Comparative study of the effects of a small-scale trout farm on the macrozoobenthos, potamoplankton, and epilithic diatom communities.

Trout farms are one of the major sources of pollution of highland streams and rivers, which are very sensitive ecosystems otherwise scarcely influenced by human activities. A trout farm with low annual production was chosen for comparative monitoring of its effects on the macrozoobenthos, epilithic diatom, and potamoplankton communities in the receiving stream. Research was conducted every 2 months during a period of 1 year (one control point and three localities downstream from the farm). The fish farm discharge induced moderate alteration of environmental factors in the receiving watercourse, viz., significant increase of the ionized and un-ionized ammonia fraction, dissolved oxygen concentration, and biological oxygen demand. Species richness and Simpson's diversity index did not change in any of the communities. Correspondent analysis of potamoplankton community composition clearly showed trout farm effects, thereby indicating the need for reevaluation of saprobic valence values for potamoplankton species. The complete lack of influence of the trout farm discharge on the epilithic diatom community is probably due to absence of significant changes of phosphorus concentration in the receiving stream. Effects of the trout farm on the macrozoobenthos community were further characterized by 14 additional indices. The percentages of Chironomidae larvae, shredders, and total collectors; ratio of shredders to collectors; and the family biotic index are suggested as candidates for future multimetric index for measuring of trout farm influence.

Optimisation of BMP-2 dosage for the osseointegration of porous titanium implants in an ovine model.

In clinical orthopaedics, total joint replacements and spinal fusions are routine undertakings. Many of the implicated patients suffer from osteoporosis, severe arthrosis or osteopaenia. In individuals thus afflicted, the bony bed lacks the mechanical stability that is a requisite for a firm anchorage of the implant and its functional competence. To promote the bony bondage of an implant it is necessary to induce neo-ossification by the introduction of an osteogenic agent, such as bone morphogenetic protein 2 (BMP-2). Since this growth factor is generally applied in a free form and at high dosages to maximise its osteogenicity, untoward side effects frequently ensue. We hypothesise that the administration of BMP-2 using a suitable delivery vehicle, and its gradual, low dose release therefrom in a cell-mediated manner, would avert the triggering of undesired side effects and enhance its efficacy. To test this postulate, implants of porous titanium were coated with a layer of calcium phosphate into which BMP-2 was biomimetically incorporated at dosages ranging from 0.8 to 500 µg/g of coating material (delivery system) prior to their surgical placement in the tibiae of adult sheep. The volume and the surface area of newly-formed bone were evaluated histomorphometrically after 3 and 6 weeks. The highest values were achieved using BMP-2 dosages of 20 to 100 µg/g of coating: The deposition of bone was confined to the immediate vicinity of the implant and was observed deep within the interstices of its meshwork, to the walls of which it bonded well. The findings of the study attest to the validity of our hypothesis.

Failure to detect viral RNA in bat samples collected in the Balkan region.

Bats represent a known reservoir of emerging viruses, yet no molecular data are found about the occurrence of zoonotic viruses in bats in the Balkans. The aim of this study was to determine the presence of paramyxo- and hanta-viruses in bats, examined by PCR in 95 deceased bats, that were collected in Serbia and Montenegro, during the period 2002 to 2009. All samples tested positive for beta-actin mRNA, confirming successful RNA isolation and amplification. However, no sample tested positive for virus specific RNA. Our findings might reflect tissue degradation in carcass samples and do not exclude bats as potential viral reservoir in the surveyed geographic area.

Characterisation and preliminary lipid-lowering evaluation of Lactobacillus isolated from a traditional Serbian dairy product.

We investigated the potential probiotic properties of indigenous lactic acid bacteria (LAB) isolated from Serbian homemade cheese. Seventeen LAB strains were isolated and characterised using standard protocols. One of the strains showed several probiotic properties: survival at low pH and in bile salts solution, antimicrobial activity, susceptibility to antibiotics and adhesion to hexodecane. DNA analysis identified the isolate as Lactobacillus casei, hereafter named L. casei 5s. The lipid lowering effect of L. casei 5s was evaluated in vivo using a hyperlipidemic rat model. Orally administered L. casei 5s significantly decreased the elevated total serum cholesterol and triglycerides, and attenuated macro vesicular steatosis in the liver. Moreover, L. casei 5s improved the intestinal microbial balance in favour of lactobacilli, while decreasing the number of Escherichia coli cells. The bacteria were re-isolated and identified from the surface of the intestinal mucosa and from the faecal samples of treated animals, indicating adhesiveness and colonisation ability. The results of an acute oral toxicity study in mice and the absence of translocation to other organs demonstrated the safety of the strain. In conclusion, L. casei 5s demonstrated promising probiotic potential and might be a good candidate for more detailed investigations.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Inclusion of chemotherapy in addition to anthracycline in the treatment of acute promyelocytic leukaemia does not improve outcomes: results of the MRC AML15 trial.

Two hundred eighty-five patients, median age 42, with PML-RARα-positive acute promyelocytic leukaemia were randomised to Ara-C-containing 'Medical Research Council (MRC) Chemotherapy'+ATRA (All-trans-retinoic acid) or anthracycline+ATRA (modified 'Spanish') therapy. MRC treatment comprised four courses with ATRA in courses 1-2. Spanish treatment comprised four anthracycline-based courses with ATRA in courses 1-3. In course 3 patients were randomised to gemtuzumab ozogamicin (GO) or not. The Spanish arm received 24-month maintenance. Patients were sequentially molecularly monitored. Quality of life was assessed at baseline, 3, 6, 9, 12, 24 months. Remission rates were similar in both arms (93%): cumulative incidence of haematological relapse (CIHR) was 6% at 5 years; 5 patients relapsed molecularly. Survival post relapse was 80%. There were more deaths in remission in the MRC arm (4% vs 10%: P=0.2). The overall 5-year relapse-free and overall survival was similar between arms (81% vs 82% and 84% vs 83%, respectively). More supportive care and hospitalisation (81.8 vs 63 days, P<0.0001) was required in the MRC arm. GO did not provide benefit. High white blood cell count (>10 × 10(9)/l) was not prognostic overall, or within treatment arms. Both approaches deliver similar results with minor differences in quality of life. MRC treatment required more hospitalisation. This suggests that additional chemotherapy, Ara-C in particular, is not required.

Development of standardized approaches to reporting of minimal residual disease data using a reporting software package designed within the European LeukemiaNet.

Quantitative PCR (qPCR) for detection of fusion transcripts and overexpressed genes is a promising tool for following minimal residual disease (MRD) in patients with hematological malignancies. Its widespread clinical use has to some extent been hampered by differences in data analysis and presentation that complicate multicenter clinical trials. To address these issues, we designed a highly flexible MRD-reporting software program, in which data from various qPCR platforms can be imported, processed, and presented in a uniform manner to generate intuitively understandable reports. The software was tested in a two-step quality control (QC) study; the first step involved eight centers, whose previous experience with the software ranged from none to extensive. The participants received cDNA from consecutive samples from a BCR-ABL+ chronic myeloid leukemia (CML) patient and an acute myeloid leukemia (AML) patient with both CBFß-MYH11 and WT1 target genes, they conducted qPCR on their respective hardware platforms and generated a series of reports with pre-defined features. In step two, five centers used the software to report BCR-ABL+ MRD in a harmonized manner, applying their recently obtained CML international scale conversion factors. The QC study demonstrated that this MRD-reporting software is suitable for efficient handling of qPCR data, generation of MRD reports and harmonization of MRD data.

Cytogenetic findings in Serbian patients with Turner's syndrome stigmata.

Cytogenetic findings are reported for 31 female patients with Turner's syndrome. Chromosome studies were made from lymphocyte cultures. Non-mosaicism 45,X was demonstrated in 15 of these patients, whereas only three were apparently mosaic. Eight patients showed non-mosaic and four patients showed mosaic structural aberrations of the X-chromosome. One non-mosaic case displayed a karyotype containing a small marker chromosome. Conventional cytogenetics was supplemented by fluorescence in situ hybridization (FISH) with an X-specific probe to identify the chromosomal origin of the ring and a 1q12-specific DNA probe to identify de novo balanced translocation (1;9) in one patient. To our knowledge, this is the first finding of karyotype 45,X,t(1;9)(cen;cen)/46,X,r(X),t(1;9)(cen;cen) in Turner's syndrome. The same X-specific probe was also used to identify a derivative chromosome in one patient.

