RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation.

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.

Substrate Specificities of Variants of Barley (1,3)- and (1,3;1,4)-ß-d-Glucanases Resulting from Mutagenesis and Segment Hybridization.

Barley (1,3;1,4)-ß-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-ß-d-glucanase, enabling the hydrolysis of (1,3;1,4)-ß-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-ß-d-glucan endohydrolase [(1,3;1,4)-ß-d-glucanase] isoenzyme EII (HvEII) and (1,3)-ß-d-glucan endohydrolase [(1,3)-ß-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-ß-d-glucanase and one variant that hydrolyzed both (1,3)-ß-d-glucans and (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-ß-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-ß-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-ß-d-glucans or (1,3)-ß-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-ß-d-glucans and (1,3)-ß-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.

Structures of the xyloglucans in the monocotyledon family Araceae (aroids).

MAIN CONCLUSION: The xyloglucans of all aquatic Araceae species examined had unusual structures compared with those of other non-commelinid monocotyledon families previously examined. The aquatic Araceae species Lemna minor was earlier shown to have xyloglucans with a different structure from the fucogalactoxyloglucans of other non-commelinid monocotyledons. We investigated 26 Araceae species (including L. minor), from five of the seven subfamilies. All seven aquatic species examined had xyloglucans that were unusual in having one or two of three features: < 77% XXXG core motif [L. minor (Lemnoideae) and Orontium aquaticum (Orontioideae)]; no fucosylation [L. minor (Lemnoideae), Cryptocoryne aponogetonifolia, and Lagenandra ovata (Aroideae, Rheophytes clade)]; and > 14% oligosaccharide units with S or D side chains [Spirodela polyrhiza and Landoltia punctata (Lemnoideae) and Pistia stratiotes (Aroideae, Dracunculus clade)]. Orontioideae and Lemnoideae are the two most basal subfamilies, with all species being aquatic, and Aroideae is the most derived. Two terrestrial species [Dieffenbachia seguine and Spathicarpa hastifolia (Aroideae, Zantedeschia clade)] also had xyloglucans without fucose indicating this feature was not unique to aquatic species.

The LHC Olympics 2020 a community challenge for anomaly detection in high energy physics.

A new paradigm for data-driven, model-agnostic new physics searches at colliders is emerging, and aims to leverage recent breakthroughs in anomaly detection and machine learning. In order to develop and benchmark new anomaly detection methods within this framework, it is essential to have standard datasets. To this end, we have created the LHC Olympics 2020, a community challenge accompanied by a set of simulated collider events. Participants in these Olympics have developed their methods using an R&D dataset and then tested them on black boxes: datasets with an unknown anomaly (or not). Methods made use of modern machine learning tools and were based on unsupervised learning (autoencoders, generative adversarial networks, normalizing flows), weakly supervised learning, and semi-supervised learning. This paper will review the LHC Olympics 2020 challenge, including an overview of the competition, a description of methods deployed in the competition, lessons learned from the experience, and implications for data analyses with future datasets as well as future colliders.

Heteromannans are the predominant hemicelluloses in the gametophytic stem of the umbrella moss Hypnodendron menziesii and occur in the walls of all cell types.

MAIN CONCLUSION: Heteromannans are the predominant hemicelluloses in the gametophytic stem of the moss Hypnodendron menziesii and occur in the walls of all cell types Little is known about the cell-wall polysaccharides of mosses. Monosaccharide analysis of cell walls isolated from the stem of the umbrella moss Hypnodendron menziesii was consistent with heteromannans, probably galactoglucomannans, being the predominant hemicellulosic polysaccharides in the walls. Immunofluorescence and immunogold microscopy with the monoclonal antibody LM21, specific for heteromannans, showed that these polysaccharides were present in the walls of all stem cell types. These cell types, except the hydroids, have secondary walls. Experiments in which sections were pre-treated with 0.1 M sodium carbonate and with the enzyme pectate lyase indicated that the heteromannans have O-acetyl groups that limit LM21 binding and the cell walls contain pectic homogalacturonan that masks detection of heteromannans using LM21. Therefore, to fully detect heteromannans in the cell walls, it was essential to use these pre-treatments to remove the O-acetyl groups from the heteromannans and pectic homogalacturonan from the cell walls. Fluorescence microscopy experiments with a second monoclonal antibody, LM22, also specific for heteromannans, showed similar results, but the binding was considerably weaker than with LM21, possibly as a result of subtle structural differences in the epitopes of the two antibodies. Although heteromannans occur abundantly in the cell walls of many species in basal lineages of tracheophytes, prior to the present study, research on the distribution of these polysaccharides in the walls of different cell types in mosses was confined to the model species Physcomitrium patens.

