Expert

DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the “performance specifications” needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

Sand: Monitoring and management for a sustainable future 2 March 2021 — 1:00PM TO 2:00PM Anonymous (not verified) 18 February 2021 Online

In partnership with the Global Sand Observatory Initiative, this event outlines the sand challenge, what actions are currently underway to address it, and what else needs to be done.

Please complete your registration on Zoom:

After water, sand is the raw material that the world consumes in the greatest quantity. It is no exaggeration to say that fine sand and coarser materials – the medium-to-coarse-grained pebbles, gravel and rock fragments used in construction – are the building blocks of the modern world.

When bound with cement, sand becomes concrete; when mixed with bitumen, it becomes asphalt; and when heated, it becomes glass. Without sand, we would have no highways, high-rises or high-speed trains. Yet sand – which is used here as shorthand for sand, gravel and crushed rock together – is a resource that is both abundant and finite.

In global terms, it is abundant, especially when compared with many other raw materials, albeit often not available close to where it is needed. It is finite in that the rate at which we are using it far exceeds the natural rate at which it is being replenished by the weathering of rocks by wind and water.

Industrialization, population growth and urbanization have fuelled explosive growth in the demand for sand. Precise data on sand extraction are hard to come by and the lack of data compounds the challenge of managing the resource sustainably.

However, the UN estimates that overall extraction could be in the region of 40 billion tonnes per year, driven primarily by construction sector demand. That equates to 18 kilograms of sand each day for every person on the planet and signals how strategically important these resources are for future sustainable development. Post-COVID-19 recovery investment in infrastructure, digital technologies, tourism and other economic activities are dependent on sand resources.

Current efforts to improve the management of sand resources at local, national and global levels are uneven. This is partly due to unique geological features and geography, but also differences in local manifestations of the ‘sand challenge’, national and regional demand for sand resources, as well as capacities to enforce or implement best practice assessment procedures, extractive practices, environmental management and restoration requirements.

We must put stronger conditions in place for a rapid, just and scaled transition to sustainable sand management. But where to focus our efforts for the greatest positive impact?

Angela Robinson explains the math behind the next generation of cryptographic algorithms. Whenever you log in to a website, send an email, or make an online purchase, you're counting on your data being sent securely, without hackers being able to crack the code. Our standard cryptographic systems hinge on mathematical problems that stump present-day computers, like finding the prime factors of a very large number. But in the coming decades, powerful quantum computers are expected to be able to rapidly solve some such problems, threatening the security of our online communications. To develop new methods that can withstand even the most sophisticated quantum computer, cryptographers are using a wide range of mathematical tools, many of which were originally developed without any real-life applications in mind.

Tim Chumley explains the connections between random billiards and the science of heat and energy transfer. If you've ever played billiards or pool, you've used your intuition and some mental geometry to plan your shots. Mathematicians have gone a step further, using these games as inspiration for new mathematical problems. Starting from the simple theoretical setup of a single ball bouncing around in an enclosed region, the possibilities are endless. For instance, if the region is shaped like a stadium (a rectangle with semicircles on opposite sides), and several balls start moving with nearly the same velocity and position, their paths in the region soon differ wildly: chaos. Mathematical billiards even have connections to thermodynamics, the branch of physics dealing with heat, temperature, and energy transfer.

Malena Espanol explains how she and others use linear algebra to correct blurry images. Imagine snapping a quick picture of a flying bird. The image is likely to come out blurry. But thanks to mathematics, you might be able to use software to improve the photo. Scientists often deal with blurry pictures, too. Linear algebra and clever numerical methods allow researchers to fix imperfect photos in medical imaging, astronomy, and more. In a computer, the pixels that make up an image can be represented as a column of numbers called a vector. Blurring happens when the light meant for each pixel spills into the adjacent pixels, changing the numbers in a way that can be mathematically represented as an enormous matrix. But knowing that matrix is not enough if you want to reconstruct the original (non-blurry) image.

Dr. Rekha Thomas from the University of Washington discusses three-dimensional image reconstructions from two-dimensional photos. The mathematics of image reconstruction is both simpler and more abstract than it seems. To reconstruct a 3D model based on photographic data, researchers and algorithms must solve a set of polynomial equations. Some solutions to these equations work mathematically, but correspond to an unrealistic scenario — for instance, a camera that took a photo backwards. Additional constraints help ensure this doesn't happen. Researchers are now investigating the mathematical structures underlying image reconstruction, and stumbling over unexpected links with geometry and algebra.

Daniel Le, Bao V. Le Hung, Stefano Morra, Chol Park and Zicheng Qian
Trans. Amer. Math. Soc. 377 (), 5749-5786.
Abstract, references and article information

Myelination plays an important role in cognitive development and in demyelinating diseases like multiple sclerosis (MS), where failure of remyelination promotes permanent neuro-axonal damage. Modification of cell surface receptors with branched N-glycans coordinates cell growth and differentiation by controlling glycoprotein clustering, signaling, and endocytosis. GlcNAc is a rate-limiting metabolite for N-glycan branching. Here we report that GlcNAc and N-glycan branching trigger oligodendrogenesis from precursor cells by inhibiting platelet-derived growth factor receptor-α cell endocytosis. Supplying oral GlcNAc to lactating mice drives primary myelination in newborn pups via secretion in breast milk, whereas genetically blocking N-glycan branching markedly inhibits primary myelination. In adult mice with toxin (cuprizone)-induced demyelination, oral GlcNAc prevents neuro-axonal damage by driving myelin repair. In MS patients, endogenous serum GlcNAc levels inversely correlated with imaging measures of demyelination and microstructural damage. Our data identify N-glycan branching and GlcNAc as critical regulators of primary myelination and myelin repair and suggest that oral GlcNAc may be neuroprotective in demyelinating diseases like MS.

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.

Members of the B-cell lymphoma (BCL-2) protein family regulate mitochondrial outer membrane permeabilization (MOMP), a phenomenon in which mitochondria become porous and release death-propagating complexes during the early stages of apoptosis. Pro-apoptotic BCL-2 proteins oligomerize at the mitochondrial outer membrane during MOMP, inducing pore formation. Of current interest are endogenous factors that can inhibit pro-apoptotic BCL-2 mitochondrial outer membrane translocation and oligomerization. A mitochondrial-derived peptide, Humanin (HN), was reported being expressed from an alternate ORF in the mitochondrial genome and inhibiting apoptosis through interactions with the pro-apoptotic BCL-2 proteins. Specifically, it is known to complex with BAX and BID. We recently reported the fibrillation of HN and BAX into β-sheets. Here, we detail the fibrillation between HN and BID. These fibers were characterized using several spectroscopic techniques, protease fragmentation with mass analysis, and EM. Enhanced fibrillation rates were detected with rising temperatures or pH values and the presence of a detergent. BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. BID fibers display both types of secondary structure in the fiber, whereas BAX was converted entirely to β-sheets. The data show that two distinct segments of BID are incorporated into the fiber structure, whereas other portions of BID remain solvent-exposed and retain helical structure. Similar analyses show that anti-apoptotic BCL-xL does not form fibers with humanin. These results support a general mechanism of sequestration of pro-apoptotic BCL-2 proteins into fibers by HN to inhibit MOMP.

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs −1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical −1 and −2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate −1 and −2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

6 July 2020

15 July 2020

16 July 2020

Researchers use dynamic PET imaging with target-selective tracer molecules to probe molecular processes. Kinetic models have been developed to describe these processes. The models are typically fitted to the measured PET data with the assumption that the brain is in a steady-state condition for the duration of the scan. The end results are quantitative parameters that characterize the molecular processes. The most common kinetic modeling endpoints are estimates of volume of distribution or the binding potential of a tracer. If the steady state is violated during the scanning period, the standard kinetic models may not apply. To address this issue, time-variant kinetic models have been developed for the characterization of dynamic PET data acquired while significant changes (e.g., short-lived neurotransmitter changes) are occurring in brain processes. These models are intended to extract a transient signal from data. This work in the PET field dates back at least to the 1990s. As interest has grown in imaging nonsteady events, development and refinement of time-variant models has accelerated. These new models, which we classify as belonging to the first, second, or third generation according to their innovation, have used the latest progress in mathematics, image processing, artificial intelligence, and statistics to improve the sensitivity and performance of the earliest practical time-variant models to detect and describe nonsteady phenomena. This review provides a detailed overview of the history of time-variant models in PET. It puts key advancements in the field into historical and scientific context. The sum total of the methods is an ongoing attempt to better understand the nature and implications of neurotransmitter fluctuations and other brief neurochemical phenomena.

Jessica Y. Franco
Dec 1, 2020; 19:1936-1951
Research

Ji Eun Kim
Dec 20, 2020; 0:RA120.002159v1-mcp.RA120.002159
Research

R. Greg Stacey
Nov 4, 2020; 0:RA120.002275v1-mcp.RA120.002275
Research

Laura F Fielden
Nov 11, 2020; 0:RA120.002370v1-mcp.RA120.002370
Research

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cellular stiffness in contrast to differentiated cells, which are stiffer. We have previously shown that mESCs lacking the clathrin heavy chain (Cltc), an essential component for clathrin-mediated endocytosis (CME), display a loss of pluripotency and an enhanced expression of differentiation markers. However, it is not known whether physical properties such as cellular stiffness also change upon loss of Cltc, similar to what is seen in differentiated cells, and if so, how these altered properties specifically impact pluripotency. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking Cltc display higher Young's modulus, indicative of greater cellular stiffness, compared with WT mESCs. The increase in stiffness was accompanied by the presence of actin stress fibers and accumulation of the inactive, phosphorylated, actin-binding protein cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young's modulus to values similar to those obtained with WT mESCs. However, a rescue in the expression profile of pluripotency factors was not obtained. Additionally, whereas WT mouse embryonic fibroblasts could be reprogrammed to a state of pluripotency, this was inhibited in the absence of Cltc. This indicates that the presence of active CME is essential for the pluripotency of embryonic stem cells. Additionally, whereas physical properties may serve as a simple readout of the cellular state, they may not always faithfully recapitulate the underlying molecular fate.

Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, nonalcohol fatty liver disease (NAFLD), and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum-associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation, and increased mitochondrial outer membrane permeability. Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A's activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.

Alice Santonastaso
Dec 1, 2020; 61:1687-1696
Research Articles

Tommaso Laurenzi
Dec 2, 2020; 0:jlr.RA120000843v1-jlr.RA120000843
Research Articles

The SAS 9.4M4 (TS1M4) Hot Fix D7G004 for ODS Templates installs national language support (NLS) content regardless of whether the languages were installed during the initial deployment. Having sparse

Lecithin:cholesterol-acyl-transferase (LCAT) plays a major role in cholesterol metabolism as it is the only extracellular enzyme able to esterify cholesterol. LCAT activity is required for lipoprotein remodelling and, most specifically, for the growth and maturation of HDLs. In fact, genetic alterations affecting LCAT func- tionality may cause a severe reduction in plasma levels of HDL-cholesterol with important clinical consequences. Although several hypotheses were formulated, the exact molecular recognition mechanism between LCAT and HDLs is still unknown. We employed a combination of structural bioinformatics procedures to deepen the insights into the HDL-LCAT interplay that promotes LCAT activation and cholesterol esterification. We have generated a data-driven model of reconstituted HDL (rHDL) and studied the dynamics of an assembled rHDL::LCAT supramolecular complex, pinpointing the conformational changes originating from the interaction between LCAT and apolipoprotein A-I (apoA-I) that are necessary for LCAT activation. Specifically, we propose a mechanism in which the anchoring of LCAT lid to apoA-I helices allows the formation of a hydrophobic hood that expands LCAT active site and shields it from the solvent, allowing the enzyme to process large hydrophobic substrates.

Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apo(a). The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analog, -aminocaproic acid. Interestingly, KIV-2 presents a lysine binding site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to be negligible in surface plasmon resonance experiments; and with the LBS being nonfunctional, we propose to rename it "pseudo-LBS". Further investigation of the protein by computational small-molecule docking allowed us to identify a possible heparin-binding site away from the LBS, which was confirmed by specific reverse charge mutations abolishing heparin binding. This study opens new possibilities to define the pathogenesis of Lp(a)-related diseases and to facilitate the design of specific therapeutic drugs.

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between WT IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem MS coupled with liquid chromatography (LC–MS/MS), we found 142 nonreductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2), a TCA cycle enzyme, was the only protein to show an increase in Sia content during capacitation. Interestingly, although the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A₁ activity, a decrease in the activity of ACO2 (i.e. stereospecific isomerization of citrate to isocitrate) occurred when sialylation increased (N612). The latter was confirmed by N612D recombinant protein tagged with both His and GFP. The replacement of Sia for the negatively charged Aspartic acid in the N612D mutant caused complete loss of aconitase activity compared with the WT. Computer modeling show that N612 sits atop the catalytic site of ACO2. The introduction of Sia causes a large conformational change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

Huanglongbing (HLB) is the most devastating and widespread citrus disease. All commercial citrus varieties are susceptible to the HLB-associated bacterium, Candidatus Liberibacter asiaticus (CLas), which resides in the phloem. The phloem is part of the plant vascular system and is involved in sugar transport. To investigate the plant response to CLas, we enriched for proteins surrounding the phloem in an HLB susceptible sweet orange variety, Washington navel (Citrus sinensis (L) Osbeck). Quantitative proteomics revealed global changes in the citrus proteome after CLas inoculation. Plant metabolism and translation were suppressed, whereas defense-related proteins such as peroxidases, proteases and protease inhibitors were induced in the vasculature. Transcript accumulation and enzymatic activity of plant peroxidases in CLas infected sweet orange varieties under greenhouse and field conditions were assessed. Although peroxidase transcript accumulation was induced in CLas infected sweet orange varieties, peroxidase enzymatic activity varied. Specific serine proteases were up-regulated in Washington navel in the presence of CLas based on quantitative proteomics. Subsequent activity-based protein profiling revealed increased activity of two serine proteases, and reduced activity of one protease in two C. sinensis sweet orange varieties under greenhouse and field conditions. The observations in the current study highlight global reprogramming of the citrus vascular proteome and differential regulation of enzyme classes in response to CLas infection. These results open an avenue for further investigation of diverse responses to HLB across different environmental conditions and citrus genotypes.

Colorectal cancer (CRC) arises as the consequence of progressive changes from normal epithelial cells through polyp to tumor, and thus is an useful model for studying metabolic shift. In the present study, we studied the metabolomic profiles using high analyte specific gas chromatography/mass spectrometry (GC/MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS) to attain a systems-level view of the shift in metabolism in cells progressing along the path to CRC. Colonic tissues including tumor, polyps and adjacent matched normal mucosa from 26 patients with sporadic CRC from freshly isolated resections were used for this study. The metabolic profiles were obtained using GC/MS and LC/MS/MS. Our data suggest there was a distinct profile change of a wide range of metabolites from mucosa to tumor tissues. Various amino acids and lipids in the polyps and tumors were elevated, suggesting higher energy needs for increased cellular proliferation. In contrast, significant depletion of glucose and inositol in polyps revealed that glycolysis may be critical in early tumorigenesis. In addition, the accumulation of hypoxanthine and xanthine, and the decrease of uric acid concentration, suggest that the purine biosynthesis pathway could have been substituted by the salvage pathway in CRC. Further, there was a step-wise reduction of deoxycholic acid concentration from mucosa to tumors. It appears that to gain a growth advantage, cancer cells may adopt alternate metabolic pathways in tumorigenesis and this flexibility allows them to adapt and thrive in harsh environment.

Protein tyrosine kinases (PTKs) play key roles in cellular signal transduction, cell cycle regulation, cell division, and cell differentiation. Dysregulation of PTK-activated pathways, often by receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. In this study, we have developed a parallel reaction monitoring (PRM)-based assay for quantitative profiling of 83 PTKs. The assay detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We implemented the assay in four cell models: 1) a comparison of proliferating versus epidermal growth factor (EGF)-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, and 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility (11-18) or acquired resistance (11-18R) to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We observed distinct PTK expression changes that were induced by stimuli, genomic features or drug resistance, which were consistent with previous reports. However, most of the measured expression differences were novel observations. For example, acquired resistance to erlotinib in the 11-18 cell model was associated not only with previously reported upregulation of MET, but also with upregulation of FLK2 and downregulation of LYN and PTK7. Immunoblot analyses and shotgun proteomics data were highly consistent with PRM data. Multiplexed PRM assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations. Data are available through Proteome eXchange Accession PXD002706.

Glycopeptides in peptide or digested protein samples pose a number of analytical and bioinformatics challenges beyond those posed by unmodified peptides or peptides with smaller posttranslational modifications. Exact structural elucidation of glycans is generally beyond the capability of a single mass spectrometry experiment, so a reasonable level of identification for tandem mass spectrometry, taken by several glycopeptide software tools, is that of peptide sequence and glycan composition, meaning the number of monosaccharides of each distinct mass, for example HexNAc(2)Hex(5) rather than man5. Even at this level, however, glycopeptide analysis poses challenges:  finding glycopeptide spectra when they are a tiny fraction of the total spectra; assigning spectra with unanticipated glycans, not in the initial glycan database; and finding, scoring, and labeling diagnostic peaks in tandem mass spectra.  Here we discuss recent improvements to Byonic, a glycoproteomics search program, that address these three issues. Byonic now supports filtering spectra by m/z peaks, so that the user can limit attention to spectra with diagnostic peaks, for example, at least two out of three of 204.087 for HexNAc, 274.092 for NeuAc (with water loss), and 366.139 for HexNAc-Hex, all within a set mass tolerance, for example, ± 0.01 Daltons. Also new is glycan "wildcard" search, which allows an unspecified mass within a user-set mass range to be applied to N- or O-linked glycans and enables assignment of spectra with unanticipated glycans. Finally the next release of Byonic supports user-specified peak annotations from user-defined posttranslational modifications. We demonstrate the utility of these new software features by finding previously unrecognized glycopeptides in publicly available data, including glycosylated neuropeptides from rat brain.

Biological functions emerge from complex and dynamic networks of protein-protein interactions. Because these protein-protein interaction networks, or interactomes, represent pairwise connections within a hierarchically organized system, it is often useful to identify higher-order associations embedded within them, such as multi-member protein complexes. Graph-based clustering techniques are widely used to accomplish this goal, and dozens of field-specific and general clustering algorithms exist. However, interactomes can be prone to errors, especially when inferred from high-throughput biochemical assays. Therefore, robustness to network-level noise is an important criterion for any clustering algorithm that aims to generate robust, reproducible clusters. Here, we tested the robustness of a range of graph-based clustering algorithms in the presence of noise, including algorithms common across domains and those specific to protein networks. Strikingly, we found that all of the clustering algorithms tested here markedly amplified noise within the underlying protein interaction network. Randomly rewiring only 1% of network edges yielded more than a 50% change in clustering results, indicating that clustering markedly amplified network-level noise. Moreover, we found the impact of network noise on individual clusters was not uniform: some clusters were consistently robust to injected noise while others were not. To assist in assessing this, we developed the clust.perturb R package and Shiny web application to measure the reproducibility of clusters by randomly perturbing the network. We show that clust.perturb results are predictive of real-world cluster stability: poorly reproducible clusters as identified by clust.perturb are significantly less likely to be reclustered across experiments. We conclude that graph-based clustering amplifies noise in protein interaction networks, but quantifying the robustness of a cluster to network noise can separate stable protein complexes from spurious associations.

Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver bacterial proteins, termed ‘effector proteins’ into the host cell during infection by sophisticated protein translocation systems, which manipulate cellular processes and functions. The functional contribution of individual effectors is poorly characterised, particularly in intracellular bacterial pathogens with large effector protein repertoires. Technical caveats have limited the capacity to study these proteins during a native infection, with many effector proteins having only been demonstrated to be translocated during over-expression of tagged versions. Here we developed a novel strategy to examine effector proteins in the context of infection. We coupled a broad, unbiased proteomics-based screen with organelle purification to study the host-pathogen interactions occurring between the host cell mitochondrion and the Gram-negative, Q fever pathogen Coxiella burnetii. We identify 4 novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localisation of ectopically expressed proteins confirmed their mitochondrial localisation, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein localises to the inner membrane and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to study intracellular host-pathogen interactions, providing a robust strategy to examine the sub-cellular localisation of effector proteins during native infection. This approach could be applied to a range of pathogens and host cell compartments to provide a rich map of effector dynamics throughout infection.

Urinary proteomics studies have primarily focused on identifying markers of chronic kidney disease (CKD) progression. Here, we aimed to determine urinary markers of CKD renal parenchymal injury through proteomics analysis in animal kidney tissues and cells and in the urine of patients with CKD. Label-free quantitative proteomics analysis based on liquid chromatography-tandem mass spectrometry was performed on urine samples obtained from 6 normal controls and 9, 11, and 10 patients with CKD stages 1, 3, and 5, respectively, and on kidney tissue samples from a rat CKD model by 5/6 nephrectomy. Tandem mass tag-based quantitative proteomics analysis was performed for primary cultured glomerular endothelial cells (GECs) and proximal tubular epithelial cells (PTECs) before and after inducing 24-h hypoxia injury. Upon hierarchical clustering, out of 858 differentially expressed proteins (DEPs) in the urine of CKD patients, the levels of 416 decreased and 403 increased sequentially according to the disease stage, respectively. Among 2965 DEPs across 5/6 nephrectomized and sham-operated rat kidney tissues, 86 DEPs showed same expression patterns in the urine and kidney tissue. After cross-validation with two external animal proteome datasets, 38 DEPs were organized; only 10 DEPs, including serotransferrin, gelsolin, poly ADP-ribose polymerase 1, neuroblast differentiation-associated protein AHNAK, microtubule-associated protein 4, galectin-1, protein S, thymosin beta-4, myristoylated alanine-rich C-kinase substrate, and vimentin were finalized by screening human GECs and PTECs data. Among these ten potential candidates for universal CKD marker, validation analyses for protein S and galectin-1 were conducted. Galectin-1 was observed to have a significant inverse correlation with renal function as well as higher expression in glomerulus with chronic injury than protein S. This constitutes the first multi-sample proteomics study for identifying key renal-expressed proteins associated with CKD progression. The discovered proteins represent potential markers of chronic renal cell and tissue damage and candidate contributors to CKD pathophysiology.

Libya’s War Economy: Predation, Profiteering and State Weakness Research paper sysadmin 9 April 2018

As Libya’s war economy persists, prospects for the restoration of functioning central governance become more distant.

