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Encyonema bonapartei Heudre, C.E.Wetzel & Ector is a diatom species originally documented in 2016, following its discovery in three canals in the eastern region of France. In 2021, an extensive hydrobiological survey in a mountainous part of southwestern Serbia led to the first finding of this taxon outside of France, i.e. in the Uvac Special Nature Reserve –in Lake Zlatar (Zlatarsko jezero) and in the Marić River (Marića reka). Detailed light microscopy and scanning electron microscopy observations of a large population recorded in Lake Zlatar confirmed the unique morphological characteristics of E. bonapartei, which is also the first record for the diatom flora of Serbia. The present work provides new information on the distribution and ecological preferences of E. bonapartei, taking into account physical and chemical characteristics of its habitats in Serbia.

The study of aquatic Oligochaeta in freshwaters in Serbia contributes to the oligochaete fauna inventory of the Balkans. Based on our results and literature review, 97 species (45 genera from 8 families) are listed in the running waters of Serbia. From the list of 61 species recorded during our investigation, 12 were noted for the first time. The ecological analysis of Oligochaeta communities showed the largest participation of potamal and rhithral species. As regards current preferences, rheolimnophilous species predominated, and with respect to feeding type, collectors prevailed. Most of the species are tolerant to moderate and high organic load. According to microhabitat preferences, three types of rivers stood out, as follows: (1) pelophilous (dominance of L. hoffmeisteri); (2) psammophilous (dominance of S. heringianus); (3) phytophilous (dominance of N. bretscheri). Analyses of the relation to different types of waterbodies showed regularity in the distribution of oligochaetes, with a higher species richness in main watercourses (types 1 and 2), and a lower species richness in tributaries (types 3, 4 and 6). According to the dominant taxa, the running waters of Serbia can be classified into four groups which are characterized by the prevalence of naidids (naidins and tubificins), enchytraeids and lumbriculids. Our results show that community composition is a good indicator of the horizontal distribution of Oligochaeta.

Introduction. The important indicators of the quality of work in blood transfusion banks and health care facilities in general is the ratio of the cross-matched red blood cell (RBC) units, and the number of transfused RBC, known as cross-match to transfusion ratio (C:T). The objective of this research was to provide an assessment of the quality of our work in a cross-sectional study, showing C:T ratios for certain areas of surgery or particular surgical indications. Methods. We analyzed the data related to the activities of the Department for Pre-Transfusion Testing and Blood Distribution at the Blood Transfusion Institute of Serbia during the September and November of 2017 period. In total, 341 patients were included in the study, for whom 1,067 RBC units were requested. Results. In pre-transfusion testing, 562 units were cross-matched and 249 units were transfused. The overall C:T ratio was 2.25. There are variations in C:T by departments. For the departments of abdominal surgery and reanimation, where uncrossmatched RBC units were requested, C:T was < 2. Other departments had C:T > 3 for almost all therapeutic areas. Conclusion. Our results show that the C:T ratio ranged 2.02–3.6, indicating the need to reevaluate the protocols based on which the blood is requested according to individual indications, to adequately prepare patients for surgery in order to reduce the risk of possible allogeneic transfusion, and to apply Patient Blood Management protocols, which include the use of alternatives to allogeneic blood transfusion.

Aims The aim of this study was to improve production of pentaene 32,33-didehydroroflamycoin ( DDHR) in Streptomyces durmitorensis MS405 strain to obtain quantities sufficient for in depth analysis of antimicrobial properties. Methods and Results Through classical medium optimization conditions for stable growth, DDHR production within 7 days of incubation was established. Yields of 215 mg l−1 were achieved in shake flask experiments in complex medium with mannitol as the primary carbon source. DDHR had poor antibacterial activity with minimal inhibitory concentrations ( MIC) of 400 μg ml−1 for Staphylococcus aureus and Bacillus subtilis, while MIC of 70 μg ml−1 was determined for Candida albicans. Using flow cytometry and fluorescent microscopy, it was demonstrated that DDHR induced membrane damage in C. albicans followed by cell death. Combination studies with known antifungal nystatin showed that DDHR is a promising agent for the development of novel antimycotic treatments potentially less toxic for human cells. Conclusions Pentaene didehydroroflamycoin has no antibacterial activity but can be further developed for the application in antifungal therapy. Significance and Impact of the Study This study is the first report on the stable and production in high yields of a novel pentaene family that acts on Candida cell membranes and can be used in combination with known antifungals. Polyenes are still antifungal antibiotics of choice, and therefore, isolation and production of new lead structures are highly significant. [ABSTRACT FROM AUTHOR]