A randomised controlled trial to assess the clinical effectiveness and safety of the endometrial scratch procedure prior to first-time IVF, with or without ICSI.

STUDY QUESTION: What is the clinical-effectiveness and safety of the endometrial scratch (ES) procedure compared to no ES, prior to usual first time in vitro fertilisation (IVF) treatment? SUMMARY ANSWER: ES was safe but did not improve pregnancy outcomes when performed in the mid-luteal phase prior to the first IVF cycle, with or without intracytoplasmic sperm injection (ICSI). WHAT IS KNOWN ALREADY: ES is an 'add-on' treatment that is available to women undergoing a first cycle of IVF, with or without ICSI, despite a lack of evidence to support its use. STUDY DESIGN, SIZE, DURATION: This pragmatic, superiority, open-label, multi-centre, parallel-group randomised controlled trial involving 1048 women assessed the clinical effectiveness and safety of the ES procedure prior to first time IVF, with or without ICSI, between July 2016 and October 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants aged 18-37 years undergoing their first cycle of IVF, with or without ICSI, were recruited from 16 UK fertility clinics and randomised (1:1) by a web-based system with restricted access rights that concealed allocation. Stratified block randomisation was used to allocate participants to TAU or ES in the mid-luteal phase followed by usual IVF with or without ICSI treatment. The primary outcome was live birth after completing 24 weeks gestation within 10.5 months of egg collection. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 1048 women randomised to TAU (n = 525) and ES (n = 523) were available for intention to treat analysis. In the ES group, 453 (86.6%) received the ES procedure. IVF, with or without ICSI, was received in 494 (94.1%) and 497 (95.0%) of ES and TAU participants respectively. Live birth rate was 37.1% (195/525) in the TAU and 38.6% (202/523) in the ES: an unadjusted absolute difference of 1.5% (95% CI -4.4% to 7.4%, P = 0.621). There were no statistical differences in secondary outcomes. Adverse events were comparable across groups. LIMITATIONS, REASONS FOR CAUTION: A sham ES procedure was not undertaken in the control group, however, we do not believe this would have influenced the results as objective fertility outcomes were used. WIDER IMPLICATIONS OF THE FINDINGS: This is the largest trial that is adequately powered to assess the impact of ES on women undergoing their first cycle of IVF. ES was safe, but did not significantly improve pregnancy outcomes when performed in the mid-luteal phase prior to the first IVF or ICSI cycle. We recommend that ES is not undertaken in this population. STUDY FUNDING/COMPETING INTEREST(S): Funded by the National Institute of Health Research. Stephen Walters is an National Institute for Health Research (NIHR) Senior Investigator (2018 to present) and was a member of the following during the project: National Institute for Health Research (NIHR) Health Technology Assessment (HTA) Clinical Trials and Evaluation Committee (2011-2017), NIHR HTA Commissioning Strategy Group (2012 to 2017); NIHR Programme Grants for Applied Research Committee (2020 to present); NIHR Pre doctoral Fellowship Committee (2019 to present). Dr. Martins da Silva reports grants from AstraZeneca, during the conduct of the study; and is Associate editor of Human Reproduction and Editorial Board member of Reproduction and Fertility. Dr. Bhide reports grants from Bart's Charity and grants and non-financial support from Pharmasure Pharmaceuticals outside the submitted work. TRIAL REGISTRATION NUMBER: ISRCTN number: ISRCTN23800982. TRIAL REGISTRATION DATE: 31 May 2016. DATE OF FIRST PATIENT'S ENROLMENT: 04 July 2016.