Summary

• Libya suffers from interlinked political, security and economic crises that are weakening state institutions, damaging its economy and facilitating the continued existence of non-state armed groups. As rival authorities continue to compete for power, the resulting fragmentation and dysfunction have provided a fertile environment for the development of a pervasive war economy dependent on violence.
• This war economy is dynamic and constantly in flux. Relative to earlier problems, there were signs of progress on several fronts in 2017: a reduction in human smuggling, a tripling in oil revenues, and increased local action against fuel smuggling. Yet the dynamics that have supported the war economy’s rise remain.
• Libya’s war economy is highly damaging for the future of the state for three reasons:
  • First, it provides an enabling environment for networks of armed groups, criminal networks, corrupt businessmen and political elites to sustain their activities through illicit sales and predatory practices. Their operations are closely linked to the dispensation of violence, and are thus a spur for further conflict.
  • Second, the war economy perpetuates negative incentives for those who profit from the state’s dysfunction. Only effective governance, supported by a durable political settlement, can tackle the foundations of Libya’s war economy. But neither a return to functioning central governance nor the development of a security sector that is fit for purpose is in the interests of war economy profiteers, who are therefore motivated to act as powerful spoilers of reform.
  • Third, the political contestation and resource predation practised by those engaged in the war economy are having a disastrous impact on Libya’s formal economy, undermining what remains of its institutions. As the war economy persists, therefore, the prospects for the restoration of functioning central governance become more distant. This threatens to create a vicious cycle that accelerates further state collapse.
• Due to the limited capacity for coercion available to any actor or entity connected with the state, a strategy of co-opting networks of war economy profiteers has almost exclusively prevailed. This has failed. Drawing on the lessons from these attempts, a more successful policy must pursue targeted measures to combat the enabling structures of Libya’s war economy where possible, and to co-opt war economy profiteers only where necessary.
• In this, state authorities can do more to utilize what power they have to name and shame war economy profiteers in order to weaken the local legitimacy critical to profiteers’ survival. The state must present credible alternative livelihood opportunities to those engaged in, or benefiting from, the war economy. Progress will depend in part on the creation of positive incentives to abandon such activity. Where profiteers cannot be incentivized to move towards more legitimate economic activities, greater and more effective efforts must be made to reduce the profit margins of illicit schemes.
• The international community can do more to support Libyan efforts in countering the war economy. Cooperation over the targeting of criminal groups’ overseas assets, support for increased transparency over the dispensation of state funds, and measures to reduce the viability of illicit activities can all help to strengthen the position of state authorities.

Further reading

Discover the six things you need to know about Libya’s war economy

Exploring Transatlantic Responses to Far-right Populism in Europe: Simulation Exercise Research paper sysadmin 1 May 2018

A new paper summarizes the findings of a recent simulation exercise exploring how governments on both sides of the Atlantic might respond to a descent towards populist authoritarianism in an EU member state.

Summary

• To better understand how governments on both sides of the Atlantic might respond to a descent towards populist authoritarianism in an EU member state, Chatham House organized a simulation event involving a group of experts drawn from the public sector, academia and NGOs.
• Simulation exercises enable the testing and modelling of the responses of different actors when presented with specific situations; participants’ interactions in a given set of circumstances are explored, and patterns of negotiation are captured and analysed.
• In this simulation, European, US and multilateral representatives were given the task of managing relations with Baltia, a fictional Eastern European state on the verge of electing a far-right nationalist, Eurosceptic government. They were then challenged to manage their relationship with Baltia after it had elected such a government, which was pushing for a ‘leave’ vote in a planned referendum on the country’s continued EU membership.
• The simulation highlighted a number of issues:
  • Limited instruments are available to liberal democratic governments where there is cause for concern regarding the outcome of an election in an allied country. There are relatively few tools at the disposal of governments to support political allies, or to prevent outcomes that are perceived as threatening democratic norms. The simulation reinforced the view that interventionist moves, either from the European Commission or from individual national governments, would be more likely to come in response to an unfavourable development rather than pre-emptively.
  • The EU, and caucuses of European states, are the main international interlocutors in this type of political crisis involving an EU member state. The US opted to play a limited role in the negotiations; the same was largely true for NATO, aside from its action in sharing intelligence about a potential coup in Baltia. France and Germany formed a natural working partnership, taking meetings together and coordinating policies first before discussing them with a wider European circle, although their positions did not always align.
  • The UK’s capacity to shape the outcome of collective EU discussions appeared more restricted, while Brexit also seemed to shape the response of other EU states to the developing situation in Baltia. Although member states were undoubtedly reluctant to see another country go down this route, they were also resolute in demonstrating a unity of approach and limited flexibility in the face of the new populist government’s attempt to divide them.

Exploring Public International Law and the Rights of Individuals with Chinese Scholars - Part One Other resource sysadmin 29 October 2018

As part of a roundtable series, Chatham House and China University of Political Science and Law (CUPL) jointly organized this four-day meeting at Chatham House for international lawyers to discuss a wide range of issues related to public international law and the rights of individuals.

The specific objectives were to:

• create a platform for Chinese international law academics working on international human rights law issues to present their thinking and exchange ideas with counterparts from outside China;
• build stronger understanding within the wider international law community of intellectual debates taking place in China about the international human rights system and China’s role within it;
• support networking between Chinese and non-Chinese academics working on international human rights and related areas of international law.

The roundtable forms part of a wider Chatham House project exploring China’s impact on the international human rights system and was inspired by early discussions with a burgeoning community of Chinese academics thinking, writing (mainly in Chinese) and teaching about international human rights law.