A comparative kinetics study of isothermal drug release from poly(acrylic acid) and poly(acrylic-co-methacrylic acid) hydrogels.

A comparative study of the isothermal kinetics of the release of the drug (E)-4-(4-metoxyphenyl)-4-oxo-2-butenoic acid (MEPBA) from poly(acrylic acid) (PAA) and poly(acrylic-co-methacrylic acid) (PAMA) hydrogel was performed. The isothermal kinetic curves of MEPBA release from the hydrogels in bidistilled water at different temperatures ranging from 20 to 42 degrees C were determined. The reaction rate constants of MEPBA release were determined using the initial rate, saturation rate and empirical equation developed by Peppas et.al. The so-called "model-fitting method" for determining the kinetics models of both the drug release and absorption of external solution into the hydrogel, was applied. It was found that the kinetics of the MEPBA release both from the PAA and PAMA hydrogels can be best described with the kinetics model of first order chemical reaction. The model's kinetics parameters of the investigated drug release process were calculated and significant differences for the values for PAA and PAMA hydrogels were found. The possibility to describe the kinetics of drug release with the model of reversible chemical reaction of first order was considered. It was found that kinetics of adsorption of the drug's solution can be described with kinetics model of first order chemical reaction for PAMA hydrogel, while for PAA hydrogel it can be described with the kinetics model which is characteristic for the "phase boundary controlled reaction". Based on the established dependences of the kinetic parameters (Ea and lnA) on the degree of the MEPBA released (alpha) as well as on the presence of a compensation effect a new molecular mechanism of drug delivery was established. According to that mechanism, drug release is considered as drug desorption from the xerogel/hydrogel's active desorption centers with different energies. The procedure for determining the distribution function of activation energies was developed. Different activation energy distribution function for PAA and PAMA hydrogels was established.

Detection and molecular monitoring of FIP1L1-PDGFRA-positive disease by analysis of patient-specific genomic DNA fusion junctions.

To evaluate current detection methods for FIP1L1-PDGFRA in hypereosinophilic syndrome (HES), we developed a means to rapidly amplify genomic break points. We screened 202 cases and detected genomic junctions in all samples previously identified as RT-PCR positive (n=43). Genomic fusions were amplified by single step PCR in all cases whereas only 22 (51%) were single step RT-PCR positive. Importantly, FIP1L1-PDGFRA was detected in two cases that initially tested negative by RT-PCR or fluorescence in situ hybridization. Absolute quantitation of the fusion by real-time PCR from genomic DNA (gDNA) using patient-specific primer/probe combinations at presentation (n=13) revealed a 40-fold variation between patients (range, 0.027-1.1 FIP1L1-PDGFRA copies/haploid genome). In follow up samples, quantitative analysis of gDNA gave 1-2 log greater sensitivity than RQ-PCR of cDNA. Minimal residual disease assessment using gDNA showed that 11 of 13 patients achieved complete molecular response to imatinib within a median of 9 months (range, 3-17) of starting treatment, with a sensitivity of detection of up to 1 in 10(5). One case relapsed with an acquired D842V mutation. We conclude that detection of FIP1L1-PDGFRA from gDNA is a useful adjunct to standard diagnostic procedures and enables more sensitive follow up of positive cases after treatment.

Molecular and phenotypic characteristics of patients with phenylketonuria in Serbia and Montenegro.

Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism in Caucasians. PKU is caused by mutations in the gene encoding phenylalanine hydroxylase (PAH) enzyme. Here, we report the spectrum and the frequency of mutations in the PAH gene and discuss genotype-phenotype correlation in 34 unrelated patients with PKU from Serbia and Montenegro. Using both polymerase chain reaction-restriction fragment length polymorphism and 'broad-range' denaturing-gradient gel electrophoresis/DNA sequencing analysis, 19 disease-causing mutations were identified, corresponding to mutation detection rate of 97%. The most frequent ones were L48S (21%), R408W (18%), P281L (9%), E390G (7%) and R261Q (6%), accounting for 60% of all mutant alleles. The genotype-phenotype correlation was studied in homozygous and functionally hemizygous patients. We found that the most frequent mutation, L48S, was exclusively associated with the classical (severe) PKU phenotype. The mutation E390G gave rise to mild PKU. For the mutation R261Q, patients had been recorded in two phenotype categories. Considering allele frequencies, PKU in Serbia and Montenegro is heterogeneous, reflecting numerous migrations over the Balkan Peninsula.

Occupational accidents in Serbian industries in transition.

Occupational accidents and injuries present a serious social, medical and economical problem in Serbian industries in transition. The purpose of this study is to estimate the number and average annual rate of occupational accidents and traumatic injuries in Serbian industries in transition, between 1992 and 2003, and to estimate the effects of a preventive safety program in the rubber manufacturing industry and in the production of electrical goods and equipment. This paper presents a descriptive analysis of occupational accidents and injuries which occurred in all industries over a twelve years period. In the rubber manufacturing industry and in the production of electrical goods and equipment a surveillance and prevention program has been implemented since 1997. A significant drop after 1997 in average annual accidents rate and severity ratio of injuries in these two industries has been observed, which was not the case in the other industries, and can be explained by the positive effects of the safety training program. The study documents that the safety training program showed significant effects, especially in case of workers with less than ten years of working experience.

Electron drift velocities in mixtures of helium and xenon and experimental verification of corrections to Blanc's law.

Measurements of electron drift velocities were performed in pure Xe and He and in a number of mixtures ranging up to 70% of Xe. The data were obtained by using a pulsed Townsend technique over the density-normalized electric field strength E/N between 1 and 100 Td . Even for pure gases there are no data in the entire range covered here, and these data represent an extension of accurate drift velocities to higher E/N. A selection of well-established cross sections for low energies, which was extended to higher energies, led to a reasonably good agreement of the calculated transport coefficients with the available data. At the same time we have applied the standard (common E/N) Blanc's law and two forms of common mean energy (CME, due to Chiflykian) procedures. Blanc's law fails for most mixtures at low and moderate E/N, while the CME procedure is capable of following the experimental data for the mixtures much more closely, and even predicting the negative differential conductivity region when such effect does not exist for pure gases. Thus the present paper also represents an experimental test of procedures to correct the standard Blanc's law. Finally, we have used the data for two mixtures to obtain results for the third mixture and in all cases this procedure gave excellent results even though only the standard Blanc's law was used in the process.

The influence of hydroxyapatite modification on the cross-linking of polydimethylsiloxane/HAp composites.

Hydroxyapatite (HAp) was modified by the action of various hydrophobic agents based on silicon-containing compounds. The influence of the type of applied agent on the thermodynamic and kinetic parameters of the cross-linking of poly(dimethyl siloxane)/HAp composites was investigated. All the modified HAp particles became hydrophobic and these samples were used to synthesize the polysiloxane/hydroxyapatite composites (PDMS/HAp). The possible modes of interaction between the hydroxyapatite and hydrophobing agents were discussed. The most probable interaction between hydroxyapatite and the applied hydrophobing agents is hydrogen bonding. PDMS/HAp composites were formed directly in the cell of the DSC and cross-linking was investigated in situ. It was determined that the introduction of hydroxyapatite into polysiloxane matrices changed the enthalpy of cross-linking, as well as the activation energy of cross-linking and reaction order, while the introduction of modified HAp led to thermodynamic and kinetic parameters more similar to those of the cross-linking of unfilled elastomer.

Bi-directional flow sensor with a wide dynamic range for medical applications.

This paper describes a novel three-wire thermal flow sensor for medical applications. The present innovation for low-frequency measurements involves the use of a pulsed-wire anemometer with a comparatively large wire diameter (12.5 microm and larger) together with a novel signal processing approach. A small wire is heated using a sinusoidal alternating current, and two sensing wires, acting as resistance thermometers, are set parallel to, and at a small distance on either side of, the pulsed wire. The thermal wake of the pulsed wire is convected downstream to one of the two receiving wires which detect its delayed arrival. This arrangement allows the sensing of both the direction and the flow velocity component normal to the three probes. By appropriate signal processing, the present sensor can be operated such that the phase shift between the periodic current that drives the central wire and the detected signal by either the upstream or downstream wire takes into account a combination of convection, diffusion and the finite thermal response time of both the pulsed wire and the receiving wire. Because the time constants increase as the flow velocity decreases, the time lag due to thermal inertia supplements the time lag due to the true time of flight, thus yielding an effective operating range of 0.05 m/s

Suicide in the elderly 60 years of age and over in the south-east part of Serbia.

