Identification of a 2′-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries

Ribose methylation is among the most ubiquitous modifications found in RNA. 2′-O-methyluridine is found in rRNA, snRNA, snoRNA and tRNA of Archaea, Bacteria, and Eukaryota. Moreover, 2′-O-methylribonucleosides are promising starting materials for the production of nucleic acid-based drugs. Despite the countless possibilities of practical use for the metabolic enzymes associated with methylated nucleosides, there are very few reports regarding the metabolic fate and enzymes involved in the metabolism of 2′-O-alkyl nucleosides. The presented work focuses on the cellular degradation of 2′-O-methyluridine. A novel enzyme was found using a screening strategy that employs Escherichia coli uracil auxotroph and the metagenomic libraries. A 2′-O-methyluridine hydrolase (RK9NH) has been identified together with an aldolase (RK9DPA)—forming a part of a probable gene cluster that is involved in the degradation of 2′-O-methylated nucleosides. The RK9NH is functional in E. coli uracil auxotroph and in vitro. The RK9NH nucleoside hydrolase could be engineered to enzymatically produce 2′-O-methylated nucleosides that are of great demand as raw materials for production of nucleic acid-based drugs. Moreover, RK9NH nucleoside hydrolase converts 5-fluorouridine, 5-fluoro-2′-deoxyuridine and 5-fluoro-2′-O-methyluridine into 5-fluorouracil, which suggests it could be employed in cancer therapy.This article belongs to the Section Chemical BiologyThis research was funded by the Lithuanian Research Council (LMT) grant SEN-07/2015. This research was performed in cooperation with the INMARE consortium

Mitas apie įtrauktį: kodėl technologijomis grindžiamas mokymas nėra panacėja, kaip buvo manyta

Šio straipsnio problematika pristatoma taip: studentai, turintys specialiųjų mokymosi poreikių (SUP), turi ribotas galimybes dalyvauti studijų procese kartu su negalios neturinčiaisiais, o tai gali didinti jų socialinę atskirtį. Tyrimo tikslas: įvertinti, kaip technologijomis grindžiamas mokymas (TGM) prisideda prie studentų, turinčių SUP, atskirties mažinimo studijų metu. Tyrimo objektas – studentų, turinčių SUP, socialinės įtraukties procesas, pasitelkus TGM. Šiam tikslui pasiekti buvo atliktas kokybinis tyrimas. Duomenys buvo surinkti pusiau struktūruoto interviu metu, po to analizuojami taikant kokybinio turinio analizę. Tyrimo dalyviai – antros pakopos studentai, turintys SUP, kurie šiuo metu, naudodamiesi technologijomis, studijuoja JAV universitetuose. Tyrimo rezultatai: tyrimas nepatvirtino, kad TGM gali užtikrinti visų studentų, turinčių SUP, įtrauktį ir sumažinti socialinę atskirtį, bet parodė, kad studentai, turintys SUP, siekia laisvės savo studijų procese ir ribotai bendrauti su studijų bendruomeneThe problem of the research is formulated as follows: students with special educational needs (SEN) have few chances to take part in educational activities together with non-disabled students; as a result, they become excluded from the education system. The aim of this research is to determine how technology enhanced learning (TEL) contributes to reducing the social exclusion of students with SEN from education systems and promoting social inclusion. The object of the research is the phenomenon of social inclusion of students with SEN, fostered by TEL. Qualitative research methods and methodology were applied, and data were collected during semi-structured interviews. Research participants are graduate students with SEN who are currently engaging themselves in a study process fostered by TEL at U.S. universities. The research has provided the following findings: TEL cannot ensure or promote inclusion for all students with SEN and reduce social discrimination; in fact, such students seek a more independent learning experience and limited interactionEdukologijos tyrimų institutasVytauto Didžiojo universitetasŠvietimo akademij

Contribution of technology enhanced learning to the inclusion of students with special education needs

Students with special educational needs (henceforth, SEN) are usually separated from traditional educational settings; thus, risk of being completely excluded from educational activities due to their physical or mental limitations is increased and access to education can be reduced significantly. Thus, a problem arises that students with SEN have restricted possibilities to participate in study process and to obtain desired qualifications together with non-disabled students; therefore, they may feel excluded from education system. The aim of the research is to evaluate how technology enhanced learning (henceforth, TEL) contributes to reducing exclusion of students with SEN. In order to achieve the aim, qualitative research methodology has been applied. Data has been selected during semi-structured interviews and analyzed by applying a qualitative content analysis. Research participants are graduate students with SEN who are currently engaging themselves in study process fostered by TEL at U.S. universities. The research has provided the following findings: TEL increases accessibility of education by eliminating social and physical barriers, enabling constant movement, and helping students with SEN cope with their impairments. TEL can be the only way for learners to complete their education. The research has not indicated that TEL can ensure inclusion. However, TEL enables students with SEN to receive qualifications and degrees in a much more convenient wayEdukologijos tyrimų institutasSocialinių mokslų fakultetasVytauto Didžiojo universiteta

