Biochemical Characterization of Mycobacterium tuberculosis DNA Repair Enzymes – Nfo, XthA and Nei2

Introduction: Tuberculosis (TB) is a human disease caused by Mycobacterium tuberculosis (Mtb). Treatment of TB requires long-term courses of multi-drug therapies to eliminate subpopulations of bacteria, which sometimes persist against antibiotics. Therefore, understanding of the mechanism of Mtb antibiotic-resistance is extremely important. During infection, Mtb overcomes a variety of body defense mechanisms, including treatment with the reactive species of oxygen and nitrogen. The bases in DNA molecule are susceptible to the damages caused by reactive forms of intermediate compounds of oxygen and nitrogen. Most of this damage is repaired by the base excision repair (BER) pathway. In this study, we aimed to biochemically characterize three Mtb DNA repair enzymes of BER pathway. Methods: XthA, nfo, and nei genes were identified in mycobacteria by homology search of genomic sequences available in the GenBank database. We used standard methods of genetic engineering  to clone and sequence Mtb genes, which coded Nfo, XthA and Nei2 repair enzymes. The protein products of Mtb genes were expressed and purified in Escherichia coli using affinity tags. The enzymatic activity of purified Nfo, XthA, and Nei2 proteins were measured using radioactively labeled DNA substrates containing various modified residues. Results: The genes end (Rv0670), xthA (Rv0427c), and nei (Rv3297) were PCR amplified using genomic DNA of Mtb H37Rv with primers that contain specific restriction sites. The amplified products were inserted into pET28c(+) expression vector in such a way that the recombinant proteins contain C-terminal histidine tags. The plasmid constructs were verified by sequencing and then transformed into the Escherichia coli BL21 (DE3) strain. Purification of recombinant proteins was performed using Ni2+ ions immobilized affinity column, coupled with the fast performance liquid chromatography machine AKTA. Identification of the isolated proteins was performed by protein mass spectrometry by ion trap tandem MS/MS on nLC-ESI-Ion-Trap platform. Biochemical characterization of DNA repair protein-catalyzed activity was carried out by measuring apurinic/apyrimidinic endonuclease, DNA glycosylase, exonuclease, and 3'-repair diesterase functions. In addition, effect of the opposite base and the influence of metal ion cofactors were measured. Conclusion: Results of the ongoing study will help us define the role of DNA repair enzymes in the emergence of mutations in the mycobacterial genome and, possibly, the origins of multi-drug resistance in mycobacteria

Biochemical Characterization of Mycobacterium tuberculosis DNA Repair Enzymes – Nfo, XthA and Nei2

Introduction: Tuberculosis (TB) is a human disease caused by Mycobacterium tuberculosis (Mtb). Treatment of TB requires long-term courses of multi-drug therapies to eliminate subpopulations of bacteria, which sometimes persist against antibiotics. Therefore, understanding of the mechanism of Mtb antibiotic-resistance is extremely important. During infection, Mtb overcomes a variety of body defense mechanisms, including treatment with the reactive species of oxygen and nitrogen. The bases in DNA molecule are susceptible to the damages caused by reactive forms of intermediate compounds of oxygen and nitrogen. Most of this damage is repaired by the base excision repair (BER) pathway. In this study, we aimed to biochemically characterize three Mtb DNA repair enzymes of BER pathway.Methods: XthA, nfo, and nei genes were identified in mycobacteria by homology search of genomic sequences available in the GenBank database. We used standard methods of genetic engineering  to clone and sequence Mtb genes, which coded Nfo, XthA and Nei2 repair enzymes. The protein products of Mtb genes were expressed and purified in Escherichia coli using affinity tags. The enzymatic activity of purified Nfo, XthA, and Nei2 proteins were measured using radioactively labeled DNA substrates containing various modified residues. Results: The genes end (Rv0670), xthA (Rv0427c), and nei (Rv3297) were PCR amplified using genomic DNA of Mtb H37Rv with primers that contain specific restriction sites. The amplified products were inserted into pET28c(+) expression vector in such a way that the recombinant proteins contain C-terminal histidine tags. The plasmid constructs were verified by sequencing and then transformed into the Escherichia coli BL21 (DE3) strain. Purification of recombinant proteins was performed using Ni2+ ions immobilized affinity column, coupled with the fast performance liquid chromatography machine AKTA. Identification of the isolated proteins was performed by protein mass spectrometry by ion trap tandem MS/MS on nLC-ESI-Ion-Trap platform. Biochemical characterization of DNA repair protein-catalyzed activity was carried out by measuring apurinic/apyrimidinic endonuclease, DNA glycosylase, exonuclease, and 3'-repair diesterase functions. In addition, effect of the opposite base and the influence of metal ion cofactors were measured. Conclusion: Results of the ongoing study will help us define the role of DNA repair enzymes in the emergence of mutations in the mycobacterial genome and, possibly, the origins of multi-drug resistance in mycobacteria.

