A Novel Real-Time RT-PCR-Based Methodology for the Preliminary Typing of SARS-CoV-2 Variants, Employing Non-Extendable LNA Oligonucleotides and Three Signature Mutations at the Spike Protein Receptor-Binding Domain.

Mutations resulting in amino-acid substitutions of the SARS-CoV-2 spike protein receptor-binding domain (RBD) have been associated with enhanced transmissibility and immune escape of the respective variants, namely Alpha, Beta, Gamma or Delta. Rapid identification of the aforementioned variants of concern and their discrimination of other variants is thus of importance for public health interventions. For this reason, a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed, to target signature mutations associated with amino-acid substitutions at positions 478, 484 and 501 present in the receptor-binding motif (RBM) of the spike protein RBD.

A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions.

The emergence of SARS-CoV-2 mutations resulting in the S protein amino-acid substitutions N501Y and E484K, which have been associated with enhanced transmissibility and immune escape, respectively, necessitates immediate actions, for which their rapid identification is crucial. For the simultaneous typing of both of these mutations of concern (MOCs), a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed. The assay is highly sensitive with a LOD of 117 copies/reaction, amplification efficiencies >94 % and a linear range of over 5 log copies/reaction.

Polysaccharide deacetylases serve as new targets for the design of inhibitors against Bacillus anthracis and Bacillus cereus.

Peptidoglycan N-acetylglucosamine (GlcNAc) deacetylases (PGNGdacs) from bacterial pathogens are validated targets for the development of novel antimicrobial agents. In this study we examined the in vitro inhibition of hydroxamate ligand N-hydroxy-4-(naphthalene-1-yl)benzamide (NHNB), a selective inhibitor of histone deacetylases-8 (HDAC8), against two PGNGdacs namely BC1974 and BC1960 from B. cereus, highly homologous to BA1977 and BA1961 of B.

Structures of the Peptidoglycan N-Acetylglucosamine Deacetylase Bc1974 and Its Complexes with Zinc Metalloenzyme Inhibitors.

The cell wall peptidoglycan is recognized as a primary target of the innate immune system, and usually its disintegration results in bacterial lysis. Bacillus cereus, a close relative of the highly virulent Bacillus anthracis, contains 10 polysaccharide deacetylases. Among these, the peptidoglycan N-acetylglucosamine deacetylase Bc1974 is the highest homologue to the Bacillus anthracis Ba1977 that is required for full virulence and is involved in resistance to the host's lysozyme.

Structure determination of BA0150, a putative polysaccharide deacetylase from Bacillus anthracis.

Polysaccharide deacetylases are bacterial enzymes that catalyze the deacetylation of acetylated sugars on the membranes of Gram-positive bacteria, allowing them to be unrecognized by host immune systems. Inhibition of these enzymes would disrupt such pathogenic defensive mechanisms and therefore offers a promising route for the development of novel antibiotic therapeutics. Here, the first X-ray crystal structure of BA0150, a putative polysaccharide deacetylase from Bacillus anthracis, is reported to 2.

Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases.

Alkaline phosphatases (APs) are commercially applied enzymes that catalyze the hydrolysis of phosphate monoesters by a reaction involving three active site metal ions. We have previously identified H135 as the key residue for controlling activity of the psychrophilic TAB5 AP (TAP). In this article, we describe three X-ray crystallographic structures on TAP variants H135E and H135D in complex with a variety of metal ions.

Novel endo-alpha-N-acetylgalactosaminidases with broader substrate specificity.

In an effort to identify novel endo-alpha-N-acetylgalactosaminidases (endo-alpha-GalNAcases), four potential genes were cloned. Three of the expressed proteins EngEF from Enterococcus faecalis, EngPA from Propionibacterium acnes, and EngCP from Clostridium perfringens were purified and characterized. Their substrate specificity was investigated and compared to the commercially available endo-alpha-GalNAcases from Streptococcus pneumoniae (EngSP) and Alcaligenes sp.

Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase.

Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis-Menten kinetics, activation energy and microcalorimetric parameters of unfolding.

Crystal structure of alkaline phosphatase from the Antarctic bacterium TAB5.

Alkaline phosphatases (APs) are non-specific phosphohydrolases that are widely used in molecular biology and diagnostics. We describe the structure of the cold active alkaline phosphatase from the Antarctic bacterium TAB5 (TAP). The fold and the active site geometry are conserved with the other AP structures, where the monomer has a large central beta-sheet enclosed by alpha-helices.

Expression, purification, and characterization of a cobalt-activated chitin deacetylase (Cda2p) from Saccharomyces cerevisiae.

Chitin deacetylase (Cda2p) (EC 3.5.1.