Search

Base excision repair (BER) is aimed at repair of damaged bases, which are the largest group of DNA lesions. The main steps of BER are recognition and removal of the aberrant base, cutting of the DNA sugar-phosphate backbone, gap processing (including dNMP insertion), and DNA ligation. The precise function of BER depends on the regulation of each step by regulatory/accessory proteins, the most important of which is poly(ADP-ribose) (PAR) polymerase 1 (PARP1). PARP1 plays an important role in DNA repair, maintenance of genome integrity, and regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. There is no systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells. Whole-cell extracts and RNA preparations obtained from the parental HEK293T cell line and its derivative HEK293T/P1-KD cell line with reduced PARP1 expression (shPARP1-expressing cells, a PARP1 knockdown) were used to assess the levels of mRNAs coding for BER proteins: PARP1, PARP2, uracil DNA glycosylase (UNG2), AP endonuclease 1 (APE1), DNA polymerase β (POLβ), DNA ligase III (LIG3), and XRCC1. Catalytic activities of the enzymes were evaluated in parallel. No significant effect of the PARP1 content was observed for the mRNA levels of UNG2, APE1, POLβ, LIG3, and XRCC1. The amount of the PARP2 mRNA proved to be reduced two times in HEK293T/P1-KD cells. Activities of these enzymes in whole-cell extracts did not differ significantly between HEK293T and HEK293T/P1-KD cells. No significant change was observed in the efficiencies of the reactions catalyzed by UNG2, APE1, POLβ, and LIG3 in conditions of PAR synthesis. A DNA PARylation pattern did not dramatically change in a HEK293T/P1-KD cell extract with a reduced PARP1 content as compared with an extract of the parental HEK293T cell line.

The functioning of the eukaryotic cell genome is mediated by sophisticated protein-nucleic-acid complexes, whose minimal structural unit is the nucleosome. After the damage to genomic DNA, repair proteins need to gain access directly to the lesion; therefore, the initiation of the DNA damage response inevitably leads to local chromatin reorganisation. This review focuses on the possible involvement of PARP1, as well as proteins acting nucleosome compaction, linker histone H1 and non-histone chromatin protein HMGB1. The polymer of ADP-ribose is considered the main regulator during the development of the DNA damage response and in the course of assembly of the correct repair complex.

Reaction of (ADP-ribosyl)ation catalyzed by DNA-dependent proteins of the poly(ADP-ribose)polymerase (PARP) family, PARP1, PARP2, and PARP3, comprises the cellular response to DNA damage. These proteins are involved in the base excision repair (BER) process. Despite the extensive research, it remains unknown how PARPs are involved in the regulation of the BER process and how the roles are distributed between the DNA-dependent members of the PARP family. Here, we investigated the interaction of the PARP's family DNA-dependent proteins with nucleosome core particles containing DNA intermediates of the initial stages of BER. To do that, the nucleosomes containing damage in the vicinity of one of the DNA duplex blunt ends were reconstituted based on the Widom's Clone 603 DNA sequence. Dissociation constants of the PARP complexes with nucleosomes bearing DNA contained uracil (Native), apurine/apyrimidine site (AP site), or a single-nucleotide gap with 5'-dRp fragment (Gap) were determined. It was shown that the affinity of the proteins for the nucleosomes increased in the row: PARP3<

The regulation of repair processes including base excision repair (BER) in the presence of DNA damage is implemented by a cellular signal: poly(ADP-ribosyl)ation (PARylation), which is catalysed by PARP1 and PARP2. Despite ample studies, it is far from clear how BER is regulated by PARPs and how the roles are distributed between the PARPs. Here, we investigated the effects of PARP1, PARP2 and PARylation on activities of the main BER enzymes (APE1, DNA polymerase β [Polβ] and DNA ligase IIIα [LigIIIα]) in combination with BER scaffold protein XRCC1 in the nucleosomal context. We constructed nucleosome core particles with midward- or outward-oriented damage. It was concluded that in most cases, the presence of PARP1 leads to the suppression of the activities of APE1, Polβ and to a lesser extent LigIIIα. PARylation by PARP1 attenuated this effect to various degrees depending on the enzyme. PARP2 had an influence predominantly on the last stage of BER: DNA sealing. Nonetheless, PARylation by PARP2 led to Polβ inhibition and to significant stimulation of LigIIIα activities in a NAD + -dependent manner. On the basis of the obtained and literature data, we suggest a hypothetical model of the contribution of PARP1 and PARP2 to BER.

Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn 2+ ions and accessory replicative proteins RPA and PolDIP2. The DNA displacement activity of PrimPol was found to be more efficient than the RNA displacement activity and FEN1 processed the 5'-DNA flaps generated by PrimPol in vitro.

Aim: to estimate the quality and availability of medical care for patients with ulcerative colitis (UC) and Crohn's disease (CD), to assess the impact of the economic burden of these diseases on the healthcare budget of Russia and to systematize the main problems in the organization of medical care and drug supply for patients with inflammatory bowel diseases (IBD). Regional IBD databases (2016-2018), official statistical databases, costs of treatment and results of expert interviews with specialists in IBD were used in the study. The analyzed databases showed 104,668 patients with UC in Russia in 2018 (prevalence rate 71 per 100,000 people) and 66,647 patients with CD (prevalence rate of 45 per 100,000 people). The economic burden including agents for biologic therapy (ABT) for the UC was 39.54 billion rubles a year (495 rubles per capita), and CD - 32.98 billion rubles a year (378 rubles per capita). It requires an additional 9.87 billion rubles annually for UC and 9.20 billion rubles annually for CD patients to provide the complete supply with ABT. The annual burden of IBD is 72.52 billion rubles, which is comparable to the costs of other socially significant diseases, including malignant tumors. It shows the high social and economic value of IBD for the country. The main problems of medical care and drug supply for IBD patients are the mismatch of official statistical data and real IBD prevalence in Russia due to absence of comprehensive register and the insufficient supply with ABT due to limited funding. A federal center for IBD should be founded for better quality of registration, for the precise monitoring and for the active management of personal drug supply.

DNA is constantly attacked by different damaging agents; therefore, it requires frequent repair. On the one hand, the base excision repair (BER) system is responsible for the repair of the most frequent DNA lesions. On the other hand, the formation of poly(ADP-ribose) is one of the main DNA damage response reactions that is catalysed by members of the PARP family. PARP1, which belongs to the PARP family and performs approximately 90% of PAR synthesis in cells, could be considered a main regulator of the BER process. Most of the experimental data concerning BER investigation have been obtained using naked DNA. However, in the context of the eukaryotic cell, DNA is compacted in the nucleus, and the lowest compaction level is represented by the nucleosome. Thus, the organization of DNA into the nucleosome impacts the DNA-protein interactions that are involved in BER processes. Poly(ADP-ribosyl)ation (PARylation) is thought to regulate the initiation of the BER process at the chromatin level. In this review, we focus on the mechanisms involved in BER in the nucleosomal context and the potential effect of PARylation, which is catalysed by DNA-dependent PARP1, PARP2 and PARP3 proteins, on this process.

We report on the design, synthesis and molecular modeling study of conjugates of adenosine diphosphate&nbsp;(ADP) and morpholino nucleosides as potential selective inhibitors of poly(ADP-ribose)polymerases-1, 2 and 3. Sixteen dinucleoside pyrophosphates containing natural heterocyclic bases as well as 5-haloganeted pyrimidines, and mimicking a main substrate of these enzymes, nicotinamide adenine dinucleotide (NAD+)-molecule, have been synthesized in a high yield. Morpholino nucleosides have been tethered to the β-phosphate of ADP via a phosphoester or phosphoramide bond. Screening of the inhibiting properties of these derivatives on the autopoly(ADP-ribosyl)ation of PARP-1 and PARP-2 has shown that the effect depends upon the type of nucleobase as well as on the linkage between ADP and morpholino nucleoside. The 5-iodination of uracil and the introduction of the P-N bond in NAD+-mimetics have shown to increase inhibition properties. Structural modeling suggested that the P-N bond can stabilize the pyrophosphate group in active conformation due to the formation of an intramolecular hydrogen bond. The most active NAD+ analog against PARP-1 contained 5-iodouracil 2'-aminomethylmorpholino nucleoside with IC50 126 ± 6 μM, while in the case of PARP-2 it was adenine 2'-aminomethylmorpholino nucleoside (IC50 63 ± 10 μM). In silico analysis revealed that thymine and uracil-based NAD+ analogs were recognized as the NAD+-analog that targets the nicotinamide binding site. On the contrary, the adenine 2'-aminomethylmorpholino nucleoside-based NAD+ analogs were predicted to identify as PAR-analogs that target the acceptor binding site of PARP-2, representing a novel molecular mechanism for selective PARP inhibition. This discovery opens a new avenue for the rational design of PARP-1/2 specific inhibitors., Competing Interests: The authors declare no conflict of interest.

DNA repair capacity in cells of naked mole rat (Hgl), a species known for its longevity and resistance to cancer, is still poorly characterized. Here, using the whole-cell extracts (WCEs) of Hgl, mouse and human cells, we studied the interrelation between DNA synthesis on the substrates of base excision repair and the activity of poly(ADP-ribose) polymerases (PARPs) responsible for the transfer of the ADP-ribose moieties onto different targets. The level of PAR synthesis was more than ten-fold higher in human WCE as compared to rodent WCEs, while the efficiency of DNA synthesis was comparable. Under conditions of PAR synthesis, the efficiency of DNA synthesis was only slightly enhanced in all extracts and in mouse WCEs unusual products of the primer elongation were detected. The results obtained with WCEs, recombinant proteins and recently found ability of PARPs to attach the ADP-ribose moieties to DNA allowed us to attribute these products to primer mono(ADP-ribosyl)ation (MARylation) at the 5'-terminal phosphate by PARP3 during the DNA synthesis. PARP1/PARP2 can then transfer the ADP-ribose moieties onto initial ADP-ribose. Our results suggest that MARylation/PARylation of DNA in the extracts depends on the ratios between PARPs and can be controlled by DNA-binding proteins.

The article is published based on the results of the Russian Consensus on the diagnosis and treatment of primary sclerosing cholangitis (PSC), discussed at the 44th annual Scientific Session of the CNIIG "Personalized Medicine in the Era of Standards" (March 1, 2018). The aim of the review is to highlight the current issues of classification of diagnosis and treatment of patients with PSC, which causes the greatest interest of specialists. The urgency of the problem is determined by the multivariate nature of the clinical manifestations, by often asymptomatic flow, severe prognosis, complexity of diagnosis and insufficient study of PSC, the natural course of which in some cases can be considered as a function with many variables in terms of the nature and speed of progression with numerous possible clinical outcomes. In addition to progression to portal hypertension, cirrhosis and its complications, PSC can be accompanied by clinical manifestations of obstructive jaundice, bacterial cholangitis, cholangiocarcinoma and colorectal cancer. Magnetic resonance cholangiography is the main method of radial diagnostics of PSC, which allows to obtain an image of bile ducts in an un-invasive way. The use of liver biopsy is best justified when there is a suspicion of small-diameter PSC, autoimmune cross-syndrome PSC-AIG, IgG4-sclerosing cholangitis. Currently, a drug registered to treat primary sclerosing cholangitis which can significantly change the course and prognosis of the disease does not exist. There is no unified view on the effectiveness and usefulness of ursodeoxycholic acid and its dosage in PSC. Early diagnosis and determination of the phenotype of PSC is of clinical importance. It allows to determine the tactics of treatment, detection and prevention of complications.

Background: Crohn's disease is associated with gut dysbiosis. Independent studies have shown an increase in the abundance of certain bacterial species, particularly Escherichia coli with the adherent-invasive pathotype, in the gut. The role of these species in this disease needs to be elucidated., Methods: We performed a metagenomic study investigating the gut microbiota of patients with Crohn's disease. A metagenomic reconstruction of the consensus genome content of the species was used to assess the genetic variability., Results: The abnormal shifts in the microbial community structures in Crohn's disease were heterogeneous among the patients. The metagenomic data suggested the existence of multiple E. coli strains within individual patients. We discovered that the genetic diversity of the species was high and that only a few samples manifested similarity to the adherent-invasive varieties. The other species demonstrated genetic diversity comparable to that observed in the healthy subjects. Our results were supported by a comparison of the sequenced genomes of isolates from the same microbiota samples and a meta-analysis of published gut metagenomes., Conclusions: The genomic diversity of Crohn's disease-associated E. coli within and among the patients paves the way towards an understanding of the microbial mechanisms underlying the onset and progression of the Crohn's disease and the development of new strategies for the prevention and treatment of this disease.

Introduction: The objective of the study was to evaluate the prevalence of Clostridium difficile-associated diarrhoea (CDAD) among hospitalised patients with antibiotic-associated diarrhoea (AAD) in general and by specific types of medical care and hospital units., Methods: A prospective, cross-sectional, non-interventional, multicentre study. The main inclusion criteria were: patient age ≥ 18 years, hospital stay of at least 48 h, current antibiotic therapy or antibiotic therapy within the previous 30 days, loose stools (Bristol stool types 5-7 and stool frequency ≥ 3 within ≤ 24 consecutive hours or exceeding normal for the patient) and signed informed consent form. The stool sample was taken to the local (study site) microbiology laboratory for detection of glutamate dehydrogenase (GDH) and toxins A/B using enzyme immunoassay (EIA) stool test., Results: From April 2016 to April 2017, a total of 1245 patients from 12 large hospitals were enrolled in the study. Data on 81 patients were excluded from the analysis for different reasons. Data on 1164 patients (45.2% males and 54.8% females) with a mean age of 54.9 years (range 18-95 years) were analysed. Length of hospitalisation was 2-188 days (median, 8 days). The EIA stool test showed CDAD-positive results in 21.7% (253/1164) patients. The patients were from surgery units (546/1164), internal medicine units (510/1164) and intensive care units (108/1164). The prevalence of CDAD among patients from surgery, internal medicine and intensive care units was 26.2, 17.8 and 17.6%, respectively. Oncology, gastroenterology, septic surgery, oncohaematology and general medical hospital units accounted for more than 75% of all patients included; the prevalence of CDAD by those hospital units was 11.3, 15.0, 39.2, 17.6, and 27.2%, respectively. The proportion of GDH-positive and toxin A/B-negative patients by the rapid stool test result was 16.8% (196/1164). The prevalence of CDAD varied widely between the hospitals (from 0 to 44.3%)., Conclusions: The prevalence of CDAD among hospitalised patients with AAD in this study was 21.7% (95% confidence interval: 14.8 and 28.7%). The percentage of CDAD varied widely between hospitals and by specific types of medical care and hospital units.

Many human DNA polymerases bypass DNA damage during translesion synthesis (TLS). Human primase and polymerase, PrimPol, assists fork progression by repriming DNA synthesis downstream of the lesion using its DNA primase activity. We tested the properties of PrimPol as a TLS polymerase in the presence of different metal ions in vitro. We demonstrate that in the presence of Mg 2+ ions PrimPol carries out efficient and relatively accurate synthesis past 8-oxoguanine and 5-formyluracil. It also bypasses an abasic site and O 6 -methylguanine, but is blocked by thymine glycol and 1,N 6 -ethenoadenine. Substitution of Mg 2+ with Mn 2+ stimulates the TLS activity of PrimPol and allows for efficient, but error-prone, synthesis on DNA templates containing all tested DNA lesions, including thymine glycol and 1,N 6 -ethenoadenine. The TLS activity of PrimPol has possible relevant functions in vivo; e.g., the combined primase and DNA polymerase activities of PrimPol might facilitate replication of DNA with clustered damage., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Poly(ADP-ribosyl)ation, which is catalyzed by PARP family proteins, is one of the main reactions in the cell response to genomic DNA damage. Massive impact of DNA-damaging agents (such as oxidative stress and ionizing radiation) causes numerous breaks in DNA. In this case, the development of a fast cell response, which allows the genomic DNA integrity to be retained, may be more important than the repair by more accurate but long-term restoration of the DNA structure. This is the first study to show the possibility of eliminating DNA breaks through their PARP3-dependent mono(ADP-ribosyl)ation followed by ligation and repair of the formed ribo-AP sites by the base excision repair (BER) enzyme complex. Taken together, the results of the studies on ADP-ribosylation of DNA and the data obtained in this study suggest that PARP3 may be a component of the DNA break repair system involving the BER enzyme complex.

The Russian consensus on exo- and endocrine pancreatic insufficiency after surgical treatment was prepared on the initiative of the Russian "Pancreatic Club" on the Delphi method. His goal was to clarify and consolidate the opinions of specialists on the most relevant issues of diagnosis and treatment of exo- and endocrine insufficiency after surgical interventions on the pancreas. An interdisciplinary approach is provided by the participation of leading gastroenterologists and surgeons.

Circular RNA are a family of covalently closed circular RNA molecules, formed from pre-mRNA of coding genes by means of splicing (canonical and alternative noncanonical splicing). Maturation of circular RNA is regulated by cis- and trans-elements. Complete list of biological functions of these RNA is not yet compiled; however, their capacity to interact with specific microRNA and play a role of a depot attracts the greatest interest. This property makes circular RNA active regulatory transcription factors. Circular RNA have many advantages over their linear analogs: synthesis of these molecules is conservative, they are universal, characterized by clearly determined specificity, and are resistant to exonucleases. In addition, the level of their expression is often higher than that of their linear forms. It should be noted that expression of circular RNA is tissue-specific. Moreover, some correlations between changes in the repertoire and intensity of expression of circular RNA and the development of some pathologies have been detected. Circular RNA have certain advantages and can serve as new biomarkers for the diagnosis, prognosis, and evaluation of response to therapy.

Poly(ADP-ribose) polymerases (PARPs) act as DNA break sensors and catalyze the synthesis of polymers of ADP-ribose (PAR) covalently attached to acceptor proteins at DNA damage sites. It has been demonstrated that both mammalian PARP1 and PARP2 PARylate double-strand break termini in DNA oligonucleotide duplexes in vitro. Here, we show that mammalian PARP2 and PARP3 can PARylate and mono(ADP-ribosyl)ate (MARylate), respectively, 5'- and 3'-terminal phosphate residues at double- and single-strand break termini of a DNA molecule containing multiple strand breaks. PARP3-catalyzed DNA MARylation can be considered a new type of reversible post-replicative DNA modification. According to DNA substrate specificity of PARP3 and PARP2, we propose a putative mechanistic model of PARP-catalyzed strand break-oriented ADP-ribosylation of DNA termini. Notably, PARP-mediated DNA ADP-ribosylation can be more effective than PARPs' auto-ADP-ribosylation depending on the DNA substrates and reaction conditions used. Finally, we show an effective PARP3- or PARP2-catalyzed ADP-ribosylation of high-molecular-weight (∼3-kb) DNA molecules, PARP-mediated DNA PARylation in cell-free extracts and a persisting signal of anti-PAR antibodies in a serially purified genomic DNA from bleomycin-treated poly(ADP-ribose) glycohydrolase-depleted HeLa cells. These results suggest that certain types of complex DNA breaks can be effectively ADP-ribosylated by PARPs in cellular response to DNA damage.

Most members of the poly(ADP-ribose)polymerase family, PARP family, have a catalytic activity that involves the transfer of ADP-ribose from a beta-NAD+-molecule to protein acceptors. It was recently discovered by Talhaoui et al. that DNA-dependent PARP1 and PARP2 can also modify DNA. Here, we demonstrate that DNA-dependent PARP3 can modify DNA and form a specific primed structure for further use by the repair proteins. We demonstrated that gapped DNA that was ADP-ribosylated by PARP3 could be ligated to double-stranded DNA by DNA ligases. Moreover, this ADP-ribosylated DNA could serve as a primed DNA substrate for PAR chain elongation by the purified proteins PARP1 and PARP2 as well as by cell-free extracts. We suggest that this ADP-ribose modification can be involved in cellular pathways that are important for cell survival in the process of double-strand break formation.

At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants-urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pancreatology Club Professional Medical Community, 1A.S. Loginov Moscow Clinical Research and Practical Center, Moscow Healthcare Department, Moscow; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Ministry of Health of Russia, Moscow; 3Kazan State Medical University, Ministry of Health of Russia, Kazan; 4Kazan (Volga) Federal University, Kazan; 5Far Eastern State Medical University, Ministry of Health of Russia, Khabarovsk; 6Morozov City Children's Clinical Hospital, Moscow Healthcare Department, Moscow; 7I.I. Mechnikov North-Western State Medical University, Ministry of Health of Russia, Saint Petersburg; 8Siberian State Medical University, Ministry of Health of Russia, Tomsk; 9M.F. Vladimirsky Moscow Regional Research Clinical Institute, Moscow; 10Maimonides State Classical Academy, Moscow; 11V.I. Razumovsky State Medical University, Ministry of Health of Russia, Saratov; 12I.M. Sechenov First Moscow State Medical University, Ministry of Health of Russia, Moscow; 13S.M. Kirov Military Medical Academy, Ministry of Defense of Russia, Saint Petersburg; 14Surgut State Medical University, Ministry of Health of Russia, Surgut; 15City Clinical Hospital Five, Moscow Healthcare Department, Moscow; 16Nizhny Novgorod Medical Academy, Ministry of Health of Russia, Nizhny Novgorod; 17Territorial Clinical Hospital Two, Ministry of Health of the Krasnodar Territory, Krasnodar; 18Saint Petersburg State Pediatric Medical University, Ministry of Health of Russia, Saint Petersburg; 19Rostov State Medical University, Ministry of Health of Russia, Rostov-on-Don; 20Omsk Medical University, Ministry of Health of Russia, Omsk; 21Russian Medical Academy of Postgraduate Education, Ministry of Health of Russia, Moscow; 22Novosibirsk State Medical University, Ministry of Health of Russia, Novosibirsk; 23Stavropol State Medical University, Ministry of Health of Russia, Stavropol; 24Kemerovo State Medical University, Ministry of Health of Russia, Kemerovo; 25N.I. Pirogov Russian National Research Medical University, Ministry of Health of Russia, Moscow; 26A.M. Nikiforov All-Russian Center of Emergency and Radiation Medicine, Russian Ministry for Civil Defense, Emergencies and Elimination of Consequences of Natural Disasters, Saint Petersburg; 27Research Institute for Medical Problems of the North, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk; 28S.P. Botkin City Clinical Hospital, Moscow Healthcare Department, Moscow; 29Tver State Medical University, Ministry of Health of Russia, Tver The Russian consensus on the diagnosis and treatment of chronic pancreatitis has been prepared on the initiative of the Russian Pancreatology Club to clarify and consolidate the opinions of Russian specialists (gastroenterologists, surgeons, and pediatricians) on the most significant problems of diagnosis and treatment of chronic pancreatitis. This article continues a series of publications explaining the most significant interdisciplinary consensus statements and deals with enzyme replacement therapy.

The paper presents the All-Russian consensus on the diagnosis and treatment of celiac disease in children and adults, which has been elaborated by leading experts, such as gastroenterologists and pediatricians of Russia on the basis of the existing Russian and international guidelines. The consensus approved at the 42nd Annual Scientific Session of the Central Research Institute of Gastroenterology on Principles of Evidence-Based Medicine into Clinical Practice (March 2-3, 2016). The consensus is intended for practitioners engaged in the management and treatment of patients with celiac disease. Evidence for the main provisions of the consensus was sought in electronic databases. In making recommendations, the main source was the publications included in the Cochrane Library, EMBASE, MEDLINE, and PubMed. The search depth was 10 years. Recommendations in the preliminary version were reviewed by independent experts. Voting was done by the Delphic polling system.

Real-time or quantitative loop-mediated isothermal amplification (qLAMP) is a promising technique for the accurate detection of pathogens in organisms and the environment. Here we present a comparative study of the performance of six fluorescent intercalating dyes-SYTO-9, SYTO-13, SYTO-82, SYBR Green I, SYBR Gold, EvaGreen-in three different qLAMP model systems. SYTO-9 and SYTO-82, which had the best results, were used for additional enzyme and template titration studies. SYTO-82 demonstrated the best combination of time-to-threshold (Tt) and signal-to-noise ratio (SNR).

Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

Among the set of mammalian DNA polymerases, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are involved in repair, including base excision repair and non-homologous end joining. Some of them play a crucial role during the specific process which is referred to as translesion synthesis (TLS). TLS intends for the cell surviving during the replication of damaged DNA templates. Additionally, specific activities of TLS-polymerases have to be useful for repair of double-stranded clustered lesions: if the synthesis is proceeded via base excision repair process, the role of DNA polymerases β or λ will be important. In this review we discussed the biochemical properties and functional relevance of X family DNA polymerases β and λ., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The generation and evolution of Earth's continental crust has played a fundamental role in the development of the planet. Its formation modified the composition of the mantle, contributed to the establishment of the atmosphere, and led to the creation of ecological niches important for early life. Here we show that in the Archean, the formation and stabilization of continents also controlled the location, geochemistry, and volcanology of the hottest preserved lavas on Earth: komatiites. These magmas typically represent 50-30% partial melting of the mantle and subsequently record important information on the thermal and chemical evolution of the Archean-Proterozoic Earth. As a result, it is vital to constrain and understand the processes that govern their localization and emplacement. Here, we combined Lu-Hf isotopes and U-Pb geochronology to map the four-dimensional evolution of the Yilgarn Craton, Western Australia, and reveal the progressive development of an Archean microcontinent. Our results show that in the early Earth, relatively small crustal blocks, analogous to modern microplates, progressively amalgamated to form larger continental masses, and eventually the first cratons. This cratonization process drove the hottest and most voluminous komatiite eruptions to the edge of established continental blocks. The dynamic evolution of the early continents thus directly influenced the addition of deep mantle material to the Archean crust, oceans, and atmosphere, while also providing a fundamental control on the distribution of major magmatic ore deposits.

Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1-2 helical turns (clustered lesion). Here, we explored the repair opportunity of the apurinic/apyrimidinic site (AP site) composed of the clustered lesion with 5-formyluracil (5-foU) by the base excision repair (BER) proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human apurinic/apyrimidinic endonuclease 1 (hAPE1) decreased under AP site excursion to the 3'-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process.

Background and Aims: Budesonide may be an effective therapy for mild-to-moderately active ulcerative colitis (UC). This study aimed to demonstrate non-inferiority for oral 9mg budesonide once daily (OD) versus 3g mesalazine granules OD., Methods: This was an eight-week randomised, double-blind, double-dummy, multicentre study in which patients with mild-to-moderately active UC, defined as Clinical Activity Index (CAI) ≥6 and Endoscopic Index (EI) ≥4, received budesonide (Budenofalk® 3mg capsules×3) or mesalazine (Salofalk® 1000mg granules×3). The primary endpoint was clinical remission at week 8 (CAI ≤4 with stool frequency and rectal bleeding subscores of "0")., Results: 343 patients were randomised (177 budesonide, 166 mesalazine). Fewer patients achieved the primary endpoint with budesonide versus mesalazine (70/177 [39.5%] versus 91/166 [54.8%]) with a difference in proportions of -15.3% (95% CI [-25.7%, -4.8%]; p=0.520 for non-inferiority). The median time to first resolution of symptoms was 14.0 days (budesonide) and 11.0 days (mesalazine) (hazard ratio 1.19; 95% CI [0.94, 1.51]). Mucosal healing was observed in 54/177 (30.5%) budesonide patients versus 65/166 (39.2%) mesalazine patients, a difference of -8.6% (95% CI [-18.7%, 1.4%]; p=0.093). The incidences of adverse events (budesonide 26.6%, mesalazine 25.3%) and serious adverse events (budesonide 1.7%, mesalazine 1.2%) were similar., Conclusions: Once-daily 3g mesalazine administered as granules is superior to 9mg budesonide OD administered as capsules for achieving remission in mild-to-moderately active UC. However, it is noteworthy that remission of UC was attained in about 40% of budesonide-treated patients with a rapid onset of resolution., (Copyright © 2010. Published by Elsevier B.V.)

One of the key stages of life of a cell is genome duplication. The main enzymes which lead this process are DNA-dependent DNA polymerases. At the moment, 19 DNA polymerases with striking properties are listed in the eukaryotic cells. Mitochondrial DNA polymerase gamma from A family and most of the nuclear enzymes from B family are high fidelity DNA polymerases which are participate in genome DNA replication process as well as in DNA repair. Among the other 1 5 proteins, the D N A polymerases belonging to the X and Y families have a special place. They participate in a different repair processes such as base excision repair and non-homologous end joining. Moreover, some of them play a specific role in the replication of the damaged DNA templates. This process is referred as translesion synthesis or TLS. The DNA polymerases beta and lambda members of X family are enclosed in polyfunctional enzymes, and their properties and functions will be discussed in this review.

Here we investigated the ability of the human X-family DNA polymerases beta and lambda to bypass thymine glycol (Tg) in gapped DNA substrates with the damage located in a defined position of the template strand. Maximum velocities and the Michaelis constant values were determined to study DNA synthesis in the presence of either Mg(2+) or Mn(2+). Additionally, the influence of hRPA (human replication protein A) and hPCNA (human proliferating cell nuclear antigen) on TLS (translesion synthesis) activity of DNA polymerases beta and lambda was examined. The results show that (i) DNA polymerase lambda is able to catalyze DNA synthesis across Tg, (ii) the ability of DNA polymerase lambda to elongate from a base paired to a Tg lesion is influenced by the size of the DNA gap, (iii) hPCNA increases the fidelity of Tg bypass and does not influence normal DNA synthesis catalyzed by DNA polymerase lambda, (iv) DNA polymerase beta catalyzes the incorporation of all four dNTPs opposite Tg, and (v) hPCNA as well as hRPA has no specific effect on TLS in comparison with the normal DNA synthesis catalyzed by DNA polymerase beta. These results considerably extend our knowledge concerning the ability of specialized DNA polymerases to cope with a very common DNA lesion such as Tg.

The main strategy used by pro- and eukaryotic cells for replication of damaged DNA is translesion synthesis (TLS). Here, we investigate the TLS process catalyzed by DNA polymerases beta and lambda on DNA substrates using mono- or dinucleotide gaps opposite damage located in the template strand. An analog of a natural apurinic/apyrimidinic site, the 3-hydroxy-2-hydroxymetylthetrahydrofuran residue (THF), was used as damage. DNA was synthesized in the presence of either Mg2+ or Mn2+. DNA polymerases beta and lambda were able to catalyze DNA synthesis across THF only in the presence of Mn2+. Moreover, strand displacement synthesis was not observed. The primer was elongated by only one nucleotide. Another unusual aspect of the synthesis is that dTTP could not serve as a substrate in all cases. dATP was a preferential substrate for synthesis catalyzed by DNA polymerase beta. As for DNA polymerase lambda, dGMP was the only incorporated nucleotide out of four investigated. Modified on heterocyclic base photoreactive analogs of dCTP and dUTP showed substrate specificity for DNA polymerase beta. In contrast, the dCTP analog modified on the exocyclic amino group was a substrate for DNA polymerase lambda. We also observed that human replication protein A inhibited polymerase incorporation by both DNA polymerases beta and lambda on DNA templates containing damage.

Replication of damaged DNA (translesion synthesis, TLS) is realized by specialized DNA polymerases. Additional protein factors such as replication protein A (RPA) play important roles in this process. However, details of the interaction are unknown. Here we analyzed the influence of the hRPA and its mutant hABCD lacking domains responsible for protein-protein interactions on ability of DNA polymerase lambda to catalyze TLS. The primer-template structures containing varying parts of extended strand (16 and 37 nt) were used as model systems imitating DNA intermediate of first stage of TLS. The 8-oxoguanine disposed in +1 position of the template strand in relation to 3 -end of primer was exploited as damage. It was shown that RPA stimulated TLS DNA synthesis catalyzed by DNA polymerase lambda in its globular but not in extended conformation. Moreover, this effect is dependent on the presence of p70N and p32C domains in RPA molecule.

Translesion synthesis (TLS) is one of the DNA damage tolerance strategies that has evolved to enable orga-nisms to replicate their genome despite the presence of unrepaired damage. TLS complexes are dynamic systems composed of DNA polymerases and associated protein factors. Therefore, it is hard to study these assembles by X-ray analysis or other instrumental methods. Here, we have suggested applying the photoaffinity labeling technique for studying the TLS system in nuclear/cellular extracts. As a tool we proposed to use partial DNA duplexes containing base-substituted photoreactive deoxynucleotides at the 3' end of primer opposite to DNA damage at the template strand. We demonstrated that photoreactive dNTPs can be potentially used to synthesize photoreactive DNA probes mimicking the DNA intermediates of the first stage of translesion synthesis by specialized DNA polymerases. We used synthetic apurinic/apyrimidinic site (AP-site) - tetrahydrofuran (THF) and 8 oxoguanine as damages in +1 position of the template strand with respect to 3' end of primer. Activity of human DNA polymerases beta and lambda was exploited for construction of photoreactive DNAs using photo derivatives of dNTPs. The kinetic parameters of the elongation reaction in model systems were estimated. Using photoaffinity crosslinking we found that only a few proteins in the bovine testis nuclear extract were strongly labeled by TLS probes.

The translesion synthesis (TLS) capacity of the thermostable DNA polymerases Taq, Tte and Tte-seq utilizing a synthetic abasic site, tetrahydrofuran (THF), and an 8-oxoguanine-containing DNA template was investigated. Measurements with human DNA polymerase beta were used as a "positive control". Thermostable DNA polymerases were observed to perform TLS with different specificities on both substrates. With a THF-containing template, dGMP was preferentially inserted by all the DNA polymerases. In the presence of Mn(II) as a cofactor, all the polymerases incorporated dCMP opposite 8-oxoguanine whereas, in the presence of Mg(II) ions, dAMP was incorporated. It was found that none of the thermophilic DNA polymerases utilized dTTP with either an 8-oxoguanine or a THF-containing template. In all cases, DNA duplex containing THF as damage was processed to full length less effectively than DNA duplex containing 8-oxoguanine.

A new photoreactive oligonucleotide derivative was synthesized with a perfluoroarylazido group attached to the 2'-position of the ribose fragment of the 5'-terminal nucleotide. Using this conjugate, photoreactive DNA duplexes were produced which contained single-stranded regions of different length, single-stranded breaks (nicks), and also ds duplex with a photoreactive group inside one of the chains. These structures imitate DNA intermediates generated at different stages of DNA replication and repair. The interaction of replication protein A (RPA) with the resulting DNA structures was studied using photoaffinity modification and gel retardation assay. Independently of the DNA structure, only the large subunit of RPA (p70) was crosslinked to photoreactive DNAs, and the intensity of its labeling increased with decrease in the size of the single-stranded region and was maximal in the case of the nick-containing DNA structure. By gel retardation, the most effective binding of RPA to this structure was shown, whereas the complexing of RPA with DNA containing the unmodified nick and also with the full duplex containing the photoreactive group inside the chain was significantly less effective. The data suggest that RPA should be sensitive to such damages in the double-stranded DNA structure.

There was an analysis of the results of a 5-year observation over 226 patients with the most prevailing digestive apparatus diseases: stomach ulcer and duodenal ulcer (SU and DU), chronic pancreatitis (CP), irritable bowel syndrome (IBS) in an outpatient clinic. Patients were supervised by gastroenterologists (168 patients) and therapeutists (58 patients). It was noted that supervision of the patients by gastroenterologists authentically reduces the frequency of hospitalizations and duration of their stay on the sick-list as compared with the patients being observed by therapeutists and results in higher indices of life quality (LQ) among the patients.

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase beta (Pol beta) interaction with DNA. Using Pol beta, a photoreactive dTTP analog was added to the 3' end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5' end of a nick or a flap. Photoaffinity labeling of Pol beta substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol beta-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol beta during long-patch base excision repair.

40 patients with irritable colon syndrome were treated outpatiently: 26 patients by gastroenterologist and 14 ones by therapist. Management of such patients by gastroenterologist significantly lowered frequency of their hospitalizations and sick leave duration as compared to those treated by therapist.

To introduce photoreactive dNTP residues to the 3'-end of a mononucleotide gap, base-substituted photoreactive deoxynucleoside triphosphate derivatives, (5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-trans-3-aminopropenyl-1]- and 5-(N-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]- trans-3-aminopropenyl-1)-2'-deoxyuridine 5'-triphosphates, were used as substrates in the DNA polymerase beta-catalyzed reaction. The resulting nick, containing a modified base at the 3'-end, was sealed by T4 phage DNA ligase. This approach enables the preparation of DNA duplexes bearing photoreactive groups at predetermined position(s) of the nucleotide chain. Using the generated photoreactive DNA duplexes, the photoaffinity modifications of DNA polymerase beta and human replicative protein A (hRPA) were carried out. It was shown that DNA polymerase beta and hRPA subunits were modified with the photoreactive double-stranded DNA considerably less effectively than by the nicked DNA. In the case of double-stranded DNA, the hRPA p70 subunit was preferentially labeled, implying a crucial role of this subunit in the protein-DNA interaction.

The aim of the study was to assess effects of H2-blocker of the IIId generation famotidine (quamatel) on the progression of acute and chronic pancreatitis and intensity of pain in patients with pancreatitis. 46 patients received standard therapy plus intravenous quamatel. Control patients received only standard treatment. It was found that intravenous quamatel relieves pain primarily within the first treatment week. Quamatel shortens duration of analgetics use, narcotic, in particular. It is thought valid to include quamatel in combined treatment of patients with acute and chronic pancreatitis.

Antidiarrheal action of sandostatin (octreotide) was studied in dumping syndrome, diabetic enteropathy, short-bowel syndrome, Crohn's disease and ulcerative colitis. Sandostatin proved effective in diarrhea of secretory, motor and osmotic origin. Dumping syndrome, diabetic enteropathy, Crohn's disease and short-bowel syndrome responded completely. In ulcerative colitis the effect was paradoxical: intensive diarrhea discontinued, but moderate diarrhea became more intensive. Sandostatin indications in ulcerative colitis should receive further investigations.

The authors consider different mechanisms participating in progression of diarrhea in ulcerative colitis. The involvement of osmotic component was suggested in view of incomplete hydrolysis of carbohydrates by small intestinal glucoamylase under mono- and bisubstrate digestion, whereas diarrhea secretory component develops in participation of cyclic nucleotides. Motor mechanism is also present in diarrhea as evidenced by high concentrations of blood motilin and changes in intestinal evacuation and motility. Polycomponent dysbioses detected in ulcerative colitis patients may also be essential for pathogenesis of diarrhea. Determination of the leading component in each diarrhea case is thought helpful in valid choice of individual therapeutic policy.

The concentrations of cyclic nucleotides (CN) were examined in rectal mucosal biopsy specimens from 52 patients with active ulcerative colitis (UC) and 10 healthy controls. The three degrees of inflammation, evaluated macroscopically by endoscopy and by morphometry in the specimens by estimating the cellular infiltration of the lamina propria, corresponded nicely with each other. The proliferative-regenerative morphologic changes in the rectal mucosa were graded qualitatively and quantitatively in three groups in accordance with defined criteria. A significant difference in cAMP concentration was only found between the most severe degree of inflammation (infiltration, 44.6%) in UC and healthy controls. cAMP concentration in rectal mucosal specimens was lower at the highest degree of regeneration than in specimens with lower degrees. Thus cAMP may play a role in the epithelial regeneration of rectal mucosa. The values of CN concentration did not correlate with the degree of inflammation in rectal mucosa from patients with UC.