Probiotics in the times of COVID-19.

The time of COVID-19 pandemic focused the attention of scientist to recognise the complex medical symptoms of the disease, modes of infection and possible therapies. The organisms' response towards SARS-CoV-2 infection depends on many individual factors and the course of disease is described as unprecedented and complex. Numerous symptoms from the respiratory system, abnormalities in the gastrointestinal tract, stroke, liver damage and coagulopathy, among others, are accompanied by negative side effects of the pandemic lifestyle, including immunity depletion, overall fitness impairment, skin condition worsening, psychological and psychiatric consequences. There is an urgent need to seek all possible routes for assuring favouring conditions to build and support the organisms' microbiological barriers and enhance immunity, which will also help during the ongoing vaccination action. Probiotic Lactic Acid Bacteria (LAB) and environmental Bacillus species are microorganisms typically found in food products or dietary supplements, but also applied on body surfaces or technological surfaces at home and in the industry. Since the contemporary definition of probiotics points to positive health effects, it is of highest importance to follow strict regulations and standards of product manufacturing, especially in the times of biohazard risks and rising public distrust of therapies. There is an urgent need to seek all possible routes for assuring the favouring conditions to build and support the organisms' microbiological barriers and enhance the immunity, that will serve also during the ongoing vaccination action. Probiotic LAB and environmental Bacillus species are microorganisms typically found in food products or dietary supplements, but also applied on body surface or technological surfaces in household and industry. Since the contemporary definition of probiotics points out the positive health effects, it is of highest importance to follow strict regulations and standards of product manufacturing, especially in the times of biohazard and rising public distrust of therapies.

PCR synthesis of double stranded DNA labeled with 5-bromouridine. A step towards finding a bromonucleoside for clinical trials.

Incorporation of 5-bromouridine (5BrdU) into DNA makes it sensitive to UV and ionizing radiation, which opens up a prospective route for the clinical usage of 5-bromouridine and other halonucleosides. In the present work the polymerase chain reaction (PCR) protocol, which enables a long DNA fragment (resembling DNA synthesized in the cell in the presence of halonucleosides) to be completely substituted with 5BrdU, was optimized. Using HPLC coupled to enzymatic digestion, it was demonstrated that the actual amounts of native nucleosides and 5BrdU correspond very well to those calculated from the sequence of PCR products. The synthesized DNA is photosensitive to photons of 300nm. HPLC analysis demonstrated that the photolysis of labeled PCR products leads to a significant decrease in the 5BrdU signal and the simultaneous occurrence of a uridine peak. Agarose and polyacrylamide gel electrophoresis suggest that single strand breaks and cross-links are formed as a result of UV irradiation. The PCR protocol described in the current paper may be employed for labeling DNA not only with BrdU but also with other halonucleosides.

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence.

BACKGROUND: Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. RESULTS: An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb). CONCLUSIONS: The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy.