Between good and evil: Complexation of the human cathelicidin LL-37 with nucleic acids.

The innate immune system provides a crucial first line of defense against invading pathogens attacking the body. As the only member of the human cathelicidin family, the antimicrobial peptide LL-37 has been shown to have antiviral, antifungal, and antibacterial properties. In complexation with nucleic acids, LL-37 is suggested to maintain its beneficial health effects while also acting as a condensation agent for the nucleic acid. Complexes formed by LL-37 and nucleic acids have been shown to be immunostimulatory with a positive impact on the human innate immune system. However, some studies also suggest that in some circumstances, LL-37/nucleic acid complexes may be a contributing factor to autoimmune disorders such as psoriasis and systemic lupus erythematosus. This review provides a comprehensive discussion of research highlighting the beneficial health effects of LL-37/nucleic acid complexes, as well as discussing observed detrimental effects. We will emphasize why it is important to investigate and elucidate structural characteristics, such as condensation patterns of nucleic acids within complexation, and their mechanisms of action, to shed light on the intricate physiological effects of LL-37 and the seemingly contradictory role of LL-37/nucleic acid complexes in the innate immune response.

Microfluidic Platform for the Isolation of Cancer-Cell Subpopulations Based on Single-Cell Glycolysis.

High rates of glycolysis in tumors have been associated with cancer metastasis, tumor recurrence, and poor outcomes. In this light, single cells that exhibit high glycolysis are specific targets for therapy. However, the study of these cells requires efficient tools for their isolation. We use a droplet microfluidic technique developed in our lab, Sorting by Interfacial Tension (SIFT), to isolate cancer cell subpopulations based on glycolysis without the use of labels or active sorting components. By controlling the flow conditions on chip, the threshold of selection can be modified, enabling the isolation of cells with different levels of glycolysis. Hypoxia in tumors, that can be simulated with treatment with CoCl2, leads to an increase in glycolysis, and more dangerous tumors. The device was used to enrich CoCl2 treated MDA-MB 231 breast cancer cells from an untreated population. It is also used to sort K562 human chronic myelogenous leukemia cells that have either been treated or untreated with 2-deoxy-d-glucose (2DG), a pharmaceutical that targets cell metabolism. The technique provides a facile and robust way of separating cells based on elevated glycolytic activity; a biomarker associated with cancer cell malignancy.

Comparison between conventional and frit-inlet channels in separation of biopolymers by asymmetric flow field-flow fractionation.

Asymmetric flow field-flow fractionation (AF4) is a separation technique in which a focusing/relaxation step is used after the sample is injected onto the separation channel. During the focusing/relaxation step, the sample is focused by two counter-directed flows. This allows sample components to establish a diffusion-dependent equilibrium concentration profile. The focusing step may, in some cases, cause a loss of sample due to adsorption into the accumulation wall (i.e. the membrane) or due to aggregation of the sample. In addition, the increase in sample concentration during the focusing step may prevent complete relaxation and cause overloading effects. In this study, a modified AF4 channel equipped with a frit inlet (FI-AF4) is utilized, where the sample is relaxed hydrodynamically as it enters to the channel through the frit. The main advantage of the FI-AF4 channel is to omit the focusing step. The FI-AF4 channel could also allow higher injection mass than in a conventional channel while still avoiding overloading. The purpose of the present study is to compare two channels (conventional and FI-AF4 channels) in terms of the plate height (H), resolution (Rs) and the mass recovery for analysis of a mixture of glycogen and pullulan. In addition, waxy maize (WM) starch was used to compare the mass overloading of the two channels. The results show that the type of relaxation method (i.e. focusing or hydrodynamic relaxation) had no significant effect on mass recovery. The resolution (Rs), was higher in the conventional AF4 channel than in the FI-AF4 channel for the separation of glycogen and pullulan. The results also show that it was possible to inject a higher mass of WM starch (i.e. twice the mass) onto the FI-AF4 channel, compared to a conventional AF4 channel, without observing an overloading effect.

Characterization of the molar mass distribution of macromolecules in beer for different mashing processes using asymmetric flow field-flow fractionation (AF4) coupled with multiple detectors.

