Different Enzymatic Processing of γ‐Phosphoramidate and γ‐Phosphoester‐Modified ATP Analogues

Monitoring the activity of ATP‐consuming enzymes provides the basis for elucidating their modes of action and regulation. Although a number of ATP analogues have been developed for this, their scope is restricted because of the limited acceptance by respective enzymes. In order to clarify which kind of phosphate‐modified ATP analogues are accepted by the α‐β‐phosphoanhydride‐cleaving ubiquitin‐activating enzyme 1 (UBA1) and the β‐γ‐phosphoanhydride‐cleaving focal adhesion kinase (FAK), we tested phosphoramidate‐ and phosphoester‐modified ATP analogues. UBA1 and FAK were able to convert phosphoramidate‐modified ATP analogues, even with a bulky modification like biotin. In contrast, a phosphoester‐modified analogue was poorly accepted. These results demonstrate that minor variations in the design of ATP analogues for monitoring ATP utilization have a significant impact on enzymatic acceptance.     

Detection near 1-nm with a laminar-flow,water-based condensation particle counter

Presented is a laminar-flow, water-based condensation particle counter capable of particle detection near 1 nm. This instrument employs a three-stage, laminar-flow growth tube with a “moderator” stage that reduces the temperature and water content of the output flow without reducing the peak supersaturation, and makes feasible operation at the large temperature differences necessary for achieving high supersaturations. The instrument has an aerosol flow of 0.3 L/min, and does not use a filtered sheath flow. It is referred to as a “versatile” water condensation particle counter, or vWCPC, as operating temperatures can be adjusted in accordance with the cut-point desired. When operated with wall temperatures of ～2°C, >90°C, and ～22°C for the three stages, respectively, the vWCPC detects particles generated from a heated nichrome wire with a 50% efficiency cut-point near 1.6 nm mobility diameter. At these operating temperatures, it also detects 10–20% of large molecular ions formed from passing filtered ambient air through a bipolar ion source. Decreasing the temperature difference between the first two stages, with the first and second stages operated at 10 and 90°C, respectively, essentially eliminates the response to charger ions, and raises the 50% efficiency cut-point for the nichrome wire particles to 1.9 nm mobility diameter. The time response, as measured by rapid removal of an inlet filter, yields a characteristic time constant of 195 ms.

Copyright © 2017 American Association for Aerosol Research     

Multi-photon imaging of amine-functionalized silica nanoparticles

A convenient and simple strategy for preparing water soluble, photoluminescent functionalized silica nanoparticles (M-dots) in the absence of fluorophores or metal doping is demonstrated. These M-dots can be used for bioimaging using one and two-photon microscopy. Because of their high photostability, low toxicity and high biocompatibility compared with Lumidot? CdSe/ZnS quantum dots, functionalized silica particles are superior alternatives for current bioimaging platforms. Moreover, the presence of a free amine group at the surface of the M-dots allows biomolecule conjugation (e.g. with antibodies, proteins) in a single step for converting these photoluminescent SiO(2) nanoparticles into multifunctional efficient vehicles for theragnostics.     

Effects of dietary α-linolenic acid on the conversion and oxidation of13C-α-linolenic acid

The effects of a diet rich in α-linolenic acid vs. one rich in oleic acid on the oxidation of uniformly labeled¹³C-α-linolenic acid and its conversion into longer-chain polyunsaturates (LCP) were investigatedin vivo in healthy human subjects. Volunteers received a diet rich in oleic acid (n=5) or a diet rich in α-linolenic acid (n=7; 8.3 g/d) for 6 wk before and during the study. After 6 wk, subjects were given 45 mg of¹³C-α-linolenic acid dissolved in olive oil. Blood samples were collected att=0, 5, 11, 24, 96, and 336 h. Breath was sampled and CO₂ production was measured each hour for the first 12 h. The mean (±SEM) maximal absolute amount of¹³C-eicosapentaenoic acid (EPA) in plasma total lipids was 0.04 ±0.01 mg in the α-linolenic acid group, which was significantly lower (P=0.01) than the amount of 0.12±0.03 mg¹³C-EPA in the oleic acid group. Amounts of¹³C-docosapentaenoic acid (DPA) and¹³C-docosahexaenoic acid (DHA) tended to be lower as well. The mean proportion of labeled α-linolenic acid (ALA) recovered as¹³CO₂ in breath after 12 h was 20.4% in the ALA and 15.7% in the oleic acid group, which was not significantly different (P=0.12). The cumulative recovery of¹³C from¹³C-ALA in breath during the first 12 h was negatively correlated with the maximal amounts of plasma¹³C-EPA (r=−0.58,P=0.047) and¹³C-DPA (r=−0.63,P=0.027), but not of¹³C-DHA (r=−0.49,P=0.108). In conclusion, conversion of¹³C-ALA into its LCP may be decreased on diets rich in ALA, while oxidation of¹³C-ALA is negatively correlated with its conversion into LCP. In a few pilot samples, low¹³C enrichments of n−3 LCP were observed in a diet rich in EPA/DHA as compared to oleic acid.     

Results of an inter-laboratory comparison of methods for quantitative clay analysis

In a comparison of analytical methods in clay mineralogy, two clay samples (from Hennersdorf and Freydegg) of different clay composition were analyzed by 19 laboratories mainly by XRD. Participants used different methods of pre-treatment, preparation, analysis and evaluation.In spite of the diversity of analytical methods applied, reasonable conformity in quantitative results was obtained for the associated minerals (quartz, calcite and dolomite). On the other hand, qualitative identification of the clay minerals showed considerably stronger divergencies, causing a stronger deviation of quantitative data; analysis of the Freydegg sample proved to have been particularly difficult. The evaluation methods of the different participants were examined to determine whether similar methods provided results that could be more easily compared.In addition, the authors calculated the clay mineral composition according to three different recognized methods, using the same diffractograms and same preparation and measuring conditions. In spite of this, different results were obtained.In the next phase of the programme, an inter-laboratory comparison of methods will be achieved under exactly defined conditions of preparation and analysis.     

In vitro and in vivo Staining Characteristics of Small,Fluorescent, Aβ42‐Binding D‐Enantiomeric Peptides in Transgenic AD Mouse Models

Plaque visualisation : We identified three different D ‐enantiomeric peptides that bind to Alzheimer's amyloid β (Aβ1‐42). As there is currently no definitive pre‐mortem diagnosis for Alzheimer's disease, we investigated the peptides' suitability as molecular probes for in vivo imaging in transgenic mouse models.

     

Effects of Weight Loss on Lipid Transfer Proteins in Morbidly Obese Women

Obesity is associated with lipid abnormalities leading to an increased morbidity and mortality from atherosclerotic disease. Lipid transfer proteins such as Cholesteryl Ester Transfer Protein (CETP) and Phospholipid Transfer Protein (PLTP), and lipases such as lipoprotein lipase (LPL) and hepatic lipase (HL) are involved in the pathogenesis of the obesity associated proatherogenic dyslipidemia. Nineteen severely obese female subjects undergoing laparosopic gastric banding participated in this prospective study. Subjects were examined with respect to body composition, lipid profile, CETP, PLTP, LPL and HL before and 1 year after surgical treatment. Mean weight loss was 22.2 kg, mainly due to losses in the fat depots. Triglycerides decreased and HDL₂-C increased significantly. In respect to transfer proteins mean CETP mass decreased from 1.82 to 1.71 μg mL^?1 (P = 0.043) and mean PLTP activity was reduced from 7.15 to 6.12 μmol mL^?1 h^?1 (P = 0.002), in parallel. In addition, both mean LPL activity and mean HL activity tended to decrease from 297 to 248 nmol mL^?1 h^?1 for LPL (P = 0.139) and from 371 to 319 nmol mL^?1 h^?1 for HL (P = 0.170), respectively. We conclude that weight loss induced by bariatric surgery is associated with the amelioration of the obesity-associated dyslipidemic state. This improvement may be attributable to decreased mass and action of the adipocyte tissue derived lipid transfer proteins CETP and PLTP.     

Probing enzyme promiscuity of SGNH hydrolases

Several hydrolases of the SGNH superfamily, including the lipase SrLip from Streptomyces rimosus (Q93MW7), the acyl-CoA thioesterase I TesA from Pseudomonas aeruginosa (Q9HZY8) and the two lipolytic enzymes EstA (from P. aeruginosa, O33407) and EstP (from Pseudomonas putida, Q88QS0), were examined for promiscuity. These enzymes were tested against four chemically different classes of a total of 34 substrates known to be hydrolysed by esterases, thioesterases, lipases, phospholipases, Tweenases and proteases. Furthermore, they were also analysed with respect to their amino acid sequences and structural homology, and their phylogenetic relationship was determined. The Pseudomonas esterases EstA and EstP each have an N-terminal domain with catalytic activity together with a C-terminal autotransporter domain, and so the hybrid enzymes EstA(N)-EstP(C) and EstP(N)-EstA(C) were constructed by swapping the corresponding N- and C-terminal domains, and their hydrolytic activities were compared. Interestingly, substrate specificity and kinetic measurements indicated a significant influence of the autotransporter domains on the catalytic activities of these enzymes in solution. TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p-nitrophenyl butyrate. Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities.     

BSA Hydrogel Beads Functionalized with a Specific Aptamer Library for Capturing Pseudomonas aeruginosa in Serum and Blood

Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide. Pseudomonas aeruginosa is a WHO-alerted multi-resistant pathogen of extreme importance as a cause of sepsis. Septicemia patients have significantly increased survival chances if sepsis is diagnosed in the early stages. Affinity materials can not only represent attractive tools for specific diagnostics of pathogens in the blood but can prospectively also serve as the technical foundation of therapeutic filtration devices. Based on the recently developed aptamers directed against P. aeruginosa, we here present aptamer-functionalized beads for specific binding of this pathogen in blood samples. These aptamer capture beads (ACBs) are manufactured by crosslinking bovine serum albumin (BSA) in an emulsion and subsequent functionalization with the amino-modified aptamers on the bead surface using the thiol- and amino-reactive bispecific crosslinker PEG₄-SPDP. Specific and quantitative binding of P. aeruginosa as the dedicated target of the ACBs was demonstrated in serum and blood. These initial but promising results may open new routes for the development of ACBs as a platform technology for fast and reliable diagnosis of bloodstream infections and, in the long term, blood filtration techniques in the fight against sepsis.