Elevated A20 promotes TNF-induced and RIPK1-dependent intestinal epithelial cell death.

Intestinal epithelial cell (IEC) death is a common feature of inflammatory bowel disease (IBD) that triggers inflammation by compromising barrier integrity. In many patients with IBD, epithelial damage and inflammation are TNF-dependent. Elevated TNF production in IBD is accompanied by increased expression of the TNFAIP3 gene, which encodes A20, a negative feedback regulator of NF-κB. A20 in intestinal epithelium from patients with IBD coincided with the presence of cleaved caspase-3, and A20 transgenic (Tg) mice, in which A20 is expressed from an IEC-specific promoter, were highly susceptible to TNF-induced IEC death, intestinal damage, and shock. A20-expressing intestinal organoids were also susceptible to TNF-induced death, demonstrating that enhanced TNF-induced apoptosis was a cell-autonomous property of A20. This effect was dependent on Receptor Interacting Protein Kinase 1 (RIPK1) activity, and A20 was found to associate with the Ripoptosome complex, potentiating its ability to activate caspase-8. A20-potentiated RIPK1-dependent apoptosis did not require the A20 deubiquitinase (DUB) domain and zinc finger 4 (ZnF4), which mediate NF-κB inhibition in fibroblasts, but was strictly dependent on ZnF7 and A20 dimerization. We suggest that A20 dimers bind linear ubiquitin to stabilize the Ripoptosome and potentiate its apoptosis-inducing activity.

Long-lived particles at the energy frontier: the MATHUSLA physics case.

We examine the theoretical motivations for long-lived particle (LLP) signals at the LHC in a comprehensive survey of standard model (SM) extensions. LLPs are a common prediction of a wide range of theories that address unsolved fundamental mysteries such as naturalness, dark matter, baryogenesis and neutrino masses, and represent a natural and generic possibility for physics beyond the SM (BSM). In most cases the LLP lifetime can be treated as a free parameter from the [Formula: see text]m scale up to the Big Bang Nucleosynthesis limit of [Formula: see text] m. Neutral LLPs with lifetimes above [Formula: see text]100 m are particularly difficult to probe, as the sensitivity of the LHC main detectors is limited by challenging backgrounds, triggers, and small acceptances. MATHUSLA is a proposal for a minimally instrumented, large-volume surface detector near ATLAS or CMS. It would search for neutral LLPs produced in HL-LHC collisions by reconstructing displaced vertices (DVs) in a low-background environment, extending the sensitivity of the main detectors by orders of magnitude in the long-lifetime regime. We study the LLP physics opportunities afforded by a MATHUSLA-like detector at the HL-LHC, assuming backgrounds can be rejected as expected. We develop a model-independent approach to describe the sensitivity of MATHUSLA to BSM LLP signals, and compare it to DV and missing energy searches at ATLAS or CMS. We then explore the BSM motivations for LLPs in considerable detail, presenting a large number of new sensitivity studies. While our discussion is especially oriented towards the long-lifetime regime at MATHUSLA, this survey underlines the importance of a varied LLP search program at the LHC in general. By synthesizing these results into a general discussion of the top-down and bottom-up motivations for LLP searches, it is our aim to demonstrate the exceptional strength and breadth of the physics case for the construction of the MATHUSLA detector.

Developmental changes in collenchyma cell-wall polysaccharides in celery (Apium graveolens L.) petioles.

BACKGROUND: Collenchyma cells occur widely in eudicotyledons and provide mechanical support for growing organs. At maturity, the cells are elongated and have thick, non-lignified walls, which in celery contain cellulose and pectic polysaccharides, together with xyloglucans and heteroxylans and heteromannans. A previous study suggested that at least some of the collenchyma cell wall in celery is laid down after expansion has stopped and is thus secondary. In the present study, we re-examined this. We used chemical analysis and immunomicroscopy to determine changes in the polysaccharide compositions of these walls during development. Additionally, solid-state NMR spectroscopy was used to examine changes in polysaccharide mobilities during development. RESULTS: We showed the collenchyma walls are deposited only during cell expansion, i.e. they are primary walls. During cell-wall development, analytical and immunomicroscopy studies showed that within the pectic polysaccharides there were no overall changes in the proportions of homogalacturonans, but there was a decrease in their methyl esterification. There was also a decrease in the proportions of the (1 → 5)-α-L-arabinan and (1 → 4)-ß-D-galactan side chains of rhamnogalacturonan I. The proportions of cellulose increased, and to a lesser extent those of xyloglucans and heteroxylans. Immunomicroscopy showed the homogalacturonans occurred throughout the walls and were most abundant in the middle lamellae and middle lamella junctions. Although the (1 → 4)-ß-D-galactans occurred only in the rest of the walls, some of the (1 → 5)-α-L-arabinans also occurred in the middle lamellae and middle lamella junctions. During development, the location of the xyloglucans changed, being confined to the middle lamellae and middle lamella junctions early on, but later occurred throughout the walls. The location of the heteroxylans also changed, occurring mostly in the outer walls in young cells, but were more widely distributed in mature cells. Solid-state NMR spectroscopy showed that particularly cellulose, but also homogalacturonans, decreased in mobility during development. CONCLUSIONS: Our studies showed that celery collenchyma cell walls are primary and that during their development the polysaccharides undergo dynamic changes. Changes in the mobilities of cellulose and homogalacturonans were consistent with the cell walls becoming stiffer as expansion ceases.

Changes in the orientations of cellulose microfibrils during the development of collenchyma cell walls of celery (Apium graveolens L.).

MAIN CONCLUSION: During development, cellulose microfibrils in collenchyma walls become increasingly longitudinal, as determined by small-angle X-ray scattering, despite the walls maintaining a fine structure indicative of a crossed-polylamellate structure. Collenchyma cells have thickened primary cell walls and provide mechanical support during plant growth. During their development, these cells elongate and their walls thicken considerably. We used microscopy and synchrotron small-angle X-ray scattering to study changes in the orientations of cellulose microfibrils that occur during development in the walls of collenchyma cells present in peripheral strands in celery (Apium graveolens) petioles. Transmission electron microscopy showed that the walls consisted of many lamellae (polylamellate), with lamellae containing longitudinally oriented cellulose microfibrils alternating with microfibrils oriented at higher angles. The lamellae containing longitudinally oriented microfibrils predominated at later stages of development. Nevertheless, transmission electron microscopy of specially stained, oblique sections provided evidence that the cellulose microfibrils were ordered throughout development as crossed-polylamellate structures. These results are consistent with our synchrotron small-angle X-ray scattering results that showed the cellulose microfibrils become oriented increasingly longitudinally during development. Some passive reorientation of cellulose microfibrils may occur during development, but extensive reorientation throughout the wall would destroy ordered structures. Atomic force microscopy and field emission scanning electron microscopy were used to determine the orientations of newly deposited cellulose microfibrils. These were found to vary widely among different cells, which could be consistent with the formation of crossed-polylamellate structures. These newly deposited cellulose microfibrils are deposited in a layer of pectic polysaccharides that lies immediately outside the plasma membrane. Overall, our results show that during development of collenchyma walls, the cellulose microfibrils become increasingly longitudinal in orientation, yet organized, crossed-polylamellate structures are maintained.

Commelinid Monocotyledon Lignins Are Acylated by p-Coumarate.

Commelinid monocotyledons are a monophyletic clade differentiated from other monocotyledons by the presence of cell wall-bound ferulate and p-coumarate. The Poaceae, or grass family, is a member of this group, and most of the p-coumarate in the cell walls of this family acylates lignin. Here, we isolated and examined lignified cell wall preparations from 10 species of commelinid monocotyledons from nine families other than Poaceae, including species from all four commelinid monocotyledon orders (Poales, Zingiberales, Commelinales, and Arecales). We showed that, as in the Poaceae, lignin-linked p-coumarate occurs exclusively on the hydroxyl group on the γ-carbon of lignin unit side chains, mostly on syringyl units. Although the mechanism of acylation has not been studied directly in these species, it is likely to be similar to that in the Poaceae and involve BAHD acyl-coenzyme A:monolignol transferases.

Highly Decorated Lignins in Leaf Tissues of the Canary Island Date Palm Phoenix canariensis.

The cell walls of leaf base tissues of the Canary Island date palm (Phoenix canariensis) contain lignins with the most complex compositions described to date. The lignin composition varies by tissue region and is derived from traditional monolignols (ML) along with an unprecedented range of ML conjugates: ML-acetate, ML-benzoate, ML-p-hydroxybenzoate, ML-vanillate, ML-p-coumarate, and ML-ferulate. The specific functions of such complex lignin compositions are unknown. However, the distribution of the ML conjugates varies depending on the tissue region, indicating that they may play specific roles in the cell walls of these tissues and/or in the plant's defense system.

Editorial for the Special Issue "Dietary Fibre: New Insights on Biochemistry and Health Benefits".

When the term dietary fibre was first coined, over sixty years ago, it only referred to plant cell walls in the diet. [...].

Tricin-lignins: occurrence and quantitation of tricin in relation to phylogeny.

Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as an authentic monomer in grass lignification that likely functions as a nucleation site. It is linked onto lignin as an aryl alkyl ether by radical coupling with monolignols or their acylated analogs. However, the level of tricin that incorporates into lignin remains unclear. Herein, three lignin characterization methods: acidolysis; thioacidolysis; and derivatization followed by reductive cleavage; were applied to quantitatively assess the amount of lignin-integrated tricin. Their efficiencies at cleaving the tricin-(4'-O-ß)-ether bonds and the degradation of tricin under the corresponding reaction conditions were evaluated. A hexadeuterated tricin analog was synthesized as an internal standard for accurate quantitation purposes. Thioacidolysis proved to be the most efficient method, liberating more than 91% of the tricin with little degradation. A survey of different seed-plant species for the occurrence and content of tricin showed that it is widely distributed in the lignin from species in the family Poaceae (order Poales). Tricin occurs at low levels in some commelinid monocotyledon families outside the Poaceae, such as the Arecaceae (the palms, order Arecales) and Bromeliaceae (Poales), and the non-commelinid monocotyledon family Orchidaceae (Orchidales). One eudicotyledon was found to have tricin (Medicago sativa, Fabaceae). The content of lignin-integrated tricin is much higher than the extractable tricin level in all cases. Lignins, including waste lignin streams from biomass processing, could therefore provide a large and alternative source of this valuable flavone, reducing the costs, and encouraging studies into its application beyond its current roles.

Polysaccharide compositions of collenchyma cell walls from celery (Apium graveolens L.) petioles.

BACKGROUND: Collenchyma serves as a mechanical support tissue for many herbaceous plants. Previous work based on solid-state NMR and immunomicroscopy suggested collenchyma cell walls (CWs) may have similar polysaccharide compositions to those commonly found in eudicotyledon parenchyma walls, but no detailed chemical analysis was available. In this study, compositions and structures of cell wall polysaccharides of peripheral collenchyma from celery petioles were investigated. RESULTS: This is the first detailed investigation of the cell wall composition of collenchyma from any plant. Celery petioles were found to elongate throughout their length during early growth, but as they matured elongation was increasingly confined to the upper region, until elongation ceased. Mature, fully elongated, petioles were divided into three equal segments, upper, middle and lower, and peripheral collenchyma strands isolated from each. Cell walls (CWs) were prepared from the strands, which also yielded a HEPES buffer soluble fraction. The CWs were sequentially extracted with CDTA, Na2CO3, 1 M KOH and 4 M KOH. Monosaccharide compositions of the CWs showed that pectin was the most abundant polysaccharide [with homogalacturonan (HG) more abundant than rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II)], followed by cellulose, and other polysaccharides, mainly xyloglucans, with smaller amounts of heteroxylans and heteromannans. CWs from different segments had similar compositions, but those from the upper segments had slightly more pectin than those from the lower two segments. Further, the pectin in the CWs of the upper segment had a higher degree of methyl esterification than the other segments. In addition to the anticipated water-soluble pectins, the HEPES-soluble fractions surprisingly contained large amounts of heteroxylans. The CDTA and Na2CO3 fractions were rich in HG and RG-I, the 1 M KOH fraction had abundant heteroxylans, the 4 M KOH fraction was rich in xyloglucan and heteromannans, and cellulose was predominant in the final residue. The structures of the xyloglucans, heteroxylans and heteromannans were deduced from the linkage analysis and were similar to those present in most eudicotyledon parenchyma CWs. Cross polarization with magic angle spinning (CP/MAS) NMR spectroscopy showed no apparent difference in the rigid and semi-rigid polysaccharides in the CWs of the three segments. Single-pulse excitation with magic-angle spinning (SPE/MAS) NMR spectroscopy, which detects highly mobile polysaccharides, showed the presence of arabinan, the detailed structure of which varied among the cell walls from the three segments. CONCLUSIONS: Celery collenchyma CWs have similar polysaccharide compositions to most eudicotyledon parenchyma CWs. However, celery collenchyma CWs have much higher XG content than celery parenchyma CWs. The degree of methyl esterification of pectin and the structures of the arabinan side chains of RG-I show some variation in the collenchyma CWs from the different segments. Unexpectedly, the HEPES-soluble fraction contained a large amount of heteroxylans.

