O-fucosylation stabilizes the TSR3 motif in thrombospondin-1 by interacting with nearby amino acids and protecting a disulfide bond

Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by β3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER.     

FastD: Fast detection of insecticide target‐site mutations and overexpressed detoxification genes in insect populations from RNA‐Seq data

Target‐site mutations and detoxification gene overexpression are two major mechanisms conferring insecticide resistance. Molecular assays applied to detect these resistance genetic markers are time‐consuming and with high false‐positive rates. RNA‐Seq data contains information on the variations within expressed genomic regions and expression of detoxification genes. However, there is no corresponding method to detect resistance markers at present. Here, we collected 66 reported resistance mutations of four insecticide targets (AChE, VGSC, RyR, and nAChR) from 82 insect species. Next, we obtained 403 sequences of the four target genes and 12,665 sequences of three kinds of detoxification genes including P450s, GSTs, and CCEs. Then, we developed a Perl program, FastD, to detect target‐site mutations and overexpressed detoxification genes from RNA‐Seq data and constructed a web server for FastD (http://www.insect-genome.com/fastd). The estimation of FastD on simulated RNA‐Seq data showed high sensitivity and specificity. We applied FastD to detect resistant markers in 15 populations of six insects, Plutella xylostella, Aphis gossypii, Anopheles arabiensis, Musca domestica, Leptinotarsa decemlineata and Apis mellifera. Results showed that 11 RyR mutations in P. xylostella, one nAChR mutation in A. gossypii, one VGSC mutation in A. arabiensis and five VGSC mutations in M. domestica were found to be with frequency difference >40% between resistant and susceptible populations including previously confirmed mutations G4946E in RyR, R81T in nAChR and L1014F in VGSC. And 49 detoxification genes were found to be overexpressed in resistant populations compared with susceptible populations including previously confirmed detoxification genes CYP6BG1, CYP6CY22, CYP6CY13, CYP6P3, CYP6M2, CYP6P4 and CYP4G16. The candidate target‐site mutations and detoxification genes were worth further validation. Resistance estimates according to confirmed markers were consistent with population phenotypes, confirming the reliability of this program in predicting population resistance at omics‐level.     

Drosophila sperm surface alpha-l-fucosidase interacts with the egg coats through its core fucose residues

Sperm–oocyte interaction during fertilization is multiphasic, with multicomponent events, taking place between egg's glycoproteins and sperm surface receptors. Protein–carbohydrate complementarities in gamete recognition have observed in cases throughout the whole evolutionary scale. Sperm-associated α-l-fucosidases have been identified in various organisms. Their wide distribution and known properties reflect the hypothesis that fucose and α-l-fucosidases have fundamental function(s) during gamete interactions. An α-l-fucosidase has been detected as transmembrane protein on the surface of spermatozoa of eleven species across the genus Drosophila. Immunofluorescence labeling showed that the protein is localized in the sperm plasma membrane over the acrosome and the tail, in Drosophila melanogaster. In the present study, efforts were made to analyze with solid phase assays the oligosaccharide recognition ability of fruit fly sperm α-l-fucosidase with defined carbohydrate chains that can functionally mimic egg glycoconjugates. Our results showed that α-l-fucosidase bound to fucose residue and in particular it prefers N-glycans carrying core α1,6-linked fucose and core α1,3-linked fucose in N-glycans carrying only a terminal mannose residue. The ability of sperm α-l-fucosidase to bind to the micropylar chorion and to the vitelline envelope was examined in in vitro assays in presence of α-l-fucosidase, either alone or in combination with molecules containing fucose residues. No binding was detected when α-l-fucosidase was pre-incubated with fucoidan, a polymer of α-l-fucose and the monosaccharide fucose. Furthermore, egg labeling with anti-horseradish peroxidase, that recognized only core α1,3-linked fucose, correlates with α-l-fucosidase micropylar binding. Collectively, these data support the hypothesis of the potential role of this glycosidase in sperm–egg interactions in Drosophila.     

Epidemiology of Doublet/Multiplet Mutations in Lung Cancers: Evidence that a Subset Arises by Chronocoordinate Events

Background

Evidence strongly suggests that spontaneous doublet mutations in normal mouse tissues generally arise from chronocoordinate events. These chronocoordinate mutations sometimes reflect “mutation showers”, which are multiple chronocoordinate mutations spanning many kilobases. However, little is known about mutagenesis of doublet and multiplet mutations (domuplets) in human cancer. Lung cancer accounts for about 25% of all cancer deaths. Herein, we analyze the epidemiology of domuplets in the EGFR and TP53 genes in lung cancer. The EGFR gene is an oncogene in which doublets are generally driver plus driver mutations, while the TP53 gene is a tumor suppressor gene with a more typical situation in which doublets derive from a driver and passenger mutation.

Methodology/Principal Findings

EGFR mutations identified by sequencing were collected from 66 published papers and our updated EGFR mutation database (www.egfr.org). TP53 mutations were collected from IARC version 12 (www-p53.iarc.fr). For EGFR and TP53 doublets, no clearly significant differences in race, ethnicity, gender and smoking status were observed. Doublets in the EGFR and TP53 genes in human lung cancer are elevated about eight- and three-fold, respectively, relative to spontaneous doublets in mouse (6% and 2.3% versus 0.7%).

Conclusions/Significance

Although no one characteristic is definitive, the aggregate properties of doublet and multiplet mutations in lung cancer are consistent with a subset derived from chronocoordinate events in the EGFR gene: i) the eight frameshift doublets (present in 0.5% of all patients with EGFR mutations) are clustered and produce a net in-frame change; ii) about 32% of doublets are very closely spaced (≤30 nt); and iii) multiplets contain two or more closely spaced mutations. TP53 mutations in lung cancer are very closely spaced (≤30 nt) in 33% of doublets, and multiplets generally contain two or more very closely spaced mutations. Work in model systems is necessary to confirm the significance of chronocoordinate events in lung and other cancers.     

Chemical characterisation of the released polysaccharide from the cyanobacterium Aphanothece halophytica GR02

The released polysaccharide from the halophilic cyanobacterium Aphanothece halophytica GR02 was separated into two main fractions byanion-exchange chromatography. The major fraction consisted of glucose,fucose, mannose, arabinose and glucuronic acid. Judging from thechromatography on Sepharose 2B, the major fraction was not furtherfractionated, and its apparent molecular weight was above 2.0 × 10⁶ Da.The minor fraction consisted of rhamnose, mannose, fucose,glucose, galactose and glucuronic acid, with traces of arabinose.Methylation and GC-MS spectrometry analyses of the major fractionrevealed the presence of 1-linked glucose, 1,3-linked glucose, 1,3-linkedfucose, 1,4-linked fucose, 1,3-linked arabinose, 1,2,4-linked mannose,1,3,6-linked mannose, 1-linked glucuronic acid and 1,3-linked glucuronicacid residues. The major fraction was thought to originate from capsularpolysaccharide. The released polysaccharides, obtained from cultures atdifferent age of culture, showed no striking variations in themonosaccharide composition and the relative proportions of themonosaccharides. However, the proportions of galactose and rhamnose inthe released polysaccharides, obtained from cultures under different salinity,were significantly different. The released polysaccharide also exhibitedgelling properties and strong affinity for metal ions.     

Identifying modules of cooperating cancer drivers

Identifying cooperating modules of driver alterations can provide insights into cancer etiology and advance the development of effective personalized treatments. We present Cancer Rule Set Optimization (CRSO) for inferring the combinations of alterations that cooperate to drive tumor formation in individual patients. Application to 19 TCGA cancer types revealed a mean of 11 core driver combinations per cancer, comprising 2–6 alterations per combination and accounting for a mean of 70% of samples per cancer type. CRSO is distinct from methods based on statistical co‐occurrence, which we demonstrate is a suboptimal criterion for investigating driver cooperation. CRSO identified well‐studied driver combinations that were not detected by other approaches and nominated novel combinations that correlate with clinical outcomes in multiple cancer types. Novel synergies were identified in NRAS‐mutant melanomas that may be therapeutically relevant. Core driver combinations involving NFE2L2 mutations were identified in four cancer types, supporting the therapeutic potential of NRF2 pathway inhibition. CRSO is available at https://github.com/mikekleinsgit/CRSO/.     

DNA G-quadruplexes for native mass spectrometry in potassium: a database of validated structures in electrospray-compatible conditions

G-quadruplex DNA structures have become attractive drug targets, and native mass spectrometry can provide detailed characterization of drug binding stoichiometry and affinity, potentially at high throughput. However, the G-quadruplex DNA polymorphism poses problems for interpreting ligand screening assays. In order to establish standardized MS-based screening assays, we studied 28 sequences with documented NMR structures in (usually ∼100 mM) potassium, and report here their circular dichroism (CD), melting temperature (T_m), NMR spectra and electrospray mass spectra in 1 mM KCl/100 mM trimethylammonium acetate. Based on these results, we make a short-list of sequences that adopt the same structure in the MS assay as reported by NMR, and provide recommendations on using them for MS-based assays. We also built an R-based open-source application to build and consult a database, wherein further sequences can be incorporated in the future. The application handles automatically most of the data processing, and allows generating custom figures and reports. The database is included in the g4dbr package (https://github.com/EricLarG4/g4dbr) and can be explored online (https://ericlarg4.github.io/G4_database.html).     

Reply to Bronstein and Vinogradov

The response by the author. Subject Categories: S&S: Economics & Business, S&S: Ethics

I thank Michael Bronstein and Sophia Vinogradov for their interest and comments. I would like to respond to a few of their points.First, I agree with the authors that empirical studies should be conducted to validate any approaches to prevent the spread of misinformation before their implementation. Nonetheless, I think that the ideas I have proposed may be worth further discussion and inspire empirical studies to test their effectiveness.Second, the authors warn that informing about the imperfections of scientific research may undermine trust in science and scientists, which could result in higher vulnerability to online health misinformation (Roozenbeek et al, 2020; Bronstein & Vinogradov, 2021). I believe that transparency about limitations and problems in research does not necessarily have to diminish trust in science and scientists. On the contrary, as Veit et al put it, “such honesty… is a prerequisite for maintaining a trusting relationship between medical institutions (and practitioners) and the public” (Veit et al, 2021). Importantly, to give an honest picture of scientific research, information about its limitations should be put in adequate context. In particular, the public also should be aware that “good science” is being done by many researchers; we do have solid evidence of effectiveness of many medical interventions; and efforts are being taken to address the problems related to quality of research.Third, Bronstein and Vinogradov suggest that false and dangerous information should be censored. I agree with the authors that “[c]ensorship can prevent individuals from being exposed to false and potentially dangerous ideas” (Bronstein & Vinogradov, 2021). I also recognize that some information is false beyond any doubt and its spread may be harmful. What I am concerned about are, among others, the challenges related to defining what is dangerous and false information and limiting censorship only to this kind of information. For example, on what sources should decisions to censor be based and who should make such decisions? Anyone, whether an individual or an organization, with a responsibility to censor information will likely not only be prone to mistakes, but also to abuses of power to foster their interests. Do the benefits we want to achieve by censorship outweigh the potential risks?Fourth, we need rigorous empirical studies examining the actual impact of medical misinformation. What exactly are the harms we try to protect against and what is their scale? This information is necessary to choose proportionte and effective measures to reduce the harms. Bronstein and Vinogradov give an example of a harm which may be caused by misinformation—an increase in methanol poisoning in Iran. Yet, as noticed by the authors, misinformation is not the sole factor in this case; there are also cultural and other contexts (Arasteh et al, 2020; Bronstein & Vinogradov, 2021). Importantly, the methods of studies exploring the effects of misinformation should be carefully elaborated, especially when study participants are asked to self‐report. A recent study suggests that some claims about the prevalence of dangerous behaviors, such as drinking bleach, which may have been caused by misinformation are largely exaggerated due to the presence of problematic respondents in surveys (preprint: Litman et al, 2021).Last but not least, I would like to call attention to the importance of how veracity of information is determined in empirical studies on misinformation. For example, in a study of Roozenbeek et al, cited by Bronstein and Vinogradov, the World Health Organization (WHO) was used as reliable source of information, which raises questions. For instance, Roozenbeek et al (2020) used a statement “the coronavirus was bioengineered in a military lab in Wuhan” as an example of false information, relying on the judgment of the WHO found on its “mythbusters” website (Roozenbeek et al, 2020). Yet, is there a solid evidence to claim that this statement is false? At present, at least some scientists declare that we cannot rule out that the virus was genetically manipulated in a laboratory (Relman, 2020; Segreto & Deigin, 2020). Interestingly, the WHO also no longer excludes such a possibility and has launched an investigation on this issue (https://www.who.int/health‐topics/coronavirus/origins‐of‐the‐virus, https://www.who.int/emergencies/diseases/novel‐coronavirus‐2019/media‐resources/science‐in‐5/episode‐21‐‐‐covid‐19‐‐‐origins‐of‐the‐sars‐cov‐2‐virus); the information about the laboratory origin of the virus being false is no longer present on the WHO “mythbusters” website (https://www.who.int/emergencies/diseases/novel‐coronavirus‐2019/advice‐for‐public/myth‐busters). Against this backdrop, some results of the study by Roozenbeek et al (2020) seem misleading. In particular, the perception of the reliability of the statement about bioengineered virus by study participants in Roozenbeek et al (2020) does not reflect the susceptibility to misinformation, as intended by the researchers, but rather how the respondents perceive reliability of uncertain information.I hope that discussion and research on these and related issues will continue.     

Differential Regulation of the Ten-Eleven Translocation (TET) Family of Dioxygenases by O-Linked β-N-Acetylglucosamine Transferase (OGT)

The ten-eleven translocation (TET) family of dioxygenases (TET1/2/3) converts 5-methylcytosine to 5-hydroxymethylcytosine and provides a vital mechanism for DNA demethylation. However, how TET proteins are regulated is largely unknown. Here we report that the O-linked β-GlcNAc (O-GlcNAc) transferase (OGT) is not only a major TET3-interacting protein but also regulates TET3 subcellular localization and enzymatic activity. OGT catalyzes the O-GlcNAcylation of TET3, promotes TET3 nuclear export, and, consequently, inhibits the formation of 5-hydroxymethylcytosine catalyzed by TET3. Although TET1 and TET2 also interact with and can be O-GlcNAcylated by OGT, neither their subcellular localization nor their enzymatic activity are affected by OGT. Furthermore, we show that the nuclear localization and O-GlcNAcylation of TET3 are regulated by glucose metabolism. Our study reveals the differential regulation of TET family proteins by OGT and a novel link between glucose metabolism and DNA epigenetic modification.     

The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria

1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase and NADH–cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP⁺-linked) and NADPH–cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, α-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.     

Notes: β(1-3)Glucanosyltransferase Gel4p Is Essential for Aspergillus fumigatus

The β(1-3)glucanosyltransferase GEL family of Aspergillus fumigatus contains 7 genes, among which only 3 are expressed during mycelial growth. The role of the GEL4 gene was investigated in this study. Like the other Gelps, it encodes a glycosylphosphatidylinositol (GPI)-anchored protein. In contrast to the other β(1-3)glucanosyltransferases analyzed to date, it is essential for this fungal species.β(1-3)Glucan is the main component of the fungal cell wall (11). In fungi, β(1-3)glucans are synthesized by a plasma membrane-bound glucan synthase complex. Neosynthesized glucans are then extruded into the periplasmic space (2, 3, 9), where they become branched and covalently linked to other cell wall components, resulting in the formation of three-dimensional rigid structures. In the search of transglycosidase in the filamentous fungus Aspergillus fumigatus, β(1-3)glucanosyltransferases were identified and classified as a unique family (GH72) in the Carbohydrate-Active enZYmes database (http://www.cazy.org/). These enzymes cleave the β(1-3) bond of a β(1-3)glucan oligosaccharide with at least 10 glucose units and transfer the newly formed reducing end (>5 glucose units) to the nonreducing end of another β(1-3)glucan oligosaccharide, resulting in the elongation of the β(1-3)glucans. This reaction can proceed in vitro until the neosynthetized β(1-3)glucan becomes insoluble. Initially demonstrated biochemically, the requirement for long-chain β(1-3)glucan oligosaccharide has now been confirmed by the analysis of the first crystal structure obtained in this transglycosidase family (7, 8). First discovered in Aspergillus fumigatus and named Gelp for glucan elongase, this activity has been found in all fungal species investigated to date and could be assigned to orthologous proteins, such as Gasp or Phrp, that were known to be involved in cell wall integrity but were endowed with an unknown biochemical function (12, 13, 14).     

Granular detail of β cell structures for insulin secretion

Pancreatic β cells secrete insulin in response to increased glucose concentrations. Müller et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202010039) use 3D FIB-SEM to study the architecture of these cells and to elucidate how glucose stimulation remodels microtubules to control insulin secretory granule exocytosis.

The pancreatic islet β cell is a prototypical model for regulated protein secretion, which has been studied extensively because of its importance for diabetes in humans. Upon stimulation by increased glucose concentrations, β cells mobilize insulin-containing secretory vesicles to the cell surface. These “insulin secretory granules” fuse at the plasma membrane to release insulin into the circulation. Insulin then acts on the liver to inhibit glucose production and on muscle and fat cells to stimulate glucose uptake, thus returning blood glucose concentrations to a narrow physiological range. During the development of diabetes, this negative feedback system fails. In type 1 diabetes, β cells are destroyed by an autoimmune process; in type 2 diabetes, attenuated insulin action (“insulin resistance”) occurs together with impaired β cell function. In both cases, blood glucose concentrations rise above the normal range. Overall, glucose homeostasis requires a delicate balance between glucose-stimulated insulin secretion from β cells and insulin-stimulated effects on glucose metabolism in liver, muscle, fat, and other cell types.Each β cell contains 5,000 to ≥10,000 insulin secretory granules, and acute glucose stimulation causes exocytosis of only 1–2% of this pool (1, 2). Both readily releasable and reserve pools of granules contribute to insulin secretion. Classically, the readily releasable pool has been considered as vesicles that are docked at the plasma membrane; the reserve pool, which is larger and more important, resides deeper within the cell and relies on microtubule-based transport to reach the cell surface. Yet, other data show that newly synthesized insulin is preferentially released, and aged insulin granules are targeted for degradation in lysosomes, implying that microtubules play a more complicated role in granule trafficking (3). In the setting of insulin resistance, the flux of insulin through the secretory pathway is increased. Demands are placed upon the machinery for folding of the insulin precursor, proinsulin, for its proteolytic conversion to produce insulin, and for insulin secretory granule maturation. As well, in type 2 diabetes, lipids and other metabolites act directly on β cells to impair glucose-stimulated insulin secretion. What steps are affected by this critical pathophysiologic insult is not well understood, in part because basic mechanisms by which glucose stimulation remodels microtubules to promote insulin release remain undefined.To better understand these processes, Müller et al. used focused ion beam scanning electron microscopy (FIB-SEM) to image microtubules, insulin secretory granules, and other organelles in whole primary mouse β cells and to study the effects of glucose stimulation on these structures (4). Together with advances in sample preparation, image segmentation, and analysis, FIB-SEM is uniquely suited to this task. The resulting 3D images have a voxel size of 4 × 4 × 4 nm and encompass volumes of 20–30 µm in x, y, and z dimensions. This is sufficient to image microtubules, which have an outer diameter of ~25 nm, in whole β cells, which are 10–20 µm in diameter. Moreover, the imaging was performed on intact islets, rather than on dissociated cells, which may better preserve insulin secretory granule dynamics. The images captured seven β cells in all, three in a low-glucose (unstimulated) condition and four in the glucose-stimulated state. Finally, the images were quantified in a way that controlled for the overall geometry of the cells.The data show that the β cell microtubule network is nonradial, dense, tortuous, and mostly not connected to either centrioles or the Golgi complex, so that microtubules appear to be freely positioned in the cytosol (Fig. 1). This is similar to other differentiated cells (5) but in contrast to previous data suggesting that in β cells most microtubules originate at the Golgi (6). The microtubule cytoskeleton negatively regulates insulin granule exocytosis in unstimulated cells (7). Yet, the FIB-SEM data suggest that the effect of glucose is not simply to disinhibit this effect. Glucose stimulated an approximately threefold increase in the number of microtubules and an approximately threefold decrease in the average length of each microtubule, so that total tubulin polymer density remained unchanged (4). The images also show that microtubule ends and insulin secretory granules are enriched near the plasma membrane and indicate an important role for microtubules in positioning the granules for exocytosis. Intriguingly, glucose stimulation did not cause any marked change in the association of secretory granules with microtubules, suggesting that it may act by other mechanisms to increase microtubule-based transport (3). The data also demonstrate an increase in the number of secretory granules near the Golgi in glucose-stimulated cells, raising the possibility that glucose may promote budding of nascent secretory vesicles at the trans-Golgi network. If so, it is not yet clear whether this is a direct effect or if it is secondary to increased flux through the secretory pathway.Open in a separate windowFigure 1.Regulation of β cell architecture by glucose. In low-glucose conditions (left panel), microtubules form a dense, nonradial network. The tubules are mostly not anchored to the Golgi complex or to centrosomes but are associated with insulin secretory granules near the plasma membrane (PM). After stimulation with high-glucose concentrations (right panel), the microtubules are shorter and more numerous, and an increased density of vesicles near the Golgi was observed.The observation that glucose stimulation results in shorter, more numerous microtubules, without changing tubulin polymer density, suggests a possible role for severing enzymes such as katanin, spastin, or fidgetin (8). Whether and how such enzymes may be stimulated by glucose is not known. Glucose stimulates insulin secretion by increasing the ATP/ADP ratio, in part due to local ATP generation by pyruvate kinase, as well as by oxidative phosphorylation (9). Although these microtubule-severing enzymes are AAA ATPases, it is not clear that ATP availability acts physiologically to regulate their activities. Other data show that in β cells, glucose-responsive kinases phosphorylate tau, causing it to dissociate from microtubule plus ends to destabilize microtubules and to promote remodeling (10). Could such kinases regulate severing enzymes as well?The technological achievements of Müller et al. are impressive, and the work will serve as a model for the analysis of cellular architecture in other cell types. In β cells, the results may be extended by using FIB-SEM to study effects of various genetic manipulations, by using EM-visible tags, and by examining diabetes models. It may be informative to image granules of different ages or to use drugs to manipulate microtubules or membrane lipids, or that act on β cells to enhance insulin secretion (3). In this study, Müller and colleagues used a 1-h high-glucose stimulation, but it may be interesting to test other time points to determine the effects of more acute versus chronic glucose exposure. Although FIB-SEM can image only fixed, static cells, the work will complement other studies using super-resolution imaging and live cell microscopy. Aside from these future directions, it is worthwhile to pause and celebrate the present work, which is the first to reconstruct the entire 3D architecture of the microtubule network in a primary mammalian cell during interphase. The movies in the supplement are gorgeous. The work bodes well for the future of FIB-SEM and will stimulate new directions to understand both diabetes physiology and regulated protein secretion.     

