Three-Component Glycosylation of Transient Hemiacetals Toward Tunable Aryl-Bisacetal Substrates.

An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems

EXPERIENCE OF THE DATABASE SCIENCE ONLINE USAGE IN MONITORING OF THE RESEARCH ACTIVITY OF THE UNIVERSITYLesovoy V.N., Kapustnyk V.A., Hetman V.A., Myasoedov V.V.Kharkiv National Medical University, UkraineAbstract. The article describes the experience of the online database Science Online usage in monitoring of research activity of the research and educational staff of the University and the impact of this rating on the effectiveness of their scientific work. Rating evaluation is a type of quantitative and qualitative monitoring of the research activity carried out by teaching staff.Key words: rating of research and educational staff activity, adapted information system, monitoring of scientific activity. Досвід використання інтерактивної бази Наука онлайн у моніторингу наукової діяльності вищого навчального закладуЛісовий В.М., Капустник В.А., Гетьман В.О., М’ясоєдов В.В.Харківський національний медичний університет, УкраїнаРезюме. У статті розглядається досвід використання інтерактивної бази Наука онлайн у моніторингу наукової діяльності викладачів та вплив рейтингового оцінювання діяльності науково­педагогічнихпрацівників університету на ефективність їхньої наукової роботи. Рейтингове оцінювання є одним з видів кількісного та якісного моніторингу діяльності викладача. Ключові слова: рейтингове оцінювання діяльності викладача, адаптована інформаційна система, моніторинг наукової діяльності. Опыт использования интерактивной базы Наука онлайн в мониторинге научной деятельности вузаЛесовой В.Н., Капустник В.А., Гетьман В.А., Мясоедов В.В.Харьковский национальный медицинский университет, УкраинаРезюме.В статье рассматривается опыт использования интерактивной базы Наука онлайн в мониторинге научной деятельности преподавателей и влияние рейтинговой оценки деятельности научно-педагогических работников университета на эффективность их научной работы. Рейтинговая оценка является одним из видов количественного и качественного мониторинга деятельности преподавателя.Ключевые слова:рейтинговая оценка деятельности преподавателя, адаптируемая информационная система, мониторинг научной деятельности.

Heavy Water Labeling of Keratin as a Non-Invasive Biomarker of Skin Turnover In Vivo in Rodents and Humans

roteolytic enzymes are molecular scissors that are responsible for the amide bond breakdown in peptide and protein substrates. Over the years, the view on proteases has been considerably changed from non-specific digestive enzymes to sophisticated biocatalysts, which by performing limited proteolysis control virtually all biological processes. In order to better understand how proteases work and what are their biologically relevant target substrates, it is indispensable to determine their catalytic preferences. This knowledge can be further utilized to develop selective substrates, inhibitors and activity-based probes (ABPs) enabling the monitoring of proteases activity in various settings, from in vitro analysis on recombinant enzymes or cell lysates to ex vivo and in vivo imaging at the single cell level. Among many chemical-based approaches that have been developed and applied over the years, the Hybrid Combinatorial Substrate Library (HyCoSuL) technology has emerged as one of the most powerful one. HyCoSuL is a combinatorial peptide-based library of fluorogenic substrates, that comprise natural and unnatural amino acids, that can deeply explore the chemical space in proteases active site, providing a structural framework for the development of highly-selective chemical tools. In this review we present the most prominent examples of proteolytic enzymes that have been profiled with HyCoSuL approach yielding selective substrates, potent inhibitors, and very sensitive activity-based probes.

