Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli : purification , biochemical and kinetic characterisation

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl-aminopeptidase I. The amino acid sequence, deduced from the nucleotide sequence, revealed that it consists of 209 amino acid residues and showed to have 98% homology with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary among the two sequences. The bovine enzyme was expressed in XL10-gold Esherichia coli cells. Immobilizied Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 3.3mg of PAP1 per litre culture. The purified enzyme had a specific activity of 1700 units/ml. SDS-PAGE produced a single band for bovine PAP1 with a molecular weight of ~23-24 kDa which is in good agreement with previously reported data on PAP1. Kinetic constants Km and Kcat were 59μΜ and 3.5s-1, respectively. It possessed an optimum pH between 9-9.5, a temperature of 37°C and showed an absolute requirement for a thiol-reducing agent (10mM DTT). EDTA didn’t prove to have an effect on enzyme activity. Competitive inhibition was seen with pyroglutamyl peptides pGlu-His-Pro-NH2 (TRH; Ki= 44.1 uM), pGlu-Ala- OH (Ki=141 uM) and pGlu-Val-OH (Ki=652.17)

Hard-Object Feeding in Sooty Mangabeys (Cercocebus atys) and Interpretation of Early Hominin Feeding Ecology

Morphology of the dentofacial complex of early hominins has figured prominently in the inference of their dietary adaptations. Recent theoretical analysis of craniofacial morphology of Australopithecus africanus proposes that skull form in this taxon represents adaptation to feeding on large, hard objects. A modern analog for this specific dietary specialization is provided by the West African sooty mangabey, Cercocebus atys. This species habitually feeds on the large, exceptionally hard nuts of Sacoglottis gabonensis, stereotypically crushing the seed casings using their premolars and molars. This type of behavior has been inferred for A. africanus based on mathematical stress analysis and aspects of dental wear and morphology. While postcanine megadontia, premolar enlargement and thick molar enamel characterize both A. africanus and C. atys, these features are not universally associated with durophagy among living anthropoids. Occlusal microwear analysis reveals complex microwear textures in C. atys unlike those observed in A. africanus, but more closely resembling textures observed in Paranthropus robustus. Since sooty mangabeys process hard objects in a manner similar to that proposed for A. africanus, yet do so without the craniofacial buttressing characteristic of this hominin, it follows that derived features of the australopith skull are sufficient but not necessary for the consumption of large, hard objects. The adaptive significance of australopith craniofacial morphology may instead be related to the toughness, rather than the hardness, of ingested foods

Portuguese propolis disturbs glycolytic metabolism of human colorectal cancer in vitro

Propolis is a resin collected by bees from plant buds and exudates, which is further processed through the activity of bee enzymes. Propolis has been shown to possess many biological and pharmacological properties, such as antimicrobial, antioxidant, immunostimulant and antitumor activities. Due to this bioactivity profile, this resin can become an alternative, economic and safe source of natural bioactive compounds.Antitumor action has been reported in vitro and in vivo for propolis extracts or its isolated compounds; however, Portuguese propolis has been little explored. The aim of this work was to evaluate the in vitro antitumor activity of Portuguese propolis on the human colon carcinoma cell line HCT-15, assessing the effect of different fractions (hexane, chloroform and ethanol residual) of a propolis ethanol extract on cell viability, proliferation, metabolism and death. METHODS: Propolis from Angra do Heroísmo (Azores) was extracted with ethanol and sequentially fractionated in solvents with increasing polarity, n-hexane and chloroform. To assess cell viability, cell proliferation and cell death, Sulforhodamine B, BrDU incorporation assay and Anexin V/Propidium iodide were used, respectively. Glycolytic metabolism was estimated using specific kits. RESULTS: All propolis samples exhibited a cytotoxic effect against tumor cells, in a dose- and time-dependent way. Chloroform fraction, the most enriched in phenolic compounds, appears to be the most active, both in terms of inhibition of viability and cell death. Data also show that this cytotoxicity involves disturbance in tumor cell glycolytic metabolism, seen by a decrease in glucose consumption and lactate production. CONCLUSION: Our results show that Portuguese propolis from Angra do Heroísmo (Azores) can be a potential therapeutic agent against human colorectal cancer.We thank the Portuguese Science and Technology Foundation (FCT) for VMG fellowship (ref. SFRH/BI/33503/2008). The authors thank Mr. Antonio Marques from Frutercoop - Azores, who kindly collected and provided the propolis sample for the study

Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

XPB, also known as ERCC3 and RAD25, is a 3′→5′ DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3′→5′ DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg2+/Mn2+. Consistent with the 3′→5′ polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3′ overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3′ DNA tail, it was not active on substrates containing a 3′ RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB

Inhibition of alpha-synuclein fibrillization by dopamine is mediated by interactions with five C-terminal residues and with E83 in the NAC region

The interplay between dopamine and alpha-synuclein (AS) plays a central role in Parkinson's disease (PD). PD results primarily from a severe and selective devastation of dopaminergic neurons in substantia nigra pars compacta. The neuropathological hallmark of the disease is the presence of intraneuronal proteinaceous inclusions known as Lewy bodies within the surviving neurons, enriched in filamentous AS. In vitro, dopamine inhibits AS fibril formation, but the molecular determinants of this inhibition remain obscure. Here we use molecular dynamic (MD) simulations to investigate the binding of dopamine and several of its derivatives onto conformers representative of an NMR ensemble of AS structures in aqueous solution. Within the limitations inherent to MD simulations of unstructured proteins, our calculations suggest that the ligands bind to the (125)YEMPS(129) region, consistent with experimental findings. The ligands are further stabilized by long-range electrostatic interactions with glutamate 83 (E83) in the NAC region. These results suggest that by forming these interactions with AS, dopamine may affect AS aggregation and fibrillization properties. To test this hypothesis, we investigated in vitro the effects of dopamine on the aggregation of mutants designed to alter or abolish these interactions. We found that point mutations in the (125)YEMPS(129) region do not affect AS aggregation, which is consistent with the fact that dopamine interacts non-specifically with this region. In contrast, and consistent with our modeling studies, the replacement of glutamate by alanine at position 83 (E83A) abolishes the ability of dopamine to inhibit AS fibrillization

Environmentally induced changes in antioxidant phenolic compounds levels in wild plants

[EN] Different adverse environmental conditions cause oxidative stress in plants by generation of reactive oxygen species (ROS). Accordingly, a general response to abiotic stress is the activation of enzymatic and non-enzymatic antioxidant systems. Many phenolic compounds, especially flavonoids, are known antioxidants and efficient ROS scavengers in vitro, but their exact role in plant stress responses in nature is still under debate. The aim of our work is to investigate this role by correlating the degree of environmental stress with phenolic and flavonoid levels in stress-tolerant plants. Total phenolic and antioxidant flavonoid contents were determined in 19 wild species. Meteorological data and plant and soil samples were collected in three successive seasons from four Mediterranean ecosystems: salt marsh, dune, semiarid and gypsum habitats. Changes in phenolic and flavonoid levels were correlated with the environmental conditions of the plants and were found to depend on both the taxonomy and ecology of the investigated species. Despite species-specific differences, principal component analyses of the results established a positive correlation between plant phenolics and several environmental parameters, such as altitude, and those related to water stress: temperature, evapotranspiration, and soil water deficit. The correlation with salt stress was, however, very weak. The joint analysis of all the species showed the lowest phenolic and flavonoid levels in the halophytes from the salt marsh. This finding supports previous data indicating that the halophytes analysed here do not undergo oxidative stress in their natural habitat and therefore do not need to activate antioxidant systems as a defence against salinity.This work has been funded by the Spanish Ministry of Science and Innovation (Project CGL2008-00438/BOS), with contribution from the European Regional Development Fund. Thanks to Dr. Rafael Herrera for critical reading of the manuscript.Bautista, I.; Boscaiu, M.; Lidón, A.; Llinares Palacios, JV.; Lull, C.; Donat-Torres, MP.; Mayoral García-Berlanga, O.... (2016). Environmentally induced changes in antioxidant phenolic compounds levels in wild plants. Acta Physiologiae Plantarum. 38(1):1-15. https://doi.org/10.1007/s11738-015-2025-2S115381Agati G, Biricolti S, Guidi L, Ferrini F, Fini A, Tattini M (2011) The biosynthesis of flavonoids is enhanced similarly by UV radiation and root zone salinity in L. vulgare leaves. J Plant Physiol 168:204–212Agati G, Brunetti C, Di Ferdinando M, Ferrini F, Pollastri S, Tattini M (2013) Functional roles of flavonoids in photoprotection: new evidence, lessons from the past. Plant Physiol Biochem 72:35–45Albert A, Sareedenchai V, Heller W, Seidlitz HK, Zidorn C (2009) Temperature is the key to altitudinal variation of phenolics in Arnica montana L. cv. ARBO. Oecologia 160:1–8Appel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399Bachereau F, Marigo G, Asta J (1998) Effect of solar radiation (UV and visible) at high altitude on CAM-cycling and phenolic compounds biosynthesis in Sedum album. Physiol Plant 104:203–210Ballizany WL, Hofmann RV, Jahufer MZZ, Barrett BB (2012) Multivariate associations of flavonoid and biomass accumulation in white clover (Trifolium repens) under drought. Funct Plant Biol 39:167–177Bieza K, Lois R (2001) An Arabidopsis mutant tolerant to lethal ultraviolet-B levels shows constitutively elevated accumulation of flavonoids and other phenolics. Plant Physiol 126:1105–1115Bilger W, Rolland M, Nybakken L (2007) UV screening in higher plants induced by low temperature in the absence of UV-B radiation. Photochem Photobiol Sci 6:190–195Blumthaler M, Ambach M, Ellinger R (1997) Increase in solar UV radiation with altitude. J Photochem Photobiol B 39:130–134Boscaiu M, Lull C, Llinares J, Vicente O, Boira H (2013) Proline as a biochemical marker in relation to the ecology of two halophytic Juncus species. J Plant Ecol 6:177–186Bose J, Rodrigo-Moreno A, Shabala S (2013) ROS homeostasis in halophytes in the context of salinity stress tolerance. J Exp Bot 65:1241–1257Brown DE, Rashotte AM, Murphy AS, Normanly J, Tague BW, Peer WA, Taiz L, Muday GK (2001) Flavonoids act as a negative regulators of auxin transport in vivo in Arabidopsis. Plant Physiol 126:524–535Burchard P, Bilger W, Weissenböck G (2000) Contribution of hydroxycinnamates and flavonoids to epidermal shielding of UV-A and UV-B radiation in developing rye primary leaves as assessed by ultraviolet-induced chlorophyll fluorescence measurements. Plant Cell Environ 23:1373–1380Burriel F, Hernando V (1947) Nuevo método para determinar el fósforo asimilable en los suelos. Anales de Edafología Fisiología Vegetal 9:611–622Cheynier V, Comte G, Davies KM, Lattanzio V, Martens S (2013) Plant phenolics: recent advances on their biosynthesis, genetics, and ecophysiology. Plant Physiol Biochem 72:1–20Coman C, Rugina OD, Socaciu C (2012) Plants and natural compounds with antidiabetic action. Not Bot Horti Agrobo 40:314–325Di Ferdinando M, Brunetti C, Fini A, Tattini M (2012) Flavonoids as antioxidants in plants under abiotic stresses. In: Ahmad P, Prasad MNV (eds) Abiotic stress responses in plants: metabolism, productivity and sustainability. Springer, New York, pp 159–179Di Ferdinando M, Brunetti C, Agati G, Tattini M (2014) Multiple functions of polyphenols in plants inhabiting unfavourable Mediterranean areas. Environ Exper Bot 103:107–116FAO (1990) Management of gypsiferous soils. FAO Soils Bull, p 62Fini A, Brunetti C, Di Ferdinando M, Ferrini F, Tattini M (2011) Stress-induced flavonoid biosynthesis and the antioxidant machinery of plants. Plant Signal Behav 6:709–711Gil R, Lull C, Boscaiu M, Bautista I, Lidón A, Vicente O (2011) Soluble carbohydrates as osmolytes in several halophytes from a Mediterranean salt marsh. Not Bot Horti Agrobo 39:9–17Gil R, Bautista I, Boscaiu M, Lidón A, Wankhade S, Sánchez H, Llinares J, Vicente O (2014) Responses of five Mediterranean halophytes to seasonal changes in environmental conditions. AoB Plants 6: plu049Gould KS, Lister C (2006) Flavonoid function in plants. In: Andersen ØM, Marham KR (eds) Flavonoids, chemistry, biochemistry and application. CRC Press, Boca Raton, pp 397–442Hajimahmoodi M, Moghaddam G, Ranjbar AM, Khazani H, Sadeghi N, Oveisi MR, Jannat B (2013) Total phenolic, flavonoids, tannin content and antioxidant power of some Iranian pomegranate flower cultivars (Punica granatum L.). Am J Plant Sci 4:1815–1820Halliwell B (2006) Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life. Plant Physiol 141:312–322Harborne JB, Williams C (2000) Advances in flavonoid research since 1992. Phytochemistry 55:481–504Hernández I, Alegre L, Munné-Bosch S (2004) Drought-induced changes in flavonoids and other low molecular weight antioxidants in Cistus clusii grown under Mediterranean field conditions. Tree Physiol 24:1303–1311Hernández I, Alegre L, Van Breusegem F, Munné-Bosch S (2008) How relevant are flavonoids as antioxidants in plants? Trends Plant Sci 14:125–132Iwashina T (2000) The structure and distribution of the flavonoids in plants. J Plant Res 113:287–299Jaakola L, Määttä-Riihinen K, Kärenlampi S, Hohtola A (2004) Activation of flavonoid biosynthesis by solar radiation in bilberry (Vaccinium myrtillus L.) leaves. Planta 218:721–728Jenkins GI (2009) Signal transduction in responses to UB-B radiation. Annu Rev Plant Biol 60:407–431Jenkins GI, Long JC, Wade HK, Shenton MR, Bibikova TN (2001) UV and blue light signalling: pathways regulating chalcone synthase gene expression in Arabidopsis. New Phytol 151:121–131Kaulen H, Schell J, Kreuzaler F (1986) Light-induced expression of the chimeric chalcone synthase-NPTII gene in tobacco cells. EMBO J 5:1–8Kim DO, Jeong SW, Lee CY (2003) Antioxidant capacity of phenolic phytochemicals from various cultivars of plums. Food Chem 81:321–326Kirakosyan A, Seymour E, Kaufman PB, Warber S, Bolling S, Chang SC (2003) Antioxidant capacity of polyphenolic extracts from leaves of Crataegus laevigata and Crataegus monogyna (Hawthorn) subjected to drought and cold stress. J Agr Food Chem 51:3973–3976Knudssen D, Peterson GA, Pratt PF (1982) Lithium, Sodium and Potassium. In: Page AL et al (eds) Methods of soil analysis, chemical and microbiological properties. American Society of Agronomy, Madison, pp 225–246Koes RE, Spelt CE, Mol JNM (1989) The chalcone synthase multigene family of Petunia hybrida (V30): differential, light-regulated expression during flower development and UV light induction. Plant Mol Biol 12:213–225Körner C (1999) Alpine plant life. Functional plant ecology of high mountain ecosytems, BerlinKumar S, Pandey AK (2013) Chemistry and biological activities of flavonoids: an overview. Sci World J 2013:1–16Kuo S (1996) Phosphorus. In: Spark D (ed) Methods of soil analysis: chemical methods, part 3. American Society of Agronomy, Madison, pp 869–919Lavola A (1998) Accumulation of flavonoids and related compounds in birch induced by UV-B irradiance. Tree Physiol 18:53–58Li J, Ou-Lee TM, Raba R, Amundson RG, Last RL (1993) Arabidopsis flavonoid mutants are hypersensitive to UV-B radiation. Plant Cell 5:171–179Llinares JV, Bautista I, Donat MP, Lidón A, Lull C, Mayoral O, Wankhade S, Boscaiu M, Vicente O (2015) Responses to environmental stress in plants adapted to Mediterranean gypsum habitats. Not Sci Biol 7:34–44Marinova D, Ribarova F, Atanassova M (2005) Total phenolics and total flavonoids in Bulgarian fruits and vegetables. J Univ Chem Technol Metall 40:255–260Martens H, Naes T (1989) Multivariate calibration. Wiley, New YorkMurai Y, Takemura S, Takeda K, Kitajima K, Iwashina T (2009) Altitudinal variation of UV-absorbing compounds in Plantago asiatica. Biochem Syst Ecol 37:78–384Nakabayashi R, Yonekura-Sakakibara K, Urano K, Suzuki M, Yamada Y, Nishizawa T, Matsuda F, Kojima M, Sakakibara H, Shinozaki K, Michael AJ, Tohge T, Yamazaki M, Saito K (2014) Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids. Plant J 77:367–379Napoli CA, Fahy D, Wang HY, Taylor LP (1999) white anther: a petunia mutant that abolishes pollen flavonoid accumulation, induces male sterility, and is complemented by a chalcone synthase transgene. Plant Physiol 120:615–622Nechita A, Cotea VV, Nechita CB, Pincu RR, Mihai CT, Colibaba CL (2012) Study of cytostatic and cytotoxic activity of several polyphenolic extracts obtained from Vitis vinifera. Not Bot Horti Agrobo 40:216–221Nelson DW, Sommers LE (1982) Total carbon, organic carbon, and organic matter. In: Page AL et al (eds) Methods of soil analysis, chemical and microbiological properties. Soil Science Society of America, Madison, pp 539–577Nelson RE, Klameth LC, Nettleton WD (1978) Determining soil gypsum content and expressing properties of gypsiferous soils. Soil Sci Soc Am J 42:659–661Nile SH, Khobragade CN (2010) Antioxidant activity and flavonoid derivatives of Plumbago zeylanica. J Nat Prod 3:130–133Park HL, Lee SW, Jung KH, Hahn TR, Cho MH (2013) Transcriptomic analysis of UV-treated rice leaves reveals UV-induced phytoalexin biosynthetic pathways and their regulatory networks in rice. Phytochemistry 96:57–71Pękal A, Pyrzynska K (2014) Evaluation of aluminium complexation reaction for flavonoid content assay. Food Anal Method 7:1776–1782Pollastri S, Tattini M (2011) Flavonols: old compounds for old roles. Ann Bot 108:1225–1233Ravishankar D, Rajora AK, Greco F, Osborn HM (2013) Flavonoids as prospective compounds for anti-cancer therapy. Int J Biochem Cell B 45:2821–2831Rice-Evans CA, Miller NJ, Paganga G (1996) Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Bio Med 20:933–956Rieger G, Müller M, Guttenberger H, Bucar F (2008) Influence of altitudinal variation on the content of phenolic compounds in wild populations of Calluna vulgaris, Sambucus nigra, and Vaccinium myrtillus. J Agric Food Chem 58:9080–9086Rivas-Martínez S, Rivas-Saenz S (1996–2009) Worldwide bioclimatic classification system. Phytosociological Research Center, Spain. http://www.globalbioclimatics.org . Accessed 1 July 2013Rohman A, Riyanto S, Yuniarti N, Saputra WR, Utami R, Mulatsih W (2010) Antioxidant activity, total phenolic, and total flavonoid of extracts and fractions of red fruit (Pandanus conoideus Lam). Int Food Res J 17:97–106Romano B, Pagano E, Montanaro V, Fortunato AL, Milic N, Borrelli F (2013) Novel insights into the pharmacology of flavonoids. Phytother Res 27:1588–1596Rozema J, van de Staaij J, Björn LO, Caldwell MM (1997) UV-B as an environmental factor in plant life: stress and regulation. Trends Ecol Evol 12:22–28Rozema J, Bjorn LO, Bornman JF, Gaberščik A, Häder DP, Trošt T, Germ M, Klisch M, Gröniger A, Sinha RP, Lebert M, He YY, Buffoni-Hall R, de Bakker NVJ, van de Staaij J, Meijkamp BB (2002) The role of UV-B radiation in aquatic and terrestrial ecosystems—an experimental and functional analysis of the evolution of UV-absorbing compounds. Photochem Photobiol B Biol 66:2–12Schulze-Lefert P, Dangl JL, Becker-André M, Hahlbrock K, Schulz W (1989) Inducible in vivo DNA footprints define sequences necessary for UV light activation of the parsley chalcone synthase gene. EMBO J 8:651–656Sena MM, Frighetto RTS, Valarini PJ, Tokeshi H, Poppi RJ (2002) Discrimination of management effects on soil parameters by using principal component analysis: a multivariate analysis case study. Soil Till Res 67:171–181Shulaev V, Oliver DJ (2006) Metabolic and proteomic markers for oxidative stress. New tools for reactive oxygen species research. Plant Physiol 141:367–372Singleton VL, Rossi JA Jr (1965) Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am J EnolVitic 16:144–158Spitaler R, Winkler A, Lins I, Yanar S, Stuppner H, Zidorn C (2008) Altitudinal variation of phenolic contents in flowering heads of Arnica montana cv. ARBO: a 3-year comparison. J Chem Ecol 34:369–375Stapleton AE, Walbot V (1994) Flavonoids can protect maize DNA from the induction of UV radiation damage. Plant Physiol 105:881–889Takahashi M, Asada K (1988) Superoxide production in aprotic interior of chloroplast thylakoids. Arch Biochem Biophys 267:714–722Tattini M, Gravano E, Pinelli P, Mulinacci N, Romani A (2000) Flavonoids accumulate in leaves and glandular trichomes of Phillyrea latifolia exposed to excess solar radiation. New Phytol 148:69–77Tattini M, Galardi C, Pinelli P, Massai R, Remorini D, Agati G (2004) Differential accumulation of flavonoids and hydroxycinnamates in leaves of Ligustrum vulgare under excess light and drought stress. New Phytol 163:547–561Treutter D (2005) Significance of flavonoids in plant resistance and enhancement of their biosynthesis. Plant Biol 7:581–591Treutter D (2006) Significance of flavonoids in plant resistance: a review. Environ Chem Lett 4:147–157Van Breusegem F, Dat JF (2006) Reactive oxygen species in plant cell death. Plant Physiol 141:384–390Williams CA, Grayer RJ (2004) Anthocyanins and other flavonoids. Nat Prod Rep 21:539–573Winkel-Shirley B (2002) Biosynthesis of flavonoids and effect of stress. Curr Opin Plant Biol 5:218–223Ylstra B, Touraev A, Benito Moreno RM, Stöger E, van Tunen AA, Vicente O, Mol JNM, Heberle-Bors E (1992) Flavonols stimulate development, germination and tube growth of tobacco pollen. Plant Physiol 100:902–907Zhishen J, Mengcheng T, Jianming W (1999) The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem 64:555–559Zidorn C, Schubert B, Stuppner H (2005) Altitudinal differences in the contents of phenolics in flowering heads of three members of the tribe Lactuceae (Asteraceae) occurring as introduced species in New Zealand. Biochem Syst Ecol 33:855–87

Recurrent SARS-CoV-2 mutations in immunodeficient patients

Long-term severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in immunodeficient patients are an important source of variation for the virus but are understudied. Many case studies have been published which describe one or a small number of long-term infected individuals but no study has combined these sequences into a cohesive dataset. This work aims to rectify this and study the genomics of this patient group through a combination of literature searches as well as identifying new case series directly from the COVID-19 Genomics UK (COG-UK) dataset. The spike gene receptor-binding domain and N-terminal domain (NTD) were identified as mutation hotspots. Numerous mutations associated with variants of concern were observed to emerge recurrently. Additionally a mutation in the envelope gene, T30I was determined to be the second most frequent recurrently occurring mutation arising in persistent infections. A high proportion of recurrent mutations in immunodeficient individuals are associated with ACE2 affinity, immune escape, or viral packaging optimisation.There is an apparent selective pressure for mutations that aid cell–cell transmission within the host or persistence which are often different from mutations that aid inter-host transmission, although the fact that multiple recurrent de novo mutations are considered defining for variants of concern strongly indicates that this potential source of novel variants should not be discounted

Genome-wide meta-analysis of cerebral white matter hyperintensities in patients with stroke.

OBJECTIVE: For 3,670 stroke patients from the United Kingdom, United States, Australia, Belgium, and Italy, we performed a genome-wide meta-analysis of white matter hyperintensity volumes (WMHV) on data imputed to the 1000 Genomes reference dataset to provide insights into disease mechanisms. METHODS: We first sought to identify genetic associations with white matter hyperintensities in a stroke population, and then examined whether genetic loci previously linked to WMHV in community populations are also associated in stroke patients. Having established that genetic associations are shared between the 2 populations, we performed a meta-analysis testing which associations with WMHV in stroke-free populations are associated overall when combined with stroke populations. RESULTS: There were no associations at genome-wide significance with WMHV in stroke patients. All previously reported genome-wide significant associations with WMHV in community populations shared direction of effect in stroke patients. In a meta-analysis of the genome-wide significant and suggestive loci (p < 5 × 10(-6)) from community populations (15 single nucleotide polymorphisms in total) and from stroke patients, 6 independent loci were associated with WMHV in both populations. Four of these are novel associations at the genome-wide level (rs72934505 [NBEAL1], p = 2.2 × 10(-8); rs941898 [EVL], p = 4.0 × 10(-8); rs962888 [C1QL1], p = 1.1 × 10(-8); rs9515201 [COL4A2], p = 6.9 × 10(-9)). CONCLUSIONS: Genetic associations with WMHV are shared in otherwise healthy individuals and patients with stroke, indicating common genetic susceptibility in cerebral small vessel disease.Funding for collection, genotyping, and analysis of stroke samples was provided by Wellcome Trust Case Control Consortium-2, a functional genomics grant from the Wellcome Trust (DNA-Lacunar), the Stroke Association (DNA-lacunar), the Intramural Research Program of National Institute of Ageing (Massachusetts General Hospital [MGH] and Ischemic Stroke Genetics Study [ISGS]), National Institute of Neurological Disorders and Stroke (Siblings With Ischemic Stroke Study, ISGS, and MGH), the American Heart Association/Bugher Foundation Centers for Stroke Prevention Research (MGH), Deane Institute for Integrative Study of Atrial Fibrillation and Stroke (MGH), National Health and Medical Research Council (Australian Stroke Genetics Collaborative), and Italian Ministry of Health (Milan). Additional support for sample collection came from the Medical Research Council, National Institute of Health Research Biomedical Research Centre and Acute Vascular Imaging Centre (Oxford), Wellcome Trust and Binks Trust (Edinburgh), and Vascular Dementia Research Foundation (Munich). MT is supported by a project grant from the Stroke Association (TSA 2013/01). HSM is supported by an NIHR Senior Investigator award. HSM and SB are supported by the NIHR Cambridge University Hospitals Comprehensive Biomedical Research Centre. VT and RL are supported by grants from FWO Flanders. PR holds NIHR and Wellcome Trust Senior Investigator Awards. PAS is supported by an MRC Fellowship. CML’s research is supported by the National Institute for Health Research Biomedical Research Centre (BRC) based at Guy's and St Thomas' NHS Foundation Trust and King's College London, and the BRC for Mental Health at South London and Maudsley NHS Foundation Trust and King’s College London. This is the final version of the article. It first appeared from Wolters Kluwer via http://dx.doi.org/10.1212/WNL.000000000000226

The impact of viral mutations on recognition by SARS-CoV-2 specific T cells.

We identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.This work is supported by the UK Medical Research Council (MRC); Chinese Academy of Medical Sciences(CAMS) Innovation Fund for Medical Sciences (CIFMS), China; National Institute for Health Research (NIHR)Oxford Biomedical Research Centre, and UK Researchand Innovation (UKRI)/NIHR through the UK Coro-navirus Immunology Consortium (UK-CIC). Sequencing of SARS-CoV-2 samples and collation of data wasundertaken by the COG-UK CONSORTIUM. COG-UK is supported by funding from the Medical ResearchCouncil (MRC) part of UK Research & Innovation (UKRI),the National Institute of Health Research (NIHR),and Genome Research Limited, operating as the Wellcome Sanger Institute. T.I.d.S. is supported by a Well-come Trust Intermediate Clinical Fellowship (110058/Z/15/Z). L.T. is supported by the Wellcome Trust(grant number 205228/Z/16/Z) and by theUniversity of Liverpool Centre for Excellence in Infectious DiseaseResearch (CEIDR). S.D. is funded by an NIHR GlobalResearch Professorship (NIHR300791). L.T. and S.C.M.are also supported by the U.S. Food and Drug Administration Medical Countermeasures Initiative contract75F40120C00085 and the National Institute for Health Research Health Protection Research Unit (HPRU) inEmerging and Zoonotic Infections (NIHR200907) at University of Liverpool inpartnership with Public HealthEngland (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford.L.T. is based at the University of Liverpool. M.D.P. is funded by the NIHR Sheffield Biomedical ResearchCentre (BRC – IS-BRC-1215-20017). ISARIC4C is supported by the MRC (grant no MC_PC_19059). J.C.K.is a Wellcome Investigator (WT204969/Z/16/Z) and supported by NIHR Oxford Biomedical Research Centreand CIFMS. The views expressed are those of the authors and not necessarily those of the NIHR or MRC