The macromolecular composition of beer is largely determined by the brewing and the mashing process. It is known that the physico-chemical properties of proteinaceous and polysaccharide molecules are closely related to the mechanism of foam stability. Three types of "American pale ale" style beer were prepared using different mashing protocols. The foam stability of the beers was assessed using the Derek Rudin standard method. Asymmetric flow field-flow fractionation (AF4) in combination with ultraviolet (UV), multiangle light scattering (MALS) and differential refractive index (dRI) detectors was used to separate the macromolecules present in the beers and the molar mass (M) and molar mass distributions (MD) were determined. Macromolecular components were identified by enzymatic treatments with ß-glucanase and proteinase K. The MD of ß-glucan ranged from 106 to 108 g/mol. In addition, correlation between the beer's composition and foam stability was investigated (increased concentration of protein and ß-glucan was associated with increased foam stability).

Droplet Microfluidic Technology for the Early and Label-Free Isolation of Highly-Glycolytic, Activated T-Cells.

A label-free, fixation-free and passive sorting method is presented to isolate activated T-cells shortly after activation and prior to the display of activation surface markers. It uses a recently developed sorting platform dubbed "Sorting by Interfacial Tension" (SIFT) that sorts droplets based on pH. After polyclonal (anti-CD3/CD28 bead) activation and a brief incubation on chip, droplets containing activated T-cells display a lower pH than those containing naive cells due to increased glycolysis. Under specific surfactant conditions, a change in pH can lead to a concurrent increase in droplet interfacial tension. The isolation of activated T-cells on chip is hence achieved as flattened droplets are displaced as they encounter a micro-fabricated trench oriented diagonally with respect to the direction of flow. This technique leads to an enrichment of activated primary CD4+ T-cells to over 95% from an initial mixed population of naive cells and cells activated for as little as 15 min. Moreover, since the pH change is correlated to successful activation, the technique allows the isolation of T-cells with the earliest activation and highest glycolysis, an important feature for the testing of T-cell activation modulators and to determine regulators and predictors of differentiation outcomes.

Method for Passive Droplet Sorting after Photo-Tagging.

We present a method to photo-tag individual microfluidic droplets for latter selection by passive sorting. The use of a specific surfactant leads to the interfacial tension to be very sensitive to droplet pH. The photoexcitation of droplets containing a photoacid, pyranine, leads to a decrease in droplet pH. The concurrent increase in droplet interfacial tension enables the passive selection of irradiated droplets. The technique is used to select individual droplets within a droplet array as illuminated droplets remain in the wells while other droplets are eluted by the flow of the external oil. This method was used to select droplets in an array containing cells at a specific stage of apoptosis. The technique is also adaptable to continuous-flow sorting. By passing confined droplets over a microfabricated trench positioned diagonally in relation to the direction of flow, photo-tagged droplets were directed toward a different chip exit based on their lateral movement. The technique can be performed on a conventional fluorescence microscope and uncouples the observation and selection of droplets, thus enabling the selection on a large variety of signals, or based on qualitative user-defined features.

Interaction between cereal ß-glucan and proteins in solution and at interfaces.

Cereal ß-glucan is well known for its beneficial health effects, such as lowering of blood cholesterol values and a reduced risk of coronary heart disease. These effects are often discussed in relation to the dissolution and aggregation behavior of the ß-glucan during human digestion. Furthermore, potential proteinaceous material present is believed to have an important impact on the formation of viscous slurries during digestion and might influence the aggregation behavior of the ß-glucan. Therefore, the interaction and aggregation behavior of a ß-glucan isolate (OBC90) with two different proteins (gliadin and whey protein) was investigated in solution at different pH with regards to kinetics of aggregation and protein/ß-glucan ratio and at interfaces. Aggregates were found at low pH and the aggregation and composition of aggregates seems to depend on the type of protein. Furthermore, phosphate was found at low concentrations in the ß-glucan, most likely being the reason for the net negative charge at pH≤4. Therefore, electrostatic interaction is suggested to play an important role for the aggregation between ß-glucan and proteins.

Co-elution phenomena in polymer mixtures studied by asymmetric flow field-flow fractionation.

Most polymers generally have complex characteristics. Analysis and understanding of these characteristics is crucial as they, for instance, influence functionality. Separation and analysis of samples of polymers, biopolymers in particular, is challenging since they often display broad distributions in size, structure and molar mass (M) and/or a tendency to form aggregates. Only few analytical techniques are suitable for the task. AF4-MALS-dRI is highly suited for the task, but the analysis can nevertheless be especially challenging for heterogeneous mixtures of polymers that exhibit wide size distributions or aggregation. For such systems, systematic and thorough method development is clearly a requirement. This is the purpose of the present work, where we approach the problem of heterogeneous polymer samples systematically by analyzing mixtures of two different polymers which are also characterized individually. An often observed phenomenon in AF4 of samples with a high polydispersity is a downturn in M vs. elution time, especially common at high retention. This result is often dismissed as an artifact attributed to various errors in detection and data processing. In this work, we utilize AF4-MALS-dRI to separate and analyze binary mixtures of the well-known polysaccharides pullulan and glycogen, or pullulan and poly(ethylene oxide), respectively, in solution. The results show that an observed downturn - or even an upturn - in M can be a correct result, caused by inherent properties of the analyzed polymers.

Co-elution effects can influence molar mass determination of large macromolecules with asymmetric flow field-flow fractionation coupled to multiangle light scattering.

Starch and hence, amylopectin is an important biomacromolecule in both the human diet as well as in technical applications. Therefore, accurate and reliable analytical methods for its characterization are needed. A suitable method for analyzing macromolecules with ultra-high molar mass, branched structure and high polydispersity is asymmetric flow field-flow fractionation (AF4) in combination with multiangle light scattering (MALS) detection. In this paper we illustrate how co-elution of low quantities of very large analytes in AF4 may cause disturbances in the MALS data which, in turn, causes an overestimation of the size. Furthermore, it is shown how pre-injection filtering of the sample can improve the results.

Analysis of ß-glucan molar mass from barley malt and brewer's spent grain with asymmetric flow field-flow fractionation (AF4) and their association to proteins.

ß-Glucan benefits are related with its molar mass and it would be of interest to better understand how this parameter can be changed by processing and variety for design of food with specific health effects. For this purpose, extracts from barley malts and brewers' spent grain, processed at different conditions, were analysed regarding ß-glucan content, molar mass, and protein content. Molar mass distribution was assessed using asymmetric flow field-flow fractionation (AF4) with multiangle light scattering (MALS), differential refractive index (dRI) and fluorescence (FL) detection. ß-Glucan was detected in a wide molar mass range, <2000 to approximately 6.7×106g/mol. Differences in molar masses were more noticeable between barley varieties and steeping malting conditions than by mashing of malt. Barley products processed to preserve ß-glucan contained more ß-glucan of high molar mass with potential to shift the fermentation site to the distal colon. Enzymatic degradation of proteins indicated presence of aggregates containing ß-glucan and protein.

Characterization of cereal ß-glucan extracts from oat and barley and quantification of proteinaceous matter.

An extraction method for mixed-linkage ß-glucan from oat and barley was developed in order to minimize the effect of extraction on the ß-glucan structure. ß-Glucan were characterized in terms of molecular size and molar mass distributions using asymmetric flow field-flow fractionation (AF4) coupled to multiangle light scattering (MALS), differential refractive index (dRI) and fluorescence (FL) detection. The carbohydrate composition of the extracts was analysed using polysaccharide analysis by carbohydrate gel electrophoresis (PACE) and high-performance anion-exchange chromatography (HPAEC). Whether there were any proteinaceous moieties linked to ß-glucan was also examined. Purified extracts contained 65% and 53% ß-glucan for oats and barley, respectively. The main impurities were degradation products of starch. The extracts contained high molecular weight ß-glucan (105-108 g/mol) and large sizes (root-mean-square radii from 20 to 140 nm). No proteins covalently bound to ß-glucan were detected; therefore, any suggested functionality of proteins regarding the health benefits of ß-glucan can be discounted.

The effect of baking and enzymatic treatment on the structural properties of wheat starch.

In this study, bread was baked with and without the addition of α-amylase. Starch was extracted from the baked bread and its molecular properties were characterized using (1)H NMR and asymmetric flow field-flow fractionation (AF4) connected to multi-angle light scattering (MALS) and other detectors. The approach allows determination of molar mass, root- mean-square radius and apparent density as well as the average degree of branching of amylopectin. The results show that starch size and structure is affected as a result of the baking process. The effect is larger when α-amylase is added. The changes include both a decrease molar mass and size as well as an increase in apparent density. Moreover, an increase in average degree of branching and the number of reducing ends H-1(ß-r) and H-1(α-r) can be observed.