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that “simple” organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic subpool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a “bisecting” β-galactose, which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Our data, showing the presence of novel N-glycan modifications, will enable more targeted studies to understand the biological functions and interactions of nematode glycans.Nematodes represent, along with arthropods, one of the largest groups of animals to exist on the planet; 25.000 species are described, but the existence of up to one million has been estimated (1, 2). They have various ecological niches and include free-living “worms” in the soil, fungivorous, entomopathogenic, and necromenic species as well as parasites of plants and mammals, which share the basic conserved body plan (more-or-less a digestive tube surrounded with muscle, whether larger or smaller). There are five major clades (Rhabditina, Enoplia, Spirurina, Tylenchina, and Dorylaimia) (2), yet the glycosylation of only a few nematode species has been studied with an inevitable focus on the model nematode Caenorhabditis elegans and parasitic species (3). Thereby, the use of C. elegans mutants has been highly valuable in dissecting aspects of nematode N-glycan biosynthesis and revealing the in vivo substrates for certain glycosyltransferases (4).As many nematodes are parasites, their interactions with the immune systems of their hosts have attracted attention; particularly, there are relationships between autoimmunity, allergy, vaccination, and helminth infections. The “old friends” hypothesis seeks to understand the evolutionary factors that have shaped the immune system and to explain correlations between lifestyles in the developed world and modern “epidemics,” which are due to immunological misbalance (5–7). Promising data have suggested that “worm therapy” may bring advantages to some patients with Crohn''s disease or allergies (8, 9); however, such approaches are controversial. Nevertheless, crude extracts even of Caenorhabditis elegans were shown to induce a glycan-dependent Th2 response (10), whereas the excretory-secretory products of some nematodes also have immunomodulatory activity (11). Furthermore, the native glycoproteins of some nematodes have proven effective in vaccination trials, whereas recombinant forms are not, which is suggestive that post-translational modifications may have a role in an efficacious immune response (12).As at least some of the molecules relevant to nematode immunomodulation or vaccination are glycoproteins, a proper understanding of nematode glycosylation is of biomedical and veterinary relevance. Over the years, it has become apparent that the core chitobiosyl region of nematode N-glycans is subject to a range of modifications, with up to three core fucose residues being present (α1,3- and α1,6-linked on the reducing-terminal “proximal” GlcNAc and α1,3-linked on the second “distal” GlcNAc). However, up to four fucose residues have been detected on C. elegans N-glycans and the exact nature of the linkage of the fourth fucose has remained obscure despite work in our own and other laboratories (3, 13–15). Combined with the latest knowledge regarding the specificity of C. elegans core fucosyltransferases (13, 16, 17) as well as our recent data regarding the exact structures of N-glycans from the C. elegans double hexosaminidase mutant and other nematodes (18–20), we concluded that some models for the tri- and tetrafucosylated N-glycans were incorrect. By preparing a triple mutant unable to core fucosylate its N-glycans, we generated a C. elegans strain containing maximally one fucose residue on the N-linked oligosaccharides. Thereby a pool of unusual mannosidase-resistant N-glycans was identified and, using mass spectrometry (MS) and NMR, we reveal their modification with bisecting galactose frequently capped with fucose or methylfucose.     

Structure-guided affinity maturation of a single-chain variable fragment antibody against the Fu-bc epitope of the dengue virus envelope protein

Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (K_D) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (K_D = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy.     

Positron emission tomography and magnetic resonance imaging in experimental human malaria to identify organ-specific changes in morphology and glucose metabolism: A prospective cohort study

BackgroundPlasmodium vivax has been proposed to infect and replicate in the human spleen and bone marrow. Compared to Plasmodium falciparum, which is known to undergo microvascular tissue sequestration, little is known about the behavior of P. vivax outside of the circulating compartment. This may be due in part to difficulties in studying parasite location and activity in life.Methods and findingsTo identify organ-specific changes during the early stages of P. vivax infection, we performed 18-F fluorodeoxyglucose (FDG) positron emission tomography/magnetic resonance imaging (PET/MRI) at baseline and just prior to onset of clinical illness in P. vivax experimentally induced blood-stage malaria (IBSM) and compared findings to P. falciparum IBSM. Seven healthy, malaria-naive participants were enrolled from 3 IBSM trials: {"type":"clinical-trial","attrs":{"text":"NCT02867059","term_id":"NCT02867059"}}NCT02867059, ACTRN12616000174482, and ACTRN12619001085167. Imaging took place between 2016 and 2019 at the Herston Imaging Research Facility, Australia. Postinoculation imaging was performed after a median of 9 days in both species (n = 3 P. vivax; n = 4 P. falciparum). All participants were aged between 19 and 23 years, and 6/7 were male. Splenic volume (P. vivax: +28.8% [confidence interval (CI) +10.3% to +57.3%], P. falciparum: +22.9 [CI −15.3% to +61.1%]) and radiotracer uptake (P. vivax: +15.5% [CI −0.7% to +31.7%], P. falciparum: +5.5% [CI +1.4% to +9.6%]) increased following infection with each species, but more so in P. vivax infection (volume: p = 0.72, radiotracer uptake: p = 0.036). There was no change in FDG uptake in the bone marrow (P. vivax: +4.6% [CI −15.9% to +25.0%], P. falciparum: +3.2% [CI −3.2% to +9.6%]) or liver (P. vivax: +6.2% [CI −8.7% to +21.1%], P. falciparum: −1.4% [CI −4.6% to +1.8%]) following infection with either species. In participants with P. vivax, hemoglobin, hematocrit, and platelet count decreased from baseline at the time of postinoculation imaging. Decrements in hemoglobin and hematocrit were significantly greater in participants with P. vivax infection compared to P. falciparum. The main limitations of this study are the small sample size and the inability of this tracer to differentiate between host and parasite metabolic activity.ConclusionsPET/MRI indicated greater splenic tropism and metabolic activity in early P. vivax infection compared to P. falciparum, supporting the hypothesis of splenic accumulation of P. vivax very early in infection. The absence of uptake in the bone marrow and liver suggests that, at least in early infection, these tissues do not harbor a large parasite biomass or do not provoke a prominent metabolic response. PET/MRI is a safe and noninvasive method to evaluate infection-associated organ changes in morphology and glucose metabolism.

John Woodford and co-authors use positron emission tomography/magnetic resonance imaging (PET/MRI) to describe unique splenic morphology and metabolism in early P. vivax infection.     

Chromatin-Associated Proteins HP1 and Mod(mdg4) Modify Y-Linked Regulatory Variation in the Drosophila Testis

Chromatin remodeling is crucial for gene regulation. Remodeling is often mediated through chemical modifications of the DNA template, DNA-associated proteins, and RNA-mediated processes. Y-linked regulatory variation (YRV) refers to the quantitative effects that polymorphic tracts of Y-linked chromatin exert on gene expression of X-linked and autosomal genes. Here we show that naturally occurring polymorphisms in the Drosophila melanogaster Y chromosome contribute disproportionally to gene expression variation in the testis. The variation is dependent on wild-type expression levels of mod(mdg4) as well as Su(var)205; the latter gene codes for heterochromatin protein 1 (HP1) in Drosophila. Testis-specific YRV is abolished in genotypes with heterozygous loss-of-function mutations for mod(mdg4) and Su(var)205 but not in similar experiments with JIL-1. Furthermore, the Y chromosome differentially regulates several ubiquitously expressed genes. The results highlight the requirement for wild-type dosage of Su(var)205 and mod(mdg4) in enabling naturally occurring Y-linked regulatory variation in the testis. The phenotypes that emerge in the context of wild-type levels of the HP1 and Mod(mdg4) proteins might be part of an adaptive response to the environment.     

Differences Between Lipopolysaccharide Compositions of Plant Pathogenic and Saprophytic Pseudomonas Species

Lipopolysaccharides (LPS) were obtained by washing cells of plant pathogenic and saprophytic Pseudomonas species with saline (fraction 1) and then with saline-EDTA (fraction 2). The cells subsequently were extracted with phenol to yield a third aqueous preparation (fraction 3). Each fraction type contained the LPS components, lipid A, heptose, 2-keto-3-deoxy sugar, and neutral and amino sugars. The neutral sugar compositions of fractions 1, 2, and 3, although similar within a species, differed between the Pseudomonas species. The LPS of two pathovars (pv.) of Pseudomonas syringae had glucose and rhamnose as major components: 13 (±3)% glucose and 87 (±3)% rhamnose for P. syringae pv. pisi and 18 (±5)% glucose and 76 (±2)% rhamnose for P. syringae pv. syringae. Fucose was present in addition to glucose and rhamnose for P. syringae pv. phaseolicola (68 [±8]% rhamnose, 14 [±1]% fucose, and 14 [±5]% glucose) and P. syringae pv. tabaci (24 [±2]% rhamnose, 54 [±3]% fucose, and 17 [±1]% glucose). The LPS from different races of P. syringae pv. pisi and P. syringae pv. phaseolicola could not be distinguished by neutral sugar composition. Three saprophytic species, P. aeruginosa, P. fluorescens, and P. putida, also produced LPS which had different proportions of rhamnose, fucose, and glucose. The LPS from three isolates of P. putida were distinct in possessing a high proportion of amino sugar and containing glucose as the major neutral sugar component (86 to 100%). The LPS fractions from plant pathogenic and saprophytic Pseudomonas species did not elicit browning or phytoalexin production in treated dark red kidney bean cotyledons or red Mexican bean leaves. Rather, chlorosis of the LPS-treated leaf tissue was observed.     

The fucomic potential of mosquitoes: Fucosylated N-glycan epitopes and their cognate fucosyltransferases

Fucoconjugates are key mediators of protein-glycan interactions in prokaryotes and eukaryotes. As examples, N-glycans modified with the non-mammalian core α1,3-linked fucose have been detected in various organisms ranging from plants to insects and are immunogenic in mammals. The rabbit polyclonal antibody raised against plant horseradish peroxidase (anti-HRP) is able to recognize the α1,3-linked fucose epitope and is also known to specifically stain neural tissues in the fruit fly Drosophila melanogaster. In this study, we have detected and localized the anti-HRP cross-reactivity in another insect species, the malaria mosquito vector Anopheles gambiae. We were able to identify and structurally elucidate fucosylated N-glycans including core mono- and difucosylated structures (responsible for anti-HRP cross reactivity) as well as a Lewis-type antennal modification on mosquito anionic N-glycans by applying enzymatic and chemical treatments. The three mosquito fucosyltransferase open reading frames (FucT6, FucTA and FucTC) required for the in vivo biosynthesis of the fucosylated N-glycan epitopes were identified in the Anopheles gambiae genome, cloned and recombinantly expressed in Pichia pastoris. Using a robust MALDI-TOF MS approach, we characterised the activity of the three recombinant fucosyltransferases in vitro and demonstrate that they share similar enzymatic properties as compared to their homologues from D. melanogaster and Apis mellifera. Thus, not only do we confirm the neural reactivity of anti-HRP in a mosquito species, but also demonstrate enzymatic activity for all its α1,3- and α1,6-fucosyltransferase homologues, whose specificity matches the results of glycomic analyses.