Characterization of CIM monoliths as enzyme reactors

The immobilization of the enzymes citrate lyase, malate dehydrogenase, isocitrate dehydrogenase and lactate dehydrogenase to CIM monolithic supports was performed. The long-term stability, reproducibility, and linear response range of the immobilized enzyme reactors were investigated along with the determination of the kinetic behavior of the enzymes immobilized on the CIM monoliths. The Michaelis-Menten constant K(m) and the turnover number k(3) of the immobilized enzymes were found to be flow-unaffected. Furthermore, the K(m) values of the soluble and immobilized enzyme were found to be comparable. Both facts indicate the absence of a diffusional limitation in immobilized CIM enzyme reactors.     

Speciation of Al in human serum by convective-interaction media fast-monolithic chromatography with inductively coupled plasma mass spectrometric detection

A new analytical procedure using anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of Al in human serum. The separation of proteins was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic disk. To prevent co-elution of low molecular mass (LMM) Al species with high molecular mass (HMM) Al compounds on CIM disk serum proteins were first separated from LMM-Al species by the use of size exclusion chromatography (SEC). For this purpose 1 mL of serum was injected onto SEC (Superdex 75 HR 10/30) column. Isocratic elution using 0.05 M TRIS-HCl+0.03 M NaHCO(3) was applied and separation of proteins was followed by UV detection at 278 nm. It was experimentally proven that proteins were eluted in 5.5 mL peak that was collected into a polyethylene cup. A 0.1 mL of the sample aliquot was then injected onto the CIM DEAE disk. The separation of serum proteins was obtained in 10 min by applying linear gradient elution from 100% buffer A (0.05 M TRIS-HCl+0.03 M NaHCO(3)) to 100% buffer B (A+1M NH(4)Cl) and followed by UV detection at 278 nm. Separated Al species were detected on-line by inductively coupled plasma mass spectrometry (ICP-MS). Well-resolved protein peaks were obtained. It was experimentally proven that 90+/-3% of Al in spiked serum of renal patient was eluted under the transferrin peak. The proposed speciation procedure removes LMM-Al species and enables reliable determination of the concentration and composition of Al bound to proteins by CIM DEAE-ICP-MS when the concentration of Al in serum is higher than 5 ng mL(-1). In comparison to chromatographic columns CIM disks enable faster separation and simpler manipulation during cleaning procedure and coupling to ICP-MS.     

Selective recording of electroneurograms from the left vagus nerve of a dog during stimulation of cardiovascular or respiratory systems

Selective electroneurograms (ENGs) from superficial regions of the left vagus nerve of a dog were recorded with a 33-electrode spiral cuff (cuff) implanted on the nerve at the neck in an adult Beagle dog. The electrodes in the cuff were arranged in thirteen groups of three electrodes (GTE 1-13). To identify the relative positions of the particular nerve regions that innervated the heart and lungs, stimulating pulses (2 mA, 200 micros, 20 Hz) were individually delivered to all thirteen GTEs. It was shown that by delivering stimulating pulses to GTEs 4 and 9, heart rate, blood pressure and respiratory rate were modulated. Precisely, only when the stimuli were delivered to GTE 9, the heart rate began to fall and only when the stimuli were delivered to GTE 4 the rate of breathing decreased. To test the selectivity of recording the above-defined groups GTEs 4 and 9 and randomly chosen GTEs 1 and 7 were simultaneously used as recording GTEs while cardio-vascular or respiratory systems were stimulated by carotid artery compression, epinephrine injection and non-invasive, positive end-pressure ventilation. Results showed that stimulations elicited site-specific changes in ENG power spectra recorded from the superficial regions of the vagus nerve. Power spectrum of the ENG recorded with GTE 9, contained frequencies belonging to the neural activity elicited by compression of the carotid artery and injection of epinephrine. The power spectrum of the ENG recorded with GTE 4, contained frequencies belonging to the neural activity elicited by non-invasive, positive end-expiratory pressure ventilation. We concluded that the multi-electrode nerve cuff enables selective stimulation and recording of nerve activity from internal organs.     

Metal binding of metallothioneins in human astrocytomas (U87 MG,IPDDC-2A)

Astroglia cells structurally and nutritionally support neurons in the central nervous system. They play an important role in guiding the construction of the nervous system and controlling the chemical and ionic environment of neurons. They also represent the major sites for accumulation and immobilisation of toxic metal ions most probably connected with metallothioneins. For this reason astroglia cells possess high cytosolic levels of metallothioneins I, II and III (MT-I,II,III). Our aim was to establish the inducibility and metal binding of MTs in two human astrocytoma cell lines, U87 MG (astrocytoma–glioblastoma, grade IV) and IPDDC-2A (astrocytoma, grade II), on exposure to cadmium chloride (1 μM). MTs were identified by molecular weight (size exclusion chromatography) and their metal content (Cd, Zn and Cu) to follow the interactions between metals. We showed that MTs are constitutively expressed in both human astrocytoma cell lines. In accordance with the higher malignancy grade of U87 MG, the amount of MTs was higher in U87 MG than in IPDDC-2A cells. After 24 hours of exposure to Cd their expression greatly increased in both cell lines and they were capable of immobilising almost all water soluble Cd. Induction of MTs in U87 MG cells was additionally followed up to 48 hours with exposure to different concentrations of CdCl₂ (1, 10 μM). Induction was a time dependent process throughout the period. Isoform III (identified by chromatographic separation of isoform III from I/II) was present at all exposure times, but only in traces with respect to the prevailing amounts of MT-I/II isoforms. So induction can be attributed to isoform I/II only.     

Potential of promotion of alleles by genome editing to improve quantitative traits in livestock breeding programs

Background

Genome editing (GE) is a method that enables specific nucleotides in the genome of an individual to be changed. To date, use of GE in livestock has focussed on simple traits that are controlled by a few quantitative trait nucleotides (QTN) with large effects. The aim of this study was to evaluate the potential of GE to improve quantitative traits that are controlled by many QTN, referred to here as promotion of alleles by genome editing (PAGE).

Methods

Multiple scenarios were simulated to test alternative PAGE strategies for a quantitative trait. They differed in (i) the number of edits per sire (0 to 100), (ii) the number of edits per generation (0 to 500), and (iii) the extent of use of PAGE (i.e. editing all sires or only a proportion of them). The base line scenario involved selecting individuals on true breeding values (i.e., genomic selection only (GS only)-genomic selection with perfect accuracy) for several generations. Alternative scenarios complemented this base line scenario with PAGE (GS + PAGE). The effect of different PAGE strategies was quantified by comparing response to selection, changes in allele frequencies, the number of distinct QTN edited, the sum of absolute effects of the edited QTN per generation, and inbreeding.

Results

Response to selection after 20 generations was between 1.08 and 4.12 times higher with GS + PAGE than with GS only. Increases in response to selection were larger with more edits per sire and more sires edited. When the total resources for PAGE were limited, editing a few sires for many QTN resulted in greater response to selection and inbreeding compared to editing many sires for a few QTN. Between the scenarios GS only and GS + PAGE, there was little difference in the average change in QTN allele frequencies, but there was a major difference for the QTN with the largest effects. The sum of the effects of the edited QTN decreased across generations.

Conclusions

This study showed that PAGE has great potential for application in livestock breeding programs, but inbreeding needs to be managed.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0135-3) contains supplementary material, which is available to authorized users.     

Thrombin inhibitors with lipid peroxidation and lipoxygenase inhibitory activities

Vascular oxidative stress, endothelial injury, and thrombosis are intertwined processes that display a synergistic pathological effect in many cardiovascular diseases. Antithrombotic therapy with anticoagulant and/or antiplatelet agents, combined with interventions against vascular oxidative stress and/or inflammation, both boosting endothelial antithrombotic potential, could display a synergistic action in the treatment of thrombosis. Of the compounds 10a-h and 11a-d, shown to possess thrombin inhibitory activity, 11a-d were found to display radical scavenging activity, 10a, 10d, and 10f were demonstrated to inhibit lipid peroxidation of linoleic acid, and 10b and 10h inhibited soybean lipoxygenase. The observed combination of thrombin inhibition with lipid peroxidation and/or lipoxygenase inhibitory activity makes compounds 10 and 11 interesting candidates for further investigations towards multiple antithrombotic drugs.     

Ellipticines and 9-acridinylamines as inhibitors of D-alanine:D-alanine ligase

D-Alanine:D-alanine ligase (Ddl), an intracellular bacterial enzyme essential for cell wall biosynthesis, is an attractive target for development of novel antimicrobial drugs. This study focused on an extensive evaluation of two families of Ddl inhibitors encountered in our previous research. New members of both families were obtained through similarity search and synthesis. Ellipticines and 9-acridinylamines were both found to possess inhibitory activity against Ddl from Escherichia coli and antimicrobial activity against E. coli and Staphylococcus aureus. Ellipticines with a quaternary methylpyridinium moiety were the most potent among all studied compounds, with MIC values as low as 2 mg/L in strains with intact efflux mechanisms. Antimicrobial activity of the studied compounds was connected to membrane damage, making their development as antibacterial drug candidates unlikely unless analogues devoid of this nonspecific effect can be discovered.     

Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase

Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone, all had IC(50) values between 1 and 5microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.     

Chromatographic detection of residual cellular DNA on short monolithic columns

Analysis of crude samples from biotechnological processes is often required to demonstrate that residual host cell impurities are reduced or eliminated during purification. Current knowledge suggests that a continuous-cell-line DNA can be considered a cellular contaminant rather than a significant risk factor requiring removal to extremely low levels. Anion-exchange chromatography is one of the most important methods used in the downstream processing and analysis of different biomolecules. In this article, an application using Convective Interaction Media monolithic columns to improve the detection of residual cellular DNA is described.     

Selective formation of glycosidic linkages of N-unsubstituted 4-hydroxyquinolin-2-(1H)-ones

A comparative study for selective glucosylation of N-unsubstituted 4-hydroxyquinolin-2(1H)-ones into 4-(tetra-O-acetyl-β-d-glucopyranosyloxy)quinolin-2(1H)-ones is reported. Four glycosyl donors including tetra-O-acetyl-α-d-glucopyranosyl bromide, β-d-glucose pentaacetate, glucose tetraacetate and tetra-O-acetyl-α-d-glucopyranosyl trichloroacetimidate were tested, along with different promoters and reaction conditions. The best results were obtained with tetra-O-acetyl-α-d-glucopyranosyl bromide with Cs₂CO₃ in CH₃CN. In some cases the 4-O-glucosylation of the quinolinone ring was accompanied by 2-O-glucosylation yielding the corresponding 2,4-bis(tetra-O-acetyl-β-d-glucopyranosyloxy)quinoline. Next, 4-(tetra-O-acetyl-β-d-glucopyranosyloxy)quinolin-2(1H)-ones were deacetylated into 4-(β-d-glucopyranosyloxy)quinolin-2(1H)-ones with Et₃N in MeOH. In some instances the deacetylation was accompanied by the sugar-aglycone bond cleavage. Structure elucidation, complete assignment of proton and carbon resonances as well as assignment of anomeric configuration for all the products under investigation were performed by 1D and 2D NMR spectroscopy.