Clinical performance of a low cost near infrared sensor for continuous glucose monitoring applied with subcutaneous microdialysis.

In this work we present a low cost, minimally invasive, and chip-based near infrared (NIR) sensor, combined with subcutaneous microdialysis, for continuous glucose monitoring (CGM). The sensor principle is based on difference absorption spectroscopy in the 1st overtone band known to be dominated by glucose-specific absorption features. The device comprises a multi-emitter LED and InGaAs-photodiodes, which are located on a single electronic board (non-disposable part), connected to a personal computer via Bluetooth. The disposable part consists of a chip containing the fluidic connections for microdialysis, two fluidic channels acting as optical transmission cells and total internally reflecting mirrors for in- and out-coupling of the light to the chip and to the detectors. The use of the sensor in conjunction with a subcutaneous microdialysis catheter to separate the glucose from the cells and proteins has been demonstrated to be extremely useful and advantageous for obtaining continuous glucose monitoring data and detecting glycemic levels in real time for a long period. Several in vitro and in vivo experiments were conducted to test the reliability of the device. In vitro measurements showed a linear relationship between glucose concentration and the integrated difference signal with a coefficient of determination of 99 % at the physiological concentration range. Clinical trial on 6 subjects with Type 1 diabetes showed that the NIR-CGM sensor data reflects the blood reference values adequately, if a proper calibration and signal drift compensation is applied. The MARD (mean absolute relative difference) value taken on retrospective data over all subjects is 8.5 % (range 6-11.5 %).

Sequencing and analysis of the biosynthetic gene cluster of the lipopeptide antibiotic Friulimicin in Actinoplanes friuliensis.

Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin, which is a cyclic peptide with one exocyclic amino acid linked to a branched-chain fatty acid acyl residue. The structural relationship to daptomycin and the excellent antibacterial performance of friulimicin make the antibiotic an attractive drug candidate. The complete friulimicin biosynthetic gene cluster of 24 open reading frames from A. friuliensis was sequenced and analyzed. In addition to genes for regulation, self-resistance, and transport, the cluster contains genes encoding peptide synthetases, proteins involved in the synthesis and linkage of the fatty acid component of the antibiotic, and proteins involved in the synthesis of the nonproteinogenic amino acids pipecolinic acid, methylaspartic acid, and 2,3-diaminobutyric acid. By using heterologous gene expression in Escherichia coli, we provide biochemical evidence for the stereoselective synthesis of L-pipecolinic acid by the deduced protein of the lysine cyclodeaminase gene pip. Furthermore, we show the involvement of the dabA and dabB genes in the biosynthesis of 2,3-diaminobutyric acid by gene inactivation and subsequent feeding experiments.

Biosynthetic gene cluster for the polyenoyltetramic acid alpha-lipomycin.

The gram-positive bacterium Streptomyces aureofaciens Tü117 produces the acyclic polyene antibiotic alpha-lipomycin. The entire biosynthetic gene cluster (lip gene cluster) was cloned and characterized. DNA sequence analysis of a 74-kb region revealed the presence of 28 complete open reading frames (ORFs), 22 of them belonging to the biosynthetic gene cluster. Central to the cluster is a polyketide synthase locus that encodes an eight-module system comprised of four multifunctional proteins. In addition, one ORF shows homology to those for nonribosomal peptide synthetases, indicating that alpha-lipomycin belongs to the classification of hybrid peptide-polyketide natural products. Furthermore, the lip cluster includes genes responsible for the formation and attachment of d-digitoxose as well as ORFs that resemble those for putative regulatory and export functions. We generated biosynthetic mutants by insertional gene inactivation. By analysis of culture extracts of these mutants, we could prove that, indeed, the genes involved in the biosynthesis of lipomycin had been cloned, and additionally we gained insight into an unusual biosynthesis pathway.

Characterization of a new extracellular hydrolase from Thermobifida fusca degrading aliphatic-aromatic copolyesters.

The paper describes the purification, biochemical characterization, sequence determination, and classification of a novel thermophilic hydrolase from Thermobifida fusca (TfH) which is highly active in hydrolyzing aliphatic-aromatic copolyesters. The secretion of the extracellular enzyme is induced by the presence of aliphatic-aromatic copolyesters but also by adding several other esters to the medium. The hydrophobic enzyme could be purified applying a combination of (NH(4))SO(4)-precipitation, cation-exchange chromatography, and hydrophobic interaction chromatography. The 28 kDa enzyme exhibits a temperature maximum of activity between 65 and 70 degrees C and a pH maximum between pH 6 and 7 depending on the ion strength of the solution. According to the amino sequence determination, the enzyme consists of 261 amino acids and was classified as a serine hydrolase showing high sequence similarity to a triacylglycerol lipase from Streptomyces albus G and triacylglycerol-aclyhydrolase from Streptomyces sp. M11. The comparison with other lipases and esterases revealed the TfH exhibits a catalytic behavior between a lipase and an esterase. Such enzymes often are named as cutinases. However, the results obtained here show, that classifying enzymes as cutinases seems to be generally questionable.

Exploiting the genetic potential of polyketide producing streptomycetes.

Streptomycetes are the most important bacterial producers of bioactive secondary metabolites such as antibiotics or cytostatics. Due to the emerging resistance of pathogenic bacteria to all commonly used antibiotics, new and modified natural compounds are required for the development of novel drugs. In addition to the classical screening for natural compounds, genome driven approaches like combinatorial biosynthesis are permanently gaining relevance for the generation of new structures. This technology utilizes the combination of genes from different biosynthesis pathways resulting in the production of novel or modified metabolites. The basis for this strategy is the access to a significant number of genes and the knowledge about the activity and specificity of the enzymes encoded by them. A joint initiative was started to exploit the biosynthesis gene clusters from streptomycetes. In this publication, an overview of the strategy for the identification and characterization of numerous biosynthesis gene clusters for polyketides displaying interesting functions and particular structural features is given.

Biosynthetic gene cluster of simocyclinone, a natural multihybrid antibiotic.

The entire simocyclinone biosynthetic cluster (sim gene cluster) from the producer Streptomyces antibioticus Tü6040 was identified on six overlapping cosmids (1N1, 5J10, 2L16, 2P6, 4G22, and 1K3). In total, 80.7 kb of DNA from these cosmids was sequenced, and the analysis revealed 49 complete open reading frames (ORFs). These ORFs include genes responsible for the formation and attachment of four different moieties originating from at least three different pools of primary metabolites. Also in the sim gene cluster, four ORFs were detected that resemble putative regulatory and export functions. Based on the putative function of the gene products, a model for simocyclinone D8 biosynthesis was proposed. Biosynthetic mutants were generated by insertional gene inactivation experiments, and culture extracts of these mutants were analyzed by high-performance liquid chromatography. Production of simocyclinone D8 was clearly detectable in the wild-type strain but was not detectable in the mutant strains. This indicated that indeed the sim gene cluster had been cloned.

Biosynthesis of the orthosomycin antibiotic avilamycin A: deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces viridochromogenes Tü57 and production of new antibiotics.

BACKGROUND: Streptomyces viridochromogenes Tü57 is the producer of avilamycin A. The antibiotic consists of a heptasaccharide side chain and a polyketide-derived dichloroisoeverninic acid as aglycone. Molecular cloning and characterization of the genes governing the avilamycin A biosynthesis is of major interest as this information might set the direction for the development of new antimicrobial agents. RESULTS: A 60-kb section of the S. viridochromogenes Tü57 chromosome containing genes involved in avilamycin biosynthesis was sequenced. Analysis of the DNA sequence revealed 54 open reading frames. Based on the putative function of the gene products a model for avilamycin biosynthesis is proposed. Inactivation of aviG4 and aviH, encoding a methyltransferase and a halogenase, respectively, prevented the mutant strains from producing the complete dichloroisoeverninic acid moiety resulting in the accumulation of new antibiotics named gavibamycins. CONCLUSIONS: The avilamycin A biosynthetic gene cluster represents an interesting system to study the formation and attachment of unusual deoxysugars. Several enzymes putatively responsible for specific steps of this pathway could be assigned. Two genes encoding enzymes involved in post-PKS tailoring reactions were deleted allowing the production of new analogues of avilamycin A.

Construction and characterization of a gridded cattle BAC library.

A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de).

A 5x genome coverage bovine BAC library: production, characterization, and distribution.

A bovine large-insert DNA library has been constructed in a Bacterial Artificial Chromosome (BAC) vector. The source DNA was derived from lymphocytes of a Jersey male. High-molecular-weight DNA fragments were produced by treatment with EcoRI/EcoRI methylase and cloned into the EcoRI site of pBACe3.6. In total, 157,240 individual BACs have been picked into 384-well plates. Approximately 190 randomly chosen clones have been characterized by Pulsed Field Gel Electrophoresis (PFGE) and have an average insert size of 105 kb, suggesting library coverage representing 5-6 genome equivalents. The frequency of clones without inserts is 4%. The chromosomal location of 51 BACs was studied by FISH; 3 showed more than one signal, indicating a chimerism frequency of roughly 6%. Approximately 50% of the clones in the library contain Simple Repeat Sequences (microsatellites), and 4% of the clones contain centromeric repeats. Insert stability was assessed by restriction digestion of DNA prepared from 20 clones after serial culture for one and three nights. Only one clone showed any evidence of an altered restriction pattern. Clones from 360 x 384-well plates (138,240 colonies) were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Both membranes and superpools are available from the RZPD, Berlin (http://www.rzpd.de). PCR 4-D superpools have been prepared from an additional 23,000 clones. The library has been screened for a total of 24 single-copy sequences; positive clones have been obtained in all cases.

Gridded genomic libraries of different chordate species: a reference library system for basic and comparative genetic studies of chordate genomes.

The use of genomic libraries maintained in arrayed format is becoming a more and more popular tool for the analysis of molecular evolution and comparative molecular development. Being able to use already existing reference libraries considerably reduces the work load, and if results are made publicly available, it will facilitate in silica experiments in the future. Here we describe the construction and preliminary characterization of six cosmid libraries of different chordate species, Ciona intestinalis (Hemichordate), Branchiostoma floridae (Cephalochordate), Lampetra fluviatilis (Cyclostoma), Xiphophorus maculatus, and Danio rerio (Osteichthyes) in Lawrist7 and Fugu rubripes in Lawrist4.

Patients' profiles in end stage coronary artery disease. Indications for treatment with transmyocardial laser revascularisation.

There is a subgroup of patients with coronary artery disease who are refractory to the therapeutical methods so far applied. We report on 128 patients who fulfill this definition and have therefore undergone pure transmyocardial laser revascularisation (TMLR) or transmyocardial laser revascularisation in combination with coronary bypass surgery at our institution. The patients can be characterized by a long history of coronary artery disease with multiple revascularizing procedures, e.g. bypass surgery or percutaneous transluminal coronary angioplasty (PTCA), pronounced symptoms of coronary artery disease and chronic heart failure in the presence of markedly reduced left ventricular ejection fractions and intense antiischemic medical therapy. The patients were 62.2 +/- 9.8 (SD) years of age, in 89.9% of them at least one bypass operation and in 44.5% up to more than three percutaneous transluminal coronary angioplasties (PTCAs) had been performed prior to TMLR. There was a history of myocardial infarction in 90.7% of patients and 89.8% were in the Canadian Cardiovascular Society (CCS) classes III or IV and 94.5% of them were in the NYHA classes III or IV. The left ventricular ejection fraction was 49.5 +/- 16.4% and all of the patients were under intense antiischemic medical treatment which included nitrates or molsidomine in 96.9%, beta blockers in 53.1%, angiotensin converting enzyme inhibitors (ACE inhibitors) in 44.5%, digitalis in 22.7% and diuretics in 52.3% of patients. The preoperative data on myocardial viability, inducible ischemia and coronary morphology provided important clinical information for the decision, which revascularizing method would be the most appropriate for each vessel or myocardial region. This had to be weighed against the patient's operative risk, which is predominantly determined by the left ventricular ejection fraction, the arteriosclerotic involvement of the remaining vascular system and concomitant diseases, particularly of pulmonary origin.

Increase of pain threshold by calcitonin during electrical stimulation of the dental pulp. Investigations in healthy volunteers with regard to plasma concentration.

A randomized, double-blind study was performed to test the analgesic effect of salmon calcitonin (sCT). The pain threshold of ten healthy subjects was measured during electrical stimulation of the dental pulp. Each subject underwent four different tests, whereby sCT at doses of 50 IU, 100 IU and 200 IU or placebo was administered subcutaneously. For all subjects, four curves were obtained that showed the time course of the intensity of electrical stimulation needed to attain the pain threshold, up to 240 min post-application. Parallel to these studies, radioimmunoassay was performed to determine the plasma level at the time at which the maximum concentration was expected, i.e. 15 min after the injection. The pain threshold was raised by 10 mA or more with all three doses of calcitonin tested. The latent period before the threshold had risen by 10 mA declined in parallel with the doses of sCT administered (from 82 to 40+min), whereas the duration of action was increased (from 95 to over 182 min). The maximal threshold change was also significantly dependent on the dosage: with placebo the maximal change was 4 mA, while with sCT 50, sCT 100, and sCT 200 it was 14, 17 and 18 mA, respectively. The plasma levels of sCT and its analgesic activity were significantly correlated, as was demonstrated by means of linear regression based upon the bilogarithmic transformation of the plasma concentration. Altogether, the results prove conclusively that calcitonin given systemically possesses primary analgesic efficacy, a property that fits well into its spectrum of activity in the treatment of (painful) bone diseases.