Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera

1. Download : Download high-res image (217KB)
2. Download : Download full-size image

     

Enantioselectivity of Candida rugosa lipases (Lip1, Lip3, and Lip4) towards 2-bromo phenylacetic acid octyl esters controlled by a single amino acid

Enantiomer discrimination by enzymes is a very accurate mechanism, which often involves few amino acids located at the active site. Lipase isoforms from Candida rugosa are very good enzymatic models to study this phenomenon as they display high sequence homology (>80%) and their enantioselectivity is often pointed out. In the present work, we investigated three lipases from C. rugosa (Lip1, Lip3, and Lip4, respectively) towards the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. All exhibited a high enantioselectivity, with Lip4 preferring the R-enantiomer (E-value = 15), while Lip1 and Lip3 showed an S-enantioselectivity >200. A combination of sequence and structure analysis of the three C. rugosa lipases suggested that position 296 could play a role in S- or R-enantiomer preference of C. rugosa lipases. This led to the construction by site-directed mutagenesis of Lip1 and Lip4 variants in which position 296 was, respectively, exchanged by a Gly, Ala, Leu, or Phe amino acid. Screening of these variants for their enantioselectivity toward 2-bromo phenyl acetic acid octyl esters revealed that steric hindrance of the amino acid residue introduced at position 296 controls both the enantiopreference and the enantioselectivity value of the lipase: bulkier is the amino acid at position 296, larger is the selectivity towards the S-enantiomer. To investigate further these observations at an atomic level, we carried out a preliminary modeling study of the tetrahedral intermediates formed by Lip1 and Lip4 with the (R, S)-2-bromo-phenylacetic acid octyl ester enantiomers that provides some insight regarding the determinants responsible for lipase enantiodiscrimination.     

<Emphasis Type="Italic">Sporidesmioides thailandica</Emphasis> gen. et sp. nov. (Dothideomycetes) from northern Thailand

Sporidesmioides thailandica gen. et sp. nov., isolated from herbaceous litter in northern Thailand, is described and illustrated. The new genus is characterised by sporidesmium-like, septate, solitary, acrogenous, pale brown to brown, obclavate, conidia, which taper towards the apex and are truncate at the base and septate conidiophores similar to those of Sporidesmium species in Sporidesmiaceae (Sordariomycetes). It differs from similar genera by the presence of a stromatic base, polyblastic conidiogenous cells and a distinct, colourless sheath at the conidial apex. Phylogenetic analyses of combined LSU, SSU, TEF1-α and RPB2 sequence data show that the new taxon, Sporidesmioides thailandica, forms a separate, single clade with Torulaceae in Pleosporales, Dothideomycetes, and is distinct from Sporidesmiaceae (Sordariomycetes).     

<Emphasis Type="Italic">Diatrypella tectonae</Emphasis> and <Emphasis Type="Italic">Peroneutypa mackenziei</Emphasis> spp. nov. (Diatrypaceae) from northern Thailand

Two novel species of Diatrypaceae, Diatrypella tectonae and Peroneutypa mackenziei, were collected from dead wood in northern Thailand. The new species are introduced in this paper, with evidence from morphology and phylogenetic analyses. The new taxa are described, illustrated and compared with related taxa. Phylogenetic analyses of combined ITS and partial β-tubulin (β-tub) sequence data support their natural placements in the genera Diatrypella and Peroneutypa as new species.     

Phylogenetic characterization of two novel <Emphasis Type="Italic">Kamalomyces</Emphasis> species in Tubeufiaceae (Tubeufiales)

Two novel species of bambusicolous fungi in the genus Kamalomyces, collected from northern Thailand, are described and illustrated herein. Kamalomyces bambusicola and K. thailandicus spp. nov. are typical of the genus Kamalomyces (Tubeufiaceae, Tubeufiales) and are morphologically distinct from known species with respect to their size of ascomata, asci and ascospores, ascospore septation and peridium structure, including the subiculum comprising hyphae on the host surface. Morphological examination reveals that the asexual morph of K. bambusicola is associated with its sexual morph in a subiculum forming dictyochlamydosporous conidia, which are similar to the asexual morph of Chlamydotubeufia. Phylogenetic analyses of combined LSU, ITS and TEF1-α sequence data also support these two species as distinct and confirm their phylogenetic affinities within the Tubeufiaceae. In particular, Kamalomyces shares a close phylogenetic relationship to Helicoma.     

Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera

1. Download : Download high-res image (240KB)
2. Download : Download full-size image

     

Rationally engineered double substituted variants of Yarrowia lipolytica lipase with enhanced activity coupled with highly inverted enantioselectivity towards 2‐bromo phenyl acetic acid esters

Inverting enzyme enantioselectivity by protein engineering is still a great challenge. Lip2p lipase from Yarrowia lipolytica, which demonstrates a low S‐enantioselectivity (E‐value = 5) during the hydrolytic kinetic resolution of 2‐bromo‐phenyl acetic acid octyl esters (an important class of chemical intermediates in the pharmaceutical industry), was converted, by a rational engineering approach, into a totally R‐selective enzyme (E‐value > 200). This tremendous change in selectivity is the result of only two amino acid changes. The starting point of our strategy was the prior identification of two key positions, 97 and 232, for enantiomer discrimination. Four single substitution variants were recently identified as exhibiting a low inversion of selectivity coupled to a low‐hydrolytic activity. On the basis of these results, six double substituted variants, combining relevant mutations at both 97 and 232 positions, were constructed by site‐directed mutagenesis. This work led to the isolation of one double substituted variant (D97A‐V232F), which displays a total preference for the R‐enantiomer. The highly reversed enantioselectivity of this variant is accompanied by a 4.5‐fold enhancement of its activity toward the preferred enantiomer. The molecular docking of the R‐ and S‐enantiomers in the wild‐type enzyme and the D97A‐V232F variant suggests that V232F mutation provides a more favorable stacking interaction for the phenyl group of the R‐enantiomer, that could explain both the enhanced activity and the reversal of enantioselectivity. These results demonstrate the potential of rationally engineered mutations to further enhance enzyme activity and to modulate selectivity. Biotechnol. Bioeng. 2010;106: 852–859. © 2010 Wiley Periodicals, Inc.     

Chronic Opisthorchis viverrini Infection Changes the Liver Microbiome and Promotes Helicobacter Growth

Adults of Opisthorchis viverrini reside in the biliary system, inducing inflammation of bile ducts and cholangitis, leading to hepatobiliary disease (HBD) including cholangiocarcinoma. O. viverrini infection also has major implications for the bacterial community in bile ducts and liver. To investigate this in chronic O. viverrini infection (≥ 8 months p.i.), bacterial genomic DNA from livers of hamsters and from worms was investigated using culture techniques, PCR for Helicobacter spp. and high-throughput next-generation sequencing targeting the V3-V4 hypervariable regions of prokaryotic 16S rRNA gene. Of a total of 855,046 DNA sequence reads, 417,953 were useable after filtering. Metagenomic analyses assigned these to 93 operational taxonomic units (OTUs) consisting of 80 OTUs of bacteria, including 6 phyla and 42 genera. In the chronic O. viverrini-infected group, bacterial community composition and diversity were significantly increased compared to controls. Sequences of Fusobacterium spp. were the most common (13.81%), followed by Streptococcus luteciae (10.76%), Escherichia coli (10.18%), and Bifidobacterium spp. (0.58%). In addition, Helicobacter pylori (0.17% of sequences) was also identified in the liver of chronic O. viverrini infections, but not in normal liver. The presence of H. pylori was confirmed by PCR and by use of an antibody against bacterial antigen, supporting the metagenomics data. The identities of bacteria cultured for enrichment suggested that chronic O. viverrini infection changes the liver microbiome and promotes Helicobacter spp. growth. There may be synergy between O. viverrini and the liver microbiome in enhancing immune response-mediated hepatobiliary diseases.