A protein methylation pathway in Chlamydomonas flagella is active during flagellar resorption

Author Posting. © American Society for Cell Biology, 2008. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 19 (2008): 4319-4327, doi:10.1091/mbc.E08-05-0470.During intraflagellar transport (IFT), the regulation of motor proteins, the loading and unloading of cargo and the turnover of flagellar proteins all occur at the flagellar tip. To begin an analysis of the protein composition of the flagellar tip, we used difference gel electrophoresis to compare long versus short (i.e., regenerating) flagella. The concentration of tip proteins should be higher relative to that of tubulin (which is constant per unit length of the flagellum) in short compared with long flagella. One protein we have identified is the cobalamin-independent form of methionine synthase (MetE). Antibodies to MetE label flagella in a punctate pattern reminiscent of IFT particle staining, and immunoblot analysis reveals that the amount of MetE in flagella is low in full-length flagella, increased in regenerating flagella, and highest in resorbing flagella. Four methylated proteins have been identified in resorbing flagella, using antibodies specific for asymmetrically dimethylated arginine residues. These proteins are found almost exclusively in the axonemal fraction, and the methylated forms of these proteins are essentially absent in full-length and regenerating flagella. Because most cells resorb cilia/flagella before cell division, these data indicate a link between flagellar protein methylation and progression through the cell cycle.This work was supported by National Institutes of Health Grant DK071720 (R.D.S.) and National Science Foundation Grant MCB 0418877 (R.D.S.)

Geometric and Electronic Structures of the NiI and Methyl−NiIII Intermediates of Methyl-Coenzyme M Reductase†

ABSTRACT: Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in the formation of biological methane from methyl-coenzyme M (Me-SCoM) and coenzyme B (CoBSH). The active site in MCR contains a Ni-F430 cofactor, which can exist in different oxidation states. The catalytic mechanism of methane formation has remained elusive despite intense spectroscopic and theoretical investigations. On the basis of spectroscopic and crystallographic data, the first step of the mechanism is proposed to involve a nucleophilic attack of the NiI active state (MCRred1) on Me-SCoM to form a NiIII-methyl intermediate, while computational studies indicate that the first step involves the attack of NiI on the sulfur of Me-SCoM, forming a CH3 radical and a NiII-thiolate species. In this study, a combination of Ni K-edge X-ray absorption spectroscopic (XAS) studies and density functional theory (DFT) calculations have been performed on the NiI (MCRred1), NiII (MCRred1-silent), and NiIII-methyl (MCRMe) states of MCR to elucidate the geometric and electronic structures of the different redox states. Ni K-edge EXAFS data are used to reveal a five-coordinate active site with an open upper axial coordination site in MCRred1. Ni K-pre-edge and EXAFS data and time-dependent DFT calculations unambiguously demonstrate the presence of a long Ni-C bond (∼2.04 Å) in the NiIII-methyl state of MCR. The formation and stability of this species support mechanism I, and the Ni-C bond length suggests a homolytic cleavage of the NiIII-methyl bon

Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach

Background: Fruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening. [br/] Methodology/Principal Findings: To engineer melon fruit with improved shelf-life, we conducted a translational research experiment. We set up a TILLING platform in a monoecious and climacteric melon line, cloned genes that control ethylene production and screened for induced mutations that lead to fruits with enhanced shelf life. Two missense mutations, L124F and G194D, of the ethylene biosynthetic enzyme, ACC oxidase 1, were identified and the mutant plants were characterized with respect to fruit maturation. The L124F mutation is a conservative mutation occurring away from the enzyme active site and thus was predicted to not affect ethylene production and thus fruit ripening. In contrast, G194D modification occurs in a highly conserved amino acid position predicted, by crystallographic analysis, to affect the enzymatic activity. Phenotypic analysis of the G194D mutant fruit showed complete delayed ripening and yellowing with improved shelf life and, as predicted, the L124F mutation did not have an effect. [br/] Conclusions/Significance: We constructed a mutant collection of 4023 melon M2 families. Based on the TILLING of 11 genes, we calculated the overall mutation rate of one mutation every 573 kb and identified 8 alleles per tilled kilobase. We also identified a TILLING mutant with enhanced fruit shelf life. This work demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. As cucurbits are model species in different areas of plant biology, we anticipate that the developed tool will be widely exploited by the scientific community

Euclid: Identifying the reddest high-redshift galaxies in the Euclid Deep Fields with gradient-boosted trees

Context. ALMA observations show that dusty, distant, massive (M* & 1011 M ) galaxies usually have a remarkable star-formation activity, contributing of the order of 25% of the cosmic star-formation rate density at z 3 5, and up to 30% at z ∼ 7. Nonetheless, they are elusive in classical optical surveys, and current near-IR surveys are able to detect them only in very small sky areas. Since these objects have low space densities, deep and wide surveys are necessary to obtain statistically relevant results about them. Euclid will potentially be capable of delivering the required information, but, given the lack of spectroscopic features at these distances within its bands, it is still unclear if Euclid will be able to identify and characterise these objects. Aims. The goal of this work is to assess the capability of Euclid, together with ancillary optical and near-IR data, to identify these distant, dusty, and massive galaxies based on broadband photometry. Methods. We used a gradient-boosting algorithm to predict both the redshift and spectral type of objects at high z. To perform such an analysis, we made use of simulated photometric observations that mimic the Euclid Deep Survey, derived using the state-of-the-art Spectro-Photometric Realizations of Infrared-selected Targets at all-z (SPRITZ) software. Results. The gradient-boosting algorithm was found to be accurate in predicting both the redshift and spectral type of objects within the simulated Euclid Deep Survey catalogue at z > 2, while drastically decreasing the runtime with respect to spectral-energy-distribution-fitting methods. In particular, we studied the analogue of HIEROs (i.e. sources selected on the basis of a red H - [4:5] > 2:25), combining Euclid and Spitzer data at the depth of the Deep Fields. These sources include the bulk of obscured and massive galaxies in a broad redshift range, 3 < z < 7. We find that the dusty population at 3 . z . 7 is well identified, with a redshift root mean squared error and catastrophic outlier fraction of only 0:55 and 8:5% (HE = 26), respectively. Our findings suggest that with Euclid we will obtain meaningful insights into the impact of massive and dusty galaxies on the cosmic star-formation rate over time

Disease-specific and general health-related quality of life in newly diagnosed prostate cancer patients: The Pros-IT CNR study

Background: The National Research Council (CNR) prostate cancer monitoring project in Italy (Pros-IT CNR) is an observational, prospective, ongoing, multicentre study aiming to monitor a sample of Italian males diagnosed as new cases of prostate cancer. The present study aims to present data on the quality of life at time prostate cancer is diagnosed. Methods: One thousand seven hundred five patients were enrolled. Quality of life is evaluated at the time cancer was diagnosed and at subsequent assessments via the Italian version of the University of California Los Angeles-Prostate Cancer Index (UCLA-PCI) and the Short Form Health Survey (SF-12). Results: At diagnosis, lower scores on the physical component of the SF-12 were associated to older ages, obesity and the presence of 3+ moderate/severe comorbidities. Lower scores on the mental component were associated to younger ages, the presence of 3+ moderate/severe comorbidities and a T-score higher than one. Urinary and bowel functions according to UCLA-PCI were generally good. Almost 5% of the sample reported using at least one safety pad daily to control urinary loss; less than 3% reported moderate/severe problems attributable to bowel functions, and sexual function was a moderate/severe problem for 26.7%. Diabetes, 3+ moderate/severe comorbidities, T2 or T3-T4 categories and a Gleason score of eight or more were significantly associated with lower sexual function scores at diagnosis. Conclusions: Data collected by the Pros-IT CNR study have clarified the baseline status of newly diagnosed prostate cancer patients. A comprehensive assessment of quality of life will allow to objectively evaluate outcomes of different profile of care

Small shelly fossils and carbon isotopes from the early Cambrian (Stage 3-4) Mural Formation of western Laurentia

The extraordinary window of phosphatised and phosphatic Small Shelly Fossils (SSFs) during the early and middle Cambrian is an important testament to the radiation of biomineralising metazoans. While SSF are well known from most Cambrian palaeocontinents during this time interval, western Laurentia has relatively few SSF faunas. Here we describe a diverse SSF fauna from the early Cambrian (Stage 3-4) Mural Formation at three localities in Alberta and British Columbia, Canada, complemented by carbon isotope measurements to aid in a potential future bio-chemostratigraphic framework. The fauna expands the recorded SSF assemblage diversity in western Laurentia and includes several brachiopods, four bradoriids, three chancelloriids, two hyoliths, a tommotiid and a helcionellid mollusc as well as echinoderm ossicles and specimens of Microdictyon, Volborthella and Hyolithellus. New taxa include the tommotiid genus Canadiella gen. nov., the new bradoriid species Hipponicharion perforata sp. nov. and Pseudobeyrichona taurata sp. nov. Compared to contemporaneous faunas from western Laurentia, the fauna is relatively diverse, particularly in taxa with originally phosphatic shells, which appear to be associated with archaeocyathid buildups. This suggests that the generally low faunal diversity in western Laurentia may be at least partly a consequence of poor sampling of suitable archaeocyathan reef environments. In addition, the tommotiid Canadiella filigrana appears to be of biostratigraphic significance in Cambrian Stage 3 strata of western Laurentia and the unexpected high diversity of bradoriid arthropods in the fauna also suggests that this group may prove useful for biostratigraphic resolution in the region