Interactions of TiO2 Nanoparticles with Ingredients from Modern Lifestyle Products and Their Effects on Human Skin Cells.

The number of consumer products containing nanoparticles (NPs) experienced a rapid increase during the past decades. However, most studies of nanosafety have been conducted using only pure NPs produced in the laboratory, while the interactions with other ingredients in consumer products have rarely been considered so far. In the present study, we investigated such interactions-with a special focus on modern lifestyle products (MLPs) used by adolescents. An extensive survey was undertaken at different high schools all over Austria to identify MLPs that either contain NPs or that could come easily in contact with NPs from other consumer products (such as TiO2 from sunscreens). Based on the results from a survey among secondary schools students, we focused on ingredients from Henna tattoos (2-hydroxy-1,4-naphtoquinone, HNQ, and p-phenylenediamine, PPD), fragrances (butylphenyl methylpropional, known as Lilial), cosmetics and skin-care products (four different parabens). As a cellular model, we decided to use neonatal normal human dermal fibroblasts (nNHDF), since skin contact is the main route of exposure for these compounds. TiO2 NPs interacted with these compounds as evidenced by alterations in their hydrodynamic diameter observed by nanoparticle tracking analysis. Combinations of TiO2 NPs with the different MLP components did not show altered cytotoxicity profiles compared to MLP components without TiO2 NPs. Nevertheless, altered cellular glutathione contents were detected after incubation of the cells with Lilial. This effect was independent of the presence of TiO2 NPs. Testing mixtures of NPs with other compounds from consumer products is an important approach to achieve a more reliable safety assessment.

The structure and function of Iristatin, a novel immunosuppressive tick salivary cystatin.

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

In silico target network analysis of de novo-discovered, tick saliva-specific microRNAs reveals important combinatorial effects in their interference with vertebrate host physiology.

The hard tick Ixodes ricinus is an important disease vector whose salivary secretions mediate blood-feeding success on vertebrate hosts, including humans. Here we describe the expression profiles and downstream analysis of de novo-discovered microRNAs (miRNAs) expressed in I. ricinus salivary glands and saliva. Eleven tick-derived libraries were sequenced to produce 67,375,557 Illumina reads. De novo prediction yielded 67 bona fide miRNAs out of which 35 are currently not present in miRBase. We report for the first time the presence of microRNAs in tick saliva, obtaining furthermore molecular indicators that those might be of exosomal origin. Ten out of these microRNAs are at least 100 times more represented in saliva. For the four most expressed microRNAs from this subset, we analyzed their combinatorial effects upon their host transcriptome using a novel in silico target network approach. We show that only the inclusion of combinatorial effects reveals the functions in important pathways related to inflammation and pain sensing. A control set of highly abundant microRNAs in both saliva and salivary glands indicates no significant pathways and a far lower number of shared target genes. Therefore, the analysis of miRNAs from pure tick saliva strongly supports the hypothesis that tick saliva miRNAs can modulate vertebrate host homeostasis and represents the first direct evidence of tick miRNA-mediated regulation of vertebrate host gene expression at the tick-host interface. As such, the herein described miRNAs may support future drug discovery and development projects that will also experimentally question their predicted molecular targets in the vertebrate host.

Microbial Degradation of Phenanthrene in Pristine and Contaminated Sandy Soils.