Monitoring in situ microbial activity in anoxic submerged soils and aquatic sediments can be labor intensive and technically difficult, especially in dynamic environments in which a record of changes in microbial activity over time is desired. Microbial fuel cell concepts have previously been adapted to detect changes in the availability of relatively high concentrations of organic compounds in waste water but, in most soils and sediments, rates of microbial activity are not linked to the concentrations of labile substrates, but rather to the turnover rates of the substrate pools with steady state concentrations in the nM–μM range. In order to determine whether levels of current produced at a graphite anode would correspond to the rates of microbial metabolism in anoxic sediments, small graphite anodes were inserted in sediment cores and connected to graphite brush cathodes in the overlying water. Currents produced were compared with the rates of [2-14C]-acetate metabolism. There was a direct correlation between current production and the rate that [2-14C]-acetate was metabolized to 14CO2 and 14CH4 in sediments in which Fe(III) reduction, sulfate reduction, or methane production was the predominant terminal electron-accepting process. At comparable acetate turnover rates, currents were higher in the sediments in which sulfate-reduction or Fe(III) reduction predominated than in methanogenic sediments. This was attributed to reduced products (Fe(II), sulfide) produced at distance from the anode contributing to current production in addition to the current that was produced from microbial oxidation of organic substrates with electron transfer to the anode surface in all three sediment types. The results demonstrate that inexpensive graphite electrodes may provide a simple strategy for real-time monitoring of microbial activity in a diversity of anoxic soils and sediments.

A passive, resonant sensor was developed that can be embedded in closed systems for wireless monitoring of hydrolytic enzyme activity. The resonators are rapidly prototyped from copper coated polyimide substrates that are masked using an indelible marker with an XY plotter and subsequently etched. The resonator's frequency response window is designed by the Archimedean coil length and pitch and is tuned for the 1-100 MHz range for better penetration through soil, water, and tissue. The resonant frequency is measured up to 5 cm stand-off distance by a coplanar, two-loop coil reader antenna attached to a vector network analyzer monitoring the S21 scattering parameter. The resonant frequency is modulated (up to 50 MHz redshift) by changing the relative permittivity of the medium in contact with the resonator (e.g., air to water). The resonant sensors are coated by an enzyme substrate, which, when degraded, causes a change in dielectric and a shift in resonant frequency (up to 7 MHz redshift). The activity (turnover rate, or kcat) of the enzyme is calculated by fitting the measured data via a custom transport and reaction model which simulates the radial digestion profile. This is used to test purified Subtilisin A and unpurified bacterial protease samples at concentrations of 30 mg/mL to 200 mg/mL with kcat ranges of 0.003-0.002 and 0.008-0.004 gsubstrate/ genzyme per second. The sensor response rate can be tuned by substrate composition (e.g., gelatin and glycerol plasticizer weight percentage). Finally, the utility of these sensors is demonstrated by wirelessly measuring the proteolytic activity of farm soil with a measured kcat of 0.00152 gsubstrate/( gsoil·s).

We investigated the influence of tillage (conventional, minimum and reduced) on selected soil microbial properties of a fine-sandy loamy Haplic Chernozem over a period of 8 years. The microbial biomass and soil microbial processes were affected mostly by type of tillage and to a lesser extent by the date of soil sampling. Whereas xylanase activity was significantly higher in the 0 to 10-cm soil layer of the reduced and minimum tillage systems within the first year of the experiment (protease and phosphatase activities were significantly higher in the second year), significant treatment effects on microbial biomass, N mineralisation and potential nitrification were observed after a 4-year period. The slow response of substrate-induced respiration to the change in type of tillage may have been due to the differences in the biomass C turnover rates. After a 4-year period, the stratification of the soil microbial biomass within the profile of reduced and minimum tillage systems was probably responsible for the more intensive soil microbial processes near the soil surface compared with conventional tillage. In the 20 to 30-cm layer, N mineralisation, potential nitrification and xylanase activity in the conventional treatment were significantly higher than in the minimum and reduced tillage plots due to buried organic materials. Discriminant analysis underlined the similarity of the enzyme activity patterns in the top layer of the reduced and minimum tillage treatments, and in both layers of the conventional tillage system. The trend towards a significant increase in functional diversity caused by reduced tillage became obvious within the first year of the experiment, and this effect was still manifest after 8 years. All relationships suggested that there were differences in available resources (e.g. organic matter) along the sequence of different tillage systems; this was reflected in part by enhanced enzymatic and microbial activities in the soil layers. In conclusion, this study showed that soils affected by tillage may be classified on the basis of their functional diversity. Therefore, the soil microbial properties chosen for microbiological soil monitoring (microbial biomass, N mineralisation and enzyme activities involved in C, N and P cycling) provide a reliable tool with which to estimate early changes in the dynamics and distribution of soil microbial processes within soil profiles.

We report the development and characterization of digital microfluidic (DMF) immobilized enzyme reactors (IMERs) for studying cytochrome P450 (CYP)-mediated drug metabolism on droplet scale. The on-chip IMERs consist of porous polymer (thiol-ene) monolith plugs prepared in situ by photopolymerization and functionalized with recombinant CYP1A1 isoforms (an important detoxification route for many drugs and other xenobiotics). The DMF devices also incorporate inexpensive, inkjet-printed microheaters for on-demand regio-specific heating of the IMERs to physiological temperature, which is crucial for maintaining the activity of the temperature-sensitive CYP reaction. For on-chip monitoring of the CYP activity, the DMF devices were combined with a commercial well-plate reader, and a custom fluorescence quantification method was developed for detection of the chosen CYP1A1 model activity (ethoxyresorufin-O-deethylation). The reproducibility of the developed assay was examined with the help of ten parallel CYP-IMERs. All CYP-IMERs provided statistically significant difference (in fluorescence response) compared to any of the negative controls (including room-temperature reactions). The average (n = 10) turnover rate was 20.3 ± 9.0 fmol resorufin per minute. Via parallelization, the concept of the droplet-based CYP-IMER developed in this study provides a viable approach to rapid and low-cost prediction of the metabolic clearance of new chemical entities in vitro.

In order to control tuberculosis, the Brazilian Ministry of Health recommends the decentralization of control actions directed to the Primary Health Care, and there are few studies on the performance of the Tuberculosis Control Program in decentralized contexts. To evaluate the performance of Primary Health Care services in tuberculosis treatment. This is an evaluative study with cross-sectional approach conducted in 2011. Two hundred and thirty-nine health professionals from Primary Health Care units were interviewed using a structured instrument based on the evaluation reference of the health services quality (structure - process - results). The performance of these services was analyzed applying techniques of descriptive statistics, validation, and construction of indicators and by determining the reduced variable "Z". The indicators "participation of professionals in tuberculosis patients' care" (structure) and "reference and counterreference" (process) had the best evaluations, whereas "professional training" (structure) and "external actions for tuberculosis control" (process) had the worst results. The decentralization of tuberculosis control actions has been taking place in a vertical manner in Primary Health Care. The challenge of controlling tuberculosis involves overcoming constraints related to the engagement, training, and turnover rates among health professionals, which is a coordination between services and monitoring of control actions in Primary Health Care.

A shuffled CYP2C library with a high degree of structural integrity and functional versatility

The role of Phe213 in the allosteric mechanism of human cytochrome P450 CYP3A4 was studied using a combination of progesterone (PGS) and carbamazepine (CBZ) as probe substrates. We expressed, purified, and incorporated into POPC Nanodiscs three mutants, F213A, F213S, and F213Y, and compared them with wild-type (WT) CYP3A4 by monitoring spectral titration, the rate of NADPH oxidation, and steady-state product turnover rates with pure substrates and substrate mixtures. All mutants demonstrated higher activity with CBZ, lower activity with PGS, and a reduced level of activation of CBZ epoxidation by PGS, which was most pronounced in the F213A mutant. Using all-atom molecular dynamics simulations, we compared the dynamics of WT CYP3A4 and the F213A mutant incorporated into the lipid bilayer and the effect of the presence of the PGS molecule at the allosteric peripheral site and evaluated the critical role of Phe213 in mediating the heterotropic allosteric interactions in CYP3A4.

The ability to measure changes in neuronal activity in a quantifiable and precise manner is of fundamental importance to understand neuron development and function. Repeated monitoring of neuronal activity of the same population of neurons over several days is challenging and, typically, low-throughput. Here, we describe a new biochemical reporter assay that allows for repeated measurements of neuronal activity in a cell type-specific manner. We coupled activity-dependent elements from the Arc/Arg3.1 gene with a secreted reporter, Gaussia luciferase (Gluc), to quantify neuronal activity without sacrificing the neurons. The reporter predominantly senses calcium and NMDA receptor (NMDAR)-dependent activity. By repeatedly measuring the accumulation of the reporter in cell media, we can profile the developmental dynamics of neuronal activity in cultured neurons from male and female mice. The assay also allows for longitudinal analysis of pharmacological treatments, thus distinguishing acute from delayed responses. Moreover, conditional expression of the reporter allows for monitoring cell type-specific changes. This simple, quantitative, cost-effective, automatable, and cell type-specific activity reporter is a valuable tool to study the development of neuronal activity in normal and disease-model conditions, and to identify small molecules or protein factors that selectively modulate the activity of a specific population of neurons.

In vitro cardiac tissue model holds great potential as a powerful platform for drug screening. Respiratory activity, contraction frequency, and extracellular H2O2 levels are the three key parameters for determining the physiological functions of cardiac tissues, which are technically challenging to be monitored in an in situ and quantitative manner. Herein, we constructed an in vitro cardiac tissue model on polyacrylamide gels and applied a pulsatile electrical field to promote the maturation of the cardiac tissue. Then, we built a scanning electrochemical microscopy (SECM) platform with programmable pulse potentials to in situ characterize the dynamic changes in the respiratory activity, contraction frequency, and extracellular H2O2 level of cardiac tissues under both normal physiological and drug (isoproterenol and propranolol) treatment conditions using oxygen, ferrocenecarboxylic acid (FcCOOH), and H2O2 as the corresponding redox mediators. The SECM results showed that isoproterenol treatment induced enhanced oxygen consumption, accelerated contractile frequency, and increased released H2O2 level, while propranolol treatment induced dynamically decreased oxygen consumption and contractile frequency and no obvious change in H2O2 levels, suggesting the effects of activation and inhibition of β-adrenoceptor on the metabolic and electrophysiological activities of cardiac tissues. Our work realizes the in situ and quantitative monitoring of respiratory activity, contraction frequency, and secreted H2O2 level of living cardiac tissues using SECM for the first time. The programmable SECM methodology can also be used to real-time and quantitatively monitor electrochemical and electrophysiological parameters of cardiac tissues for future drug screening studies.

The analysis of receptor activity, especially in its native cellular environment, has always been of great interest to evaluate its intrinsic but also downstream biological activity. An important group of cellular receptors are ion channels. Since they are involved in a broad range of crucial cell functions, they represent important therapeutic targets. Thus, novel analytical techniques for the quantitative monitoring and screening of biological receptor activity are of great interest. In this context, we developed an impedance spectroscopy-based label-free and non-invasive monitoring system that enabled us to analyze the activation of the transient receptor potential channel Vanilloid 1 (TRPV1) in detail. TRPV1 channel activation by capsaicin resulted in a reproducible impedance decrease. Moreover, concentration response curves with an EC50 value of 0.9μM could be determined. Control experiments with non TRPV1 channel expressing HEK cells as well as experiments with the TRPV1 channel blocker ruthenium red validated the specificity of the observed impedance decrease. More strikingly, through correlative studies with a cytoskeleton restructuring inhibitor mixture and equivalent circuit analysis of the acquired impedance spectra, we could quantitatively discriminate between the direct TRPV1 channel activation and downstream-induced biological effects. In summary, we developed a quantitative impedimetric monitoring system for the analysis of TRPV1 channel activity as well as downstream-induced biological activity in living cells. It has the capabilities to identify novel ion channel activators as well as inhibitors for the TRPV1 channel but could also easily be applied to other ion channel-based receptors.

Parasites have had a significant impact on domestic ruminant health and production for a long time, but the emerging threat of drug resistance urgently requires an improved approach to parasite monitoring and control activities. The study reviewed the international literature to analyze the different proposals for the sampling approach and the quantitative estimation of parasite burdens in groups of animals. Moreover, the use of thresholds to decide when and which animal to treat was also investigated. The findings of the study highlighted the presence of a wide-ranging literature on quantitative monitoring for gastrointestinal nematodes (GIN), while more limited data were found for coccidia, and no specific indications were reported for tapeworms. Concerning liver flukes, bronchopulmonary nematodes (BPN) and permanent ectoparasites (lice and mange mites), the diagnostic process is usually aimed at the detection of the parasite rather than at the burden estimation. The main research gaps that need further investigation were also highlighted. For some groups of parasites (e.g., GIN and coccidia) the quantitative approach requires an improved standardization, while its usefulness needs to be confirmed for others (e.g., BPN and lice). The development of practical guidelines for monitoring is also encouraged.