Optimization of RNA storage in a biobank, as well as methods for manual and automated isolation of RNA from whole blood and leukocyte fraction

Aim. To optimize the technique for the isolation and storage of ribonucleic acid (RNA) from whole blood and leukocyte fraction.Materials and methods. Comparison of isolation quality was carried out for RNA samples obtained from 228 leukocyte samples and 198 whole blood samples. Isolation was performed from fresh and frozen samples using ExtractRNA™ reagent and a MagNA Pure Compact automated system. Various methods of removing erythrocytes (centrifugation and treatment with hemolytic agents from two manufacturers) were tested, as well as freezing with and without preservatives for subsequent RNA isolation.Results. Twenty-one combinations of conditions were tested. The highest quality RNA was isolated by manual extraction using the ExtractRNA™ reagent from a fresh leukocyte fraction, purified by the Amplisens hemolytic agent (successful extraction — 94%, median RIN=8,4); frozen in IntactRNA™, purified by leukocyte fraction centrifugation (successful extraction — 100%, median RIN=8); frozen in ExtractRNA™, purified by leukocyte fraction centrifugation (successful extraction — 100%, median RIN=9,3).Conclusion. RNA can be isolated from frozen blood fractions, which is not inferior in quality to that isolated from fresh samples. Thus, it is not necessary to isolate RNA immediately after the receipt of biological material.

CATTLE NECROBACTERIOSIS IN THE LIVESTOCK FACILITIES OF THE ALMATY REGION

Necrobacteriosis affects many species of animals. The most susceptible and sensitive to Fusobacterium necrophorum are reindeer, cattle and small cattle, pigs, and rabbits. A constant carriership of the causative agent of necrobacteriosis in the rumen and intestines of ruminants has been established, causative agent is found in food particles during chewing, as well as in feces. The causative agent of necrobacteriosis is widespread in the environment (livestock buildings, walking yards, manure, soil, pastures, stagnant reservoirs, etc.). Infestation of animals occurs when the pathogen enters the injured areas of the skin or mucous membranes of animals. Disturbed blood circulation, cracks and peeling of the horn happen as a result of long-term keeping of animals in damp premices, grazing them in damp, swampy areas, and also maceration of the limb tissues. Four cultures of the causative agent of cattle necrobacteriosis Fusobacterium necrophorum were isolated from sick animals with symptoms of lameness, their biological properties were studied. The pathogenicity of the isolated cultures was studied in laboratory animals. The work was conducted in laboratory and production conditions in "KazSRVI" LLP and at the dairy farm at "Arkabay" human settlement (village) of Talgar district of Almaty region, where stall keeping of animals is practiced. Slices from the diseased hoof of cows were taken at the border of the diseased and healthy tissue. Samples of the selected biological material were plated on Kitt-Tarozzi medium at the sampling site on the farm. The biological material taken from sick animals was studied within several hours after sampling in accordance with the guidelines for laboratory diagnosis of necrobacteriosis. Material for laboratory research (sections from the horny tissue of the hoof on the border with the healthy one) were taken fresh and inoculated on a nutrient medium for anaerobes. The results of cultivation of the necrobacteriosis causative agent on liquid and solid nutrient media under anaerobic conditions are presented. To get rid of the accompanying microflora and obtain a pure culture of F. necrophorum, a bioassay was set on laboratory animals - rabbits. All isolated cultures were highly pathogenic for rabbits. On the 14-15th day after infection, the experimental rabbits died. A pure culture of F. necrophorum, not contaminated with extraneous microflora, was sown from the internal organs of rabbits. It was found that rabbits are the optimal biomodel for purification of the F. necrophorum culture. The biochemical properties of the isolated cultures have been studied. It was found that epizootic cultures of the causative agent of necrobacteriosis emitted hydrogen sulfide and had hemolytic properties. In experiments in vitro and in vivo, it was found that the isolated cultures of F. necrophorum showed hyaluronidase activity. Cultures of F. necrophorum had a high catalase activity, they split hydrogen peroxide with the formation of oxygen (gas bubbles). When studying biochemical properties, it was found that F. necrophorum releases ammonia within 2-3 hours. Four cultures of F. necrophorum isolated from biological material from cattle were identical in biological properties.

Identification of benzydamine and its metabolit in the presence of certain anti-inflammatory non-steroidal drugs

The aim of the work. Currently, a large number of cases of non-medical use of benzydamine hydrochloride have been described. The identification of benzydamine and its metabolite, benzydamine N-oxide, in the presence of some non-steroidal anti-inflammatory drugs, has been insufficiently studied. Therefore, the development of a method for its identification in biological material is an urgent task. Materials and methods. The subjects of the study were benzydamine hydrochloride and its metabolite, as well as some non-steroidal anti-inflammatory drugs, which are its analogues in terms of pharmacological action. The studies were carried out by methods of thin layer chromatography and high-performance liquid chromatography. Results. At the first stage a screening method for benzydamine identification was studied using the extraction in acidic and basic conditions. It was shown that benzydamine can be isolated in both medias with subsequent development with a solution of iodoplatinate and Dragendorff's reagent according to Munier or with Mandelin reagent respectively. The mobile phase was selected and respective hRf for the target molecule were defined. After a preliminary identification of benzydamine a reference method for the final confirmation of the drug that had led to poisoning was proposed. A robust, specific and accurate reversed phase HPLC method was chosen. It was shown that benzydamine exists in biological material mainly in a form of metabolite – benzydamine N-oxide. The selected method was able to separate and determine key analytes in biological samples after a preparative isolation by TLC method. The comparison with UV spectra of the reference standard of benzydamine hydrochloride was proposed to avoid false positive conclusion of drug identification. Conclusions. Proposed methodology can be applied for routine identification of benzydamine poisoning in toxicological laboratories

Application of Accu-Chek® Test Strips as inexpensive microelectrodes for BIA research

In the midst of an investigation to perform bioelectrical impedance analysis (BIA), we were faced with the need to search for optimally adjusted electrodes to perform reading in small biological samples. The best option to carry out these readings is the well-known gold microelectrodes; however, these are very expensive for our research purposes. For this reason, we found an alternative using Accu-Chek Performa test strips as reading microelectrodes due to their low cost and ease of disposal. This article contains an in-depth detail of the components of the Accu-Chek Performa test strip and the process that was used so that they could be suitable for taking measurements on biological material. In addition, a measurement scheme is shown in conclusion to the operation of the test strip as a microelectrode and the possible problems to consider if it is to be used for future research.

L’héritage d’Henrietta Lacks

Many developments in biology and biotechnology have relied on the HeLa cell line, originally derived in 1951 from a Black cancer patient without her knowledge. This origin became generally known at the turn of the century, and the patient’s descendants have sought and obtained some recognition and some control but little compensation. They have now retained two famous attorneys to sue a biotech firm for very extensive damages, with more legal action planned against other companies. This may have important repercussions for the biotech industry, and raises complex issues regarding ownership of biological material and compensation to patients from whom these materials have been obtained.

Biosorption of Zn(II) from Seawater Solution by the Microalgal Biomass of Tetraselmis marina AC16-MESO

Biosorption refers to a physicochemical process where substances are removed from the solution by a biological material (live or dead) via adsorption processes governed by mechanisms such as surface complexation, ion exchange, and precipitation. This study aimed to evaluate the adsorption of Zn2+ in seawater using the microalgal biomass of Tetraselmis marina AC16-MESO “in vivo” and “not alive” at different concentrations of Zn2+ (0, 5, 10, and 20 mg L−1) at 72 h. Analysis was carried out by using the Langmuir isotherms and by evaluating the autofluorescence from microalgae. The maximum adsorption of Zn2+ by the Langmuir model using the Qmax parameter in the living microalgal biomass (Qmax = 0.03051 mg g−1) was more significant than the non-living microalgal biomass of T. marine AC16-MESO (Qmax = 0.02297 mg g−1). Furthermore, a decrease in fluorescence was detected in cells from T. marina AC16-MESO, in the following order: Zn2+ (0 < 20 < 5 < 10) mg L−1. Zn2+ was adsorbed quickly by living cells from T. marine AC16-MESO compared to the non-living microalgal biomass, with a decrease in photosystem II activities from 0 to 20 mg L−1 Zn2+ in living cells.