Tracheid cell-wall structures and locations of (1 → 4)-ß-D-galactans and (1 → 3)-ß-D-glucans in compression woods of radiata pine (Pinus radiata D. Don).

BACKGROUND: Compression wood (CW) forms on the underside of tilted stems of coniferous gymnosperms and opposite wood (OW) on the upperside. The tracheid walls of these wood types differ structurally and chemically. Although much is known about the most severe form of CW, severe CW (SCW), mild CWs (MCWs), also occur, but less is known about them. In this study, tracheid wall structures and compositions of two grades of MCWs (1 and 2) and SCW were investigated and compared with OW in slightly tilted radiata pine (Pinus radiata) stems. RESULTS: The four wood types were identified by the distribution of lignin in their tracheid walls. Only the tracheid walls of OW and MCW1 had a S3 layer and this was thin in MCW1. The tracheid walls of only SCW had a S2 layer with helical cavities in the inner region (S2i). Using immunomicroscopy, (1 → 4)-ß-D-galactans and (1 → 3)-ß-D-glucans were detected in the tracheid walls of all CWs, but in only trace amounts in OW. The (1 → 4)-ß-D-galactans were located in the outer region of the S2 layer, whereas the (1 → 3)-ß-D-glucans were in the inner S2i region. The areas and intensities of labelling increased with CW severity. The antibody for (1 → 4)-ß-D-galactans was also used to identify the locations and relative amounts of these galactans in whole stem cross sections based on the formation of an insoluble dye. Areas containing the four wood types were clearly differentiated depending on colour intensity. The neutral monosaccharide compositions of the non-cellulosic polysaccharides of these wood types were determined on small, well defined discs, and showed the proportion of galactose was higher for CWs and increased with severity. CONCLUSION: The presence of an S3 wall layer is a marker for very MCW and the presence of helical cavities in the S2 wall layer for SCW. The occurrence and proportions of (1 → 4)-ß-D-galactans and (1 → 3)-ß-D-glucans can be used as markers for CW and its severity. The proportions of galactose were consistent with the labelling results for (1 → 4)-ß-D-galactans.

Cutting Edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice.

RIP1 (RIPK1) kinase is a key regulator of TNF-induced NF-κB activation, apoptosis, and necroptosis through its kinase and scaffolding activities. Dissecting the balance of RIP1 kinase activity and scaffolding function in vivo during development and TNF-dependent inflammation has been hampered by the perinatal lethality of RIP1-deficient mice. In this study, we generated RIP1 kinase-dead (Ripk1(K45A)) mice and showed they are viable and healthy, indicating that the kinase activity of RIP1, but not its scaffolding function, is dispensable for viability and homeostasis. After validating that the Ripk1(K45A) mice were specifically protected against necroptotic stimuli in vitro and in vivo, we crossed them with SHARPIN-deficient cpdm mice, which develop severe skin and multiorgan inflammation that has been hypothesized to be mediated by TNF-dependent apoptosis and/or necroptosis. Remarkably, crossing Ripk1(K45A) mice with the cpdm strain protected against all cpdm-related pathology. Together, these data suggest that RIP1 kinase represents an attractive therapeutic target for TNF-driven inflammatory diseases.