Phenanthrene mineralisation studies in both pristine and contaminated sandy soils were undertaken through detailed assessment of the activity and diversity of the microbial community. Stable isotope probing (SIP) was used to assess and identify active 13C-labelled phenanthrene degraders. Baseline profiling indicated that there was little difference in fungal diversity but a significant difference in bacterial diversity dependent on contamination history. Identification of dominant fungal and bacterial species highlighted the presence of organisms capable of degrading various petroleum-based compounds together with other anthropogenic compounds, regardless of contamination history. Community response following a simulated contamination event (14C-phenanthrene) showed that the microbial community in deep pristine and shallow contaminated soils adapted most to the presence of phenanthrene. The similarity in microbial community structure of well-adapted soils demonstrated that a highly adaptable fungal community in these soils enabled a rapid response to the introduction of a contaminant. Ten fungal and 15 bacterial species were identified as active degraders of phenanthrene. The fungal degraders were dominated by the phylum Basidiomycota including the genus Crypotococcus, Cladosporium and Tremellales. Bacterial degraders included the genera Alcanivorax, Marinobacter and Enterococcus. There was little synergy between dominant baseline microbes, predicted degraders and those that were determined to be actually degrading the contaminant. Overall, assessment of baseline microbial community in contaminated soils provides useful information; however, additional laboratory assessment of the microbial community's ability to degrade pollutants allows for better prediction of the bioremediation potential of a soil.

[Description of contributing factors in adverse events related to patient safety and their preventability]. / Descripción de factores contribuyentes en sucesos adversos relacionados con la seguridad del paciente y su evitabilidad.

OBJECTIVE: To assess the extent of healthcare related adverse events (AEs), their effect on patients, and their seriousness. To analyse the factors leading to the development of AEs, their relationship with the damage caused, and their degree of preventability. DESIGN: Retrospective descriptive study. LOCATION: Porriño, Pontevedra, Spain, Primary Care Service, from January-2014 to April-2016. PARTICIPANTS AND/OR CONTEXT: Reported AEs were entered into the Patient Safety Reporting and Learning System (SiNASP). METHOD: The variables measured were: Near Incident (NI) an occurrence with no effect or harm on the patient; Adverse Event (AE) an occurrence that affects or harms a patient. The level of harm is classified as minimal, minor, moderate, critical, and catastrophic. Preventability was classified as little evidence of being preventable, 50% preventable, and sound evidence of being preventable. DATA ANALYSIS: percentages and Chi-squared test for qualitative variables; P<.05 with SPSS.15. DATA SOURCE: SiNASP. Ethical considerations: approved by the Research Ethics Committee (2016/344). RESULTS: There were 166 recorded AEs (50.6% in males, and 46.4% in women. The mean age was 60.80years). Almost two-thirds 62.7% of AEs affected the patient, with 45.8% causing minimal damage, while 2.4% caused critical damages. Healthcare professionals were a contributing factor in 71.7% of the AEs, with the trend showing that poor communication and lack of protocols were related to the damage caused. Degree of preventability: 96.4%. CONCLUSIONS: Most AEs affected the patient, and were related to medication, diagnostic tests, and laboratory errors. The level of harm was related to communication problems, lack of, or deficient, protocols and a poor safety culture.

Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets.

Nitric oxide (NO), S-nitrosoglutathione (GSNO) and S-nitrosocysteine are highly potent signaling molecules, acting both by cGMP-dependent and cGMP-independent mechanisms. The NO metabolite nitrite (NO2 (-)) is a major NO reservoir. Hemoglobin, xanthine oxidoreductase and carbonic anhydrase (CA) have been reported to reduce/convert nitrite to NO. We evaluated the role and the physiological importance of CA for an extra-platelet CA/nitrite/NO/cGMP pathway in human platelets. Authentic NO was analyzed by an NO-sensitive electrode. GSNO and GS(15)NO were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). cGMP was determined by LC-MS/MS or RIA. In reduced glutathione (GSH) containing aqueous buffer (pH 7.4), human and bovine erythrocytic CAII-mediated formation of GSNO from nitrite and GS(15)NO from (15)N-nitrite. In the presence of L-cysteine and GSH, this reaction was accompanied by NO release. Incubation of nitrite with bovine erythrocytic CAII and recombinant soluble guanylyl cyclase resulted in cGMP formation. Upon incubation of nitrite with bovine erythrocytic CAII and washed human platelets, cGMP and P-VASP(S239) were formed in the platelets. This study provides the first evidence that extra-platelet nitrite and erythrocytic CAII may modulate platelet function in a cGMP-dependent manner. The new nitrite-dependent CA activity may be a general principle and explain the cardioprotective effects of inorganic nitrite in the vasculature. We propose that nitrous acid (ONOH) is the primary CA-catalyzed reaction product of nitrite.

