Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

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Digestion of restriction enzyme for the detection of single-base mismatch in DNA

DNA hybridization and enzymatic digestion for the detection of mutation was investigated on the gold nanoparticles-calf thymus DNA (AuNPs-ctDNA) modified glassy carbon electrode (GCE). The thiol modified probe oligonucleotides (SH-ssDNA) were assembled on the surface of AuNPs-ctDNA modified GCE. The electrochemical response of the electrode was measured by differential pulse voltammetry and cyclic voltammetry. Methylene blue (MB) was used as the electroactive indicator. AuNPs were then dispersed effectively on the GCE surface in the presence of ct-DNA. When hybridization occurred, a decrease in the signal of MB current was observed. The modified electrode was used for the detection of mutations during the enzymatic digestion reaction in DNA. During this reaction, an increase in the signal of MB current was observed. So, the modified SH-ssDNA had a higher electrochemical response on the AuNPs-ctDNA/GCE because of the strong affinity of MB for guanine residues in it. The electrochemical detection of restriction enzyme digestion can provide a simple and practical method for observing single-base mismatches that can help in distinguishing mismatch sequences of DNA from the complementary ones.     

Dehydroquinate synthase: the role of divalent metal cations and of nicotinamide adenine dinucleotide in catalysis

The cofactor requirements of dehydroquinate synthase from Escherichia coli have been characterized. The homogeneous enzyme, purified from the overproducing strain RB791 (pJB14), is a monomeric metalloenzyme of Mr = 39,000 that contains 1 mol of tightly bound Co(II) according to atomic absorption analysis. The holoenzyme rapidly loses activity upon incubation with EDTA, giving rise to a stable but catalytically inactive apoenzyme. Activity is fully restored by reconstitution with Co(II) and partially restored with other divalent cations. Reconstitution of the apoenzyme with Zn(II) (which is probably the functioning metal in vivo) restores activity to 53% of the level observed with the Co(II)-holoenzyme. The presence of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) blocks the inactivation by EDTA. Dehydroquinate synthase also binds 1 mol of NAD+, the presence of which is essential for catalytic activity. The rate constant for the dissociation of NAD+ from the Co(II)-holoenzyme was found to be 0.024 min-1. Under turnover conditions with saturating levels of substrate, the dissociation rate of NAD+ increases by a factor of 40, to 1 min-1. Under these conditions (pH 7.5, 20 degrees C), the Km for NAD+ was determined to be 80 nM.     

Development of coral and zooxanthella‐specific microsatellites in three species of Pocillopora (Cnidaria,Scleractinia) from French Polynesia

Since the building of coral reefs results from the association of corals and zooxanthellae, their intracellular algal symbionts, genetic markers for both organisms are essential for studying the contribution of their respective dispersal to the resilience of endangered reef ecosystems. Very few microsatellites have been obtained in corals thus far. Here we report the successful cloning of six polymorphic microsatellites (allele number: 5–15) from Pocillopora verrucosa, P. meandrina and P. damicornis. Four of them amplified coral, and two amplified zooxanthella DNA.     

Virtual screening,docking, and dynamics of potential new inhibitors of dihydrofolate reductase from Yersinia pestis

In the present work, we propose to design drugs that target the enzyme dihydrofolate redutase (DHFR) as a means of a novel drug therapy against plague. Potential inhibitors of DHFR from Yersinia pestis (YpDHFR) were selected by virtual screening and subjected to docking, molecular dynamics (MD) simulations, and Poisson–Boltzmann surface area method, in order to evaluate their interactions in the active sites of YpDHFR and human DHFR (HssDHFR). The results suggested selectivity for three compounds that were further used to propose the structures of six new potential selective inhibitors for YpDHFR.     

Immunomodulatory agents for COVID-19 treatment: possible mechanism of action and immunopathology features

Molecular and Cellular Biochemistry - The novel coronavirus pandemic has emerged as one of the significant medical-health challenges of the current century. The World Health Organization has named...     

Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity

Heat shock protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by cochaperones but also by distinct posttranslational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α helix-1 of the yeast Hsp90 N-domain both in?vitro and in?vivo. This α helix participates in?a hydrophobic interaction with the catalytic loop in Hsp90's middle domain, helping to stabilize the chaperone's ATPase-competent state. Phosphomimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function and impacts interaction with the cochaperones Aha1 and Cdc37. Overexpression of Aha1 stimulates the ATPase activity, restores cochaperone interactions, and compensates for the functional defects of these Hsp90 mutants.     

Comparative ontogeny of perfect and pistillate florets in Senecio vernalis (Asteraceae)

We studied the inflorescence, and in particular ontogeny and development of the florets in Senecio vernalis as a representative member of Asteraceae, using epi-illumination microscopy. Initiation and subsequent development of florets on the highly convex inflorescence apex occur acropetally, except for pistillate ray florets, which show a lag in initiation. Receptacular bracts derive from the receptacular surface after development of all florets. The order of whorl initiation in both disc and ray florets include corolla, androecium and finally the pappus, together with the gynoecium. Development of corolla lobes from a ring meristem occurs in bidirectional order starting from the lateral side, whereas stamens incept unidirectionally from the abaxial side. Concurrently with the inception of two median carpel primordia, a ring meristem develops at the base of the corolla from which pappus bristles differentiate in later stages. Pistillate ray florets show significant differences from perfect disc florets as reflected by the zygomorphic shape of the floral apex and a shift of floral merosity from pentamery to tetramery. Loss of stamens in ray florets occurs due to abortion of primordia after initiation.