Spatially resolved in situ determination of reaction progress using microfluidic systems and FT-IR spectroscopy as a tool for biocatalytic process development.

A concept for the determination of concentrations in microchannels using FT-IR spectroscopy in transmission is presented. The fundamental idea of spatially resolved measurements along several measuring points was implemented in a single-channel microreactor. Compared to existing microreactor setups for the analysis of fast chemical reactions or mixing processes, the presented concept enables longer residence times at appropriate resolution. Once steady-state conditions were reached in the reactor, mid-infrared spectra were collected at different locations. Information throughout the considered conversion range is available, which is of great importance to analyze inhibitory effects, next to the kinetic constants (vmax and KM). Therefore, this technology enables a rapid screening of (bio-)catalysts, substrate specificity and process conditions. In particular, the analysis of real substrates instead of model substrates and the possibility to follow side reactions and follow-up reactions during enzymatic catalysis open a broad field of application.

Novel µ-membrane module for online determination of the free fatty acid content in the dispersed phase of oil-in-water emulsions.

Monitoring the dispersed phase of an oil-in-water (O-W) emulsion by means of Fourier transform infrared (FTIR) spectroscopy is a challenging task, restricted to the continuous phase that is in contact with the FTIR probe. Nonetheless, real-time measurement and kinetic analysis by FTIR, including analysis of the dispersed, often non-polar phase containing substrates and/or products, is desirable. Enzymatic hydrolysis of sunflower oil was performed in an O-W emulsion. After separation of the oil phase by use of a newly developed µ-membrane module, infrared spectra were collected using an attenuated total reflectance (ATR) cell. Different chemometric models were calibrated using the partial least squares (PLS) algorithm. Online application of a chemometric model based on the FTIR spectra enabled real-time monitoring of free fatty acid concentrations in the oil phase.

Elucidating the mechanism of lipid membrane-induced IAPP fibrillogenesis and its inhibition by the red wine compound resveratrol: a synchrotron X-ray reflectivity study.

The islet amyloid polypeptide (IAPP) or amylin is a pancreatic hormone and crucially involved in the pathogenesis of type-II diabetes mellitus (T2DM). Aggregation and amyloid formation of IAPP is considered as the primary culprit for pancreatic beta-cell loss in T2DM patients. In this study, first X-ray reflectivity (XRR) measurements on IAPP at lipid interfaces have been carried out, providing a molecular level characterization of the first steps of the lipid-induced fibrillation process of IAPP, which is initiated by lipid-induced nucleation, oligomerization, followed by detachment of larger IAPP aggregate structures from the lipid membrane, and terminated by the formation of mature fibrils in the bulk solution. The adsorption process of IAPP at lipid interfaces in the absence and presence of negatively charged lipid has also been studied by complementary ATR-FTIR spectroscopic measurements. The morphological properties were followed by atomic force microscopy (AFM). Moreover, we show that the polyphenolic red wine compound resveratrol is able to inhibit IAPP aggregation also in the presence of aggregation-fostering negatively charged lipid interfaces, revealing its potential as a drug candidate for T2DM.

Inhibiting islet amyloid polypeptide fibril formation by the red wine compound resveratrol.

GRAPES FOR AMYLOIDS: The red wine compound resveratrol can effectively inhibit the formation of IAPP amyloid that is found in type II diabetes. Our in vitro inhibition results do not depend on the antioxidant activity of resveratrol. Further, the markedly enhanced cell survival in the presence of resveratrol also indicates that the small oligomeric structures that are observed during beta-sheet formation are not toxic and could be off-pathway assembly products.

Suppression of IAPP fibrillation at anionic lipid membranes via IAPP-derived amyloid inhibitors and insulin.

Aggregation of human islet amyloid polypeptide (hIAPP) into cytotoxic beta-sheet oligomers and amyloid plaques is considered a key event in pancreatic beta-cell degeneration in type 2 diabetes (T2D). hIAPP is synthesized in the pancreatic beta-cells and it is stored, co-processed in the secretory granules, and co-secreted to the extracellular matrix together with insulin. In vivo, hIAPP aggregation may start and proceed at the water-cell membrane interface and anionic lipid membranes strongly enhance the process of hIAPP fibrillization which is causally linked to membrane disintegration and cell degeneration. In this study we explored the amyloidogenic propensity and conformational properties of hIAPP in the presence of negatively charged membrane (DOPC/DOPG phospholipid bilayers) surfaces upon addition of two recently designed potent hIAPP-derived inhibitors of hIAPP amyloidogenesis, the hexapeptide NF(N-Me)GA(N-Me)IL (NFGAIL-GI) and the 37-residue non-amyloidogenic hIAPP analog [(N-Me)G24, (N-Me)I26]-IAPP (IAPP-GI). For comparison, the effects of insulin, which is a natively occurring hIAPP aggregation inhibitor, rat IAPP (rIAPP), which is a natively non-amyloidogenic hIAPP analog, and the hIAPP amyloid core peptide hIAPP(22-27) or NFGAIL were also studied. The aim of our study was to test whether and how the above peptides which have been shown to completely block or suppress hIAPP amyloidogenesis in bulk solution in vitro would also affect these processes in the presence of lipid membranes. To this end, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) was applied. We find that IAPP-GI, NFGAIL-GI, insulin, and rIAPP are potent inhibitors of hIAPP fibrillization. Importantly, our data also suggest that the hetero-complexes of IAPP-GI, rIAPP, and insulin with hIAPP although non-amyloidogenic per se are still able to adsorb at the lipid membrane. By contrast, in the presence of NFGAIL-GI, interaction of hIAPP with the lipid membrane is completely abolished, consistent with NFGAIL-GI mediated sequestration of hIAPP via hetero-complexation in the aqueous phase mainly accounting for the observed strong effect of NFGAIL-GI on hIAPP fibrillogenesis at the lipid membrane interface. Finally, our studies show that once hIAPP is fibrillized at the water-lipid membrane interface with fibrils being attached to the lipid membrane, it cannot be disaggregated by all above peptides.

Amyloidogenic propensities and conformational properties of ProIAPP and IAPP in the presence of lipid bilayer membranes.

Human islet amyloid polypeptide (hIAPP), which is considered the primary culprit for beta-cell loss in type 2 diabetes mellitus patients, is synthesized in beta-cells of the pancreas from its precursor pro-islet amyloid polypeptide (proIAPP), which may be important in early intracellular amyloid formation as well. We compare the amyloidogenic propensities and conformational properties of proIAPP and hIAPP in the presence of negatively charged lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. Circular dichroism studies verify the initial secondary structures of proIAPP and hIAPP to be predominantly unordered with small amounts of ordered secondary structure elements, and exhibit minor differences between these two peptides only. Using attenuated total reflection-Fourier transform infrared spectroscopy and thioflavin T fluorescence spectroscopy, as well as atomic force microscopy, we show that in the presence of negatively charged membranes, proIAPP exhibits a much higher amyloidogenic propensity than in bulk solvent. Compared to hIAPP, it is still much less amyloidogenic, however. Although differences in the secondary structures of the aggregated species of hIAPP and proIAPP at the lipid interface are small, they are reflected in morphological changes. Unlike hIAPP, proIAPP forms essentially oligomeric-like structures at the lipid interface. Besides the interaction with anionic membranes [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)+x1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]], interaction with zwitterionic homogeneous (DOPC) and heterogeneous (1,2-dipalmitoyl-sn-glycero-3-phosphocholine:DOPC:cholesterol 1:2:1 model raft mixture) membranes has also been studied. Both peptides do not aggregate significantly at DOPC bilayers. In the presence of the model raft membrane, hIAPP aggregates markedly as well. Conversely, proIAPP clusters into less ordered structures and to a minor extent at raft membranes only. The addition of proIAPP to hIAPP retards the hIAPP fibrillation process also in the presence of negatively charged lipid bilayers. In excess proIAPP, increased aggregation levels are finally observed, however, which could be attributed to seed-induced cofibrillation of proIAPP.