss of hepatic GSH in TrxR1/Gsr-null livers but not in wildtype controls. Dehydroascorbate (0.3 mg/g i.p.) also depleted hepatic GSH in TrxR1/Gsr-null livers, whereas GSH levels were not significantly affected by either treatment in wildtype livers. Curiously, however, despite depleting GSH, ascorbate treatment diminished basal DNA damage and oxidative stress markers in TrxR1/Gsr-null livers. This suggests that, although ascorbate supplementation can prevent oxidative damage, it also can deplete GSH and compromise already stressed livers

Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms

Background: Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4 % of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be “RAS-pathway alteration negative”, using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations. Methods: Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously. Results: We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9 %) and mucinous borderline tumors (MBOT) (92.3 %). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8 % and 11.5 %, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68 % of MC and as many as 20 % of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell populations, while consistency of KRAS allelic frequency in both ERBB2 amplified and non-amplified regions suggests this mutation occurred in advance of the amplification event. Conclusions: Overall, the prevalence of RAS-alteration and striking co-occurrence of pathway “double-hits” supports a critical role for tumor progression in this ovarian malignancy. Given the spectrum of RAS-activating mutations, it is clear that targeting this pathway may be a viable therapeutic option for patients with recurrent or advanced stage mucinous ovarian carcinoma, however caution should be exercised in selecting one or more personalized therapeutics given the frequency of non-redundant RAS-activating alterations

Copper binding to the Alzheimer’s disease amyloid precursor protein

Alzheimer’s disease is the fourth biggest killer in developed countries. Amyloid precursor protein (APP) plays a central role in the development of the disease, through the generation of a peptide called Aβ by proteolysis of the precursor protein. APP can function as a metalloprotein and modulate copper transport via its extracellular copper binding domain (CuBD). Copper binding to this domain has been shown to reduce Aβ levels and hence a molecular understanding of the interaction between metal and protein could lead to the development of novel therapeutics to treat the disease. We have recently determined the three-dimensional structures of apo and copper bound forms of CuBD. The structures provide a mechanism by which CuBD could readily transfer copper ions to other proteins. Importantly, the lack of significant conformational changes to CuBD on copper binding suggests a model in which copper binding affects the dimerisation state of APP leading to reduction in Aβ production. We thus predict that disruption of APP dimers may be a novel therapeutic approach to treat Alzheimer’s disease

Digital Gene Expression Profiling by 5′-End Sequencing of cDNAs during Reprogramming in the Moss Physcomitrella patens

Stem cells self-renew and repeatedly produce differentiated cells during development and growth. The differentiated cells can be converted into stem cells in some metazoans and land plants with appropriate treatments. After leaves of the moss Physcomitrella patens are excised, leaf cells reenter the cell cycle and commence tip growth, which is characteristic of stem cells called chloronema apical cells. To understand the underlying molecular mechanisms, a digital gene expression profiling method using mRNA 5′-end tags (5′-DGE) was established. The 5′-DGE method produced reproducible data with a dynamic range of four orders that correlated well with qRT-PCR measurements. After the excision of leaves, the expression levels of 11% of the transcripts changed significantly within 6 h. Genes involved in stress responses and proteolysis were induced and those involved in metabolism, including photosynthesis, were reduced. The later processes of reprogramming involved photosynthesis recovery and higher macromolecule biosynthesis, including of RNA and proteins. Auxin and cytokinin signaling pathways, which are activated during stem cell formation via callus in flowering plants, are also activated during reprogramming in P. patens, although no exogenous phytohormone is applied in the moss system, suggesting that an intrinsic phytohormone regulatory system may be used in the moss

The Selaginella Genome Identifies Genetic Changes Associated with the Evolution of Vascular Plants

Vascular plants appeared ~410 million years ago then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes (1). We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first non-seed vascular plant genome reported. By comparing gene content in evolutionary diverse taxa, we found that the transition from a gametophyte- to sporophyte- dominated life cycle required far fewer new genes than the transition from a non-seed vascular to a flowering plant, while secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in post- transcriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the tasiRNA pathway and extensive RNA editing of organellar genes

From Prototyping to Allotyping. The invention of change of use and the crisis of building types

The chapter analyses the invention and the form of the discourse on building conversion as one particular instance of redefining what a technology is and how it operates. I describe a shift from expert defined closure to lay based openness and tinkering as a shift from prototyping to allotyping: Since the early 1970s, change of use and building conversion have become a central and fashionable discourse among architects and architectural theorists. Before the 1970s, buildings were understood as technologies, as ‘society made durable’. The notion of building type was central to link a building to a given use. A bank was a bank because architects applied existing templates, prototypes, to turn a building into a bank. In the 1970s, suddenly buildings became flexible – discursively, since building conversion always existed: ‘Building type’ no longer was a meaningful link between a building and its use. A bank should not stay a bank, but become a hotel, a theatre or a flat, in short: an allotype. The chapter elucidate this central shift in thinking about buildings and reflects on the special case of allotyping buildings and how it continues to vex thinking about buildings