The investigation of the Despotovica River was carried out in April 2017 with the aim to provide an indicative ecological status assessment. Phytobenthos and aquatic macroinvertebrate communities were followed as well as physico-chemical parameters of water. Ecological status assessment is given based on biological (epilithic diatoms and macroinvertebrates) and supporting physico-chemical water quality elements, according to the Serbian National regulation on parameters of ecological and chemical status of surface waters and parameters of chemical and quantitative status of ground waters. The ecological status was determined by the worst evaluated water quality element. Based on the analysis of investigated parameters of biological and physico-chemical water quality elements, the indicative ecological status of the Despotovica River could be assessed as bad (class V), which is the result of the river's load of organic and inorganic origin but also of heavy metals.

The aim of the study was to evaluate and adapt the PCR-based protocol that utilizes the developed serotype-specific primers to identify Salmonella enterica species and its serotypes that are most frequently isolated from poultry samples in Vojvodina. Using the slide agglutination test, 64 and 33 out of 107 Salmonella isolates were identified as S. Infantis and S. Enteritidis, respectively, while ten isolates were identified as eight different Salmonella serovars. Using the same isolates, presence of 993-bp (bcfC gene), 636-bp (steB gene) and 293-bp (sdf locus) amplicons in multiplex PCR unambiguously identified 31 isolates as S. Enteritidis. Two isolates identified as Enteritidis in slide agglutination test were not identified as such in PCR-based approach since they both were missing 293-bp long PCR product. Thirty-nine isolates produced a 727-bp amplicon in the specific simplex PCR, and thus were identified as S. Infantis. The greatest discrepancy in comparison to the results of conventional serotyping has been observed in the case of S. Infantis, since 25 more isolates were noted as S. Infantis by conventional serotyping. Seven isolates, with unexpected PCR profiles stayed unidentified by molecular typing, although they were serotyped as S. Typhimurium (1) and S. Infantis (6). S. Gallinarum serovar has to be additionally confirmed, since it shares the same PCR profile with S. Livingstone. Clearly, PCR-based identification has to be thoroughly checked, verified and adapted if it is to be applied as the routine identification protocol.

Extremely acidic environments are frequently formed in areas impacted by mining activities, and Lake Robule is such an ecosystem. Although an extreme environment, Lake Robule is inhabited by acidophilic microorganisms. We investigated biodiversity of the macroscopic structure known as a microbial mat formed on the lake bottom in shallow waters. Microbial mats are common in acidic environments, but their composition can differ significantly from site to site. Microbial diversity of the mat from Lake Robule was investigated using both cultivation-dependent and metagenomic approaches. The results showed the mat to be mostly inhabited by heterotrophic acidophilic bacteria. When compared to the microbial community of Lake Robule’s surface water, the microbial mat proved to be a more complex community. A biogeochemical model of the mat of Lake Robule is proposed on the basis of our results and available published data.

Young plants of celery, parsley, parsnip and carrot, grown in nutrient solution, were treated with sodium naphthenate (10−7 mol dm−3), applying foliar and root treatments. Both treatments affected the root content of all investigated elements present in the nutrient solution, but in a different way, depending on the plant species. An average change (increase/decrease) in the contents of investigated essential elements was about 35%. Our experiments with naphthenate showed that this treatment may enhance the efficiency of essential elements uptake and increase its content in plants without changing concentration of these elements in the nutrient solution. Especially interesting results were obtained in the case of carrot, as increased contents were observed in the elements that are usually deficient in nutrition (Fe, Zn, Mn), whereas the other remained unchanged.

Bioleaching of samples taken from depths of 10, 15, and 20 meters from old flotation tailings of the Copper Mine Bor was conducted in shaken flasks using extremely acidic water of Lake Robuleas lixiviant. Yield of copper after five weeks of the bioleaching experiment was 68.34±1.21% for 15 m sample, 72.57±0.57% for 20 m sample and 97.78±5.50% for 10 m sample. The obtained results were compared to the results of acid leaching of the same samples and it was concluded that bioleaching was generally more efficient for the treatment of samples taken from depths of 10 m and 20 m. The content of pyrite in the 20 m sample, which contained the highest amount of this mineral, was reduced after bioleaching. Benefits of this approach are: recovery of substantial amounts of copper, reducing the environmental impact of flotation tailings and the application of abundant and free water from the Robule acidic lake as lixiviant. Results of the experiment showed that bioleaching can be more efficient than acid leaching for copper extraction from flotation tailings with higher sulfide contents. [Projekat Ministarstva nauke Republike Srbije, br. 176016 i br. 173048]

The influence of naphthenic acids (NAs) mixture and their narrow fractions (called NA pH 4, pH 8 and pH 10) onto permeability of beetroot cell membrane is examined. The results showed that the effect depends on treatment duration, concentration and NAs structure. Longer treatment of plant cell membranes with sodium naphthenate (Na-naph) resulted in the increase of membrane permeability (e.g. 4-hour treatment with Na-naph (C=100 μmol L-1) increased membrane permeability about 3 times, while prolongation of treatment to 24 hour resulted in the 18 times increasing of the effect). NAs in the concentration range from 0.1 to 10 μmol L-1 does not change membrane permeability, while membrane permeability is increasing linearly with concentration increasing from 10-100 μmol L-1. The strongest effect expressed fraction pH 8, where bi- and tricyclic carboxylic acids are the most abundant. These structures are predominant in the total NAs mixture as well. Thereby could be explained their closest, but a little bit weaker effect, comparing to NAs present in fraction pH 8. The effect of NAs onto beetroot cell membrane is between the effects of anionic (SDS and LS) and non-ionic surfactants (Triton X-100). [Projekat Ministarstva nauke Republike Srbije, br. 172006. i br. TR31036]

An investigation of the microbial diversity of the extremely acidic, metal-rich Lake Robule was carried out using culture-dependant and culture-independent (T-RFLP) methods, and the ability of indigenous bacteria from the lake water to leach copper from a mineral concentrate was tested. T-RFLP analysis revealed that the dominant bacteria in lake water samples were the obligate heterotroph Acidiphilium cryptum (~50% of total bacteria) and the iron-oxidizing autotroph Leptospirillum ferrooxidans (~40%) The iron/sulfur-oxidizing autotroph Acidithiobacillus ferrooxidans had been reported to be the most abundant bacteria in the lake in an earlier study by other authors, but it was not detected in the present study using T-RFLP. Although it was isolated on solid media and detected in enrichment (bioleaching) cultures. The presence of the two bacterial species detected by T-RFLP (L. ferrooxidans and A. cryptum) was also confirmed by cultivation on solid media. The presence and relative abundance of bacteria inhabiting Lake Robule was explained by the physiological characteristics of the bacteria and the physico-chemical characteristics of the lake water. [Projekat Ministarstva nauke Republike Srbije, br. 176016 i br.173048]

This is a reply on the Commentary (hereafter: Commentary) by V. Beškoski and M. Vrvić (Commentary on the article entitled “Investigation of the microbial diversity of an extremely acidic, metal-rich water body (Lake Robule, Bor, Serbia)” by Srđan Stanković, Ivana Morić, Aleksandar Pavić, Branka Vasiljević, D. Barrie Johnson and Vladica Cvetković, published in the Journal of the Serbian Chemical Society, Volume 79, Issue 6, Pages: 729-741; V. P. Beškoski, M. M. Vrvić, J. Serb. Chem. Soc. /2014/, doi: 10.2298/JSC131228100B).

The bacterial diversity of the surface and deep sediment of the Copper Mining and Smelting Complex Bor, Serbia, was investigated using culture-dependent and culture-independent approaches. Sequencing analysis of 16S rDNA libraries revealed greater bacterial diversity in the surface sediment of the mining complex (MS) in comparison to deeper mine sediment (MU). While in the MS sample members of seven different phylogenetic groups were detected, in the MU sample library representatives of only three different groups were detected. The use of a culture-dependent approach revealed the presence of only three bacterial groups in both samples: Actinobacteria, Firmicutes and Proteobacteria, while six isolates exhibiting the highest metal tolerance were members of Arthrobacter and Staphylococcus genera. The most promising isolate, MSI08, was able to grow in the presence of high concentrations of Cd2+ (535 μM), Ni2+(17 mM) and Cr6+ (38.5 mM) and as such this indigenous strain has potential in the bioremediation of the contaminated surrounds of the city of Bor.

Background/Aim. Diagnosis of perinatal hypoxic-ischemic encephalopathy (HIE) and early prediction neurological outcome is important and difficult. The aim of this study was to determine the prognostic value of amplitude integrated electroencephalography (aEEG) for abnormal neurodevelopment outcome in a neonate with HIE. Methods. A total of 90 neonates > 32 gestational age (GA) with HIE were enrolled prospectively. All neonates with HIE were categorized into three grades according to the Sarnat and Sarnat clinical scoring system (mild HIE, moderate HIE and severe HIE). aEEG traces were recorded with a cerebral function monitor (CFM) during the first 72 h of life. The neurodevelopment outcome was assessed at 12 months of age of corrected gestational age. Results. The pattern of aEEG correlated with the severity of HIE (p < 0.0001) and subsequent neurodevelopment outcome (p < 0.001). We found that aEEG background patterns exhibited superior prediction of abnormal outcomes at 12 months of age (sensitivity of 91.7% and specificity of 94.3%, positive predictive value of 78.6% and negative predictive value of 98.1%) when compared to aEEG seizure (sensitivity of 94% and specificity of 48%, positive predictive value of 57% and a negative predictive value of 92%). Electroclinical dissociation seizure was detected in 28% of the neonates with HIE. Conclusions. Our results confirm that aEEG is simple and accurate bedside diagnostic method for assessing extension of hypoxic-ischemic brain damage and early identification of neonates with perinatal HIE who are at high risk of neurodevelopmental impairment.

Introduction. The pathogenesis of perinatal hypoxic-ischemic brain damage is highly complex. Objective. The aim of this study was to assess the role of oxidative stress in hypoxic-ischemic brain injury and subsequent abnormal neurological outcome in infants with perinatal hypoxic-ischemic encephalopathy (HIE). We estimated perinatal oxidative brain damage measuring activity of glutathione peroxidase (GPX) in cerebrospinal fluid (CSF) as an indirect biomarker of free radical production during cerebral hypoxia-ischemia in correlation with the level of intracellular enzyme neuron specific enolase (NSE) in CSF as a biomarker of extend of brain injury. Methods. Ninety neonates (>32 GA) with perinatal HIE were enrolled prospectively. HIE was categorized into three stages according Sarnat and Sarnat clinical scoring system and changes seen on amplitude integrated EEG. CSF for GPX analysis and NSE analysis was taken in the first 72 hours of life. Neurodevelopment outcome was assessed at 12 months of corrected gestational age. Results. GPX activity in CSF was in good relation with clinical stage of HIE (p

In this study, we investigated the level of sanitary pollution and organic contamination of the Velika Morava River, the largest river in central Serbia. Samples of water for microbiological analysis were collected at 5 sites, monthly, from April 2010 to February 2011. Sanitary analysis, i.e. enumeration of total and fecal coliforms and intestinal enterococci, indicated moderate to critical fecal contamination, while organic load assessment (oligotroph to heterotroph ratio, index of phosphatase activity) revealed the category of moderately polluted water. We also investigated seasonal variations of these groups of bacteria and the factors that could contribute to these differences. Our results showed that the micro- biological quality of the water in the Velika Morava River during different seasons is affected by numerous factors such as unequal loading of wastewaters, solar irradiation, and relations of flow/dilution and rainfall/runoff. [Acknowledgments. This study was financially supported by the Ministry of Education and Science of the Republic of Serbia: projects No 173025, No 177045 and No 043002.]

We report the discovery of Bothrioneurum vejdovskyanum Štolc, 1886 (Annelida: Oligochaeta: Tubificidae: Rhyacodrilinae) in Serbian freshwaters. Fifteen specimens were found in the middle course of the River Ibar (southern Serbia), at a locality where the macrolithal type of substrate and strong currents prevail. These organisms were found together with Stylodrilus heringianus (Claparede, 1862). They were among the most abundant Oligochaeta species. The species are described and its biology and distribution are discussed. The present record contributes to the knowledge on the general distribution of this rare species, particularly in the Balkans (southeastern Europe). [Acknowledgments. This work was supported by the Ministry of Education and Science of the Republic of Serbia, Projects No. ON 173025 and III 43002]

Aggregatibacter actinomycetemcomitans is considered one of the bacterial species of etiological importance in periodontitis. The aim of this study was to evaluate the serotype of A. actinomycetemcomitans in the subgingival biofilm in subjects with periodontal health and disease. Pooled samples of subgingival plaque were taken for culture-based identification of microorganisms. Colonies suspected to be A. actinomycetemcomitans were selected for molecular identification using either multiplex or conventional PCR in serotype-specific genotyping and 16S rRNA gene sequencing. In silico analysis showed that most selected colonies belong to the genus Campylobacter, although positive signals for serotypes of A. actinomycetemcomitans were obtained with these samples. Identification of A. actinomycetemcomitans by conventional PCR for 16S rRNA with one species-specific and one universal primer was inconclusive because an almost identical signal with Campylobacter gracilis was obtained. Although PCR-based methods for the identification of A. actinomycetemcomitans are more rapid, sequencing should not be omitted. [Projekat Ministarstva nauke Republike Srbije, br. 41008 and br. 173048]

Two novel Bacillus sp. were isolated from a soil sample from a bank of the Tamiš river in close proximity to a petrochemical facility. They were capable of utilizing a broad range of aromatic compounds as a sole source of carbon and energy (including phenol, benzene, toluene, biphenyl, naphthalene). The isolates were designated as Bacillus sp. TN41 and TN42, based on their 16S rDNA sequence. Their catabolic potential was compared to two Bacillus sp. strains (PS1 and PS11) isolated from the rhizosphere of the endemorelict plant Ramonda serbica. Specific activities of phenol hydroxylase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase were analyzed from crude cell extracts of the isolates, as well as the temperature and pH effects on enzyme activity. Although all four isolates had the ability to degrade a similar range of aromatic compounds, the specific activities of the enzymes indicative of aromatic compound catabolism of TN isolates were 2 to 90-fold lower compared to the PS isolates. Phenol hydroxylase and catechol dioxygenases exhibited broad temperature (10°C-80°C) and pH (4-9) activity ranges in all four Bacillus isolates. While phenol inhibited both phenol hydroxylase and catechol dioxygenases in the TN strains, it was an inducer for phenol hydroxylase in the PS strains.

Background/Aim. The use of color Doppler neurosonography (cD-US) allows simultaneous examination of parenchymal and vascular cerebral structures. Evaluation of cerebral blood flow velocities (CBFV) and vascular resistance are important in assessment of cerebral circulation in neonates with hypoxic ischemic encephalopathy (HIE). The aim of this study was to evaluate the predictive value of cD-US for abnormal neurodevelopmental outcome in the neonates with HIE. Methods. A total of 90 neonates (>32 weeks gestational age) with HIE were enrolled prospectively. All the neonates with HIE were categorized into three grades according to the Sarnat and Sarnat clinical staging system: mild HIE, moderate HIE, and severe HIE. cD-US was performed simultaneously during the first 24 h of life. Neurodevelopment outcome was assessed at 12 months of age in all the neonates. Resluts. The values of CBFV and the values of index resistance (RI) correlated with the severity of HIE (p < 0.0001) and subsequent neurodevelopmental outcome (p < 0.001). We detected a significant difference in values of CBFV and in values of RI between preterm and full-term neonates (p < 0.01). The cut-off value of RI for poor neurodevelopmental outcomes was 0.81. Conclusions. cD-US could be very useful and safe diagnostic tool for assessing severity of HIE and subsequent adverse neurodevelopmental outcome.