Molecular phylogenetic analysis of Tulipa (Liliaceae) from Aksu-Zhabagly Nature Reserve

Barcodes are conserved sequences of genomic, plastid and mitochondrial DNA that can be utilized to uniquely identify an unidentified specimen to its species when conventional identification methods are inapplicable. Among prokaryotic and eukaryotic species, nuclear ribosomal internal transcribed spacer (ITS) sections are one of the most often utilized DNA markers in DNA barcoding and phylogenetic research. In addition to the ribosomal genes, the plastid genes are the most suitable for identifying plant species. The Aksu-Zhabagly Nature Reserve is the oldest nature reserve in Central Asia and is home to 1,312 vascular plant species, 44 of which are categorized as threatened or endangered in Kazakhstan's red data book. In this study, a collection of specimens of uncommon tulip species was compiled, along with their morphological identification and DNA barcoding. The ITS region and parts of the matK and ycf1b genes of tulip plastid DNA were sequenced. The evolutionary link between species of tulips was investigated. Phylogenetic study predicted two Tulipa subclades. Tulipa species have substantially preserved MatK genes. Tulips' ycf1b gene has evolved more slowly than other Liliaceae family members. Nuclear and plastid DNA sequences investigated Tulipa species evolutionary relationships. The findings about the ITS region of nuclear DNA were more definite. Overall, our work shows that genetic data will be important in determining species concepts in this genus, however, even with a molecular perspective pulling apart closely related taxa can be extremely challenging

Introduction on Using the FastPCR Software and the Related Java Web Tools for PCR and Oligonucleotide Assembly and Analysis

This chapter introduces the FastPCR software as an integrated tool environment for PCR primer and probe design, which predicts properties of oligonucleotides based on experimental studies of the PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations. These include the standard PCR as well as the multiplex, long-distance, inverse, real-time, group-specific, unique, overlap extension PCR for multi-fragments assembling cloning and loop-mediated isothermal amplification (LAMP). It also contains a built-in program to design oligonucleotide sets both for long sequence assembly by ligase chain reaction and for design of amplicons that tile across a region(s) of interest. The software calculates the melting temperature for the standard and degenerate oligonucleotides including locked nucleic acid (LNA) and other modifications. It also provides analyses for a set of primers with the prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, linguistic complexity as well as a primer dilution and resuspension calculator. The program consists of various bioinformatical tools for analysis of sequences with the GC or AT skew, CG% and GA% content, and the purine–pyrimidine skew. It also analyzes the linguistic sequence complexity and performs generation of random DNA sequence as well as restriction endonucleases analysis. The program allows to find or create restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It performs efficient and complete detection of various repeat types with visual display. The FastPCR software allows the sequence file batch processing that is essential for automation. The program is available for download at http://primerdigital.com/fastpcr.html, and its online version is located at http://primerdigital.com/tools/pcr.html.Peer reviewe

Дослідження впливу підводного електричного вибуху на вибірковість руйнування кварцової сировини