A systems level analysis reveals transcriptomic and proteomic complexity in Ixodes ricinus midgut and salivary glands during early attachment and feeding.

Although pathogens are usually transmitted within the first 24-48 h of attachment of the castor bean tick Ixodes ricinus, little is known about the tick's biological responses at these earliest phases of attachment. Tick midgut and salivary glands are the main tissues involved in tick blood feeding and pathogen transmission but the limited genomic information for I. ricinus delays the application of high-throughput methods to study their physiology. We took advantage of the latest advances in the fields of Next Generation RNA-Sequencing and Label-free Quantitative Proteomics to deliver an unprecedented, quantitative description of the gene expression dynamics in the midgut and salivary glands of this disease vector upon attachment to the vertebrate host. A total of 373 of 1510 identified proteins had higher expression in the salivary glands, but only 110 had correspondingly high transcript levels in the same tissue. Furthermore, there was midgut-specific expression of 217 genes at both the transcriptome and proteome level. Tissue-dependent transcript, but not protein, accumulation was revealed for 552 of 885 genes. Moreover, we discovered the enrichment of tick salivary glands in proteins involved in gene transcription and translation, which agrees with the secretory role of this tissue; this finding also agrees with our finding of lower tick t-RNA representation in the salivary glands when compared with the midgut. The midgut, in turn, is enriched in metabolic components and proteins that support its mechanical integrity in order to accommodate and metabolize the ingested blood. Beyond understanding the physiological events that support hematophagy by arthropod ectoparasites, we discovered more than 1500 proteins located at the interface between ticks, the vertebrate host, and the tick-borne pathogens. Thus, our work significantly improves the knowledge of the genetics underlying the transmission lifecycle of this tick species, which is an essential step for developing alternative methods to better control tick-borne diseases.

A Deep Insight Into the Sialotranscriptome of the Chagas Disease Vector, Panstrongylus megistus (Hemiptera: Heteroptera).

Saliva of blood-sucking arthropods contains a complex cocktail of pharmacologically active compounds that assists feeding by counteracting their hosts' hemostatic and inflammatory reactions. Panstrongylus megistus (Burmeister) is an important vector of Chagas disease in South America, but despite its importance there is only one salivary protein sequence publicly deposited in GenBank. In the present work, we used Illumina technology to disclose and publicly deposit 3,703 coding sequences obtained from the assembly of >70 million reads. These sequences should assist proteomic experiments aimed at identifying pharmacologically active proteins and immunological markers of vector exposure. A supplemental file of the transcriptome and deducted protein sequences can be obtained from http://exon.niaid.nih.gov/transcriptome/P_megistus/Pmeg-web.xlsx.

Salivary antigen-5/CAP family members are Cu2+-dependent antioxidant enzymes that scavenge O2₋. and inhibit collagen-induced platelet aggregation and neutrophil oxidative burst.

The function of the antigen-5/CAP family of proteins found in the salivary gland of bloodsucking animals has remained elusive for decades. Antigen-5 members from the hematophagous insects Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation, ATP secretion, and thromboxane A2 generation by low doses of collagen (<1 µg/ml) but no other agonists. DMAV did not interact with collagen, glycoprotein VI, or integrin α2ß1. This inhibitory profile resembles the effects of antioxidants Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in platelet function. Accordingly, DMAV was found to inhibit cytochrome c reduction by O2[Symbol: see text] generated by the xanthine/xanthine oxidase, implying that it exhibits antioxidant activity. Moreover, our results demonstrate that DMAV blunts the luminescence signal of O2[Symbol: see text] generated by phorbol 12-myristate 13-acetate-stimulated neutrophils. Mechanistically, inductively coupled plasma mass spectrometry and fluorescence spectroscopy revealed that DMAV, like Cu,Zn-SOD, interacts with Cu(2+), which provides redox potential for catalytic removal of O2[Symbol: see text]. Notably, surface plasmon resonance experiments (BIAcore) determined that DMAV binds sulfated glycosaminoglycans (e.g. heparin, KD ~100 nmol/liter), as reported for extracellular SOD. Finally, fractions of the salivary gland of D. maxima with native DMAV contain Cu(2+) and display metal-dependent antioxidant properties. Antigen-5/CAP emerges as novel family of Cu(2+)-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and negatively modulate platelet aggregation by a unique salivary mechanism.

Understanding the evolutionary structural variability and target specificity of tick salivary Kunitz peptides using next generation transcriptome data.

BACKGROUND: Ticks are blood-sucking arthropods and a primary function of tick salivary proteins is to counteract the host's immune response. Tick salivary Kunitz-domain proteins perform multiple functions within the feeding lesion and have been classified as venoms; thereby, constituting them as one of the important elements in the arms race with the host. The two main mechanisms advocated to explain the functional heterogeneity of tick salivary Kunitz-domain proteins are gene sharing and gene duplication. Both do not, however, elucidate the evolution of the Kunitz family in ticks from a structural dynamic point of view. The Red Queen hypothesis offers a fruitful theoretical framework to give a dynamic explanation for host-parasite interactions. Using the recent salivary gland Ixodes ricinus transcriptome we analyze, for the first time, single Kunitz-domain encoding transcripts by means of computational, structural bioinformatics and phylogenetic approaches to improve our understanding of the structural evolution of this important multigenic protein family. RESULTS: Organizing the I. ricinus single Kunitz-domain peptides based on their cysteine motif allowed us to specify a putative target and to relate this target specificity to Illumina transcript reads during tick feeding. We observe that several of these Kunitz peptide groups vary in their translated amino acid sequence, secondary structure, antigenicity, and intrinsic disorder, and that the majority of these groups are subject to a purifying (negative) selection. We finalize by describing the evolution and emergence of these Kunitz peptides. The overall interpretation of our analyses discloses a rapidly emerging Kunitz group with a distinct disulfide bond pattern from the I. ricinus salivary gland transcriptome. CONCLUSIONS: We propose a model to explain the structural and functional evolution of tick salivary Kunitz peptides that we call target-oriented evolution. Our study reveals that combining analytical approaches (transcriptomes, computational, bioinformatics and phylogenetics) improves our understanding of the biological functions of important salivary gland mediators during tick feeding.

De novo Ixodes ricinus salivary gland transcriptome analysis using two next-generation sequencing methodologies.

Tick salivary gland (SG) proteins possess powerful pharmacologic properties that facilitate tick feeding and pathogen transmission. For the first time, SG transcriptomes of Ixodes ricinus, an important disease vector for humans and animals, were analyzed using next-generation sequencing. SGs were collected from different tick life stages fed on various animal species, including cofeeding of nymphs and adults on the same host. Four cDNA samples were sequenced, discriminating tick SG transcriptomes of early- and late-feeding nymphs or adults. In total, 441,381,454 pyrosequencing reads and 67,703,183 Illumina reads were assembled into 272,220 contigs, of which 34,560 extensively annotated coding sequences are disclosed; 8686 coding sequences were submitted to GenBank. Overall, 13% of contigs were classified as secreted proteins that showed significant differences in the transcript representation among the 4 SG samples, including high numbers of sample-specific transcripts. Detailed phylogenetic reconstructions of two relatively abundant SG-secreted protein families demonstrated how this study improves our understanding of the molecular evolution of hematophagy in arthropods. Our data significantly increase the available genomic information for I. ricinus and form a solid basis for future tick genome/transcriptome assemblies and the functional analysis of effectors that mediate the feeding physiology and parasite-vector interaction of I. ricinus.

Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket.