For China University of Political Science and Law, one of the largest and most prestigious law schools in China and perhaps the only university in the world with an entire faculty of international law, the initiative is part of a drive to forge partnerships beyond China in the international law field.

The roundtable had a total of 22 participants, 10 Chinese (from universities and other academic institutions in Beijing and Shanghai) and 12 non-Chinese (from Australia, Germany, the Netherlands, Switzerland, the United Kingdom and the United States).

All discussions were held in English under the Chatham House Rule.

Exploring Public International Law and the Rights of Individuals with Chinese Scholars - Part Two Other resource sysadmin 30 October 2018

As part of a roundtable series, Chatham House and China University of Political Science and Law (CUPL) held a two-day roundtable meeting in Beijing on public international law and the rights of individuals.

The specific objectives were to:

• create a platform for Chinese international law academics working on international human rights law issues to present their thinking and exchange ideas with counterparts from outside China;
• build stronger understanding within the wider international law community of intellectual debates taking place in China about the international human rights system and China’s role within it;
• support networking between Chinese and non-Chinese academics working on international human rights and related areas of international law.

The roundtable forms part of a wider Chatham House project exploring China’s impact on the international human rights system and was inspired by early discussions with a burgeoning community of Chinese academics thinking, writing (mainly in Chinese) and teaching about international human rights law.

For CUPL, one of the largest and most prestigious law schools in China and perhaps the only university in the world with an entire faculty of international law, the initiative is part of a drive to forge partnerships beyond China in the international law field.

The meeting in Beijing was hosted by CUPL and involved 20 participants, 10 Chinese (from universities and other academic institutions in Beijing) and 10 non-Chinese (from Australia, the Netherlands, South Africa, Switzerland, the United Kingdom and the United States).

To ensure continuity while also expanding the experts network being built, the second meeting included a mix of participants from the first meeting and some new participants.

All discussions were held in English under the Chatham House Rule.

Exploring Public International Law and the Rights of Individuals with Chinese Scholars - Part Three Other resource sysadmin 30 October 2018

As part of a roundtable series, Chatham House, China University of Political Science and Law (CUPL) and the Graduate Institute Geneva held a two-day roundtable meeting in Geneva on public international law and the rights of individuals.

The specific objectives were to:

• create a platform for Chinese international law academics working on international human rights law issues to present their thinking and exchange ideas with counterparts from outside China;
• build stronger understanding within the wider international law community of intellectual debates taking place in China about the international human rights system and China’s role within it;
• support networking between Chinese and non-Chinese academics working on international human rights and related areas of international law.

The roundtable forms part of a wider Chatham House project exploring China’s impact on the international human rights system and was inspired by early discussions with a burgeoning community of Chinese academics thinking, writing (mainly in Chinese) and teaching about international human rights law.

For CUPL, one of the largest and most prestigious law schools in China and perhaps the only university in the world with an entire faculty of international law, the initiative is part of a drive to forge partnerships beyond China in the international law field.

The meeting in Geneva was co-hosted by the Graduate Institute Geneva and involved 19 participants, 9 Chinese (from six research institutions in Beijing and Shanghai) and 11 non-Chinese (from eight research institutions in Australia, Germany, the Netherlands, Switzerland, the United Kingdom and the United States).

To ensure continuity while also expanding the expert network being built, the third meeting included a mix of participants from the first two meetings and some new participants

All discussions were held in English under the Chatham House Rule.

Exploring Public International Law Issues with Chinese Scholars – Part Four Other resource sysadmin 30 October 2018

As part of a roundtable series, Chatham House and the China University of Political Science and Law (CUPL) held a two-day roundtable in Beijing on emerging issues of public international law.

The specific objectives were to:

• create a platform for Chinese international law academics working on international human rights law issues to present their thinking and exchange ideas with counterparts from outside China;
• build stronger understanding within the wider international law community of intellectual debates taking place in China about the international human rights system and China’s role within it;
• support networking between Chinese and non-Chinese academics working on international human rights and related areas of international law.

The roundtable forms part of a wider Chatham House project exploring China’s impact on the international human rights system and was inspired by early discussions with a burgeoning community of Chinese academics thinking, writing (mainly in Chinese) and teaching about international human rights law.

For CUPL, one of the largest and most prestigious law schools in China and perhaps the only university in the world with an entire faculty of international law, the initiative is part of a drive to forge partnerships beyond China in the international law field.

The meeting was co-hosted with CUPL and involved 28 participants, consisting of 19 Chinese participants (from six leading research institutions in Beijing and Shanghai) and nine nonChinese participants (from eight leading research institutions in Australia, the Netherlands, the UK, Switzerland, Canada and Singapore).

To ensure continuity while also expanding the expert network being built, the fifth meeting included a mix of participants from the previous meetings and some new participants.

All discussions were held in English under the Chatham House Rule.

Delivering Sustainable Food and Land Use Systems: The Role of International Trade Research paper sysadmin 20 September 2019

This paper explores a set of core trade-related issues affecting the food and land use system, and proposes constructive ways forward in reconfiguring the global trading system towards delivering a more sustainable and healthy diet for all.

• Meeting future global food security requirements is not just about quantity; it is also about meeting growing needs in a way that safeguards human as well as planetary health. But national priorities and policies often remain out of sync with aspirations for more sustainable and healthy food systems.
• International trade and trade policies play an ambiguous role in the current food system. With 80 per cent of the world’s population depending on imports to meet at least part of their food and nutritional requirements, trade has a unique function in offsetting imbalances between supply and demand. However, in the absence of effective regulatory frameworks or pricing frameworks that internalize environmental, social or health costs, trade can exacerbate and globalize challenges associated with food production and land use trends such as deforestation, land degradation, greenhouse gas emissions, biodiversity loss and the shift to unhealthy diets.
• Over the last two decades, trade in agricultural products (excluding intra-EU flows) has more than tripled in value, to reach $1.33 trillion. The geography of global food trade flows has also shifted, primarily towards South–South trade, which now accounts for roughly a quarter of total agricultural trade flows. The nature of global trade has changed drastically, with traditional exports such as wheat and coffee growing slowly at around 2 per cent per year, while products such as palm oil, fruit juice, soft drinks and other processed products have grown at 8 per cent or more annually.
• This overall increase in trade in agricultural products raises questions about the growing utilization of resources, such as water or soil nutrients, that are embedded in those products through production and processing. Trade itself also causes negative environmental impacts, starting with greenhouse gas emissions associated with transport and storage. If the environmental cost associated with production and trade is not reflected in the final price of goods, trade may accelerate the depletion of resources or their unsustainable use.
• It is critical to ensure that trade policy options pursued by producing and consuming countries alike will support a transition to more sustainable and healthier food and land use systems. The first step in addressing trade-related food systems challenges must involve rebuilding trust among policy actors. There is a need for new spaces for informal dialogue among actors, and ‘soft’ governance mechanisms that can help rebuild consensus on the best ways forward. Meeting these challenges also requires an appreciation of the complex interactions between sectoral policies (e.g. on water, land, food, etc.) and their multiple interfaces with trade policies.
• Conditioning the use of subsidies on their sustainability and/or health impacts encourages the delivery of essential public goods in ways that are consistent with sustainability and health goals. A first step therefore is the removal of perverse incentives (e.g. subsidies encouraging the overuse of fertilizers or pesticides or the overproduction of certain commodities, as well as certain biofuels subsidies) and replacing them with market-correcting subsidies.
• Trade facilitation measures for fruits and vegetables that are aimed at easing transit at the border, by cutting unnecessary bureaucracy and reducing waiting times, can improve their availability, reduce costs and improve food quality and safety for consumers. Similarly, measures aimed at improving sustainable cold storage and upgrading value chains can support better diets and consumption by increasing the availability of fresh produce on markets, especially in developing countries.
• A global food stamps programme developed through the G20 and facilitated by the UN’s food agencies could address purchasing power imbalances and tackle malnutrition in developing countries. If carefully designed, such ‘safety net’ schemes can not only contribute to improving calorific intakes but also help deliver more balanced and healthier diets. Careful attention must be given to how such a scheme would work in practice, building on experience to date with similar initiatives.
• Integrating the notion of sustainable food and inputs trade in the post-2020 global biodiversity framework can help to deliver more sustainable and healthier food and land use systems. This could be achieved by likeminded countries introducing a set of goals or targets aimed at mitigating the role of trade in placing indirect pressure on biodiversity, and to encourage trade in biodiversity-based products including natural ingredients produced ethically and following sustainability principles and criteria.
• An SDG-oriented agenda for agricultural trade is needed. It could be formed by countries seeking to remove perverse incentives, guaranteeing a safe harbour for market-correcting measures, clarifying existing rules and establishing plurilateral negotiations among subsets of the WTO membership, or sectoral approaches, to address specific challenges.
• Greenhouse gas emissions resulting from trade need to be addressed. Governments could seek to achieve this through ensuring the carbon neutrality of existing and new trade deals, either by connecting carbon markets among contracting parties or by developing joint initiatives to tax international maritime and air transport emissions.

Centenary Series: Exploring the International Affairs Archive dora.popova 14 September 2020

International Affairs has been a central part of the institute’s history, both as a record of speeches made by dignitaries such as Mahatma Gandhi and Henry Kissinger, and as a forum for policy-relevant academic research.

Delving into the International Affairs archive brings out stories behind some of the most significant players of the last century.