Utilization of Carrier-Frequency Offset Measurements in UWB TDoA Positioning with Receiving Tag.

High-capacity impulse-radio ultra-wideband (IR-UWB) indoor localization systems are typically based on the time difference of arrival (TDoA) principle. When the fixed and synchronized localization infrastructure, the anchors, transmit precisely timestamped messages, a virtually unlimited number of user receivers (tags) are able to estimate their position based on differences in the time of arrival of those messages. However, the drift of the tag clock causes systematic errors at a sufficiently high magnitude to effectively deny the positioning, if left uncorrected. Previously, the extended Kalman filter (EKF) has been used to track and compensate for the clock drift. In this article, the utilization of a carrier frequency offset (CFO) measurement for suppressing the clock-drift related error in anchor-to-tag positioning is presented and compared to the filtered solution. The CFO is readily available in the coherent UWB transceivers, such as Decawave DW1000. It is inherently related to the clock drift, since both carrier and timestamping frequencies are derived from the identical reference oscillator. The experimental evaluation shows that the CFO-aided solution performs worse than the EKF-based solution in terms of accuracy. Nonetheless, with CFO-aiding it is possible to obtain a solution based on measurements from a single epoch, which is favorable especially for power-constrained applications.

Crucial moments in Czechoslovak radiobiology history.

If we consider the development of any specialization in our region in an historical context, we cannot separate the Czech and Slovak Republics. The 75 years together (with the exception of 1939-1945) were a period of narrow cooperation between Czech and Slovak Universities and Scientific Institutes, and Radiobiology is no exception, which is why the discussion falls to Czechoslovak Radiobiology, as opposed to Czech Radiobiology alone. Czechoslovak Radiobiology has gone through three important stages in its history, influenced also by international political-military situations. Here it is necessary to emphasize that the results obtained by Czechoslovak Radiobiology were and are comparable with foreign institutions. The first period, which dates back to 1895-1939, represents the initial discovery period of the mechanism and consequences of ionizing radiation on the body. The second period in 1939-1990 is considered to be the phase of peaceful uses of nuclear energy, their misuse for military purposes, along with detailed studies of radiation sickness and radiation protection, the majority of the results which were kept secret and could not be published at the time. The third period after 1990 is dedicated to the detailed study of post-radiation changes at the intracellular level with the aim to be used especially in radiotherapy and radiation protection, as well as the dangers of misuse of ionizing radiation by terrorists. Increased attention is also devoted to the effect of ionizing radiation on plants and their possible subsequent use.

Investigation of flexibility of neuraminidase 150-loop using tamiflu derivatives in influenza A viruses H1N1 and H5N1.

This study focuses on design, synthesis and in vitro evaluation of inhibitory potency of two series of sialylmimetic that target an exosite ("150-cavity") adjacent to the active site of influenza neuraminidases from A/California/07/2009 (H1N1) pandemic strain and A/chicken/Nakorn-Patom/Thailand/CU-K2-2004 (H5N1). The structure-activity analysis as well as 3-D structure of the complex of parental compound with the pandemic neuraminidase p09N1 revealed high flexibility of the 150-cavity towards various modification of the neuraminidase inhibitors. Furthermore, our comparison of two methods for inhibition constant determination performed at slightly different pH values suggest that the experimental conditions of the measurement could dramatically influence the outcome of the analysis in the compound-dependent manner. Therefore, previously reported Ki values determined at non-physiological pH should be carefully scrutinized.

DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors.

Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.

DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening.

Human diseases are often diagnosed by determining levels of relevant enzymes and treated by enzyme inhibitors. We describe an assay suitable for both ultrasensitive enzyme quantification and quantitative inhibitor screening with unpurified enzymes. In the DNA-linked Inhibitor ANtibody Assay (DIANA), the target enzyme is captured by an immobilized antibody, probed with a small-molecule inhibitor attached to a reporter DNA and detected by quantitative PCR. We validate the approach using the putative cancer markers prostate-specific membrane antigen and carbonic anhydrase IX. We show that DIANA has a linear range of up to six logs and it selectively detects zeptomoles of targets in complex biological samples. DIANA's wide dynamic range permits determination of target enzyme inhibition constants using a single inhibitor concentration. DIANA also enables quantitative screening of small-molecule enzyme inhibitors using microliters of human blood serum containing picograms of target enzyme. DIANA's performance characteristics make it a superior tool for disease detection and drug discovery.