Virtually all DNA viruses including hepatitis B viruses (HBV) replicate their genome inside the nucleus. In non-dividing cells, the genome has to pass through the nuclear pore complexes (NPCs) by the aid of nuclear transport receptors as e.g. importin beta (karyopherin). Most viruses release their genome in the cytoplasm or at the cytosolic face of the NPC, as the diameter of their capsids exceeds the size of the NPC. The DNA genome of HBV is derived from reverse transcription of an RNA pregenome. Genome maturation occurs in cytosolic capsids and progeny capsids can deliver the genome into the nucleus causing nuclear genome amplification. The karyophilic capsids are small enough to pass the NPC, but nuclear entry of capsids with an immature genome is halted in the nuclear basket on the nuclear side of the NPC, and the genome remains encapsidated. In contrast, capsids with a mature genome enter the basket and consequently liberate the genome. Investigating the difference between immature and mature capsids, we found that mature capsids had to disintegrate in order to leave the nuclear basket. The arrest of a karyophilic cargo at the nuclear pore is a rare phenomenon, which has been described for only very few cellular proteins participating in nuclear entry. We analyzed the interactions causing HBV capsid retention. By pull-down assays and partial siRNA depletion, we showed that HBV capsids directly interact with nucleoporin 153 (Nup153), an essential protein of the nuclear basket which participates in nuclear transport via importin beta. The binding sites of importin beta and capsids were shown to overlap but capsid binding was 150-fold stronger. In cellulo experiments using digitonin-permeabilized cells confirmed the interference between capsid binding and nuclear import by importin beta. Collectively, our findings describe a unique nuclear import strategy not only for viruses but for all karyophilic cargos.

Renal carbonic anhydrases are involved in the reabsorption of endogenous nitrite.

Nitrite (ONO(-)) exerts nitric oxide (NO)-related biological actions and its concentration in the circulation may be of particular importance. Nitrite is excreted in the urine. Hence, the kidney may play an important role in nitrite/NO homeostasis in the vasculature. We investigated a possible involvement of renal carbonic anhydrases (CAs) in endogenous nitrite reabsorption in the proximal tubule. The potent CA inhibitor acetazolamide was administered orally to six healthy volunteers (5 mg/kg) and nitrite was measured in spot urine samples before and after administration. Acetazolamide increased abruptly nitrite excretion in the urine, strongly suggesting that renal CAs are involved in nitrite reabsorption in healthy humans. Additional in vitro experiments support our hypothesis that nitrite reacts with CO(2), analogous to the reaction of peroxynitrite (ONOO(-)) with CO(2), to form acid-labile nitrito carbonate [ONOC(O)O(-)]. We assume that this reaction is catalyzed by CAs and that nitrito carbonate represents the nitrite form that is actively transported into the kidney. The significance of nitrite reabsorption in the kidney and the underlying mechanisms, notably a direct involvement of CAs in the reaction between nitrite and CO(2), remain to be elucidated.

Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation.

Capsids of hepatitis B virus and other hepadnaviruses contain a cellular protein kinase, which phosphorylates the capsid protein. Some phosphorylation sites are shown to be essential for distinct steps of viral replication as pregenome packaging or plus strand DNA synthesis. Although different protein kinases have been reported to phosphorylate the capsid protein, varying experimental approaches do not allow direct comparison. Furthermore, the activity of a specific protein kinase has not yet been correlated to steps in the hepadnaviral life cycle. In this study we show that capsids from various sources encapsidate active protein kinase Calpha, irrespective of hepatitis B virus genotype and host cell. Treatment of a virion expressing cell line with a pseudosubstrate inhibitor showed that inhibition of protein kinase C phosphorylation did not affect genome maturation but resulted in capsid accumulation and inhibited virion release to the medium. Our results imply that different protein kinases have distinct functions within the hepadnaviral life cycle.

Obesity in children and adolescents: relationship to growth, pubarche, menarche, and voice break.

OBJECTIVE: The relationships between obesity, pubertal development, and height are controversial. Therefore, we compared the prevalence of pubarche, menarche, and voice break between a large collective of obese and normal-weight children and adolescents aged 10-16 years. METHODS: We assessed weight, height, pubarche, menarche, and voice break in 1383 obese German children and in 6615 children of a representative national German cohort aged 10-16 years. In all obese children, gonadotropins were determined and birth weight data were collected. RESULTS: Independently of gender, the height standard deviation score (SDS) was significantly greater (0.3-1.0) in obese children <14 years compared to the reference cohort. Independently of age, the percentage of obese boys with pubarche was significantly lower compared to age-matched normal-weight boys. In girls <13 years, the prevalence of obese girls with pubarche was significantly lower compared to age-matched normal-weight girls. In boys > or =11 years, the percentage of obese boys with change of voice was significantly lower compared to age-matched normal-weight boys. In girls > or =11 years, the prevalence of obese girls with menarche was significantly lower compared to age-matched normal-weight girls. Birth weight had no impact on pubarche in the obese children. Luteinizing hormone was > 0.3 IU/L in 86% of the children > or =10 years with pubarche. CONCLUSIONS: Obese children are taller than normal-weight children up to the age of 14 years. Since obese children demonstrated pubarche, menarche, and voice break later than their normal-weight peers, the increase in height in obese children does not seem to be attributable to earlier onset of puberty.

"Short-chain" alpha-1,4-glucan phosphorylase having a truncated N-terminal domain: functional expression and characterization of the enzyme from Sulfolobus solfataricus.

All known alpha-1,4-glucan phosphorylases (GPs) are active as homodimers and use their N-terminal domains for oligomerisation. Structure-based sequence comparison of a putative phosphorylase from the thermophilic crenarchaeon Sulfolobus solfataricus (SsGP) with the well characterized GP from Escherichia coli reveals that SsGP totally lacks the otherwise conserved regions for building the dimer interface. Because all efforts of producing functional SsGP in E. coli failed, we used heterologous gene expression in the hyperthermophilic archaeon Thermococcus kodakaraensis and isolated, in low amounts, SsGP harboring Strep-Tag II fused to the C-terminal Tyr-465 of the enzyme. The recombinant protein eluted in size exclusion chromatography with an apparent molecular mass of approximately 69 kDa, consistent with neither the mass expected for a monomer (55 kDa) nor that of a homodimer (110 kDa). The biochemical properties of SsGP were similar to those seen for other GPs containing the N-terminal elements for dimerisation, suggesting that the "short-chain" format of SsGP is fully appropriate for phosphorylase catalytic function and stability. However, the substrate specificity of SsGP differed from that reported for GPs from other thermophilic microorganisms.

Simultaneous measurement of [15N]nitrate and [15N]nitrite enrichment and concentration in urine by gas chromatography mass spectrometry as pentafluorobenzyl derivatives.

Quantification of (15)N-labeled nitrate and determination of the (15)N-enrichment in urine upon administration of (15)N-labeled precursors such as L-[guanidino-(15)N(2)]arginine is a suitable approach to study formation and metabolism of nitric oxide (NO) and its metabolites in vivo. Previously, we have reported on the simultaneous derivatization and accurate quantification of nitrite and nitrate in various biological fluids using pentafluorobenzyl bromide and GC/MS. We report here on a modification of this method that allows for the simultaneous determination of (15)N-enrichment of [(15)N]nitrate and [(15)N]nitrite and the simultaneous quantification of [(15)N]nitrate, [(14)N]nitrate, [(15)N]nitrite, and [(14)N]nitrite in human urine. In a pilot study, using the carbonic anhydrase inhibitor drug acetazolamide at therapeutical oral doses (5.4 and 5 mg per kg bodyweight) and by oral intake of [(15)N]nitrite (0.31 and 0.5 micromol per kg bodyweight) by two healthy volunteers, we demonstrate for the first time that renal carbonic anhydrase activity is mainly responsible for the reabsorption of nitrite from the primary urine and confirm previous findings on nitrate.

Enzymatic synthesis of beta-glucosylglycerol using a continuous-flow microreactor containing thermostable beta-glycoside hydrolase CelB immobilized on coated microchannel walls.

beta-Glucosylglycerol (betaGG) has potential applications as a moisturizing agent in cosmetic products. A stereochemically selective method of its synthesis is kinetically controlled enzymatic transglucosylation from a suitable donor substrate to glycerol as acceptor. Here, the thermostable beta-glycosidase CelB from Pyrococcus furiosus was used to develop a microstructured immobilized enzyme reactor for production of betaGG under conditions of continuous flow at 70 degrees C. Using CelB covalently attached onto coated microchannel walls to give an effective enzyme activity of 30 U per total reactor working volume of 25 microL, substrate conversion and formation of transglucosylation product was monitored in dependence of glucosyl donor (2-nitrophenyl-beta-D-glucoside (oNPGlc), 3.0 or 15 mM; cellobiose, 250 mM), the concentration of glycerol (0.25-1.0 M), and the average residence time (0.2-90 s). Glycerol caused a concentration-dependent decrease in the conversion of the glucosyl donor via hydrolysis and strongly suppressed participation of the substrate in the reaction as glucosyl acceptor. The yields of betaGG were > or =80% and approximately 60% based on oNPGlc and cellobiose converted, respectively, and maintained up to near exhaustion of substrate (> or =80%), giving about 120 mM (30 g/L) of betaGG from the reaction of cellobiose and 1 M glycerol. The structure of the transglucosylation products, 1-O-beta-D-glucopyranosyl-rac-glycerol (79%) and 2-O-beta-D-glucopyranosyl-sn-glycerol (21%), was derived from NMR analysis of the product mixture of cellobiose conversion. The microstructured reactor showed conversion characteristics similar to those for a batchwise operated stirred reactor employing soluble CelB. The advantage of miniaturization to the microfluidic format lies in the fast characterization of full reaction time courses for a range of process conditions using only a minimum amount of enzyme.

Analysis of the distribution of the tick Ixodes ricinus L. (Acari: Ixodidae) in a nature reserve of western Germany using Geographic Information Systems.

From May to November 2003, at five selected sites of the 'Siebengebirge', a nature reserve near Bonn, Germany, data on microclimate, pedology, plant sociology and tick abundance were collected weekly and correlated. A total of 2832 host-seeking Ixodes ricinus (2660 nymphs and 172 adults) were collected, with maximal abundance in June and a minor second increase in abundance in August. The spatial and temporal variation in tick abundance was closely related to the air temperature, relative humidity, soil water content and specific vegetation type. Increases in tick abundances in individual habitats were most likely caused by climate change and/or anthropogenic interventions such as increased numbers of wild boar, a host of I. ricinus. When combined with data from previous investigations a correlation of plant communities with six semi-qualitative tick abundance categories (ranging from 'no ticks' to 'very high tick abundance') was revealed, and GIS analysis indicated that 56.5% of the 'Siebengebirge' is made up of plant communities belonging to the three highest tick abundance categories. Therefore, the risk of visitors in the 'Siebengebirge' of being exposed to tick bites and being infected by Borrelia burgdorferi sensu lato had increased.

In situ proton NMR study of acetyl and formyl group migration in mono-O-acyl D-glucose.

Acetyl and formyl group migration, mutarotation, and hydrolysis of mono-O-acylated glucose are studied by in situ 1D and 2D (1)H NMR spectroscopy. Alpha-D-glucosyl-1-acetate and alpha-D-glucosyl-1-formate serve as sole starting materials. They are generated in situ by configuration retaining glucosyltransfer from alpha-D-glucosyl-1-phosphate to formate and acetate, which is catalyzed by the Glu-237 --> Gln mutant of Leuconostoc mesenteroides sucrose phosphorylase. Temporary accumulated regio-isomeric mono-O-acyl D-glucoses are identified, characterized, and quantified directly from the reaction mixture. Time courses of the transformations give insight into pH dependence of acyl group migration and mutarotation as well as into the stability of various regioisomers.