Introduction. There was used color Doppler ultrasonography (cD-USI), allowing simultaneous examination of parenchymal and vascular cerebral structures. The evaluation of blood flow velocities in cerebral arteries is important in the assessment of cerebral circulation in hypoxic-ischaemic and haemorrhagic brain damage in neonates. Objective. The aim of this study was to estimate normal values of cerebral blood flow velocities (CBFV) and Doppler indices - pulsatility index (PI) and resistance index (RI) - in the anterior cerebral artery (ACA) during the first days of life in infants. Methods. CBFV, PI and RI were obtained during the first week of life with cD-US in 70 infants divided in four groups of gestational age (GA): ≤28 gestational weeks (GW); 29-32 GW; 33-36 GW; and ≥37 GW. Infants with congenital malformations, severe perinatal asphyxia, cerebral haemorrhagic lesion, DAP or severe hypotension were excluded. Results. The mean GA of infants was 34.5±5.5 GW (range 26-40 GW) and the mean birth weight (BW) was 2540±950 g (range 750-4000 g). In the 1st group of 10 infants, ≤28 GW, the mean BW was 950±110 g and values of RI were 0.59±0.10 and PI 1.06±0.080. In the 2nd group of 20 infants, 29-32 GW, the mean BW was 1350±290 g and values of RI were 0.60±0.10 and PI 1.10±0.15. In the 3rd group of 20 infants, 33-36 GW, the mean BW was 1950±750 g and values of RI were 0.63±0.08 and PI 1.15±0.30. In the 4th group of 20 infants, ≥37 GW, the mean BW was 3540±950 g and values of RI were 0.65±0.05 and PI 1.18±0.35. Conclusion. Values of CBFV progressively increase with GA and BW due to progressive increase of cardiac output, blood pressure and closing of ductus arteriosus. Values of RI and PI gradually increase with GA and BW as a result of progressive maturation and opening of vascular cerebral bed with a reduction of the cerebrovascular resistance.

Sgm methyltransferase from Micromonospora zionensis and KgmB methyltransferase from Streptoalloteichus tenebrarius are resistant to aminoglycoside antibiotics as a result of their ability to specifically methylate G1405 within the bacterial 16S rRNA A-site. The (C)CGCCC motif, assumed to be a regulatory sequence responsible for the autoregulation of the sgm gene, could most likely also be responsible for the autoregulation of the kgmB gene. This sequence, found within the 5' untranslated region of both sgm and kgmB mRNAs, as indicated by in silico prediction, may be involved in the formation of a specific stem-loop structure. Sgm and KgmB are mutually down-regulated and it is likely that they share the same cis-acting elements. Structure probing experiments confirmed the existence of a stable secondary structure within the 5' UTR of the sgm mRNA, while the analysis of kgmB mRNA failed to confirm the predicted structure. .

Methyltransferases (MTases), a large protein superfamily, commonly use S-adenosyl-L-methionine (SAM) as the methyl group donor. SAM-dependant MTases methylate both nucleic acids (DNA, RNA) and proteins, and thus modulate their activity, function and folding. Methylation of G1405 or A1408 nucleotides of 16S rRNA in aminoglycoside-producing microorganisms confers the resistance to their own toxic product(s). This mechanism of resistance has been considered as unique to antibiotics producers until recently. Since 2003, methylation of 16S rRNA as a mechanism of resistance is increasingly emerging in pathogenic bacteria. This represents a major threat towards the usefulness of aminoglycosides in the clinical practice. A potential solution to the problem involves the design of novel compounds that would act against new ribosomal targets. The second approach to the issue includes the development of resistance MTases' inhibitors, with the idea to prevent them from modifying the bacterial rRNA, and thus reinstate the therapeutic power of existing aminoglycosides. As the latter approach has considerable potential, it is obvious that fundamental research related to protein expression, in-depth understanding of the mechanism of action and resolving a tertiary structure of 16S rRNAs MTases are prerequisites for application in medicine.

Ramonda serbica and Ramonda nathaliae, rare resurrection plants growing in the Balkan Peninsula, produce a high amount of phenolic compounds as a response to stress. The composition and size of bacterial communities in two rhizosphere soil samples of these plants were analyzed using a metagenomic approach. Fluorescent in situ hybridization (FISH) experiments together with DAPI staining showed that the metabolically active bacteria represent only a small fraction, approximately 5%, of total soil bacteria. Using universal bacteria - specific primers 16S rDNA genes were amplified directly from metagenomic DNAs and two libraries were constructed. The Restriction Fragment Length Polymorphism (RLFP) method was used in library screening. Amongst 192 clones, 35 unique operational taxonomic units (OTUs) were determined from the rhizosphere of R. nathaliae, and 13 OTUs out of 80 clones in total from the library of R. serbica. Representative clones from each OTU were sequenced. The majority of sequences from metagenomes showed very little similarity to any cultured bacteria. In conclusion, the bacterial communities in the studied soil samples showed quite poor diversity. .

Penicillin G acylase (PAC) is one of the most widely used enzymes in industrial synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (α and β) of the enzyme were N-glycosylated, while the β-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view.

INTRODUCTION C-reactive protein (CRP) is the most common diagnostic marker of infection. OBJECTIVE Objectives of this study were to determine the serum CRP level in neonates with sepsis and establish the influence of gestational age (GA) on the CRP level in the first few weeks after birth. METHOD Diagnosis of neonatal sepsis was established by the presence of clinical signs of sepsis, isolation of the causative agent of sepsis and abnormal hematological parameters. All neonates were divided into two groups: early onset sepsis (EOS) and late onset sepsis (LOS). According to GA all neonates were divided into three groups: 10 mg/l for term and near term neonates and >5 mg/l for preterm neonates. The maximum concentrations of CRP were reached 48 hr after the first symptoms of neonatal sepsis. CONCLUSION CRP levels are proportional with increasing GA and body weight in EOS. The effects of gestational age do not influence CRP levels in LOS. Maturation changes in the immune system are the most likely explanation for this and partly the organisms responsible for an infection may be different at different gestational ages and also in EOS and LOS. There is no correlation with serum CRP levels and with the severity of the disease and bad prognosis in EOS.

The 16S rRNA methylases are expressed by most of the antibiotic producing bacteria in order to protect themselves against antibiotics by methylation of 16S rRNA at positions which are crucial for their action. The sgm sisomicin-gentamicin resistance gene from Micromonospora zionensis methylates G1405 positioned in the A site of 16S rRNA, which includes a CCGCCC hexanucleotide. The same hexanucleotide is also present 14 nucleotides in front of the ribosome binding site of sgm mRNA. The model proposed for translational regulation of sgm assumes that Sgm binds to this motif, both on 16S rRNA and on the 5’ untranslated region (UTR) of its own mRNA. The 5’ UTR mRNA sequence was overexpressed on 3’-truncated sgm mRNA, and the effect on gentamicin resistance conferred by Sgm was tested in Escherichia coli and in Micromonospora melanosporea. Overexpression of the sgm mRNA regulatory region decreases the resistance to gentamicin in both E. coli and M. melanosporea. This effect is likely to be due to titration of Sgm molecules by the overexpressed 5’ UTR.

In this paper we describe the effect of the kanamycin resistance gene (Kmr) on 2μm-based plasmid maintenance in Saccharomyces cerevisiae. The influence of this marker gene on the loss of the stable model-vectors proved to be constant, as well as independent of carbon source and culture growth rates. In strains for GALUAS - driven heterologous protein production introduction of Kmr resulted in curing of the yeast episomal plasmid (YEp) from the population in a small number of generations. Application of selective pressure on the strain producing recombinant penicillin G amidase (rPGA) did not provide the expected increase of protein yield. The influence of genetic elements for heterologous protein production on vector stability was examined, and the most destabilizing factors prove to be the presence and expression of the foreign gene.

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

The Sgm gene from Micromonospora zionensis, the producer of the aminoglycoside antibiotic G-52, encodes for Sgmmethylasewhich modifies the target site on 16S rRNAand thus protects the producer against its own toxic product. The sgm gene wasmodified by polymerase chain reaction (PCR) and cloned in the QIA express pQE-30 vector in order to make a construct that places the (His)6 tag at the N-terminus of the protein. The resulting expression construct was transformed in the E. coli strain NM522 and the functional activity of the Sgm-His fusion protein was confirmed in vivo. Purification of the (His)6-tagged Sgm protein by Ni-NTA affinity chromatography was performed under native conditions and the protein was detected on a sodium dodecyl sulfate polyacrylamide gel. Sgm methylase was purified to homogeneity > 95 %. Polyclonal antibodies raised to purified (His)6-tagged Sgm protein were used to identify this protein by Western blot analysis.

The article presents a case study of a 29-year-old patient who underwent in vitro fertilization due to secondary sterility following a spontaneous but molar pregnancy. There was a failure of spontaneous conception for five years. The couple consented to an in vitro fertilization which used an ovulation induction method and the intracytoplasmic sperm injection (ICSI).