Structural and biochemical characterization of a novel aminopeptidase from human intestine.

N-acetylated α-linked acidic dipeptidase-like protein (NAALADase L), encoded by the NAALADL1 gene, is a close homolog of glutamate carboxypeptidase II, a metallopeptidase that has been intensively studied as a target for imaging and therapy of solid malignancies and neuropathologies. However, neither the physiological functions nor structural features of NAALADase L are known at present. Here, we report a thorough characterization of the protein product of the human NAALADL1 gene, including heterologous overexpression and purification, structural and biochemical characterization, and analysis of its expression profile. By solving the NAALADase L x-ray structure, we provide the first experimental evidence that it is a zinc-dependent metallopeptidase with a catalytic mechanism similar to that of glutamate carboxypeptidase II yet distinct substrate specificity. A proteome-based assay revealed that the NAALADL1 gene product possesses previously unrecognized aminopeptidase activity but no carboxy- or endopeptidase activity. These findings were corroborated by site-directed mutagenesis and identification of bestatin as a potent inhibitor of the enzyme. Analysis of NAALADL1 gene expression at both the mRNA and protein levels revealed the small intestine as the major site of protein expression and points toward extensive alternative splicing of the NAALADL1 gene transcript. Taken together, our data imply that the NAALADL1 gene product's primary physiological function is associated with the final stages of protein/peptide digestion and absorption in the human digestive system. Based on these results, we suggest a new name for this enzyme: human ileal aminopeptidase (HILAP).

iBodies: Modular Synthetic Antibody Mimetics Based on Hydrophilic Polymers Decorated with Functional Moieties.

Antibodies are indispensable tools for biomedicine and anticancer therapy. Nevertheless, their use is compromised by high production costs, limited stability, and difficulty of chemical modification. The design and preparation of synthetic polymer conjugates capable of replacing antibodies in biomedical applications such as ELISA, flow cytometry, immunocytochemistry, and immunoprecipitation is reported. The conjugates, named "iBodies", consist of an HPMA copolymer decorated with low-molecular-weight compounds that function as targeting ligands, affinity anchors, and imaging probes. We prepared specific conjugates targeting several proteins with known ligands and used these iBodies for enzyme inhibition, protein isolation, immobilization, quantification, and live-cell imaging. Our data indicate that this highly modular and versatile polymer system can be used to produce inexpensive and stable antibody substitutes directed toward virtually any protein of interest with a known ligand.

[Preparedness of health system in Israel for mass emergencies]. / Pripravenost izraelského zdravotnictví na krizové situace.

The vast number of outbreaks of tension on the planet shows that it is impossible to underestimate the preparation level of population protection in emergencies. The possibility of a terrorist attack, including the use of particularly toxic or biological substances cannot be excluded or totally prevented at this point. In fact, there may not only be a terrorist attack. Rampant population migration increases the risk of the transmission of infectious diseases, even to considerable distances from the states where the epidemiological situation might not be completely under control. The current state of the Czech healthcare system in terms of preparedness for mass emergencies is insufficient and requires prompt correction, though not through hastily adopted measures. Ideally, looking into the success of the Israeli preparedness system, where the public has been exposed to high levels of threat from a variety of causes for decades, could greatly aid the Czech Republic in moving forward effectively. The number of victims of terrorist attacks there outpaces 10000, a fact that shows Israel is experienced in responding to emergency incidents.

Detection and quantitation of glutamate carboxypeptidase II in human blood.

BACKGROUND: Glutamate carboxypeptidase II (GCPII) is a transmembrane enzyme that cleaves N-acetyl-L-aspartyl-L-glutamate (NAAG) in the brain. GCPII is highly expressed in the prostate and prostate cancer and might be associated with prostate cancer progression. Another exopeptidase, plasma glutamate carboxypeptidase (PGCP), was reported to be similar to GCPII and to share its NAAG-hydrolyzing activity. METHODS: We performed a radioenzymatic assay with [(3) H]NAAG as a substrate to detect and quantify the enzymatic activity of GCPII in plasma. Using a specific antibody raised against native GCPII (2G7), we immunoprecipitated GCPII from human plasma. We also cloned two PGCP constructs, expressed them in insect cells, and tested them for their NAAG-hydrolyzing activity. RESULTS: We detected GCPII protein in human plasma and found that its concentration ranges between 1.3 and 17.2 ng/ml in volunteers not diagnosed with prostate cancer. Recombinant PGCP was enzymatically active but exhibited no NAAG-hydrolyzing activity. CONCLUSION: GCPII is present in human blood, and its concentration within a healthy population varies. Recombinant PGCP does not hydrolyze NAAG, suggesting that GCPII alone is responsible for the NAAG-hydrolyzing activity observed in human blood. The potential correlation between GCPII serum levels and the disease status of prostate cancer patients will be further investigated.

Comprehensive treatment of temporomandibular joint disorders.

AIMS: Changing lifestyles, decreasing physical activity, which is increasing the number of degenerative joint diseases of various etiology, and certain dental procedures are increasing the number of patients complaining of pain in their temporomandibular joints. The aim of the study was to assess the benefits of comprehensive physiotherapy sessions in order to decrease the number of temporomandibular joint problems, thereby improving the patient's quality of life. METHODOLOGY: An examination by a dentist determined each patient's treatment plan, which consisted of a medical exam, physical therapy and education. Each form of treatment was applied 10 times at intervals of 7-14 days. The main goal of the therapeutic physical education was to redress the muscle imbalance in the mandibular joint. This was achieved by restoring balance between the masticatory muscles, along with releasing the spastic shrouds found in the masticatory muscles. The aim of education was to teach the patient exercises focused on the temporomandibular joint and masticatory muscles. The intensity of the exercises and their composition were individually adjusted and adapted to their current state. Physical therapy consisted of the application of pulsed magnetic therapy, laser therapy, and non-invasive positive thermotherapy. RESULTS: The above procedure was conducted on a therapeutic group of 24 patients (3 men and 20 women). In the course of therapy, there were no complications, and all patients adhered to the prescribed regime. None reported any side effects. The mean treatment duration was 123 +/- 66 days. The outcome of the therapy was evaluated as described in the methodology, the degree of pain affecting the joint, and the opening ability of the mouth. In both parameters, there was a significant decline in patient pain. CONCLUSIONS: In a study devoted to tactics of rehabilitation treatment for temporomandibular joint disorders, the need for comprehensive long-term therapy, involving education, and learning proper chewing habits was made apparent for recovery and pain reduction. A priority in physical therapy, and combinations of pulsed magnetic therapy and hyperthermia-positive peloids, are also beneficial.

Theoretical aspects of hydrolysis of peptide bonds by zinc metalloenzymes.

Activation and reaction energies for four model systems capturing the essential physicochemical features of the hydrolysis of the peptide bond have been calculated at various level of theory, including the presumably accurate CCSD(T) calculations. The models studied covered a part of the spectrum encountered in biological systems: the hydrolysis in the absence of metal ions (represented by formamide and Ala-Ala) and the hydrolysis in the presence of one and two zinc(II) ions, mimicking the active sites of mono- and dizinc metallopeptidases, respectively (by using thermolysin and glutamate carboxypeptidase II as the model catalytic systems and formamide as the model substrate). The results obtained using CCSD(T)/def2-TZVP and CCSD(T)/aug-cc-pVTZ calculations were used as the benchmark values to which the set of cheaper methods, such as (RI-)DFT, (RI-)MP2, and SCS-MP2, were referenced. It was shown that deviations of 3-5 kcal mol(-1) (translating to 2-3 orders in reaction constants) with respect to the reference CCSD(T) barriers are frequently encountered for many correlated methods and most of studied DFT functionals. It has been concluded that from the set of wave-function methods, both MP2 and SCS-MP2 methods can be recommended for smaller models (measured by the mean absolute deviation of the activation barriers over the four systems studied), whereas among the popular DFT functionals, B3LYP and especially M06-2X are likely to be reasonable choices for calculating the activation barriers of zinc metallopeptidases. Finally, with the model of glutamate carboxypeptidase II, issues related to the convergence of the calculated barriers with the size of the model system used as the representative of the enzyme active site were addressed. The intricacies related to system truncation are demonstrated, and suggest that the correlated wave-function methods may suffer from problems, such as intramolecular BSSE, which make their usage for the larger system questionable. Altogether, the presented data should contribute to efforts to understand enzymatic catalysis more deeply and to gain control of the accuracy and deficiencies of the available theoretical methods and computational approaches.

Predicting Effects of Site-Directed Mutagenesis on Enzyme Kinetics by QM/MM and QM Calculations: A Case of Glutamate Carboxypeptidase II.

Quantum and molecular mechanics (QM/MM) and QM-only (cluster model) modeling techniques represent the two workhorses in mechanistic understanding of enzyme catalysis. One of the stringent tests for QM/MM and/or QM approaches is to provide quantitative answers to real-world biochemical questions, such as the effect of single-point mutations on enzyme kinetics. This translates into predicting the relative activation energies to 1-2 kcal·mol-1 accuracy; such predictions can be used for the rational design of novel enzyme variants with desired/improved characteristics. Herein, we employ glutamate carboxypeptidase II (GCPII), a dizinc metallopeptidase, also known as the prostate specific membrane antigen, as a model system. The structure and activity of this major cancer antigen have been thoroughly studied, both experimentally and computationally, which makes it an ideal model system for method development. Its reaction mechanism is quite well understood: the reaction coordinate comprises a "tetrahedral intermediate" and two transition states and experimental activation Gibbs free energy of ∼17.5 kcal·mol-1 can be inferred for the known kcat ≈ 1 s-1. We correlate experimental kinetic data (including the E424H variant, newly characterized in this work) for various GCPII mutants (kcat = 8.6 × 10-5 s-1 to 2.7 s-1) with the energy profiles calculated by QM/MM and QM-only (cluster model) approaches. We show that the near-quantitative agreement between the experimental values and the calculated activation energies (ΔH⧧) can be obtained and recommend the combination of the two protocols: QM/MM optimized structures and cluster model (QM) energetics. The trend in relative activation energies is mostly independent of the QM method (DFT functional) used. Last but not least, a satisfactory correlation between experimental and theoretical data allows us to provide qualitative and fairly simple explanations of the observed kinetic effects which are thus based on a rigorous footing.

Factors, associated with elevated concentration of soluble carbonic anhydrase IX in plasma of women with cervical dysplasia.

Precancerous lesions of human cervix uteri have a tendency for regression or progression. In cervical intraepithelial neoplasia grade 2 (CINII) case there is an uncertainty if a lesion will progress or regress. The carbonic anhydrase IX (CAIX) enzyme is overexpressed in cervical cancer which is more sensitive to radiotherapy. CAIX is associated with poor prognosis in solid hypoxic tumors. The aim of this study was to determine factors related to elevated soluble CAIX (s-CAIX) in high-grade intraepithelial lesion (HSIL) cases. METHODS: Patients diagnosed with HSIL (N = 77) were included into the research group whereas without HSIL (N = 72)-the control group. Concentration of the soluble CAIX (s-CAIX) in plasma was determined by the DIANA ligand-antibody-based method. C. trachomatis was detected from cervical samples by PCR. Primary outcomes were risk factors elevating s-CAIX level in HSIL group. Non-parametric statistical analysis methods were used to calculate correlations. RESULTS: The s-CAIX level in patients with HSIL was elevated among older participants (rs = 0.27, p = 0.04) and with C. trachomatis infection (p = 0.028). Among heavy smokers with HSIL, the concentration of s-CAIX was higher in older women (rs = 0.52, p = 0.005), but was not related to the age of heavy smokers' controls (τ = 0.18 p = 0.40). CONCLUSION: The concentration of s-CAIX was higher among older, heavy smoking and diagnosed with C. trachomatis patients. All these factors increased the risk for HSIL progression.

Identification of Novel Carbonic Anhydrase IX Inhibitors Using High-Throughput Screening of Pooled Compound Libraries by DNA-Linked Inhibitor Antibody Assay (DIANA).

The DNA-linked inhibitor antibody assay (DIANA) has been recently validated for ultrasensitive enzyme detection and for quantitative evaluation of enzyme inhibitor potency. Here we present its adaptation for high-throughput screening of human carbonic anhydrase IX (CAIX), a promising drug and diagnostic target. We tested DIANA's performance by screening a unique compound collection of 2816 compounds consisting of lead-like small molecules synthesized at the Institute of Organic Chemistry and Biochemistry (IOCB) Prague ("IOCB library"). Additionally, to test the robustness of the assay and its potential for upscaling, we screened a pooled version of the IOCB library. The results from the pooled screening were in agreement with the initial nonpooled screen with no lost hits and no false positives, which shows DIANA's potential to screen more than 100,000 compounds per day.All DIANA screens showed a high signal-to-noise ratio with a Z' factor of >0.89. The DIANA screen identified 13 compounds with Ki values equal to or better than 10 µM. All retested hits were active also in an orthogonal enzymatic assay showing zero false positives. However, further biophysical validation of identified hits revealed that the inhibition activity of several hits was caused by a single highly potent CAIX inhibitor, being present as a minor impurity. This finding eventually led us to the identification of three novel CAIX inhibitors from the screen. We confirmed the validity of these compounds by elucidating their mode of binding into the CAIX active site by x-ray crystallography.

Mouse glutamate carboxypeptidase II (GCPII) has a similar enzyme activity and inhibition profile but a different tissue distribution to human GCPII.

Glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA) or folate hydrolase, is a metallopeptidase expressed predominantly in the human brain and prostate. GCPII expression is considerably increased in prostate carcinoma, and the enzyme also participates in glutamate excitotoxicity in the brain. Therefore, GCPII represents an important diagnostic marker of prostate cancer progression and a putative target for the treatment of both prostate cancer and neuronal disorders associated with glutamate excitotoxicity. For the development of novel therapeutics, mouse models are widely used. However, although mouse GCPII activity has been characterized, a detailed comparison of the enzymatic activity and tissue distribution of the mouse and human GCPII orthologs remains lacking. In this study, we prepared extracellular mouse GCPII and compared it with human GCPII. We found that mouse GCPII possesses lower catalytic efficiency but similar substrate specificity compared with the human protein. Using a panel of GCPII inhibitors, we discovered that inhibition constants are generally similar for mouse and human GCPII. Furthermore, we observed highest expression of GCPII protein in the mouse kidney, brain, and salivary glands. Importantly, we did not detect GCPII in the mouse prostate. Our data suggest that the differences in enzymatic activity and inhibition profile are rather small; therefore, mouse GCPII can approximate human GCPII in drug development and testing. On the other hand, significant differences in GCPII tissue expression must be taken into account when developing novel GCPII-based anticancer and therapeutic methods, including targeted anticancer drug delivery systems, and when using mice as a model organism.

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities.

UNLABELLED: Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave ß-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates. We observed a high efficiency of BCG cleavage by GCPIII but not GCPII. We also identified a strong modulation of GCPIII enzymatic activity by divalent cations, while we did not observe this effect for GCPII. Additionally, we used X-ray crystallography and computational modelling (quantum and molecular mechanical calculations) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, respectively. Finally, we took advantage of the substantial differences in the enzymatic efficiencies of GCPII and GCPIII towards their substrates, using enzymatic assays for specific detection of these proteins in human tissues. Our findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiological function related to BCG metabolism in the human body. DATABASE: The X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09.

Design of highly potent urea-based, exosite-binding inhibitors selective for glutamate carboxypeptidase II.

We present here a structure-aided design of inhibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, glutamate carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells.