Ulrich Rothbauer
Feb 1, 2008; 7:282-289
Research

Invitation Only Research Event

Event participants

Niels Engelschiøn, Director-General, Department for European Affairs, Norwegian Ministry of Foreign Affairs
Chair: Dr Robin Niblett, Director; Chief Executive, Chatham House

Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable, with T50 values of ∼30–40 °C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared with related extant forms, but they also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. The mammalian ancestor of the cytochrome P450 1B subfamily was herein characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. Whereas extant human CYP1B1 has one molecule of α-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17β-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability. To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral cytochrome P450, revealing key features that appear to contribute to its thermostability.

4 March 2020 , Volume 96, Number 2

W Gamble
Nov 1, 1978; 19:1068-1070
Articles

M. Burstein
Nov 1, 1970; 11:583-595
Articles

William R. Morrison
Oct 1, 1964; 5:600-608
Articles

JE Hixson
Mar 1, 1990; 31:545-548
Articles

17 April 2020

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene–Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H₂S has been shown to persulfidate redox sensitive cysteine residues resulting in an H₂S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H₂S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTS^GS). We propose that the RTS^GS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general <100x) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ~2500 proteins and precise quantification of ~1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.

HNF4α is a nuclear receptor produced as 12 isoforms from two promoters by alternative splicing. To characterize the transcriptional capacities of all 12 HNF4α isoforms, stable lines expressing each isoform were generated. The entire transcriptome associated with each isoform was analyzed as well as their respective interacting proteome. Major differences were noted in the transcriptional function of these isoforms. The α1 and α2 isoforms were the strongest regulators of gene expression whereas the α3 isoform exhibited significantly reduced activity. The α4, α5, and α6 isoforms, which use an alternative first exon, were characterized for the first time, and showed a greatly reduced transcriptional potential with an inability to recognize the consensus response element of HNF4α. Several transcription factors and coregulators were identified as potential specific partners for certain HNF4α isoforms. An analysis integrating the vast amount of omics data enabled the identification of transcriptional regulatory mechanisms specific to certain HNF4α isoforms, hence demonstrating the importance of considering all isoforms given their seemingly diverse functions.

The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remain poorly understood. To investigate the hRSV-associated apical and basolateral secretomes, a proteomics approach was combined with an ex vivo pediatric human airway epithelial (HAE) model of hRSV infection (data are available via ProteomeXchange and can be accessed at https://www.ebi.ac.uk/pride/ with identifier PXD013661). Following infection, a skewing of apical/basolateral abundance ratios was identified for several individual proteins. Novel modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) were detected in infected, but not in uninfected cultures. Importantly, CXCL6, CXCL16, CSF3 were also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not healthy controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells. hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome and was associated with immune modulating proteins (CXCL6, CXCL16, CSF3) never linked with this virus before. In addition, the antiviral activity of CEACAM1 against hRSV had also never been previously characterized. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.

Autoimmune thyroid diseases (AITD) are the most common group of autoimmune diseases, associated with lymphocyte infiltration and the production of thyroid autoantibodies, like thyroid peroxidase antibodies (TPOAb), in the thyroid gland. Immunoglobulins and cell-surface receptors are glycoproteins with distinctive glycosylation patterns that play a structural role in maintaining and modulating their functions. We investigated associations of total circulating IgG and peripheral blood mononuclear cells glycosylation with AITD and the influence of genetic background in a case-control study with several independent cohorts and over 3,000 individuals in total. The study revealed an inverse association of IgG core fucosylation with TPOAb and AITD, as well as decreased peripheral blood mononuclear cells antennary α1,2 fucosylation in AITD, but no shared genetic variance between AITD and glycosylation. These data suggest that the decreased level of IgG core fucosylation is a risk factor for AITD that promotes antibody-dependent cell-mediated cytotoxicity previously associated with TPOAb levels.

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. Single-stranded DNA–binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5'- and 3'-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB.

Invitation Only Research Event

Event participants

Richard King, Senior Research Fellow, Energy, Environment and Resources Department, Chatham House
Chair: Duncan Brack, Associate Fellow, Energy, Environment and Resources Department, Chatham House

29 January 2020

Policymakers are in danger of sleepwalking into ineffective carbon dioxide removal solutions in the quest to tackle climate change. This paper warns against overreliance on bioenergy with carbon capture and storage (BECCS). 

Research Event

26 March 2020

Lipid rafts are highly ordered regions of the plasma membrane that are enriched in cholesterol and sphingolipids and play important roles in many cells. In hematopoietic stem and progenitor cells (HSPCs), lipid rafts house receptors critical for normal hematopoiesis. Lipid rafts also can bind and sequester kinases that induce negative feedback pathways to limit proliferative cytokine receptor cycling back to the cell membrane. Modulation of lipid rafts occurs through an array of mechanisms, with optimal cholesterol efflux one of the major regulators. As such, cholesterol homeostasis also regulates hematopoiesis. Increased lipid raft content, which occurs in response to changes in cholesterol efflux in the membrane, can result in prolonged receptor occupancy in the cell membrane and enhanced signaling. In addition, certain diseases, like diabetes, may contribute to lipid raft formation and affect cholesterol retention in rafts. In this review, we explore the role of lipid raft-related mechanisms in hematopoiesis and CVD (specifically, atherosclerosis) and discuss how defective cholesterol efflux pathways in HSPCs contribute to expansion of lipid rafts, thereby promoting myelopoiesis and thrombopoiesis. We also discuss the utility of cholesterol acceptors in contributing to lipid raft regulation and disruption, and highlight the potential to manipulate these pathways for therapeutic gain in CVD as well as other disorders with aberrant hematopoiesis.

Expert

K. D. Cherednichenko, Yu. Yu. Ershova, A. V. Kiselev and S. N. Naboko
Trans. Moscow Math. Soc. 80 (2020), 251-294.
Abstract, references and article information

I. V. Astashova, D. A. Lashin and A. V. Filinovskii
Trans. Moscow Math. Soc. 80 (2020), 221-234.
Abstract, references and article information

V. V. Vlasov and N. A. Rautian
Trans. Moscow Math. Soc. 80 (2020), 169-188.
Abstract, references and article information

N. F. Valeev, Ya. T. Sultanaev and É. A. Nazirova
Trans. Moscow Math. Soc. 80 (2020), 153-167.
Abstract, references and article information

S. A. Nazarov
Trans. Moscow Math. Soc. 80 (2020), 1-51.
Abstract, references and article information

Publication Date: Apr 10 2020 Funds listed under Nasdaq Rule 5704 are required to submit an annual certification regarding the funds compliance with Rule 6c-11 during the most recent fiscal year. The certification is required within 30 calendar days of a fund’s fiscal year end. The certification can be found here....

Publication Date: Apr 10 2020 On or before December 22, 2020, all ETFs that meet the definition of "Exchange Traded Fund" in Nasdaq Rule 5704(a)(1)(A) will need to be compliant with Rule 6c-11. If it is determined that an ETF no longer complies with Rule 6c-11 and therefore no longer complies Nasdaq Rule 5704, Nasdaq will generally issue a letter of deficiency. The ETF will generally be given 45 days to submit a plan to regain compliance. If the plan is accepted, Nasdaq Staff can grant an...

Publication Date: Apr 10 2020 New Fund Launches In addition to completing the Listing Application, new funds are required to complete a certification prior to receiving approval of an initial listing application. The certification can be found here. Listing Transfers In addition to completing the Listing Application, funds switching from another market to Nasdaq are required to complete a certification regarding compliance with SEC Rule 6c-11. The certification must be completed prior to...

Firefox's latest version is out, with new password management features and a raft of security fixes.

17 April 2020

Sea ice is one of the least understood components of our climate. Naturally its abundance or scarcity is a telling sign of climate change, but sea ice is also an important actor in change as well, insulating the ocean and reflecting sunlight. A branch of mathematics called percolation theory helps explain how salt water travels through sea ice, a process that is crucial both to the amount of sea ice present and to the microscopic communities that sustain polar ecosystems. By taking samples, doing on-site experiments, and then incorporating the data into models of porous materials, mathematicians are working to understand sea ice and help refine climate predictions. Using probability, numerical analysis, and partial differential equations, researchers have recently shown that the permeability of sea ice is similar to that of some sedimentary rocks in the earth.s crust, even though the substances are otherwise dissimilar. One major difference between the two is the drastic changes in permeability of sea ice, from total blockage to clear passage, that occur over a range of just a few degrees. This difference can have a major effect on measurements by satellite, which provide information on the extent and thickness of sea ice. Results about sea ice will not only make satellite measurements more reliable, but they can also be applied to descriptions of lung and bone porosity, and to understanding ice on other planets. Image: Pancake ice in Antarctica, courtesy of Ken Golden. For More Information: "Thermal evolution of permeability and microstructure in sea ice," K. M. Golden, et al., Geophysical Research Letters, August 28, 2007.