The work is devoted to the study of the effect of an electric explosion on the selectivity of the destruction of quartz raw materials. The object of the study is quartz ore of the Nadyrbay deposit of the Republic of Kazakhstan. An electrohydroimpulse plant for crushing and grinding quartz raw materials has been developed and assembled. Using the electrohydroimpulse method, the granulometric composition of quartz can be adjusted. This makes it possible to adjust the magnitude of the voltage in the discharge channel and time. In this technology, quartz ore processing was carried out with an increase in the discharge voltage of the storage from 14 kV to 25 kV, the length of the interelectrode distance from 8 to 12, the capacitance of the capacitor 0.5 µF, 0.75 µF and the processing time of 5 min. Using the electrohydroimpulse method, quartz ore particles with an initial fraction of 5 mm, 10 mm and 1 mm were crushed to 0.8. The results of the grinding of quartz raw materials with the influence of an underwater electric explosion in a liquid medium allowed us to determine the degree of grinding of the material. The obtained results can be used in the course of studying the characteristics of crushing and grinding of ores. In the food industry, quartz sand within 0.25–0.5 millimeters can be used as a filler to create filters for water purification, as well as products from oil, industrial effluents, etc. Particles ranging in size from 0.5 to 1 millimeter can be used for rough processing of metal, stone and glass. The structural and quantitative analysis of powdered quartzite samples was made using a scanning electron microscope and the stoichiometry of the elements was calculatedРобота присвячена вивченню впливу електричного вибуху на вибірковість руйнування кварцової сировини. Об'єктом дослідження є кварцова руда родовища Надирбай Республіки Казахстан. Для дроблення та подрібнення кварцової сировини розроблена та зібрана електрогідроімпульсна установка. За допомогою електрогідроімпульсного методу можна регулювати гранулометричний склад кварцу. Це дозволяє регулювати величину напруги в розрядному каналі та час. За даною технологією проводилася переробка кварцової руди зі збільшенням напруги розряду накопичувача з 14 кВ до 25 кВ, довжини міжелектродної відстані з 8 до 12, ємністю конденсатора 0,5 мкФ, 0,75 мкФ і часом обробки 5 хв. З використанням електрогідроімпульсного методу частинки кварцової руди з початковою фракцією 5 мм, 10 мм та 1 мм подрібнювали до 0,8. Результати подрібнення кварцової сировини з впливом підводного електричного вибуху в рідкому середовищі дозволили визначити ступінь подрібнення матеріалу. Отримані результати можуть бути використані при вивченні особливостей дроблення і подрібнення руд. У харчовій промисловості кварцовий пісок розміром 0,25–0,5 міліметра може використовуватися в якості наповнювача для створення фільтрів очищення води, а також продуктів з нафти, промислових стоків тощо. Частинки розміром від 0,5 до 1 міліметра можуть бути використані для грубої обробки металу, каменю та скла. За допомогою скануючого електронного мікроскопа було проведено структурно-кількісний аналіз зразків порошкоподібного кварциту та розраховано стехіометрію елементі

Insight into DNA substrate specificity of PARP1-catalysed DNA poly(ADP-ribosyl)ation

DNA-dependent poly(ADP-ribose) polymerases (PARPs) PARP1, PARP2 and PARP3 act as DNA break sensors signalling DNA damage. Upon detecting DNA damage, these PARPs use nicotine adenine dinucleotide as a substrate to synthesise a monomer or polymer of ADP-ribose (MAR or PAR, respectively) covalently attached to the acceptor residue of target proteins. Recently, it was demonstrated that PARP1–3 proteins can directly ADP-ribosylate DNA breaks by attaching MAR and PAR moieties to terminal phosphates. Nevertheless, little is still known about the mechanisms governing substrate recognition and specificity of PARP1, which accounts for most of cellular PARylation activity. Here, we characterised PARP1-mediated DNA PARylation of DNA duplexes containing various types of breaks at different positions. The 3′-terminal phosphate residue at double-strand DNA break ends served as a major acceptor site for PARP1-catalysed PARylation depending on the orientation and distance between DNA strand breaks in a single DNA molecule. A preference for ADP-ribosylation of DNA molecules containing 3′-terminal phosphate over PARP1 auto-ADP-ribosylation was observed, and a model of DNA modification by PARP1 was proposed. Similar results were obtained with purified recombinant PARP1 and HeLa cell-free extracts. Thus, the biological effects of PARP-mediated ADP-ribosylation may strongly depend on the configuration of complex DNA strand breaks

Дослідження впливу підводного електричного вибуху на вибірковість руйнування кварцової сировини

