The potentials of electrochemical processes in ideal aqueous media are related to the potential of a normal hydrogen electrode (NHE). However, in non-ideal media, the potentials of a metallocene redox couple are used as a reference. Such measurements with free metallocene in solution are complicated by adsorption and mass transport phenomena and solvation effects. Herein, a platinum electrode with an anchored ferrocene moiety (Pt,Fc) was used for cyclic voltammetric measurements of the potential of ferrocene/ferrocenium (Fc/Fc+) redox transformation in not only non-aqueous but, for the first time, aqueous solutions as well. This enabled us to eliminate the aforementioned problems associated with the application of free metallocene molecules in solution and, thus, to relate the midpoint potential (Epm) of the Fc/Fc+ redox couple to a NHE. After elimination of the liquid junction potential in an aqueous 0.1 M KCl solution at 25 °C, the average intraday Epm value obtained with freshly prepared Pt,Fc electrodes was found to be 0.312 ± 0.008 V versus the secondary Ag|AgCl electrode. The Pt,Fc electrode can be applied for the standardization of electrochemical measurements and investigation of solvation phenomena at interfaces in non-ideal media.

• Publikační typ
• časopisecké články MeSH

The aim of the project is to evaluate the applicability of high throughput sequencing (HTS) as a tool for complex characterization of oral microbiome dynamics and thus for early diagnosis of risk and onset of periodontitis and for monitoring of treatment. The 7-year long-term study exploring the dynamics of changes in the oral microbiome composition in already established group of 20 initially healthy subjects and a control group of patients with chronic periodontitis will document in detail the transformation of "healthy" bacterial biofilm into the periodontal one. The HTS will be employed also for monitoring of oral microbiome during and after the therapy of a group of 60 patients with aggressive periodontitis treated by either of two standard procedures. Comparison of outputs of both experiments will reveal, whether the composition of subgingival biofilm differs in chronic and aggressive periodontitis. The complex methodological procedure for HTS integration into system of early diagnosis and treatment of patients with periodontitis will be developed.

Cílem projektu je posoudit využitelnost vysokoúčinných metod paralelního sekvenování (HTS – high-throughput sequencing) jako nástroje pro komplexní charakterizaci dynamiky mikrobiomu dutiny ústní a tím pro včasnou diagnózu rizika i nástupu parodontitidy a pro sledování postupu léčby. V dlouhodobé studii, sledující dynamiku změn složení orálního mikrobiomu v již ustaveném souboru 20 původně zdravých osob i kontrolní skupiny pacientů s chronickou parodontitidou, bude podrobně zdokumentována transformace „zdravého“ bakteriálního biofilmu na parodontální v průběhu celkem sedmiletého monitorovacího období. Na souboru 60 pacientů s agresivní parodontitidou léčených dvěma různými standartními postupy bude HTS využita pro monitorování orálního mikrobiomu v průběhu vlastní terapie i v následném období. Porovnání výstupů obou experimentů poskytne informaci, zda a jak se liší kompozice subgingiválního biofilmu u chronické a agresivní parodontitidy. Pro uživatelská medicínská pracoviště bude vypracován komplexní metodický postup pro včasnou diagnózu i monitorování průběhu léčby.

• MeSH
• agresivní parodontitida mikrobiologie   MeSH
• biofilmy MeSH
• chronická parodontitida mikrobiologie   MeSH
• mikrobiota MeSH
• stomatologie MeSH
• ústa mikrobiologie   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH

• Konspekt
• Stomatologie
• NLK Obory
• zubní lékařství
• genetika, lékařská genetika
• bakteriologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Here we provide a review on TldD/TldE family proteins, summarizing current knowledge and outlining further research perspectives. Despite being widely distributed in bacteria and archaea, TldD/TldE proteins have been escaping attention for a long time until several recent reports pointed to their unique features. Specifically, TldD/TldE generally act as peptidases, though some of them turned out to be N-deacetylases. Biological function of TldD/TldE has been extensively described in bacterial specialized metabolism, in which they participate in the biosynthesis of lincosamide antibiotics (as N-deacetylases), and in the biosynthesis of ribosomally synthesized and post-translationally modified bioactive peptides (as peptidases). These enzymes possess special position in the relevant biosynthesis since they convert non-bioactive intermediates into bioactive metabolites. Further, based on a recent study of Escherichia coli TldD/TldE, these heterodimeric metallopeptidases possess a new protein fold exhibiting several structural features with no precedent in the Protein Data Bank. The most interesting ones are structural elements forming metal-containing active site on the inner surface of the catalytically active subunit TldD, in which substrates bind through β sheet interactions in the sequence-independent manner. It results in relaxed substrate specificity of TldD/TldE, which is counterbalanced by enclosing the active centre within the hollow core of the heterodimer and only appropriate substrates can entry through a narrow channel. Based on the published data, we hypothesize a yet unrecognized central metabolic function of TldD/TldE in the degradation of (partially) unfolded proteins, i.e., in protein quality control.

• MeSH
• antibakteriální látky metabolismus   MeSH
• Bacteria genetika   metabolismus   MeSH
• Escherichia coli * genetika   metabolismus   MeSH
• linkosamidy metabolismus   MeSH
• metaloproteasy metabolismus   MeSH
• peptidy chemie   MeSH
• proteasy * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Úvod a cíl práce: Parodontitida, která u dospělé populace patří mezi onemocnění s hromadným výskytem, ve svých důsledcích představuje u osob starších než 35 let téměř stejně častý důvod ztráty zubu jako následky zubního kazu. Cílem této studie bylo zhodnotit využitelnost sekvenačních metod nové generace jako nástroje pro komplexní charakterizaci subgingiválního bakteriálního biofilmu u pacientů s pokročilou parodontitidou a pro sledování dynamiky změn složení subgingiválního bakteriálního biofilmu v průběhu terapie. Předpokládá se, že sekvenační metody nové generace poskytnou novou kvalitu sledování průběhu léčby a nabídnou parodontologům nástroj pro objektivní hodnocení účinnosti zvoleného terapeutického postupu a volbu další léčby. Metody: Do studie vstoupilo 43 pacientů s diagnózou pokročilé parodontitidy. Pacienti byli rozděleni do dvou podskupin a léčeni jednou ze dvou standardně schválených metod – pouze deep scaling a root planing (podskupina A – 20 pacientů) a deep scaling a root planing doplněný o antibiotickou terapii (podskupina B – 23 pacientů). Stav jejich parodontu byl hodnocen parodontálními indexy PPD (periodontal pocket depth), BOP (bleeding on probing), CAL (clinical attachment loss) před léčbou a dva týdny, tři měsíce a 18 měsíců po léčbě. Každému pacientovi byla ve shodných časových intervalech odebrána sada čtyř vzorků z nejhlubšího parodontálního chobotu pro stanovení složení mikrobiomu. Výsledky: V průběhu léčby došlo k významnému poklesu parodontálních indexů BOP, CAL a PPD u obou podskupin pacientů. V průběhu léčby došlo také k významnému poklesu indexu parodontálního rizika R/G. U podskupiny pacientů léčených deep scaling a root planing navíc s aplikací antibiotik (podskupina B) došlo 14 dní po léčbě k významně vyššímu poklesu R/G indexu v porovnání s podskupinou A léčenou pouze deep scaling a root planing. Tři měsíce a 18 měsíců po léčbě byly hodnoty indexu parodontálního rizika R/G u obou podskupin pacientů obdobné. Závěr: Taxonomická charakterizace mikrobiomu vyjádřená formou R/G indexu umožňuje monitorovat dynamické změny v průběhu léčby, na rozdíl od pouhého sledování klinických parametrů ukazuje jednoznačné rozdíly mezi skupinami s použitím a bez použití antibiotik v čase 14 dnů po terapii deep scaling a root planing. S ohledem na klinické parametry obou skupin, které se po třech ani 18 měsících v podstatě neliší, je ovšem na místě velmi pečlivě zvážit, zda je antibiotická podpora terapie deep scaling a root planing přínosem, neboť existuje nezanedbatelné riziko rozvoje a šíření rezistencí. Použití antibiotik v parodontologii by mělo být podloženo podrobným klinickým, mikrobiologickým a celkově medicínským vyšetřením.

Introduction, aim: Periodontitis, which is one of the most common diseases in the adult population, is almost as common cause of tooth loss in people over the age of 35 as it is due to tooth decay. The aim of this study was to evaluate the utility of new-generation sequencing methods as a tool for complex characterization of subgingival bacterial biofilm in patients with advanced periodontitis and for monitoring the dynamics of changes in the composition of subgingival bacterial biofilm during therapy. We assume that next-generation sequencing methods will provide a new quality of monitoring the course of treatment and offer periodontologists a tool for objective evaluation of the effectiveness of the chosen therapeutic procedure and choice of further treatment. Methods: The study included 43 patients with a diagnosis of advanced periodontitis. Patients were divided into two subgroups and treated with one of two standard approved methods – deep scaling and root planing only (subgroup A – 20 patients) and deep scaling and root planing supplemented with antibiotic therapy (subgroup B – 23 patients) – and their periodontal status was assessed by periodontal indices BOP (bleeding on probing), CAL (clinical attachment loss) a PPD (periodontal pocket depth) before treatment and two weeks, three months and 18 months after therapy. A set of four samples was taken from each deepest periodontal pocket at equal time intervals to determine the microbiome composition. Results: During treatment, there was a significant decrease in the periodontal indices PPD, BOP and CAL in both subgroups of patients. There was also a significant decrease in the periodontal risk index R/G during treatment. In addition, the subgroup of patients treated with deep scaling and root planing with antibiotics (subgroup B) had a significantly higher decrease in R/G index 14 days after treatment compared to subgroup A treated with deep scaling and root planing alone. Three months and 18 months after treatment, the periodontal risk index R/G values were similar in both subgroups of patients. Conclusion: Taxonomic characterization of the microbiome expressed in the form of R/G index allows to monitor dynamic changes during treatment, in contrast to mere monitoring of clinical parameters shows clear differences between groups with and without antibiotics at 14 days after deep scaling and root planing therapy. However, given the clinical parameters of the two groups, which do not differ significantly after three or 18 months, it is appropriate to carefully consider whether antibiotic support for deep scaling and root planing is beneficial, as there is a significant risk of developing and spreading resistance. The use of antibiotics in periodontology should be based on detailed clinical, microbiological and general medical examination.

• Klíčová slova
• orální mikrobiom,
• MeSH
• antibakteriální látky terapeutické užití   MeSH
• klinická studie jako téma MeSH
• lidé MeSH
• mikrobiota MeSH
• parodontitida * farmakoterapie   mikrobiologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

The estimation of oral microbiome (OM) taxonomic composition in periodontally healthy individuals can often be biased because the clinically periodontally healthy subjects for evaluation can already experience dysbiosis. Usually, they are included just based on the absence of clinical signs of periodontitis. Additionally, the age of subjects is used to be higher to correspond well with tested groups of patients with chronic periodontitis, a disorder typically associated with aging. However, the dysbiosis of the OM precedes the clinical signs of the disease by many months or even years. The absence of periodontal pockets thus does not necessarily mean also good periodontal health and the obtained image of "healthy OM" can be distorted.To overcome this bias, we taxonomically characterized the OM in almost a hundred young students of dentistry with precise oral hygiene and no signs of periodontal disease. We compared the results with the OM composition of older periodontally healthy individuals and also a group of patients with severe periodontitis (aggressive periodontitis according to former classification system). The clustering analysis revealed not only two compact clearly separated clusters corresponding to each state of health, but also a group of samples forming an overlap between both well-pronounced states. Additionally, in the cluster of periodontally healthy samples, few outliers with atypical OM and two major stomatotypes could be distinguished, differing in the prevalence and relative abundance of two main bacterial genera: Streptococcus and Veillonella. We hypothesize that the two stomatotypes could represent the microbial succession from periodontal health to starting dysbiosis. The old and young periodontally healthy subjects do not cluster separately but a trend of the OM in older subjects to periodontitis is visible. Several bacterial genera were identified to be typically more abundant in older periodontally healthy subjects.

• MeSH
• agresivní parodontitida * MeSH
• chronická parodontitida * MeSH
• dysbióza MeSH
• lidé MeSH
• mikrobiota * MeSH
• parodontální chobot MeSH
• senioři MeSH
• Check Tag
• lidé MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The dysbiosis of oral microbiome (OM) precedes the clinical signs of periodontal disease. Its simple measure thus could indicate individuals at risk of periodontitis development; however, such a tool is still missing. Up to now, numerous microbial taxa were associated with periodontal health or periodontitis. The outputs of most studies could, nevertheless, be slightly biased from following two reasons: First, the healthy group is often characterized only by the absence of the disease, but the individuals could already suffer from dysbiosis without any visible signs. Second, the healthy/diseased OM characteristics are frequently determined based on average data obtained for whole groups of periodontally healthy persons versus patients. Especially in smaller sets of tested individuals the typical individual variability can thus complicate the unambiguous assignment of oral taxa to respective state of health. In this work the taxonomic composition of OM was evaluated for 20 periodontally healthy individuals and 15 patients with chronic periodontitis. The narrowed selection set of the most diseased patients (confirmed by clinical parameters) and the most distant group of healthy individuals with the lowest probability of dysbiosis was determined by clustering analysis and used for identification of marker taxa. Based on their representation in each individual oral cavity we proposed the numeric index of periodontal health called R/G value. Its diagnostic potential was further confirmed using independent set of 20 periodontally healthy individuals and 20 patients with periodontitis with 95 percent of samples assigned correctly. We also assessed the individual temporal OM dynamics in periodontal health and we compared it to periodontitis. We revealed that the taxonomic composition of the system changes dynamically but generally it ranges within values typical for periodontal health or transient state, but far from values typical for periodontitis. R/G value tool, formulated from individually evaluated data, allowed us to arrange individual OMs into a continuous series, instead of two distinct groups, thus mimicking the gradual transformation of a virtual person from periodontal health to disease. The application of R/G value index thus represents a very promising diagnostic tool for early prediction of persons at risk of developing periodontal disease.

• MeSH
• chronická parodontitida * MeSH
• dysbióza MeSH
• lidé MeSH
• mikrobiota * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lincosamides are clinically important antibiotics originally produced as microbial specialized metabolites. The complex biosynthesis of lincosamides is coupled to the metabolism of mycothiol as a sulfur donor. Here, we elucidated the N-deacetylation of the mycothiol-derived N-acetyl-l-cysteine residue of a lincosamide intermediate, which is comprised of an amino acid and an aminooctose connected via an amide bond. We purified this intermediate from the culture broth of a deletion mutant strain and tested it as a substrate of recombinant lincosamide biosynthetic proteins in the in vitro assays that were monitored via liquid chromatography-mass spectrometry. Our findings showed that the N-deacetylation reaction is catalyzed by CcbIH/CcbQ or LmbIH/LmbQ proteins in celesticetin and lincomycin biosynthesis, respectively. These are the first N-deacetylases from the TldD/PmbA protein family, from which otherwise only several proteases and peptidases were functionally characterized. Furthermore, we present a sequence similarity network of TldD/PmbA proteins, which suggests that the lincosamide N-deacetylases are unique among these widely distributed proteins.

• MeSH
• acetylace MeSH
• bakteriální proteiny metabolismus   MeSH
• databáze proteinů MeSH
• katalýza MeSH
• linkosamidy biosyntéza   MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH

A robust and widely applicable method for sampling of aquatic microbial biofilm and further sample processing is presented. The method is based on next-generation sequencing of V4-V5 variable regions of 16S rRNA gene and further statistical analysis of sequencing data, which could be useful not only to investigate taxonomic composition of biofilm bacterial consortia but also to assess aquatic ecosystem health. Five artificial materials commonly used for biofilm growth (glass, stainless steel, aluminum, polypropylene, polyethylene) were tested to determine the one giving most robust and reproducible results. The effect of used sampler material on total microbial composition was not statistically significant; however, the non-plastic materials (glass, metal) gave more stable outputs without irregularities among sample parallels. The bias of the method is assessed with respect to the employment of a non-quantitative step (PCR amplification) to obtain quantitative results (relative abundance of identified taxa). This aspect is often overlooked in ecological and medical studies. We document that sequencing of a mixture of three merged primary PCR reactions for each sample and further evaluation of median values from three technical replicates for each sample enables to overcome this bias and gives robust and repeatable results well distinguishing among sampling localities and seasons.

• MeSH
• Bacteria klasifikace   genetika   MeSH
• biofilmy * růst a vývoj   MeSH
• mikrobiologie vody * MeSH
• mikrobiota genetika   MeSH
• monitorování životního prostředí metody   MeSH
• odběr biologického vzorku MeSH
• reprodukovatelnost výsledků MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA * MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

New sulfonamides 5/6 derived from 4-methoxyacetophenone 1 were synthesized by N-sulfonation reaction of ammonia (3) and aminopyrimidinone (4) with its sulfonyl chloride derivative 2. Sulfonamides 5 and 6 were used as precursors of two new series of chalcones 8a-f and 9a-f, which were obtained through Claisen-Schmidt condensation with aromatic aldehydes 7a-f. Compounds 5/6, 8a-d, 8f, 9a-d, and 9f were screened by the US National Cancer Institute (NCI) at 10 μM against sixty different human cancer cell lines (one-dose trial). Chalcones 8b and 9b satisfied the pre-determined threshold inhibition criteria and were selected for screening at five different concentrations (100, 10, 1.0, 0.1, and 0.01 μM). Compound 8b exhibited remarkable GI50 values ranging from 0.57 to 12.4 μM, with cytotoxic effects being observed in almost all cases, especially against the cell lines K-562 of Leukemia and LOX IMVI of Melanoma with GI50 = 0.57 and 1.28 μM, respectively. Moreover, all compounds were screened against Mycobacterium tuberculosis H37Rv, chalcones 8a-c and 9a-c were the most active showing MIC values between 14 and 42 μM, and interestingly they were devoid of antibacterial activity against Mycobacterium smegmatis and Staphylococcus aureus. These antituberculosis hits showed however low selectivity, being equally inhibitory to M. tuberculosis and mammalian T3T cells. The chalcone-sulfonamide hybrids 8a-f and 9a-f resulted to be appealing cytotoxic agents with significant antituberculosis activity.

• MeSH
• antituberkulotika chemická syntéza   chemie   farmakologie   toxicita   MeSH
• buňky 3T3 MeSH
• chalkonoidy chemická syntéza   chemie   farmakologie   toxicita   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• Mycobacterium tuberculosis účinky léků   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• protinádorové látky chemická syntéza   chemie   farmakologie   toxicita   MeSH
• screeningové testy protinádorových léčiv MeSH
• sulfonamidy chemická syntéza   chemie   farmakologie   toxicita   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• anthramycin chemie   MeSH
• bakteriální proteiny chemie   MeSH
• katalytická doména MeSH
• kyseliny fenylpyrohroznové chemie   MeSH
• linkomycin chemie   MeSH
• metylace MeSH
• molekulární struktura MeSH
• mutace MeSH
• prolin chemie   MeSH
• Streptomyces chemie   MeSH
• uhlík chemie   MeSH
• vazba proteinů MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH