A FRET-based biosensor for the detection of neutrophil elastase.

The direct and specific detection of biomarkers is crucial as it can allow monitoring the state of a tissue or wound as well as the progression of the inflammatory process. Neutrophil elastase (NE) plays an important role in many biological processes. It is involved in inflammatory diseases and is enriched in inflamed tissues, in wound exudate, and in the sputum of cystic fibrosis patients. In order to detect NE, we designed a NE-specific protein sensor whose fluorescence features are altered upon selective cleavage by NE at physiological concentration. The biosensor consists of two fluorescent GFP-derived proteins connected by a short peptide linker containing a NE-specific recognition sequence. Due to the close proximity of the two fluorescent proteins, Förster resonance energy transfer (FRET) occurs in the uncleaved form and, upon cleavage by NE, a decrease of FRET signal is observed. In this study, the construct design, the influence of the linker length, as well as the specificity for NE are described. Furthermore, the influence of biosensor immobilization on the functionality is analysed. By engineering the recognition sequence of the linker, the presented system can potentially be easily adapted to detect other proteases such as cathepsin, caspases or matrix metalloproteases.

Encapsulation of FRET-based glucose and maltose biosensors to develop functionalized silica nanoparticles.

Silicate nanoparticles with immobilized FRET-based biosensors were developed for the detection of glucose and maltose. Immobilization of the protein biosensor in the nanoparticle was achieved through specific interaction between the hexa-histidine tag of the protein and a calcium-silicate complex of the silica matrix. Encapsulation of the biosensors preserved the affinity for the respective sugar. Compared to the free biosensors, encapsulation had a stabilizing effect on the biosensor towards chemical and thermal denaturation. The demonstrated immobilization strategy for specific sensing proteins paves the way towards the development of protein-inorganic nanostructures for application in metabolite analyses.

Arxula adeninivorans recombinant adenine deaminase and its application in the production of food with low purine content.

AIMS: Construction of a transgenic Arxula adeninivorans strain that produces a high concentration of adenine deaminase and investigation into the application of the enzyme in the production of food with low purine content. METHODS AND RESULTS: The A. adeninivorans AADA gene, encoding adenine deaminase, was expressed in this yeast under the control of the strong inducible nitrite reductase promoter using the Xplor(®) 2 transformation/expression platform. The recombinant enzyme was biochemically characterized and was found to have a pH range of 5.5-7.5 and temperature range of 34-46 °C with medium thermostability. A beef broth was treated with the purified enzyme resulting in the concentration of adenine decreasing from 70.4 to 0.4 mg l(-1). CONCLUSIONS: It was shown that the production of adenine deaminase by A. adeninivorans can be increased and that the recombinant adenine deaminase can be used to lower the adenine content in the food. SIGNIFICANCE AND IMPACT OF THE STUDY: Adenine deaminase is one component of an enzymatic system that can reduce the production of uric acid from food constituents. This study gives details on the expression, characterization and application of the enzyme and thus provides evidence that supports the further development of the system.

Arxula adeninivorans xanthine oxidoreductase and its application in the production of food with low purine content.

AIMS: Isolation and characterization of xanthine oxidoreductase and its application in the production of food with low purine content. METHODS AND RESULTS: The A. adeninivorans xanthine oxidoreductase is an inducible enzyme. The best inducers were identified by enzyme activity tests and real-time PCR and used to produce large amounts of the protein. Xanthine oxidoreductase was partially purified and biochemically characterized, showing pH and temperature optimum of 8·5 and 43°C, respectively. The enzyme decreased xanthine and hypoxanthine concentrations in yeast extract and was active simultaneously with other purine-degrading enzymes so that all of the substrates for uric acid production were reduced in a single step. CONCLUSIONS: It was shown that induced A. adeninivorans can produce sufficient amount of xanthine dehydrogenase and that the enzyme is able to reduce xanthine and hypoxanthine content in food, and when used in conjunction with other enzymes of the pathway, uric acid concentration is significantly reduced. SIGNIFICANCE AND IMPACT OF THE STUDY: Reduction in dietary purines is recommended to people suffering from hyperuricemia. Elimination of most purine-rich foods may affect balanced nutrition. Food with lowered purine concentration will assist in controlling the disease. This study is a continuation of previous studies that characterized and overexpressed other enzymes of the purine degradation pathway.

Optical glucose sensing using ethanolamine-polyborate complexes.

Wound monitoring is essential to tackle chronic complications at their infancy and thus objectively scrutinize any delay in the epithelization process. Since glucose in wound exudates is recognized as key bio-marker in wound monitoring, the development of a cost-efficient detection method for glucose would aid at tackling early-stage infections in wounds. For the first time, we present a novel platform for one-step synthesis of non-enzymatic, cost-efficient optical glucose sensors. These are based on complexes formed by the interactions between polyborates and ethanolamines. The complexes, synthesized by just heating a solution of boric acid and ethanolamines at 150 °C, were characterized using 13C-NMR, 1H-NMR, 11B-NMR, analytical ultracentrifugation and DFT. The results show that the complexes in solution are extremely small (hydrodynamic diameter of around 0.5 nm) and that the polyborates species interact with the ethanolamines via both moderate and weak hydrogen bondings. These complexes were then tested on glucose concentrations ranging from 0 to 10 mM, showing significant changes in the fluorescent emission between the glucose level expressed in an healable wound (5.0-7.6 mM) and a chronic one (0.3-1.0 mM).

Simultaneous detection of pH value and glucose concentrations for wound monitoring applications.

Aging population and longer life expectancy are the main reasons for an increasing number of patients with wound problems. Although the interest in wound care increases continuously, wound management still remains a challenge mainly due to the higher occurrence of chronic wounds, which require intensive care and constant monitoring. Here, we demonstrate a fluorescent sensing system to monitor the wound status and to distinguish between an autonomously healing and a chronic wound at an early stage. The system allows monitoring two of the most relevant fluctuating wound parameters during the healing process which are pH and glucose concentration. A fluorescent pH indicator dye, carboxynaphthofluorescein, and a metabolite-sensing enzymatic system, based on glucose oxidase and horseradish peroxidase, were immobilized on a biocompatible polysaccharide matrix to develop a functional hydrogel coating for wound monitoring. The changes in metabolite and enzyme concentration in artificial wound extract were converted into a fluorescent signal.

Atypical case of sporadic Creutzfeldt-Jakob disease (CJD) in a young adult.

The great concern exists that new variant of CJD (nvCJD) developed as a result of exposure to bovine spongiform encephalopathy (BSE)-infected meat products. Therefore, all cases of CJD in the young, as the one of ours are the matter of interest. The 21-year-old female developed a rapid progression of pyramidal, extrapyramidal and cerebellar signs, visual loss and psychiatric symptoms, leading to death in 16 weeks. The microscopic features were: a neuronal loss accentuated in cerebral cortex with extensive astroglia proliferation and spongiform changes. Immunohistochemical staining, revealed the presence of "synaptic" deposits of PrP in the cerebral cortex and in the cerebellum. No florid amyloid plaques were present. The case was diagnosed as a sporadic CJD, with some features of Heidenhein variant (visual symptoms) and corticostriatocerebellar category. The pathological findings excluded a nv CJD which is linked with BSE.

[A contribution to the problem of recovery from posttraumatic apallic syndrome]. / Przyczynek do zagadnienia odwracalnosci pourazowego zespolu apalicznego.

Two cases of posttraumatic apallic syndrome were described in which despite initially severe symptoms full recovery was observed. In complex treatment lasting several months the great role plays the stimulation of various receptors by physiological stimuli. In the discussion to treat or not to treat intensively the severe posttraumatic apallic syndrome our opinion is positively yes.

[Incidence of malignant neoplasms in patients with multiple sclerosis]. / Czestosc zachorowan na nowotwory zlosliwe u chorych na stwardnienie rozsiane.

The incidence of malignant neoplasma was determined in patients with multiple sclerosis in epidemiological studies in 717 patients. No significant difference was found from the general population aged over 15 years. This seems to suggest that immunosuppression resulting from corticotherapy has no effect increasing the risk of malignant neoplasm development in multiple sclerosis.

[Comparative epidemiologic studies of multiple sclerosis and cancer of the large intestine in several provinces of western Poland]. / Porównawcze badania epidemiologiczne stwardnienia rozsianego i raka jelita grubego na terenie kilku województw zachodniej Polski.

A trial was made of finding a correlation between the incidence and prevalence of multiple sclerosis and large bowel carcinoma in several microregions (communities and areas of the former counties). The following conclusions have been reached. No correlation was found between the incidence of multiple sclerosis and large bowel carcinoma in these studied areas. A positive correlation was demonstrated between the prevalence of multiple sclerosis and large bowel carcinoma in the areas corresponding to former counties but not in communities. Since the latter index is of much lower importance for the estimation of the frequency of malignant neoplasms there is no reason to assume the existence of one or several common aetiologic factors of multiple sclerosis and large bowel carcinoma determining local differences in the frequency of these diseases.

[Changes in stroke mortality rates during the years 1977 and 1985]. / Zmiany w umieralnosci z powodu udarów mózgu w latach 1977 i 1985.

The mortality from stroke in the years 1977 and 1985 was compared in the Province of Poznan. The mortality from strokes decreased in that time period considerably in the population of males and females. The differences were significant from the age group 40-49 years. Reduced mortality from stroke in the Province of Poznan contrasted with a reverse trend in the mortality from ischaemic heart disease, which has been increasing continuously. It may be supposed that increasing effectiveness in the treatment of arterial hypertension may explain, in part at least, reduction of the mortality index from strokes. One cannot exclude, however, taht partial elimination of other risk factor due to changed way of life, changed dietary habits, may play also a considerable role in the positive trends in the epidemiology of strokes.

[Diagnostic value of regional cerebral blood flow in SPECT pattern in Alzheimer's disease]. / Wartosc diagnostyczna badania regionalnego przeplywu krwi w mózgu metoda tomografii emisyjnej w chorobie Alzheimera.

Regional cerebral blood flow in SPECT pattern was estimated in 20 cases of Alzheimer disease. In all patients diffuse hypoperfusion was found evidencing a great diagnostic value of SPECT. A special significance has the study of regional cerebral blood flow in the differential diagnosis of Alzheimer disease, frontal lobe dementia and pseudodementia in major depression.

[Changes of regional cerebral blood flow in stroke patients assessed by SPECT]. / Zmiany regionalnego przeplywu krwi u chorych z udarem mózgu badane przy pomocy tomografii emisyjnej pojedynczego fotonu.

UNLABELLED: The aim of our work was to evaluate changes in regional cerebral blood flow (rCBF) in patients with TIA and/or completed stroke and localisation of fotopenic foci in basal conditions and, in some cases, after stimulation pharmacological trial with acetazolamide. One hundred and fifty three patients participated in the study--105 with completed stroke and 48 with TIA. Regional CBF (rCBF) was evaluated using a single-headed rotating gamma camera (Diacam, Siemens) with high resolution collimator, after injection of 740 MBq 99mTc-HMPAO (Ceretec, Amersham). Sixteen patients with TIA underwent SPECT at baseline and after injection of acetazolamide (ACZ). Relative regional perfusion was expressed as the ratio of cerebral/cerebellar activity. In the completed stroke population, the perfusion ratios were significantly lower than those of controls and TIA at baseline (p < 0.001) and TIA after ACZ (p < 0.01). CONCLUSIONS: This study shows a significant difference in rCBF in patients with completed stroke and/or TIA and controls. In patients with TIA and rCBF similar to that observed in controls, perfusion deficits may be visible after ACZ injection.

[Determination of acoustic climate inside high schools in comparison with elementary schools]. / Uwarunkowania klimatu akustycznego pomieszczen szkól ogólnoksztalcacych w porównaniu ze szkolami podstawowymi.

The evaluation of acoustic conditions in four high schools showed that acoustic climate in high schools is more favourable than in elementary schools. During breaks the noise in corridors is about 77 dB (about 9 dB lower than in elementary schools). During lessons these differences are lower. In comparison to the elementary schools the noise level both in the corridors and in the classrooms is about 5 dB lower (60 i 62 dB respectively). During lessons the noise level depends mainly on number of pupils, manner of teaching, a type of task performed by schoolchildren and the day of the week. The work in groups is noisiest (average more 5-7 dB than during lectures). The more favourable acoustic conditions are in classes below 25 pupils but in classes above 30 pupils the noise level during lessons increases about 3dB. The noise in classroom depends also on period of duration of the lesson and the day of the week. However it was not observed significant differences in daily distribution of the noise level, subject of the lesson or year of schooling. Resultant daily noise exposure of schoolchildren in high schools during their school time is 73 dB and is about 7 dB lower than in elementary schools.

[Occurrence and evaluation of a law frequency noise in residential buildings]. / Wystepowanie i ocena halasów niskiej czestotliwosci w budynkach mieszkalnych.

Low-frequency noise emitted into the environment by technical equipment in the residential buildings, including equipment of workshops for services or production near these buildings, was measured. In the spectrum of noise derived from installations and equipment in residential buildings and shops low frequency (20-125 Hz) sounds and infrasounds (below 20 Hz) were detected. Their sources were mainly pumps in hydrophors, lifts, cooling machinery, central heating, air conditioning and ventilating installations. The analysed noise was in a small degree only damped by partitions in buildings and penetrated more easily than higher-frequency noise, without exceeding usually the permitted levels. Noises with dominating low-frequency sounds are regarded by the inhabitants as troublesome and causing various adverse psychosomatic effects, such as pulsation feeling, somnolence, headaches, nausea etc. The present system of noise assessment leaves low-frequency noise aside and fails to protect sufficiently the inhabitants against this nuisance.

[Noise in vocational schools. Causes of occurrence and assessment of exposure to schoolchildren]. / Halas w szkolach zawodowych. Przyczyny wystepowania i ocena narazenia uczniów.

Acoustic conditions in the classrooms of vocational schools are comparable with those in high schools. However, much worse acoustic conditions are observed in vocational school workshop. The evaluation of acoustic conditions in 712 school shops showed, that noise exposure of schoolchildren is very various and depends upon type of workshop and tasks performed by schoolchildren. The major source of noise exposure is machinery in mechanic workshops (plastic working--up to 105 dB, machine noise-from 80 to 90 dB), in textile workshops--from 90 to 100 dB and woodworking machines-from 92 to 102 dB. It should be noted that in such acoustic conditions schoolchildren stay for a relatively long time. Average exposure time of schoolchildren in mechanic or textile workshops was about 4.5 hours. At another training subjects i.e. electric, building, welding craft or gastronomy--noise exposure is usually below recommended standards (80 dB). The critical values accepted for hearing protection for occupational exposure (85 dB) were exceeded at 10% investigated training places of schoolchildren. This mostly occurred in mechanic workshops. Taking into account increased sensitivity of young people it would be worthwhile to work out separate criteria in this range for school workshops. Tentatively, problem should be solved by shortening exposure time depending on actual noise level.

[Determination of the acoustic climate inside elementary schools]. / Uwarunkowania klimatu akustycznego pomieszczen szkól podstawowych.

The evaluation of acoustic conditions in four elementary schools showed high levels of noise inside schools, especially in corridors. It refers mainly public schools, which generally are overcrowded. During breaks the most frequent noise level is 86 dB but in some parts of corridors approaches values 95-98 dB. During lessons the noise in corridors decreases to about 64 dB. However during physical exercises which take place in corridors the noise level in corridors increases up to 75-80 dB. In public schools the noise levels in corridors both during lessons and breaks are higher about 12 dB than in private schools. However during lessons in classrooms those differences are quite lower. The noise level during lessons depends mainly on number of pupils, subject of the lesson, year of schooling and period of duration of the lesson. Considerable growth of noise occurs in classes above 20 pupils and at initial and final period of the lesson. The most favourable acoustic conditions are in classes below 15 pupils but in classes above 20 pupils the noise levels increase average about 4 dB. In spite of common opinion the noise level is lower in classes with younger children than in classes with older children. In older classes the noise is higher average about 3 dB. The noise in classroom depends also on manner of teaching and type of tasks performed by schoolchildren. The work in group is noisiest (average more 8 dB than during lectures). It refers especially to gymnastics lessons during group games. In this case the noise levels usually are above 90 dB. However it was not observed significant differences in daily or weekly distribution of the noise level during lessons. Resultant daily noise exposure of schoolchildren during their school time is 80 dB (or sometimes 85 dB) in public schools and 72 dB in private schools. Average noise exposure in public schools approaches critical values accepted for hearing protection for adolescents during apprenticeship.