This article is part 4 of a series on Fantasy Fortifications by Toni Šušnjar.

Building a Fort

Build time of a castle, depending on design and available funds, may last from half a year to half a century. It also depends on the situation before the building: a ruined castle is a half-built castle after all, and rennovating (and/or updating) walls is much cheaper than building new ones. This can be seen with city of Dubrovnik, where (massive) medieval fortifications were, after the fall of Constantinople in 1453., reinforced with outer line of walls to reinforce them against cannon fire.

Both build time and extent of fortifications depend on material (financial, logistical, humane) capacities of the builder, as well as the perceived need. Many castles were never finished for lack of resources.

Builders are professionals; peasants, soldiers and other amateurs were used for muscle work only. This means that they have to be paid, and many in fact travel from a building place to a building place. Beaumaris Castle in England required 400 masons and 1,000 assistants to be built in a nearly record time (from 1278 to 1280).

Types of Castles
Motte and bailey castle

Motte and bailey castle is the earliest and simplest type of a castle.

Continue reading Fantasy Fortifications — Part 4: Types of Castles at Mythic Scribes.

David A. Mitchell
Jun 1, 2006; 47:1118-1127
Thematic Reviews

EJ Blanchette-Mackie
Jun 1, 1995; 36:1211-1226
Articles

Ronald M. Krauss
Jan 1, 1982; 23:97-104
Articles

G Lepage
Jan 1, 1986; 27:114-120
Articles

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

Invitation Only Research Event

Event participants

Ambassador Andrés Rozental, Senior Adviser, Chatham House; Founding President, Mexican Council on Foreign Relations
Dr Elena Lazarou, Associate Fellow, US and the Americas Programme, Chatham House
Chair: Dr Christopher Sabatini, Senior Research Fellow for Latin America, US and the Americas Programme, Chatham House

Invitation Only Research Event

Event participants

Dr Lindsay Newman, Senior Research Fellow, US and the Americas Programme, Chatham House
Professor Peter Trubowitz, Professor of International Relations, London School of Economics and Political Science; Associate Fellow, US and the Americas Programme, Chatham House
Amy Pope, Associate Fellow, US and the Americas Programme, Chatham House; Deputy Homeland Security Advisor, US National Security Council, 2015-17
Chair: Dr Leslie Vinjamuri, Director, US and the Americas Programme, Chatham House

Research Event

Event participants

Acute myeloid leukemia (AML) is a clonal disorder arising from hematopoietic myeloid progenitors. Aberrantly activated tyrosine kinases (TK) are involved in leukemogenesis and are associated with poor treatment outcome. Kinase inhibitor (KI) treatment has shown promise in improving patient outcome in AML. However, inhibitor selection for patients is suboptimal.

In a preclinical effort to address KI selection, we analyzed a panel of 16 AML cell lines using phosphotyrosine (pY) enrichment-based, label-free phosphoproteomics. The Integrative Inferred Kinase Activity (INKA) algorithm was used to identify hyperphosphorylated, active kinases as candidates for KI treatment, and efficacy of selected KIs was tested.

Heterogeneous signaling was observed with between 241 and 2764 phosphopeptides detected per cell line. Of 4853 identified phosphopeptides with 4229 phosphosites, 4459 phosphopeptides (4430 pY) were linked to 3605 class I sites (3525 pY). INKA analysis in single cell lines successfully pinpointed driver kinases (PDGFRA, JAK2, KIT and FLT3) corresponding with activating mutations present in these cell lines. Furthermore, potential receptor tyrosine kinase (RTK) drivers, undetected by standard molecular analyses, were identified in four cell lines (FGFR1 in KG-1 and KG-1a, PDGFRA in Kasumi-3, and FLT3 in MM6). These cell lines proved highly sensitive to specific KIs. Six AML cell lines without a clear RTK driver showed evidence of MAPK1/3 activation, indicative of the presence of activating upstream RAS mutations. Importantly, FLT3 phosphorylation was demonstrated in two clinical AML samples with a FLT3 internal tandem duplication (ITD) mutation.

Our data show the potential of pY-phosphoproteomics and INKA analysis to provide insight in AML TK signaling and identify hyperactive kinases as potential targets for treatment in AML cell lines. These results warrant future investigation of clinical samples to further our understanding of TK phosphorylation in relation to clinical response in the individual patient.

Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behcet's disease (BD). Previous studies have defined two subgroups of HLA-B*51 peptidome containing proline (Pro) or alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel subpeptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-triple knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by mass spectrometry. The characteristics of non-Pro/Ala2, Pro2, and Ala2 peptides and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Pro or Ala at P2. This unconventional group of HLA-B*51:01-bound peptides was relatively enriched for 8-mers (with relatively fewer 9-mers) compared with the Pro2 and Ala2 subpeptidomes and had similar N-terminal and C-terminal residue usages to Ala2 peptides (with the exception of the less abundant leucine at position ). Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 from 20% to ~40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell-type-dependent manner. In conclusion, we have used a novel methodology to identify an unconventional but surprisingly abundant non-Pro/Ala2 HLA-B*51:01 subpeptidome. It is increased by knockdown of ERAP1, a gene affecting the risk of developing BD. This has implications for theories of disease pathogenesis.

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.

Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fea32+ and CuB1+, and suggests that a peroxide-bound state (Fea33+–O−–O−–CuB2+) rather than an O2-bound state (Fea32+–O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fea32+–O2, whereas Fea33+–O−–O−–CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe–O distance of the ferryl center could best be described as Fea34+ = O2−, not as Fea34+–OH−. The distance suggests an ∼800-cm−1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fea33+–O−–O−–CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

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.

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD+-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH−, initially formed via NADH-mediated reduction, to the Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

• The Hunger Games: Catching Fire review
• The Wolverine Reviews
• What We Watched: jOBS, Steve Jobs: The Lost Interview, WWE For All Mankind: Life and Career of Mick Foley and The Lakota 38

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

Cholesterol/sphingolipid-rich membrane domains, known as lipid rafts or membrane rafts, play a critical role in the compartmentalization of signaling pathways. Physical segregation of proteins in lipid rafts may modulate the accessibility of proteins to regulatory or effector molecules. Thus, lipid rafts serve as sorting platforms and hubs for signal transduction proteins. Cancer cells contain higher levels of intracellular cholesterol and lipid rafts than their normal non-tumorigenic counterparts. Many signal transduction processes involved in cancer development (insulin-like growth factor system and phosphatidylinositol 3-kinase-AKT) and metastasis [cluster of differentiation (CD)44] are dependent on or modulated by lipid rafts. Additional proteins playing an important role in several malignant cancers (e.g., transmembrane glycoprotein mucin 1) are also being detected in association with lipid rafts, suggesting a major role of lipid rafts in tumor progression. Conversely, lipid rafts also serve as scaffolds for the recruitment and clustering of Fas/CD95 death receptors and downstream signaling molecules leading to cell death-promoting raft platforms. The partition of death receptors and downstream signaling molecules in aggregated lipid rafts has led to the formation of the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER, which leads to apoptosis amplification and can be pharmacologically modulated. These death-promoting rafts can be viewed as a linchpin from which apoptotic signals are launched. In this review, we discuss the involvement of lipid rafts in major signaling processes in cancer cells, including cell survival, cell death, and metastasis, and we consider the potential of lipid raft modulation as a promising target in cancer therapy.

Expert

Expert

Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fea32+ and CuB1+, and suggests that a peroxide-bound state (Fea33+–O−–O−–CuB2+) rather than an O2-bound state (Fea32+–O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fea32+–O2, whereas Fea33+–O−–O−–CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe–O distance of the ferryl center could best be described as Fea34+ = O2−, not as Fea34+–OH−. The distance suggests an ∼800-cm−1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fea33+–O−–O−–CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

pncRNA-D is an irradiation-induced 602-nt long noncoding RNA transcribed from the promoter region of the cyclin D1 (CCND1) gene. CCND1 expression is predicted to be inhibited through an interplay between pncRNA-D and RNA-binding protein TLS/FUS. Because the pncRNA-D–TLS interaction is essential for pncRNA-D–stimulated CCND1 inhibition, here we studied the possible role of RNA modification in this interaction in HeLa cells. We found that osmotic stress induces pncRNA-D by recruiting RNA polymerase II to its promoter. pncRNA-D was highly m6A-methylated in control cells, but osmotic stress reduced the methylation and also arginine methylation of TLS in the nucleus. Knockdown of the m6A modification enzyme methyltransferase-like 3 (METTL3) prolonged the half-life of pncRNA-D, and among the known m6A recognition proteins, YTH domain-containing 1 (YTHDC1) was responsible for binding m6A of pncRNA-D. Knockdown of METTL3 or YTHDC1 also enhanced the interaction of pncRNA-D with TLS, and results from RNA pulldown assays implicated YTHDC1 in the inhibitory effect on the TLS–pncRNA-D interaction. CRISPR/Cas9-mediated deletion of candidate m6A site decreased the m6A level in pncRNA-D and altered its interaction with the RNA-binding proteins. Of note, a reduction in the m6A modification arrested the cell cycle at the G0/G1 phase, and pncRNA-D knockdown partially reversed this arrest. Moreover, pncRNA-D induction in HeLa cells significantly suppressed cell growth. Collectively, these findings suggest that m6A modification of the long noncoding RNA pncRNA-D plays a role in the regulation of CCND1 gene expression and cell cycle progression.

8 January 2020 , Volume 96, Number 1

8 January 2020 , Volume 96, Number 1

Research Event

Event participants

Vasuki Shastry, Associate Fellow, Asia-Pacific Programme
Ravi Velloor, Associate Editor, The Straits Times
Chair: Yu Jie, Senior Research Fellow on China, Asia-Pacific Programme, Chatham House

There.s more mathematics involved in juggling than just trying to make sure that the number of balls (or chainsaws) that hits the ground stays at zero. Subjects such as combinatorics and abstract algebra help jugglers answer important questions, such as whether a particular juggling pattern can actually be juggled. For example, can balls be juggled so that the time period that each ball stays aloft alternates between five counts and one? The answer is Yes. Math also tells you that the number of balls needed for such a juggling pattern is the average of the counts, in this case three. Once a pattern is shown to be juggleable and the number of balls needed is known, equations of motion determine the speed with which each ball must be thrown and the maximum height it will attain. Obviously the harder a juggler throws, the faster and higher an object will go. Unfortunately hang time increases proportionally to the square root of the height, so the difficulty of keeping many objects in the air increases very quickly. Both math and juggling have been around for millennia yet questions still remain in both subjects. As two juggling mathematicians wrote, .A juggler, like a mathematician, is never finished: there is always another great unsolved problem.

There.s more mathematics involved in juggling than just trying to make sure that the number of balls (or chainsaws) that hits the ground stays at zero. Subjects such as combinatorics and abstract algebra help jugglers answer important questions, such as whether a particular juggling pattern can actually be juggled. For example, can balls be juggled so that the time period that each ball stays aloft alternates between five counts and one? The answer is Yes. Math also tells you that the number of balls needed for such a juggling pattern is the average of the counts, in this case three. Once a pattern is shown to be juggleable and the number of balls needed is known, equations of motion determine the speed with which each ball must be thrown and the maximum height it will attain. Obviously the harder a juggler throws, the faster and higher an object will go. Unfortunately hang time increases proportionally to the square root of the height, so the difficulty of keeping many objects in the air increases very quickly. Both math and juggling have been around for millennia yet questions still remain in both subjects. As two juggling mathematicians wrote, .A juggler, like a mathematician, is never finished: there is always another great unsolved problem.

Van Wedeen talks about the geometry of the brain's communication pathways.

Fishery collapses can be difficult to forecast and prevent due to hyperstability, a phenomenon where catch rates remain high even as fish abundance declines. In a recent Canadian Journal of Fisheries and Aquatic Sciences study, researchers conducted a whole-lake experiment to reveal the causes of hyperstability in recreational fisheries. Fish habitat preferences were found to leave them vulnerable to overexploitation.

New Publication: Rules, Procedures and Mechanisms Applicable to Processes under the Cartagena Protocol on Biosafety.