Identification of a 2′-O-methyluridine nucleoside hydrolase using the metagenomic libraries

Ribose methylation is among the most ubiquitous modifications found in RNA. 2'-O-methyluridine is found in rRNA, snRNA, snoRNA and tRNA of Archaea, Bacteria, and Eukaryota. Moreover, 2'-O-methylribonucleosides are promising starting materials for the production of nucleic acid-based drugs. Despite the countless possibilities of practical use for the metabolic enzymes associated with methylated nucleosides, there are very few reports regarding the metabolic fate and enzymes involved in the metabolism of 2'-O-alkyl nucleosides. The presented work focuses on the cellular degradation of 2'-O-methyluridine. A novel enzyme was found using a screening strategy that employs Escherichia coli uracil auxotroph and the metagenomic libraries. A 2'-O-methyluridine hydrolase (RK9NH) has been identified together with an aldolase (RK9DPA)-forming a part of a probable gene cluster that is involved in the degradation of 2'-O-methylated nucleosides. The RK9NH is functional in E. coli uracil auxotroph and in vitro. The RK9NH nucleoside hydrolase could be engineered to enzymatically produce 2'-O-methylated nucleosides that are of great demand as raw materials for production of nucleic acid-based drugs. Moreover, RK9NH nucleoside hydrolase converts 5-fluorouridine, 5-fluoro-2'-deoxyuridine and 5-fluoro-2'-O-methyluridine into 5-fluorouracil, which suggests it could be employed in cancer therap

Low-Temperature Virus vB_EcoM_VR26 Shows Potential in Biocontrol of STEC O26:H11

Shiga toxin-producing Escherichia coli (STEC) O26:H11 is an emerging foodborne pathogen of growing concern. Since current strategies to control microbial contamination in foodstuffs do not guarantee the elimination of O26:H11, novel approaches are needed. Bacteriophages present an alternative to traditional biocontrol methods used in the food industry. Here, a previously isolated bacteriophage vB_EcoM_VR26 (VR26), adapted to grow at common refrigeration temperatures (4 and 8 °C), has been evaluated for its potential as a biocontrol agent against O26:H11. After 2 h of treatment in broth, VR26 reduced O26:H11 numbers (p < 0.01) by > 2 log10 at 22 °C, and ~3 log10 at 4 °C. No bacterial regrowth was observed after 24 h of treatment at both temperatures. When VR26 was introduced to O26:H11-inoculated lettuce, ~2.0 log10 CFU/piece reduction was observed at 4, 8, and 22 °C. No survivors were detected after 4 and 6 h at 8 and 4 °C, respectively. Although at 22 °C, bacterial regrowth was observed after 6 h of treatment, O26:H11 counts on non-treated samples were >2 log10 CFU/piece higher than on phage-treated ones (p < 0.02). This, and the ability of VR26 to survive over a pH range of 3–11, indicates that VR26 could be used to control STEC O26:H11 in the food industry

Characterization of <i>Paenibacillus</i> sp. GKG Endo-β-1, 3-Glucanase, a Member of Family 81 Glycoside Hydrolases

Paenibacillus sp. GKG was isolated based on its ability to produce hydrolysis zones on agar plates containing yeast cell wall substrate as the single carbon source. The extracellular enzymes secreted into the culture medium were identified by LC-MS/MS proteomics. Endo-β-1,3-glucanase PsLam81A containing GH81 catalytic and the CBM56 carbohydrate-binding modules was selected for heterologous expression in Escherichia coli. The identity of the recombinant PsLam81A was confirmed by LC-MS/MS proteomics. The PsLam81A showed the highest activity at 60 °C, and the optimal pH range was between 6.5 and 8.0. The analysis of the full-length PsLam81A and truncated PsLam81AΔCBM56 enzymes showed that the CBM56 module improved the hydrolytic activity towards linear β-1,3-glucans—curdlan and pachyman but had no effect on hydrolysis of β-1,3/β1,6-branched glucans—laminarin and yeast β-glucan. The characterization of PsLam81A enzyme broadens current knowledge on the biochemical properties and substrate specificity of family 81 glycoside hydrolases and allows prediction of the necessity of CBM56 module in the process of designing new truncated or chimeric glycosidases

Exploring the enzymatic activity of depolymerase gp531 from Klebsiella pneumoniae jumbo phage RaK2

Klebsiella pneumoniae poses a major global challenge due to its virulence, multidrug resistance, and nosocomial nature. Thus, bacteriophage-derived proteins are extensively being investigated as a means to combat this bacterium. In this study, we explored the enzymatic specificity of depolymerase gp531, encoded by the jumbo bacteriophage vB_KleM_RaK2 (RaK2). We used two different methods to modify the reducing end of the oligosaccharides released during capsule hydrolysis with gp531. Subsequent acidic cleavage with TFA, followed by TLC and HPLC-MS analyses, revealed that RaK2 gp531 is a β-(1→4)-endoglucosidase. The enzyme specifically recognizes and cleaves the capsular polysaccharide (CPS) of the Klebsiella pneumoniae K54 serotype, releasing K-unit monomers (the main product), dimers, and trimers. Depolymerase gp531 remains active from 10 to 50 °C and in the pH 3–8 range, indicating its stability and versatility. Additionally, we demonstrated that gp531′s activity is not affected by CPS acetylation, which is influenced by the growth conditions of the bacterial culture. Overall, our findings provide valuable insights into the enzymatic activity of the first characterized depolymerase targeting the capsule of the clinically relevant K54 serotype of K. pneumoniae

Discovery of Bacterial Deaminases That Convert 5-Fluoroisocytosine Into 5-Fluorouracil

Cytosine is one of the four letters of a standard genetic code, found both in DNA and in RNA. This heterocyclic base can be converted into uracil upon the action of the well-known cytosine deaminase. Isocytosine (2-aminouracil) is an isomer of cytosine, yet the enzymes that could convert it into uracil were previously mainly overlooked. In order to search for the isocytosine deaminases we used a selection strategy that is based on uracil auxotrophy and the metagenomic libraries, which provide a random pool of genes from uncultivated soil bacteria. Several genes that encode isocytosine deaminases were found and two respective recombinant proteins were purified. It was established that both novel deaminases do not use cytosine as a substrate. Instead, these enzymes are able to convert not only isocytosine into uracil, but also 5-fluoroisocytosine into 5-fluorouracil. Our findings suggest that novel isocytosine deaminases have a potential to be efficiently used in targeted cancer therapy instead of the classical cytosine deaminases. Use of isocytosine instead of cytosine would produce fewer side effects since deaminases produced by the commensal E. coli gut flora are ten times less efficient in degrading isocytosine than cytosine. In addition, there are no known homologs of isocytosine deaminases in human cells that would induce the toxicity when 5-fluoroisocytosine would be used as a prodrug

Ketoreductase TpdE from Rhodococcus jostii TMP1: characterization and application in the synthesis of chiral alcohols

Production of highly pure enantiomers of vicinal diols is desirable, but difficult to achieve. Enantiomerically pure diols and acyloins are valuable bulk chemicals, promising synthones and potential building blocks for chiral polymers. Enzymatic reduction of ketones is a useful technique for the synthesis of the desired enantiomeric alcohols. Here, we report on the characterization of a ketoreductase TpdE from Rhodococcus jostii TMP1 that is a prospective tool for the synthesis of such compounds. Results. In this study, NADPH-dependent short-chain dehydrogenase/reductase TpdE from Rhodococcus jostii TMP1 was characterized. The enzyme exhibited broad substrate specificity towards aliphatic 2,3-diketones, butan-3-one-2-yl alkanoates, as well as acetoin and its acylated derivatives. TpdE stereospecifically reduced α-diketones to the corresponding diols. The GC-MS analysis of the reduction products of 2,3- and 3,4-diketones indicated that TpdE is capable of reducing both keto groups in its substrate leading to the formation of two new chiral atoms in the product molecule. Bioconversions of diketones to corresponding diols occurred using either purified enzyme or a whole-cell Escherichia coli BL21 (DE3) biocatalyst harbouring recombinant TpdE. The optimum temperature and pH were determined to be 30–35 °C and 7.5, respectively. Conclusions. The broad substrate specificity and stereoselectivity of TpdE from Rhodococcus jostii TMP1 make it a promising biocatalyst for the production of enantiomerically pure diols that are difficult to obtain by chemical routes

Looking for a generic inhibitor of amyloid-like fibril formation among flavone derivatives

A range of diseases is associated with amyloid fibril formation. Despite different proteins being responsible for each disease, all of them share similar features including beta-sheet-rich secondary structure and fibril-like protein aggregates. A number of proteins can form amyloid-like fibrils in vitro, resembling structural features of disease-related amyloids. Given these generic structural properties of amyloid and amyloid-like fibrils, generic inhibitors of fibril formation would be of interest for treatment of amyloid diseases. Recently, we identified five outstanding inhibitors of insulin amyloid-like fibril formation among the pool of 265 commercially available flavone derivatives. Here we report testing of these five compounds and of epi-gallocatechine-3-gallate (EGCG) on aggregation of alpha-synuclein and beta-amyloid. We used a Thioflavin T (ThT) fluorescence assay, relying on halftimes of aggregation as the measure of inhibition. This method avoids large numbers of false positive results. Our data indicate that four of the five flavones and EGCG inhibit alpha-synuclein aggregation in a concentration-dependent manner. However none of these derivatives were able to increase halftimes of aggregation of beta-amyloid