The work is devoted to the study of the effect of an electric explosion on the selectivity of the destruction of quartz raw materials. The object of the study is quartz ore of the Nadyrbay deposit of the Republic of Kazakhstan. An electrohydroimpulse plant for crushing and grinding quartz raw materials has been developed and assembled. Using the electrohydroimpulse method, the granulometric composition of quartz can be adjusted. This makes it possible to adjust the magnitude of the voltage in the discharge channel and time. In this technology, quartz ore processing was carried out with an increase in the discharge voltage of the storage from 14 kV to 25 kV, the length of the interelectrode distance from 8 to 12, the capacitance of the capacitor 0.5 µF, 0.75 µF and the processing time of 5 min. Using the electrohydroimpulse method, quartz ore particles with an initial fraction of 5 mm, 10 mm and 1 mm were crushed to 0.8. The results of the grinding of quartz raw materials with the influence of an underwater electric explosion in a liquid medium allowed us to determine the degree of grinding of the material. The obtained results can be used in the course of studying the characteristics of crushing and grinding of ores. In the food industry, quartz sand within 0.25–0.5 millimeters can be used as a filler to create filters for water purification, as well as products from oil, industrial effluents, etc. Particles ranging in size from 0.5 to 1 millimeter can be used for rough processing of metal, stone and glass. The structural and quantitative analysis of powdered quartzite samples was made using a scanning electron microscope and the stoichiometry of the elements was calculatedРобота присвячена вивченню впливу електричного вибуху на вибірковість руйнування кварцової сировини. Об'єктом дослідження є кварцова руда родовища Надирбай Республіки Казахстан. Для дроблення та подрібнення кварцової сировини розроблена та зібрана електрогідроімпульсна установка. За допомогою електрогідроімпульсного методу можна регулювати гранулометричний склад кварцу. Це дозволяє регулювати величину напруги в розрядному каналі та час. За даною технологією проводилася переробка кварцової руди зі збільшенням напруги розряду накопичувача з 14 кВ до 25 кВ, довжини міжелектродної відстані з 8 до 12, ємністю конденсатора 0,5 мкФ, 0,75 мкФ і часом обробки 5 хв. З використанням електрогідроімпульсного методу частинки кварцової руди з початковою фракцією 5 мм, 10 мм та 1 мм подрібнювали до 0,8. Результати подрібнення кварцової сировини з впливом підводного електричного вибуху в рідкому середовищі дозволили визначити ступінь подрібнення матеріалу. Отримані результати можуть бути використані при вивченні особливостей дроблення і подрібнення руд. У харчовій промисловості кварцовий пісок розміром 0,25–0,5 міліметра може використовуватися в якості наповнювача для створення фільтрів очищення води, а також продуктів з нафти, промислових стоків тощо. Частинки розміром від 0,5 до 1 міліметра можуть бути використані для грубої обробки металу, каменю та скла. За допомогою скануючого електронного мікроскопа було проведено структурно-кількісний аналіз зразків порошкоподібного кварциту та розраховано стехіометрію елементі

Визначення впливу імпульсних електричних розрядів на переробку побутового скла

The work was devoted to the study of the recycling of household glass waste by the electric pulse method. Cullet and glass containers were considered as the object of research. Cullet and used glass containers are considered suitable for recycling among solid household waste. The treatment of solid household waste was carried out with the formation of electrical discharges in an inhomogeneous environment (glass fragments and glass products in an aqueous environment). The experiments were carried out at different values of pulse discharge voltage (from 12 kV to 37 kV), capacitor bank capacity (from 0.4 to 1.2 µF), number of pulse discharges (from 250 to 1,000) and frequency (from 0.3 Hz to 2 Hz). The dependence of the output of the finished product on the parameters of electric pulse discharges has been revealed from the research results.  According to the main results of the research work, it was found that as the parameters of electric pulse discharges increase, the yield of the finished product increases. The data made it possible to assign effective parameters for processing cullet and glass products using the electric pulse method. The granulometric composition of the powder material obtained by this method has been determined. A product with a diameter of the largest fraction of 5-8 mm and a diameter of a small fraction of 0.4 mm and 0.7 mm was obtained. The results of the experiment can be used in research on saving natural resources, energy conservation and solving environmental problems of solid waste recycling. Powdered glass is used as a thermal insulator and decorative material in construction. In addition, crushed glass is used in the production of concrete as an additional raw material.Робота присвячена вивченню переробки побутових скляних відходів електроімпульсним методом. У якості об'єкта дослідження розглядався склобій і скляна тара. Склобій та використана скляна тара вважаються придатними для вторинної переробки серед твердих побутових відходів. Обробка твердих побутових відходів проводилася з утворенням електричних розрядів у неоднорідному середовищі (уламки скла та скляні вироби у водному середовищі). Експерименти проводилися за різних значень напруги імпульсного розряду (від 12 кВ до 37 кВ), ємності конденсаторної батареї (від 0,4 до 1,2 мкФ), кількості імпульсних розрядів (від 250 до 1000) та частоти (від 0,3 Гц до 2 Гц). За результатами досліджень виявлено залежність виходу готового продукту від параметрів електричних імпульсних розрядів. Згідно з основними результатами дослідницької роботи встановлено, що зі збільшенням параметрів електроімпульсних розрядів збільшується вихід готового продукту. Отримані дані дозволили задати ефективні параметри обробки склобою та скляних виробів електроімпульсним методом. Визначено гранулометричний склад порошкового матеріалу, одержаного даним методом. Отримано продукт з діаметром найбільшої фракції 5-8 мм та діаметром малої фракції 0,4 мм і 0,7 мм. Результати експерименту можуть бути використані в дослідженнях з економії природних ресурсів, енергозбереження та вирішення екологічних проблем переробки твердих побутових відходів. Скляний порошок використовується у якості теплоізоляційного та декоративного матеріалу у будівництві. Крім того, склобій використовується при виробництві бетону в якості додаткової сировин