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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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).</p> <p>Conclusions</p> <p>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.</p

Subjective evaluation of health behaviour of patients with chronic kidney disease

Abstract The aim of the study: The aim of the study was to evaluate of health behaviour in each of several of patients with chronic kidney disease. Material and methods: In total 116 people diagnosed with chronic kidney disease were tested. The study was conducted from February to April of 2017 in the Nephrology Department of the Regional Clinical Hospital in Zielona Góra, Poland, the Nephrology and Arterial Hypertension Department of the Regional Specialist Hospital in Lublin, Poland as well as in the Private Healthcare Centre “Diaverum” in Lublin, Poland. In the study the Inventory of the Health Behaviour (Inwentarz Zachowań Zdrowotnych, IZZ) was used. IZZ consists of four health behaviour categories: positive mental attitude, preventative actions, healthy eating habits and health practices. The patients who were research subjects determined the frequency of particular factors within the above mentioned categories. The total numerical values determine the overall health behaviour index, whose value lies between 24 and 120 points. The higher the score, the higher the declared frequency of health behaviour. Results: Patients with chronic kidney disease described their health behaviour as average. The highest rated categories were health practices and preventative actions as opposed to healthy eating habits which were rated the lowest. The educational background and place of residence do not influence the health behaviour  significantly.  The inhabitants of the Lubelskie region (with the capital city of Lublin) rated their health behaviour higher than the inhabitants of the Lubuskie region (with the capital city of Zielona Góra) in all the categories. However, this discrepancy turned out to be statistically insignificant. The age of the patients had a significant impact on the responses of the research subjects. The patients between 60 and 69 had the highest index value, and these under 49 the lowest. Conclusions: The study concludes that women rated their health behaviour higher than men, within the analysed categories except for healthy eating habits, which men rated higher than women. The place of residence did not influence the ratings of the research subjects. People living in urban areas as well as in rural areas rated their health behaviour on a similar level. In the researched group the age is the factor determining the health behaviour – the oldest patients rated their health behaviour the highest. The kind of treatment influences the health behaviour ratings. Patients who are treated with haemodialysis rated their health behaviour higher than those undergoing conservative treatment. Keywords: Health behaviour, chronic kidney diseas

Fundamental Mechanisms of DNA Radiosensitization: Damage Induced by Low-Energy Electrons in Brominated Oligonucleotide Trimers

The replacement of nucleobases with brominated analogs enhances DNA radiosensitivity. We examine the chemistry of low-energy electrons (LEEs) in this sensitization process by experiments with thin films of the oligonucleotide trimers TBrXT, where BrX = 5-BrU (5-bromouracil), 5-BrC (5-bromocytosine), 8-BrA (8-bromoadenine), or 8-BrG (8-bromoguanine). The products induced from irradiation of thin (∼ 2.5 nm) oligonucleotide films, with 10 eV electrons, under ultrahigh vacuum (UHV) are analyzed by HPLC-UV. The number of damaged brominated trimers ranges from about 12 to 15 × 10^–3 molecules per incident electron, whereas under the identical conditions, these numbers drop to 4–7 × 10^–3 for the same, but nonbrominated oligonucleotides. The results of HPLC analysis show that the main degradation pathway of trinucleotides containing brominated bases involve debromination (i.e., loss of the bromine atom and its replacement with a hydrogen atom). The electron-induced sum of products upon bromination increases by factors of 2.1 for the pyrimidines and 3.2 for the purines. Thus, substitution of any native nucleobase with a brominated one in simple models of DNA increases LEE-induced damage to DNA and hence its radiosensitivity. Furthermore, besides the brominated pyrimidines that have already been tested in clinical trials, brominated purines not only appear to be promising sensitizers for radiotherapy, but could provide a higher degree of radiosensitization