Potential Benefits of Dietary Fibre Intervention in Inflammatory Bowel Disease.

Intestinal dysbiosis is thought to be an important cause of disease progression and the gastrointestinal symptoms experienced in patients with inflammatory bowel disease (IBD). Inflammation appears to be a major contributor in perpetuating a dysregulated gut microbiota. Although current drug therapies can significantly induce and maintain disease remission, there is no cure for these diseases. Nevertheless, ongoing human studies investigating dietary fibre interventions may potentially prove to exert beneficial outcomes for IBD. Postulated mechanisms include direct interactions with the gut mucosa through immunomodulation, or indirectly through the microbiome. Component species of the microbiome may degrade dietary-fibre polysaccharides and ferment the products to form short-chain fatty acids such as butyrate. Prebiotic dietary fibres may also act more directly by altering the composition of the microbiome. Longer term benefits in reducing the risk of more aggressive disease or colorectal cancer may require other dietary fibre sources such as wheat bran or psyllium. By critically examining clinical trials that have used dietary fibre supplements or dietary patterns containing specific types or amounts of dietary fibres, it may be possible to assess whether varying the intake of specific dietary fibres may offer an efficient treatment for IBD patients.

Antioxidant Activity of 3-[N-(Acylhydrazono)ethyl]-4-hydroxy-coumarins.

A series of 3-acylhydrazono-4-hydroxycoumarins were synthesized via condensation of 3-acetyl-4-hydroxycoumarin with appropriate hydrazides. The structures of the newly-synthesized compounds were characterized by spectral and elememental analysis or HRMS measurements. Their antioxidant properties were evaluated by using scavenging effects on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical as well as inhibition of lipid peroxidation. Moreover, their ability to inhibit in vitro soybean lipoxygenase has been investigated. They were found to be capable of rapid inactivation of alkylperoxy radicals.

Family 34 glycosyltransferase (GT34) genes and proteins in Pinus radiata (radiata pine) and Pinus taeda (loblolly pine).

Using a functional genomics approach, four candidate genes (PtGT34A, PtGT34B, PtGT34C and PtGT34D) were identified in Pinus taeda. These genes encode CAZy family GT34 glycosyltransferases that are involved in the synthesis of cell-wall xyloglucans and heteromannans. The full-length coding sequences of three orthologs (PrGT34A, B and C) were isolated from a xylem-specific cDNA library from the closely related Pinus radiata. PrGT34B is the ortholog of XXT1 and XXT2, the two main xyloglucan (1→6)-α-xylosyltransferases in Arabidopsis thaliana. PrGT34C is the ortholog of XXT5 in A. thaliana, which is also involved in the xylosylation of xyloglucans. PrGT34A is an ortholog of a galactosyltransferase from fenugreek (Trigonella foenum-graecum) that is involved in galactomannan synthesis. Truncated coding sequences of the genes were cloned into plasmid vectors and expressed in a Sf9 insect cell-culture system. The heterologous proteins were purified, and in vitro assays showed that, when incubated with UDP-xylose and cellotetraose, cellopentaose or cellohexaose, PrGT34B showed xylosyltransferase activity, and, when incubated with UDP-galactose and the same cello-oligosaccharides, PrGT34B showed some galactosyltransferase activity. The ratio of xylosyltransferase to galactosyltransferase activity was 434:1. Hydrolysis of the galactosyltransferase reaction products using galactosidases showed the linkages formed were α-linkages. Analysis of the products of PrGT34B by MALDI-TOF MS showed that up to three xylosyl residues were transferred from UDP-xylose to cellohexaose. The heterologous proteins PrGT34A and PrGT34C showed no detectable enzymatic activity.