Sea ice is one of the least understood components of our climate. Naturally its abundance or scarcity is a telling sign of climate change, but sea ice is also an important actor in change as well, insulating the ocean and reflecting sunlight. A branch of mathematics called percolation theory helps explain how salt water travels through sea ice, a process that is crucial both to the amount of sea ice present and to the microscopic communities that sustain polar ecosystems. By taking samples, doing on-site experiments, and then incorporating the data into models of porous materials, mathematicians are working to understand sea ice and help refine climate predictions. Using probability, numerical analysis, and partial differential equations, researchers have recently shown that the permeability of sea ice is similar to that of some sedimentary rocks in the earth.s crust, even though the substances are otherwise dissimilar. One major difference between the two is the drastic changes in permeability of sea ice, from total blockage to clear passage, that occur over a range of just a few degrees. This difference can have a major effect on measurements by satellite, which provide information on the extent and thickness of sea ice. Results about sea ice will not only make satellite measurements more reliable, but they can also be applied to descriptions of lung and bone porosity, and to understanding ice on other planets. Image: Pancake ice in Antarctica, courtesy of Ken Golden. For More Information: "Thermal evolution of permeability and microstructure in sea ice," K. M. Golden, et al., Geophysical Research Letters, August 28, 2007.

Sea ice is one of the least understood components of our climate. Naturally its abundance or scarcity is a telling sign of climate change, but sea ice is also an important actor in change as well, insulating the ocean and reflecting sunlight. A branch of mathematics called percolation theory helps explain how salt water travels through sea ice, a process that is crucial both to the amount of sea ice present and to the microscopic communities that sustain polar ecosystems. By taking samples, doing on-site experiments, and then incorporating the data into models of porous materials, mathematicians are working to understand sea ice and help refine climate predictions. Using probability, numerical analysis, and partial differential equations, researchers have recently shown that the permeability of sea ice is similar to that of some sedimentary rocks in the earth.s crust, even though the substances are otherwise dissimilar. One major difference between the two is the drastic changes in permeability of sea ice, from total blockage to clear passage, that occur over a range of just a few degrees. This difference can have a major effect on measurements by satellite, which provide information on the extent and thickness of sea ice. Results about sea ice will not only make satellite measurements more reliable, but they can also be applied to descriptions of lung and bone porosity, and to understanding ice on other planets. Image: Pancake ice in Antarctica, courtesy of Ken Golden. For More Information: "Thermal evolution of permeability and microstructure in sea ice," K. M. Golden, et al., Geophysical Research Letters, August 28, 2007.

Sea ice is one of the least understood components of our climate. Naturally its abundance or scarcity is a telling sign of climate change, but sea ice is also an important actor in change as well, insulating the ocean and reflecting sunlight. A branch of mathematics called percolation theory helps explain how salt water travels through sea ice, a process that is crucial both to the amount of sea ice present and to the microscopic communities that sustain polar ecosystems. By taking samples, doing on-site experiments, and then incorporating the data into models of porous materials, mathematicians are working to understand sea ice and help refine climate predictions. Using probability, numerical analysis, and partial differential equations, researchers have recently shown that the permeability of sea ice is similar to that of some sedimentary rocks in the earth.s crust, even though the substances are otherwise dissimilar. One major difference between the two is the drastic changes in permeability of sea ice, from total blockage to clear passage, that occur over a range of just a few degrees. This difference can have a major effect on measurements by satellite, which provide information on the extent and thickness of sea ice. Results about sea ice will not only make satellite measurements more reliable, but they can also be applied to descriptions of lung and bone porosity, and to understanding ice on other planets. Image: Pancake ice in Antarctica, courtesy of Ken Golden. For More Information: "Thermal evolution of permeability and microstructure in sea ice," K. M. Golden, et al., Geophysical Research Letters, August 28, 2007.

Researcher: Christopher Butson, Scientific Computing and Imaging Institute, University of Utah. Christopher Butson talks about work he's done to help treat Parkinson's disease.

Researcher: Cristina Stoica, Wilfrid Laurier University
Description: Cristina Stoica talks about celestial mechanics.