Site-Specific Insertion Polymorphism of the MITE Alex-1 in the Genus Coffea Suggests Interspecific Gene Flow

Miniature Inverted-repeat Transposable Elements (MITEs) are small nonautonomous class-II transposable elements distributed throughout eukaryotic genomes. We identified a novel family of MITEs (named Alex) in the Coffea canephora genome often associated with expressed sequences. The Alex-1 element is inserted in an intron of a gene at the CcEIN4 locus. Its mobility was demonstrated by sequencing the insertion site in C. canephora accessions and Coffea species. Analysis of the insertion polymorphism of Alex-1 at this locus in Coffea species and in C. canephora showed that there was no relationship between the geographical distribution of the species, their phylogenetic relationships, and insertion polymorphism. The intraspecific distribution of C. canephora revealed an original situation within the E diversity group. These results suggest possibly greater gene flow between species than previously thought. This MITE family will enable the study of the C. canephora genome evolution, phylogenetic relationships, and possible gene flows within the Coffea genus

Microcollinearity in an ethylene receptor coding gene region of the Coffea canephora genome is extensively conserved with Vitis vinifera and other distant dicotyledonous sequenced genomes

Background: Coffea canephora, also called Robusta, belongs to the Rubiaceae, the fourth largest angiosperm family. This diploid species (2x = 2n = 22) has a fairly small genome size of approximate to 690 Mb and despite its extreme economic importance, particularly for developing countries, knowledge on the genome composition, structure and evolution remain very limited. Here, we report the 160 kb of the first C. canephora Bacterial Artificial Chromosome (BAC) clone ever sequenced and its fine analysis. Results: This clone contains the CcEIN4 gene, encoding an ethylene receptor, and twenty other predicted genes showing a high gene density of one gene per 7.8 kb. Most of them display perfect matches with C. canephora expressed sequence tags or show transcriptional activities through PCR amplifications on cDNA libraries. Twenty-three transposable elements, mainly Class II transposon derivatives, were identified at this locus. Most of these Class II elements are Miniature Inverted-repeat Transposable Elements (MITE) known to be closely associated with plant genes. This BAC composition gives a pattern similar to those found in gene rich regions of Solanum lycopersicum and Medicago truncatula genomes indicating that the CcEIN4 regions may belong to a gene rich region in the C. canephora genome. Comparative sequence analysis indicated an extensive conservation between C. canephora and most of the reference dicotyledonous genomes studied in this work, such as tomato (S. lycopersicum), grapevine (V. vinifera), barrel medic M. truncatula, black cottonwood (Populus trichocarpa) and Arabidopsis thaliana. The higher degree of microcollinearity was found between C. canephora and V. vinifera, which belong respectively to the Asterids and Rosids, two clades that diverged more than 114 million years ago. Conclusion: This study provides a first glimpse of C. canephora genome composition and evolution. Our data revealed a remarkable conservation of the microcollinearity between C. canephora and V. vinifera and a high conservation with other distant dicotyledonous reference genomes. Altogether, these results provide valuable information to identify candidate genes in C. canephora genome and serve as a foundation to establish strategies for whole genome sequencing. Future large-scale sequence comparison between C. canephora and reference sequenced genomes will help in understanding the evolutionary history of dicotyledonous plants

Characterization of three GH62 α-L-arabinofuranosidases from Talaromyces versatilis (basionym Penicillium funiculosum) and impact study with xylanases on arabinoxylan degradation

