Inhibition of nonspecific polymerase activity using Poly(Aspartic) acid as a model anionic polyelectrolyte.

DNA polymerases with strand-displacement activity allow to amplify nucleic acids under isothermal conditions but often lead to undesirable by-products. Here, we report the increase of specificity of isothermal amplification in the presence of poly (aspartic) acids (pAsp). We hypothesized that side reactions occur due to the binding of the phosphate backbone of synthesized DNA strands with surface amino groups of the polymerase, and weakly acidic polyelectrolytes could shield polymerase molecules from DNA and thereby inhibit nonspecific amplification. Suppression of nonspecific polymerase activity by pAsp was studied on multimerization as a model side reaction. It was found that a low concentration of pAsp (0.01%) provides successful amplification of specific DNA targets. The inhibitory effect of pAsp is due to its polymeric structure since aspartic acid did affect neither specific nor nonspecific amplification. Strongly acidic polyelectrolyte heparin does not possess the same selectivity since it suppresses any DNA synthesis. The applicability of pAsp to prevent nonspecific reactions and reliable detection of the specific target has been demonstrated on the genetic material of SARS-CoV-2 coronavirus using Loop-mediated isothermal amplification.

Antibacterial activity of noscapine analogs.

The antibacterial properties of close noscapine analogs have not been previously reported. We used our pDualrep2 double-reporter High Throughput Screening (HTS) platform to identify a series of noscapine derivatives with promising antibacterial activity. The platform is based on RPF (SOS-response/DNA damage) and Katushka2S (inhibition of translation) proteins and simultaneously provides information on antibacterial activity and the mechanism of action of small-molecule compounds against E. coli. The most potent compound exhibited an MIC of 13.5 µM(6.25 µg/ml) and a relatively low cytotoxicity against HEK293 cells (CC50 = 71 µM, selectivity index: ~5.5). Some compounds from this series induced average Katushka2S reporter signals, indicating inhibition of translation machinery in the bacteria; however, these compounds did not attenuate translation in vitro in a luciferase-based translation assay. The most effective compounds did not significantly arrest the mitotic cycle in HEK293 cells, in contrast to the parent compound in a flow cytometry assay. Several molecules showed activity against clinically relevant gram-negative and gram-positive bacterial strains. Compounds from the discovered series can be reasonably regarded as good templates for further development and evaluation.

The gene profile of Enterobacteriaceae virulence factors in relation to bacteriuria levels between the acute episodes of recurrent uncomplicated lower urinary tract infection.

OBJECTIVES: The pathogenic potential of uropathogens isolated between acute episodes of recurrent lower urinary tract infection (rLUTI) is studied insufficiently. The objectives were to determine the spectrum of virulence genes of Enterobacteriaceae cultured between acute episodes of rLUTI at various levels of bacteriuria. METHODS: Bacteriological examinations of 169 premenopausal women's midstream urine with rLUTI were performed between acute episodes of UTI. Sixty-two strains of Enterobacteriaceae at concentrations 102-108 CFU/ml were analyzed for the presence of papA, papE/F, papGII, afa, bmaE, iutA, feoB, fyuA, kpsMTII, and usp virulence factors genes' (VFGs) fragments. RESULTS: In all strains VFGs were found with numbers from 1 to 10. Four VFGs were found at all levels of bacteriuria (from 102 to 107-8) in most strains (>50%): papGII, feoB, fyuA, usp. In total, 28 significant Pearson contingency coefficient (PCC) were determined. Each of the genes, papA, papE/F, usp, was found more often in uropathogens from patients with a higher level of leukocyturia. CONCLUSIONS: The inter-episode period in rLUTI is associated with varying levels of bacteriuria of enterobacteria. Since enterobacteria virulent potential could be determined at all levels of bacteriuria, there is at all levels of bacteriuria a potential risk for recurrence of LUTI.