Some previous investigations indicated that economic crisis (inflation in Serbia 1993/94) have great influence on increasing suicide rates in Serbia. After that suicide decreased, despite the war and bombing in 1999, specially among the elderly people. A total of 453 suicides were registered on the territory of south-eastern Serbia during 1995-2001 years. Of them 295 (65.1%) were aged over 60 years. Generally linear trends of suicide among both genders from 1995 to 2001 decreased, but the slope of decreasing was grater among males than among females. The highest rates among the both genders were registered in the years with the maximum number of suicides, but the linear trends of rates have statistically important correlation with time (r>0.5). Average annual suicide rate among males was 42.5, and among women it was 18.7. The highest average annual suicide rate among men was observed in the age group 75 years and over (93.3), and the lowest in the age group 65-69 (20.6). Among males, in the all age group linear trends of suicide rates decreased, with the highest slope among 75 years and over and the lowest among 65-69 years. The highest suicide rate among females was registered in age group 75 years and over (25.6), the lowest in the age group 65-69 (13.5). The linear trends are similar as among males: downward trend was observed among all aged groups, with the highest slope among women 75 years and over. The most common way of suicide among men was hanging up (63%), poisoning and by firearms. There were no statistical differences between way of suicide and age groups. The most frequent way of suicide among females was hanging up (55%), poisoning (25%) and drowning (12%). There were statistically significant differences in drowning between age groups, 70-74 and 75 and over (p<0.05), and between poisoning and age groups 60-64 years and 75 years and over. Poisoning and drowning are statistically more frequent among women than among men.

[Experimental and clinical possibilities of transplantation of the injured and totally devascularized spleen]. / Eksperimentalne i klinicke mogucnosti transplantacije konkvasirane ili totalno devaskularizovane slezine.

The most severe spleen lesions with conquasation and devascularisation of entire organ, when it is practically impossible to do any preservating surgical procedure, are the true indications for the transplantation of this extremely important immunological organ. We have performed the evaluation of the surgical procedure of heterotopic auto transplantation in the 30 dogs with severe spleen lesions. Simulation of totally devascularized spleen with the lesions of V degree was performed by disrupting all segmental blood vessels with deep and long longitudinal transhilar incision. During the 3 months follow-up period, animals were subjected to numerous explorations in order to macroscopically and histologically valuate the implant. In most cases (80-85%) implants had complete vitality with the preservation of normal tissue architecture, while 15-20% of implants had partial or total fibrosis. There were no mortality and no complications after this preservation procedure. The presence of fibrosis in some implants suggests that the implant preparation should be better performed and that transplantation of larger tissue volume is needed. Enriched with this experimental experience we have performed heterotopic auto transplantation in 2 patients with spleen lesion of V degree (car accident and injury at work) with very satisfactory results.

Opposing changes in phosphorylation of specific sites in synapsin I during Ca2+-dependent glutamate release in isolated nerve terminals.

Synapsins are major neuronal phosphoproteins involved in regulation of neurotransmitter release. Synapsins are well established targets for multiple protein kinases within the nerve terminal, yet little is known about dephosphorylation processes involved in regulation of synapsin function. Here, we observed a reciprocal relationship in the phosphorylation-dephosphorylation of the established phosphorylation sites on synapsin I. We demonstrate that, in vitro, phosphorylation sites 1, 2, and 3 of synapsin I (P-site 1 phosphorylated by cAMP-dependent protein kinase; P-sites 2 and 3 phosphorylated by Ca(2+)-calmodulin-dependent protein kinase II) were excellent substrates for protein phosphatase 2A, whereas P-sites 4, 5, and 6 (phosphorylated by mitogen-activated protein kinase) were efficiently dephosphorylated only by Ca(2+)-calmodulin-dependent protein phosphatase 2B-calcineurin. In isolated nerve terminals, rapid changes in synapsin I phosphorylation were observed after Ca(2+) entry, namely, a Ca(2+)-dependent phosphorylation of P-sites 1, 2, and 3 and a Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6. Inhibition of calcineurin activity by cyclosporin A resulted in a complete block of Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6 and correlated with a prominent increase in ionomycin-evoked glutamate release. These two opposing, rapid, Ca(2+)-dependent processes may play a crucial role in the modulation of synaptic vesicle trafficking within the presynaptic terminal.