La société Adisseo produit un cocktail d’enzymes hydrolytiques appelé Rovabio® Excel sécrété par un champignon filamenteux Talaromyces versatilis. Ce cocktail est utilisé comme additif alimentaire pour augmenter la digestibilité de complexes polysaccharidiques en nutrition animale et ainsi augmenter la valeur nutritionnelle des matières premières agricoles. Une récente étude protéomique de ce champignon (Guais et al., 2008) a révélé la présence d’un grand nombre d’arabinofuranosidases (ABFs) appartenant à différentes familles de glycosides hydrolases : 5 ABFs de la famille GH54, 3 ABFs de la famille GH62 et enfin une de la famille GH51. Un des objectifs de mes travaux de thèse a été le clonage, l’expression hétérologue (hôte lévurien Pichia pastoris) des 9 gènes codant pour ces 9 enzymes et la caractérisation complète de la famille GH62. La caractérisation des capacités d’hydrolyse des ABFs 54 et 62 a également été étudiée grâce à une technique d’empreintes d’hydrolyse enzymatique sur arabinoxylane de blé. Enfin, la dernière partie de mes travaux consistait à confectionner des mélanges d’enzymes hydrolytiques des différentes familles d’ABFs associés à des xylanases et de suivre l’efficacité de la dégradation de l’arabinoxylane insoluble grâce à l’utilisation d’un réacteur torique permettant l’acquisition d’images et l’analyse en ligne de la dégradation. Ces travaux sur le réacteur ont permis de mettre en évidence une synergie entre Abfs et Xylanases.Adisseo produce and commercialize a hydrolytic enzymatic cocktail termed Rovabio and secreted by a filamentous fungus Talaromyces versatilis. This cocktail is used as feed additive for increased digestibility of complex polysaccharides in animal nutrition. A recent genomic study of this fungus revealed the presence of 5 arabinofuranosidases (Abfs) to family GH 54, 3 of GH 62 and 1 of GH51. The first aim of my thesis works was about cloning, heterologous overexpression (in pichia pastoris yeast) of this 9 genes encoding for this 9 enzymes and characterization of the family GH 62. Mode of action of ABFs 54 and 62s has been characterized by enzymatic fingerprinting analysis on wheat arabinoxylan. Then, last part was to design enzymatic cocktail with differents families of ABFs and Xylanases and test their impact on insoluble arabinoxylan hydrolysis with toric reactor. These works on reactor have bringing to light a synergy between ABFs and Xylanase

Molecular and biochemical characterization of three GH62 alpha-L-arabinofuranosidases from the soil deuteromycete Penicillium funiculosum

Penicillium funiculosum is an industrial fungus exploited for its capacity to secrete a wide array of glycosyl hydrolases (GHs) and glycosyl transferases (GTs). These enzymes are part of an enzymatic cocktail that is commercialized under the name RovabioExcel (R), which is used as feed additive in animal nutrition. The genome sequence of this filamentous fungus has revealed a remarkable richness in several accessory enzymes, and notably in alpha-L-arabinofuranosidases (alpha-L-AFases) that participate in the hydrolysis of arabinoxylans (AX) in corn/wheat fibers used in poultry feed. Here, we report on the molecular and biochemical characterization of three GH62 family alpha-L-AFases encoding genes in this filamentous fungus. Amino acids sequences showed strong similarities (>65%) between them, as well with GH62 enzymes from other filamentous fungi. Interestingly, one Of the three PfABF62, namely PfABF62c is unique in bearing at its N-terminus a canonical family 1 carbohydrate-binding module (CBM1) of 37 amino acids length, which was shown to help the protein to bind to rnicrocrystalline cellulose. Also, this PfABF62c showed optimal pH and temperature of 2.8 and 50 degrees C, respectively, whereas optimal activity for PfABF62a and PfABF62b were measured at 40 degrees C and at pH ranging between 2.6 and 4.5. Arabinan and arabinoxylan, but no other sugars or polymers were found to augment the thermal transition of the three enzymes by 3-5 degrees C as measured by differential scanning fluorimetry. Finally, enzymatic hydrolysis fingerprints of heteroxylans allowed concluding that the mode of action of the GH62 enzymes from this fungal species was to remove arabinofuranosyl residues linked in position 0-2 and 0-3 of substituted xylose units in arabinoxylan chains. (c) 2013 Elsevier Inc. All rights reserved

Characterisation of the Effect of the Spatial Organisation of Hemicellulases on the Hydrolysis of Plant Biomass Polymer

Synergism between enzymes is of crucial importance in cell metabolism. This synergism occurs often through a spatial organisation favouring proximity and substrate channelling. In this context, we developed a strategy for evaluating the impact of the geometry between two enzymes involved in nature in the recycling of the carbon derived from plant cell wall polymers. By using an innovative covalent association process using two protein fragments, Jo and In, we produced two bi-modular chimeric complexes connecting a xylanase and a xylosidase, involved in the deconstruction of xylose-based plant cell wall polymer. We first show that the intrinsic activity of the individual enzymes was preserved. Small Angle X-rays Scattering (SAXS) analysis of the complexes highlighted two different spatial organisations in solution, affecting both the distance between the enzymes (53 Å and 28 Å) and the distance between the catalytic pockets (94 Å and 75 Å). Reducing sugar and HPAEC-PAD analysis revealed different behaviour regarding the hydrolysis of Beechwood xylan. After 24 h of hydrolysis, one complex was able to release a higher amount of reducing sugar compare to the free enzymes (i.e., 15,640 and 14,549 µM of equivalent xylose, respectively). However, more interestingly, the two complexes were able to release variable percentages of xylooligosaccharides compared to the free enzymes. The structure of the complexes revealed some putative steric hindrance, which impacted both enzymatic efficiency and the product profile. This report shows that controlling the spatial geometry between two enzymes would help to better investigate synergism effect within complex multi-enzymatic machinery and control the final product

The covalent complex of Jo-In results from a long-lived, non-covalent intermediate state with near-native structure

International audienceCovalent protein complexes have been used to assemble enzymes in large scaffolds for biotechnology purposes. Although the catalytic mechanism of the covalent linking of such proteins is well known, the recognition and overall structural mechanisms driving the association are far less understood but could help further functional engineering of these complexes. Here, we study the Jo-In complex by NMR spectroscopy and molecular modelling. We characterize a transient non-covalent complex, with structural elements close to those in the final covalent complex. Using site specific mutagenesis, we further show that this non-covalent association is essential for the covalent complex to for

Microcollinearity in an ethylene receptor coding gene region of the <it>Coffea canephora </it>genome is extensively conserved with <it>Vitis vinifera </it>and other distant dicotyledonous sequenced genomes

Abstract Background Coffea canephora, also called Robusta, belongs to the Rubiaceae, the fourth largest angiosperm family. This diploid species (2x = 2n = 22) has a fairly small genome size of ≈ 690 Mb and despite its extreme economic importance, particularly for developing countries, knowledge on the genome composition, structure and evolution remain very limited. Here, we report the 160 kb of the first C. canephora Bacterial Artificial Chromosome (BAC) clone ever sequenced and its fine analysis. Results This clone contains the CcEIN4 gene, encoding an ethylene receptor, and twenty other predicted genes showing a high gene density of one gene per 7.8 kb. Most of them display perfect matches with C. canephora expressed sequence tags or show transcriptional activities through PCR amplifications on cDNA libraries. Twenty-three transposable elements, mainly Class II transposon derivatives, were identified at this locus. Most of these Class II elements are Miniature Inverted-repeat Transposable Elements (MITE) known to be closely associated with plant genes. This BAC composition gives a pattern similar to those found in gene rich regions of Solanum lycopersicum and Medicago truncatula genomes indicating that the CcEIN4 regions may belong to a gene rich region in the C. canephora genome. Comparative sequence analysis indicated an extensive conservation between C. canephora and most of the reference dicotyledonous genomes studied in this work, such as tomato (S. lycopersicum), grapevine (V. vinifera), barrel medic M. truncatula, black cottonwood (Populus trichocarpa) and Arabidopsis thaliana. The higher degree of microcollinearity was found between C. canephora and V. vinifera, which belong respectively to the Asterids and Rosids, two clades that diverged more than 114 million years ago. Conclusion This study provides a first glimpse of C. canephora genome composition and evolution. Our data revealed a remarkable conservation of the microcollinearity between C. canephora and V. vinifera and a high conservation with other distant dicotyledonous reference genomes. Altogether, these results provide valuable information to identify candidate genes in C. canephora genome and serve as a foundation to establish strategies for whole genome sequencing. Future large-scale sequence comparison between C. canephora and reference sequenced genomes will help in understanding the evolutionary history of dicotyledonous plants.</p

Respiratory support in patients with severe COVID-19 in the International Severe Acute Respiratory and Emerging Infection (ISARIC) COVID-19 study: a prospective, multinational, observational study

Background: Up to 30% of hospitalised patients with COVID-19 require advanced respiratory support, including high-flow nasal cannulas (HFNC), non-invasive mechanical ventilation (NIV), or invasive mechanical ventilation (IMV). We aimed to describe the clinical characteristics, outcomes and risk factors for failing non-invasive respiratory support in patients treated with severe COVID-19 during the first two years of the pandemic in high-income countries (HICs) and low middle-income countries (LMICs). Methods: This is a multinational, multicentre, prospective cohort study embedded in the ISARIC-WHO COVID-19 Clinical Characterisation Protocol. Patients with laboratory-confirmed SARS-CoV-2 infection who required hospital admission were recruited prospectively. Patients treated with HFNC, NIV, or IMV within the first 24 h of hospital admission were included in this study. Descriptive statistics, random forest, and logistic regression analyses were used to describe clinical characteristics and compare clinical outcomes among patients treated with the different types of advanced respiratory support. Results: A total of 66,565 patients were included in this study. Overall, 82.6% of patients were treated in HIC, and 40.6% were admitted to the hospital during the first pandemic wave. During the first 24 h after hospital admission, patients in HICs were more frequently treated with HFNC (48.0%), followed by NIV (38.6%) and IMV (13.4%). In contrast, patients admitted in lower- and middle-income countries (LMICs) were less frequently treated with HFNC (16.1%) and the majority received IMV (59.1%). The failure rate of non-invasive respiratory support (i.e. HFNC or NIV) was 15.5%, of which 71.2% were from HIC and 28.8% from LMIC. The variables most strongly associated with non-invasive ventilation failure, defined as progression to IMV, were high leukocyte counts at hospital admission (OR [95%CI]; 5.86 [4.83-7.10]), treatment in an LMIC (OR [95%CI]; 2.04 [1.97-2.11]), and tachypnoea at hospital admission (OR [95%CI]; 1.16 [1.14-1.18]). Patients who failed HFNC/NIV had a higher 28-day fatality ratio (OR [95%CI]; 1.27 [1.25-1.30]). Conclusions: In the present international cohort, the most frequently used advanced respiratory support was the HFNC. However, IMV was used more often in LMIC. Higher leucocyte count, tachypnoea, and treatment in LMIC were risk factors for HFNC/NIV failure. HFNC/NIV failure was related to worse clinical outcomes, such as 28-day mortality. Trial registration This is a prospective observational study; therefore, no health care interventions were applied to participants, and trial registration is not applicable

Respiratory support in patients with severe COVID-19 in the International Severe Acute Respiratory and Emerging Infection (ISARIC) COVID-19 study: a prospective, multinational, observational study

Background: Up to 30% of hospitalised patients with COVID-19 require advanced respiratory support, including high-flow nasal cannulas (HFNC), non-invasive mechanical ventilation (NIV), or invasive mechanical ventilation (IMV). We aimed to describe the clinical characteristics, outcomes and risk factors for failing non-invasive respiratory support in patients treated with severe COVID-19 during the first two years of the pandemic in high-income countries (HICs) and low middle-income countries (LMICs). Methods: This is a multinational, multicentre, prospective cohort study embedded in the ISARIC-WHO COVID-19 Clinical Characterisation Protocol. Patients with laboratory-confirmed SARS-CoV-2 infection who required hospital admission were recruited prospectively. Patients treated with HFNC, NIV, or IMV within the first 24 h of hospital admission were included in this study. Descriptive statistics, random forest, and logistic regression analyses were used to describe clinical characteristics and compare clinical outcomes among patients treated with the different types of advanced respiratory support. Results: A total of 66,565 patients were included in this study. Overall, 82.6% of patients were treated in HIC, and 40.6% were admitted to the hospital during the first pandemic wave. During the first 24 h after hospital admission, patients in HICs were more frequently treated with HFNC (48.0%), followed by NIV (38.6%) and IMV (13.4%). In contrast, patients admitted in lower- and middle-income countries (LMICs) were less frequently treated with HFNC (16.1%) and the majority received IMV (59.1%). The failure rate of non-invasive respiratory support (i.e. HFNC or NIV) was 15.5%, of which 71.2% were from HIC and 28.8% from LMIC. The variables most strongly associated with non-invasive ventilation failure, defined as progression to IMV, were high leukocyte counts at hospital admission (OR [95%CI]; 5.86 [4.83–7.10]), treatment in an LMIC (OR [95%CI]; 2.04 [1.97–2.11]), and tachypnoea at hospital admission (OR [95%CI]; 1.16 [1.14–1.18]). Patients who failed HFNC/NIV had a higher 28-day fatality ratio (OR [95%CI]; 1.27 [1.25–1.30]). Conclusions: In the present international cohort, the most frequently used advanced respiratory support was the HFNC. However, IMV was used more often in LMIC. Higher leucocyte count, tachypnoea, and treatment in LMIC were risk factors for HFNC/NIV failure. HFNC/NIV failure was related to worse clinical outcomes, such as 28-day mortality. Trial registration This is a prospective observational study; therefore, no health care interventions were applied to participants, and trial registration is not applicable