Direct transformation of solar energy into electrical energy by means of biological photosynthesis is considered as an attractive option for sustainable electrical energy production. Thylakoid membranes, the site of photosynthesis, are regarded as a promising biological material for the development of photoelectric biodevices, which produce electrical power consuming only light energy as oxygen evolves at photobioanode upon irradiation and biocathode converts it back to water. Therefore, in this work dual-feature photobioanode based on nanoimprinted gold substrates modified with thylakoids in combination with a capacitive part made of a planar gold substrate coated with a conductive polymer was designed and evaluated, providing open-circuit potential of -0.21 V vs. Ag vertical bar AgCl vertical bar KClsat and a capacitance of ca. 60 F m(-2) both at ambient light and artificial illumination of 400 W m(-2). Combination of thylakoid based dual-feature photobioanode with bilirubin oxidase modified transparent and capacitive indium tin oxide biocathode resulted in a photoelectric biosupercapacitor with remarkable characteristics at ambient light, viz. an open-circuit voltage as high as 0.74 V, which was stable upon charge-discharge cycles during ca. 2 h.

The crowded environment of biological systems such as the interior of living cells is occupied by macromolecules with a broad size distribution. This situation of polydispersity might influence the dependence of the diffusive dynamics of a given tracer macromolecule in a monodisperse solution on its hydrodynamic size and on the volume fraction. The resulting size dependence of diffusive transport crucially influences the function of a living cell. Here, we investigate a simplified model system consisting of two constituents in aqueous solution, namely, of the proteins bovine serum albumin (BSA) and bovine polyclonal gamma-globulin (Ig), systematically depending on the total volume fraction and ratio of these constituents. From high-resolution quasi-elastic neutron spectroscopy, the separate apparent short-time diffusion coefficients for BSA and Ig in the mixture are extracted, which show substantial deviations from the diffusion coefficients measured in monodisperse solutions at the same total volume fraction. These deviations can be modeled quantitatively using results from the short-time rotational and translational diffusion in a two-component hard sphere system with two distinct, effective hydrodynamic radii. Thus, we find that a simple colloid picture well describes short-time diffusion in binary mixtures as a function of the mixing ratio and the total volume fraction. Notably, the self-diffusion of the smaller protein BSA in the mixture is faster than the diffusion in a pure BSA solution, whereas the self-diffusion of Ig in the mixture is slower than in the pure Ig solution.

Sialic acid (SA) is a monosaccharide usually linked to the terminus of glycan chains on the cell surface. It plays a crucial role in many biological processes, and hypersialylation is a common feature in cancer. Lectins are widely used to analyze the cell surface expression of SA. However, these protein molecules are usually expensive and easily denatured, which calls for the development of alternative glycan-specific receptors and cell imaging technologies. In this study, SA-imprinted fluorescent core-shell molecularly imprinted polymer particles (SA-MIPs) were employed to recognize SA on the cell surface of cancer cell lines. The SA-MIPs improved suspensibility and scattering properties compared with previously used core-shell SA-MIPs. Although SA-imprinting was performed using SA without preference for the α2,3- and α2,6-SA forms, we screened the cancer cell lines analyzed using the lectins Maackia Amurensis Lectin I (MAL I, α2,3-SA) and Sambucus Nigra Lectin (SNA, α2,6-SA). Our results show that the selected cancer cell lines in this study presented a varied binding behavior with the SA-MIPs. The binding pattern of the lectins was also demonstrated. Moreover, two different pentavalent SA conjugates were used to inhibit the binding of the SA-MIPs to breast, skin, and lung cancer cell lines, demonstrating the specificity of the SA-MIPs in both flow cytometry and confocal fluorescence microscopy. We concluded that the synthesized SA-MIPs might be a powerful future tool in the diagnostic analysis of various cancer cells.

Substituted oligoamides are short sequences of unnatural amino acids. Oligoamides made entirely of beta(3) amino acids yield helical monomers that, if N-acylated, assemble into nanorod structures via a supramolecular assembly motif. In this work, coordination crosslinking was used to create complex nanomaterials from oligoamides WKLWEL (KE) and WELWEL (EE) (the letters denote the analogous alpha-amino acids). Upon Cu(II) addition, atomic force microscopy and small angle neutron scattering revealed morphologic changes specific to KE but absent in EE. Vibration spectroscopy measurements revealed that Cu(II) can coordinate to the amine moieties of the side chains, without direct effect on the backbone amides. While coordination in excess solvent lead to regular nanostructures, fast drying of the sample yielded oligoamide templated crystallization of CuCl2. The metal coordination crosslinking of supramolecular assemblies as reported here is the first realization of a metallosupramolecular framework structure. [GRAPHICS] .

Substituted (beta(3) oligoamides form a unique self-assembling system where each monomer folds into a helix containing approximately three (beta(3) amino acids per turn, yielding a geometrically well-defined cylindrical building block that, when N-acylated, is able to self-assemble head-to-tail into nanorods that can reach several 100 mu m length. It was shown in previous works that self-assembly can be achieved with a three residue long oligoamide as well that lacks any intramolecular H-bonds, yet it crystallizes in a helix-like conformation. The self-assembly properties of these small oligoamides are however elusive, suggesting a more complex system than the self-assembly of the H-bond stabilized helical monomers. Here we focus on the self-assembly behaviour of a three residue oligoamide, Ac-beta(3)[LIA] where the letters denote the side chain of the analogous a amino acid. Ac-beta(3)[LIA] can yield highly inhomogeneous suspensions in water with a broad range of large fibrous structures that seem to be very stable, yet occasionally fibre growth is only observed upon heating. The small size of the monomer suggests a highly dynamic equilibrium yet all previous attempts failed to clearly identify low molecular weight species. Therefore a special methodology was employed in this study to characterize the suspensions at different size ranges: SANS that is optimal to measure the small oligomers and cross sectional diameter of the assemblies, DLS that is sensitive to the large populations and therefore the length of the superstructures, and NMR that is sensitive to monomeric and small oligomeric form, in conjunction with IR spectroscopy to probe the folding and AFM to image the morphology of the assemblies. Temperature ramping was used to perturb the system to probe the dynamicity of the self-assembly. It was found that the anomalous self-assembly behaviour of Ac-beta(3)[LIA] is caused by its two stable conformations, a helix-building "horseshoe" fold and a linear conformer. The latter is exclusively found in monomeric form in solution whereas the horseshoe fold is stable in solid phase and in fibrous assemblies. Small oligomers were absent. Thus the self assembly of Ac-beta(3)[LIA] is arrested by the activation energy need of the conformation change; fibre growth might be triggered by conditions that allow increased conformational freedom of the monomers. This observation may be used to develop strategies for controlled switchable self-assembly.

In this work, an innovative and accurate affinity capillary electrophoresis (ACE) method was set up to monitor the complexation of aqueous MIP nanogels (NGs) with model cancer-related antigens. Using α2,6′- and α2,3′-sialyllactose as oversimplified cancer biomarker-mimicking templates, NGs were synthesized and characterized in terms of size, polydispersity, and overall charge. A stability study was also carried out in order to select the best storage conditions and to ensure product quality. After optimization of capillary electrophoresis conditions, injection of MIP NGs resulted in a single, sharp, and efficient peak. The mobility shift approach was applied to quantitatively estimate binding affinity, in this case resulting in an association constant of K ≈ 106 M–1. The optimized polymers further displayed a pronounced discrimination between the two sialylated sugars. The newly developed ACE protocol has the potential to become a very effective method for nonconstrained affinity screening of NG in solution, especially during the NG development phase and/or for a final accurate quantitation of the observed binding.

Bicelles are disk-shaped models of cellular membranes used to study lipid–protein interactions, as well as for structural and functional studies on transmembrane proteins. One challenge for the incorporation of transmembrane proteins in bicelles is the limited range of detergent and lipid combinations available for the successful reconstitution of proteins in model membranes. This is important, as the function and stability of transmembrane proteins are very closely linked to the detergents used for their purification and to the lipids that the proteins are embedded in. Here, we expand the toolkit of lipid and detergent combinations that allow the formation of stable bicelles. We use a combination of dynamic light scattering, small-angle X-ray scattering and cryogenic electron microscopy to perform a systematic sample characterization, thus providing a set of conditions under which bicelles can be successfully formed.

Lipid-based drug delivery systems offer the potential to enhance bioavailability, reduce dosing frequency, and improve patient adherence. In aqueous environment, initially dry lipid depots take up water and form liquid crystalline phases. Variation of lipid composition, depot size and hydration-induced phase transitions will plausibly affect the diffusion in and out of the depot. Lipid depots of soybean phosphatidylcholine (SPC) and glycerol dioleate (GDO) mixtures were hydrated for varying time durations in a phosphate-buffered saline (PBS) buffer and then analyzed with Karl Fischer titration, magnetic resonance imaging (MRI) and gravimetrically. Mathematical modeling of the swelling process using diffusion equations, was used to estimate the parameters of diffusion. Both composition of lipid mixture and depot size affect swelling kinetics… The diffusion parameters obtained in Karl Fischer titration and MRI (with temporal and spatial resolution respectively) are in good agreement. Remarkably, the MRI results show a gradient of water content within the depot even after the end of diffusion process. Apparently contradicting the first Fick's law in its classical form, these results find an explanation using the generalized Fick's law that considers the gradient of chemical potential rather than concentration as the driving force of diffusion.

This work presents the use of photoactive molecularly imprinted nanoparticles (MINs) to promote antibiotic degradation under visible light irradiation. Prototype MINs for the model antibiotic tetracycline (TC) were developed using molecular dynamics simulations to predict the TC-binding capacity of seven pre-polymerization mixtures. The studied formulations contained varying proportions of functional monomers with diverse physicochemical profiles, namely N-isopropylacrylamide (NIPAM), N-tert-butylacrylamide (TBAM), acrylic acid (AA), and (N-(3-aminopropyl)methacrylamide hydrochloride) (APMA) and a constant ratio of the cross-linker N,N '-methylene-bis-acrylamide (BIS). Two monomer formulations showed markedly higher TC-binding capacities based on template-monomer interaction energies. These mixtures were used to synthesize photoactive MINs by high-dilution radical polymerization, followed by the EDC/NHS conjugation with the organic photosensitizer toluidine blue. MINs showed higher TC-binding capacities than non-imprinted nanoparticles (nINs) of identical composition. MINs and nINs exhibited photodynamic activity under visible light irradiation, as confirmed by singlet oxygen generation experiments. TC degradation was evaluated in 50 mu mol L-1 solutions placed in microplate wells containing immobilized nanoparticles and irradiated with white LED light (150 W m(-2)) for 1 h at room temperature. Degradation followed pseudo-zero-order kinetics with accelerated profiles in MIN-containing wells. Our findings suggest a key role of molecularly imprinted cavities in bringing TC closer to the photosensitizing moieties, minimizing the loss of oxidative potential due to reactive oxygen species diffusion. This degradation strategy can potentially extend to any organic pollutants for which MINs can be synthesized and opens valuable opportunities for exploring novel applications for molecularly imprinted materials.

The fate of plastic waste is a pressing issue since it forms a visible and long-lived reminder of the environmental impact of consumer habits. In this study, we examine the structural changes in the lamellar arrangements of semicrystalline polyethylene (PE) packaging waste with the aim of understanding the physical mechanisms of embrittlement in PE exposed to the marine environment. PE microplastics and macroplastics from identifiable PE packaging were collected in the Atlantic Ocean and compared to new PE boxes. Several experimental techniques interrogate the effects of environmental exposure on their bulk and surface properties. Size exclusion chromatography determines the molecular weight distribution of the PE polymer chains and differential scanning calorimetry gives the crystallinity. Small- and wide-angle X-ray scattering examines the packing of PE chains into semicrystalline lamellae. Longitudinal acoustic mode Raman spectroscopy provides a complementary measurement of the length of PE polymer chains extending through the crystalline lamellar domains. While there is a high degree of uncertainty in the time scale for the changes, the overall picture at the molecular scale is that although PE becomes more crystalline with environmental exposure, the lamellar order present in new packing boxes is disrupted by the weathering process. This process has important implications for embrittlement and subsequent degradation.

Selective serotonin reuptake inhibitors (SSRIs) are a class of antidepressants regularly detected in the environment. This indicates that the existing wastewater treatment techniques are not successfully removing them beforehand. This study investigated the potential of molecularly imprinted polymers (MIPs) to serve as sorbents for removal of SSRIs in water treatment. Sertraline was chosen as the template for imprinting. We optimized the composition of MIPs in order to obtain materials with highest capacity, affinity, and selectivity for sertraline. We report the maximum capacity of MIP for sertraline in water at 72.6 mg g^-1, and the maximum imprinting factor at 3.7. The MIPs were cross-reactive towards other SSRIs and the metabolite norsertraline. They showed a stable performance in wastewater-relevant pH range between 6 and 8, and were reusable after a short washing cycle. Despite having a smaller surface area between 27.4 and 193.8 m²·g^-1, as compared to that of the activated carbon at 1400 m²·g^-1, their sorption capabilities in wastewaters were generally superior. The MIPs with higher surface area and pore volume that formed more non-specific interactions with the targets considerably contributed to the overall removal efficiency, which made them better suited for use in wastewater treatment.

Cluster of differentiation (CD81) is a type of protein, which is encoded by CD81 gene. Beside that CD81 is also known under other names such as Target of the Antiproliferative Antibody 1 (TAPA-1) and Tetraspanin-28 (TSPAN28). Location of CD81 is known to be on chromosome 11 (11p15.5), where it contains 15-20 bases in length. It is expressed mostly in cells of testis, ovary, endometrium, placenta, bone marrow, smooth muscles and others. The main function of the CD81 protein is to mediate signal transduction events, which are important for cells' development, activation, growth and motility. The CD81 gene is also known as a candidate for many malignancies because of its location. The characteristic feature of CD81 is that it is highly hydrophobic and contains a short N- and C-terminal cytoplasmic domains together with cytoplasmic cysteines, potential sites of palmitoylation as well as four transmembrane domains where they together hold the protein in a cell membrane. There are two CD81 isoforms, isoform 1 and isoform 2. Isoforms of CD81 are usually found in a tumor-suppressor region where they have a great impact on tumor development. There has always been a high interest in research on CD81 function in viral disease development. In fact, it is known that CD81 contributes in the development of diseases such as hepatitis C, malaria and various types of cancer. Since the complete effect of CD81 is unknown, further research and scientific methodology could potentially discover all possible functions and mechanisms regulated by the CD81 protein in human body.

HYPOTHESIS: Permeation of macromolecular drugs across biological plasma membranes is a major challenge in drug delivery. Cationic cell-penetrating peptides (CPPs) are attractive functional excipient candidates for the delivery of macromolecules across membrane barriers, due to their membrane translocating ability. The properties of CPPs can be tailored by lipidation, a promising approach to facilitate enhanced membrane insertion, potentially promoting increased translocation of the CPP and cargo.

EXPERIMENTS: To explore the impact that site and degree of lipidation have on the membrane interaction of a cationic CPP, we designed and investigated CPP conjugates with one or two fatty acid chains.

FINDINGS: Compared to the parent CPP and the single-lipidated conjugates, the double-lipidated conjugate exhibited the most pronounced membrane perturbation effects, as measured by several biophysical techniques. The experimental findings were supported by molecular dynamics (MD) simulations, demonstrating that all CPP conjugates interacted with the membrane by insertion of the lipid chain(s) into the core of the bilayer. Moreover, membrane-thinning effects and induced membrane curvature were displayed upon CPP interaction. Our results demonstrate that the impact exerted by the CPP on the membrane is notably affected by positioning and especially the degree of lipidation, which might influence the properties of CPPs as functional excipients.

Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na+/H+-exchanger 1 (NHE1) regulates intracellular pH (pHi) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interacting domain (LID), yet, the LID structure and underlying molecular mechanisms are unknown. Here we decompose these, combining structural and biophysical methods, molecular dynamics simulations, cellular biotinylation- and immunofluorescence analysis and exchanger activity assays. We find that the NHE1-LID is intrinsically disordered and, in presence of membrane mimetics, forms a helical αα-hairpin co-structure with the membrane, anchoring the regulatory domain vis-a-vis the transport domain. This co-structure is fundamental for NHE1 activity, as its disintegration reduced steady-state pHi and the rate of pHi recovery after acid loading. We propose that regulatory lipid-protein co-structures may play equally important roles in other membrane proteins.

N-Acetylneuraminic acid and its α2,3/α2,6-glycosidic linkages with galactose (Neu5Ac-Gal) are major carbohydrate antigen epitopes expressed in various pathological processes, such as cancer, influenza, and SARS-CoV-2. We here report a strategy for the synthesis and binding investigation of molecularly imprinted polymers (MIPs) toward α2,3 and α2,6 conformations of Neu5Ac-Gal antigens. Hydrophilic imprinted monoliths were synthesized from melamine monomer in the presence of four different templates, namely, N-acetylneuraminic acid (Neu5Ac), N-acetylneuraminic acid methyl ester (Neu5Ac-M), 3′-sialyllactose (3SL), and 6′-sialyllactose (6SL), in a tertiary solvent mixture at temperatures varying from −20 to +80 °C. The MIPs prepared at cryotemperatures showed a preferential affinity for the α2,6 linkage sequence of 6SL, with an imprinting factor of 2.21, whereas the α2,3 linkage sequence of 3SL resulted in nonspecific binding to the polymer scaffold. The preferable affinity for the α2,6 conformation of Neu5Ac-Gal was evident also when challenged by a mixture of other mono- and disaccharides in an aqueous test mixture. The use of saturation transfer difference nuclear magnetic resonance (STD-NMR) on suspensions of crushed monoliths allowed for directional interactions between the α2,3/α2,6 linkage sequences on their corresponding MIPs to be revealed. The Neu5Ac epitope, containing acetyl and polyalcohol moieties, was the major contributor to the sequence recognition for Neu5Ac(α2,6)Gal(β1,4)Glc, whereas contributions from the Gal and Glc segments were substantially lower.

The last decade has seen a range of studies using non-invasive neutron and X-ray techniques to probe the ultrastructure of a variety of photosynthetic membrane systems. A common denominator in this work is the lack of an explicitly formulated underlying structural model, ultimately leading to ambiguity in the data interpretation. Here we formulate and implement a full mathematical model of the scattering from a stacked double bilayer membrane system taking instrumental resolution and polydispersity into account. We validate our model by direct simulation of scattering patterns from 3D structural models. Most importantly, we demonstrate that the full scattering curves from three structurally typical cyanobacterial thylakoid membrane systems measured in vivo can all be described within this framework. The model provides realistic estimates of key structural parameters in the thylakoid membrane, in particular the overall stacking distance and how this is divided between membranes, lumen and cytoplasmic liquid. Finally, from fitted scattering length densities it becomes clear that the protein content in the inner lumen has to be lower than in the outer cytoplasmic liquid and we extract the first quantitative measure of the luminal protein content in a living cyanobacteria.

Spatial variations in the microfibril angle (MFA) of cellulose are mapped on individual radial sections of wood from the maturing stems of Australian tree species (Acacia floribunda, Acacia suaveolens, Acacia doratoxylon, Acacia havilandiorum, Brachychiton populneus and Bertya cunninghamii) from different rainfall and soil nutrient availabilities using synchrotron wide angle X-ray scattering. For the three species with the smallest radial variation there is a clear linear relationship between the MFA with Young's modulus and wood density with data published previously for these samples. The other three samples show a large radially symmetric variation in MFA across the radial section either having a continuous change from large MFA in the stem's centre to small value close to the bark or the reverse, small MFA at the centre to larger values close to the bark. For these samples, a strong linear correlation between the Young's modulus and density and the MFA is not apparent. We conclude that spatially localised measurements of MFA provide an important insight in the adaptation of wood tissue to environmental conditions.

Glycoproteins are abundant on the cell surface of mammals, providing structural support, modulating cell membrane properties, and acting as signaling agents. Variation of glycosylation patterns has been found to indicate various disease states, including cell malignancy. Sialic acid (SA) is present as a terminating group on cell-surface glycans, and its overexpression has been linked to several types of cancer. Detection of SA on the cell surface is therefore critical for detection of cancer in its early stages. In this work, a fluorescent molecularly imprinted polymer layer targeting SA was synthesized on the surface of silica-coated polystyrene (PS) particles. Compared to previous works, a PS core supplies a lighter, lower-density support for improved suspension stability and scattering properties. Moreover, their smaller size provides a higher surface-area-to-volume ratio for binding. The incorporation of a fluorescent monomer in the MIP shell allowed for simple and rapid determination of binding specificity in polar media due to a deprotonation-reprotonation interaction mechanism between the fluorescent monomer and SA, which led to spectral changes. Upon titration of the MIP particles with SA in suspension, an increase in fluorescence emission of the particles was observed, with the MIP particles binding SA more selectively compared to the nonimprinted polymer (NIP) control particles. In cell staining experiments performed by flow cytometry, the binding behavior of the MIP particles compared favorably with that of SA-binding lectins. NIPs prepared with a "dummy" template served as a better negative control in cell binding assays due to the favorable inward orientation of template-binding functional groups in the polymer shell, which reduced nonspecific binding. The results show that fluorescent MIPs targeting SA are a promising tool for in vitro fluorescence staining of cancerous cells and for future diagnosis of cancer at early stages.

Early detection of cancer is essential for successful treatment and improvement in patient prognosis. Deregulation of post-translational modifications (PTMs) of proteins, especially phosphorylation, is present in many types of cancer. Therefore, the development of materials for the rapid sensing of low abundant phosphorylated peptides in biological samples can be of great therapeutic value. In this work, we have synthesised fluorescent molecularly imprinted polymers (fMIPs) for the detection of the phosphorylated tyrosine epitope of ZAP70, a cancer biomarker. The polymers were grafted as nanometer-thin shells from functionalised submicron-sized silica particles using a reversible addition-fragmentation chain-transfer (RAFT) polymerisation. Employing the combination of fluorescent urea and intrinsically cationic bis-imidazolium receptor cross-linkers, we have developed fluorescent sensory particles, showing an imprinting factor (IF) of 5.0. The imprinted polymer can successfully distinguish between phosphorylated and non-phosphorylated tripeptides, reaching lower micromolar sensitivity in organic solvents and specifically capture unprotected peptide complements in a neutral buffer. Additionally, we have shown the importance of assessing the influence of counterions present in the MIP system on the imprinting process and final material performance. The potential drawbacks of using epitopes with protective groups, which can co-imprint with targeted functionality, are also discussed.

MUC5B is the predominant glycoprotein in saliva and is instrumental in the establishment and maintenance of multi-species eubiotic biofilms in the oral cavity. Investigations of the aciduric Lactobacillaceae family, and its role in biofilms emphasizes the diversity across different genera of the proteolytic systems involved in the nutritional utilization of mucins. We have characterized a protease from Limosilactobacillus fermentum, MdpL (Mucin degrading protease from Limosilactobacillus) with a high protein backbone similarity with commensals that exploit mucins for attachment and nutrition. MdpL was shown to be associated with the bacterial cell surface, in close proximity to MUC5B, which was sequentially degraded into low molecular weight fragments. Mapping the substrate preference revealed multiple hydrolytic sites of proteins with a high O-glycan occurrence, although hydrolysis was not dependent on the presence of O-glycans. However, since proteolysis of immunoglobulins was absent, and general protease activity was low, a preference for glycoproteins similar to MUC5B in terms of glycosylation and structure is suggested. MdpL preferentially hydrolyzed C-terminally located hydrophobic residues in peptides larger than 20 amino acids, which hinted at a limited sequence preference. To secure proper enzyme folding and optimal conditions for activity, L. fermentum incorporates a complex system that establishes a reducing environment. The importance of overall reducing conditions was confirmed by the activity boosting effect of the added reducing agents L-cysteine and DTT. High activity was retained in low to neutral pH 5.5-7.0, but the enzyme was completely inhibited in the presence of Zn2+. Here we have characterized a highly conserved mucin degrading protease from L. fermentum. MdpL, that together with the recently discovered O-glycanase and O-glycoprotease enzyme groups, increases our understanding of mucin degradation and complex biofilm dynamics.

Supported lipid bilayers (SLBs) are commonly used as model systems mimicking biological membranes. Recently, we reported a new method to produce SLBs with incorporated membrane proteins, which is based on the application of peptide discs [Luchini et al., Analytical Chemistry, 2020, 92, 1081-1088]. Peptide discs are small discoidal particles composed of a lipid core and an outer belt of self-assembled 18A peptides. SLBs including membrane proteins can be formed by depositing the peptide discs on a solid support and subsequently removing the peptide by buffer rinsing. Here, we introduce a new variant of the 18A peptide, named dark peptide (d18A). d18A exhibits UV absorption at 214 nm, whereas the absorption at 280 nm is negligible. This improves sample preparation as it enables a direct quantification of the membrane protein concentration in the peptide discs by measuring UV absorption at 280 nm. We describe the application of the peptide discs prepared with d18A (dark peptide discs) to produce SLBs with a membrane protein, synaptobrevin 2 (VAMP2). The collected data showed the successful formation of SLBs with high surface coverage and incorporation of VAMP2 in a single orientation with the extramembrane domain exposed towards the bulk solvent. Compared to 18A, we found that d18A was more efficiently removed from the SLB. Our data confirmed the structural organisation of VAMP2 as including both alpha-helical and beta-sheet secondary structure. We further verified the orientation of VAMP2 in the SLBs by characterising the binding of VAMP2 with alpha-synuclein. These results point at the produced SLBs as relevant membrane models for biophysical studies as well as nanostructured biomaterials.

Oxytocin imprinted polymer nanoparticles were synthesized by glass bead supported solid phase synthesis, with NMR and molecular dynamics studies used to investigate monomer-template interactions. The nanoparticles were characterized by dynamic light scattering, scanning- and transmission electron microscopy and X-ray photoelectron spectroscopy. Investigation of nanoparticle-template recognition using quartz crystal microbalance-based studies revealed sub-nanomolar affinity, k(d) approximate to 0.3 +/- 0.02 nM (standard error of the mean), comparable to that of commercial polyclonal antibodies, k(d) approximate to 0.02-0.2 nM.

Glycosylation plays an important role in various pathological processes such as cancer. One key alteration in the glycosylation pattern correlated with cancer progression is an increased level as well as changes in the type of sialylation. Developing molecularly-imprinted polymers (MIPs) with high affinity for sialic acid able to distinguish different glycoforms such as sialic acid linkages is an important task which can help in early cancer diagnosis. Sialyllactose with alpha 2,6 ' vs. alpha 2,3 ' sialic acid linkage served as a model trisaccharide template. Boronate chemistry was employed in combination with a library of imidazolium-based monomers targeting the carboxylate group of sialic acid. The influence of counterions of the cationic monomers and template on their interactions was investigated by means of H-1 NMR titration studies. The highest affinities were afforded using a combination of Br- and Na+ counterions of the monomers and template, respectively. The boronate ester formation was confirmed by MS and H-1/B-11 NMR, indicating 1 : 2 stoichiometries between sialyllactoses and boronic acid monomer. Polymers were synthesized in the form of microparticles using boronate and imidazolium monomers. This combinatorial approach afforded MIPs selective for the sialic acid linkages and compatible with an aqueous environment. The molecular recognition properties with respect to saccharide templates and glycosylated targets were reported.

Aberrant glycosylation has been proven to correlate with various diseases including cancer. An important alteration in cancer progression is an increased level of sialylation, making sialic acid one of the key constituents in tumor-specific glycans and an interesting biomarker for a diversity of cancer types. Developing molecularly imprinted polymers (MIPs) with high affinity toward sialic acids is an important task that can help in early cancer diagnosis. In this work, the glycospecific MIPs are produced using cooperative covalent/noncovalent imprinting. We report here on the fundamental investigation of this termolecular imprinting approach. This comprises studies of the relative contribution of orthogonally interacting functional monomers and their synergetic behavior and the choice of different counterions on the molecular recognition properties for the sialylated targets. Combining three functional monomers targeting different functionalities on the template led to enhanced imprinting factors (IFs) and selectivities. This apparent cooperative effect was supported by H-1 NMR and fluorescence titrations of monomers with templates or template analogs. Moreover, highlighting the role of the template counterion use of tetrabutylammonium (TBA) salt of sialic acid resulted in better imprinting than that of sodium salts supported by both in solution interaction studies and in MIP rebinding experiments. The glycospecific MIPs display high affinity for sialylated targets, with an overall low binding of other nontarget saccharides.

Supramolecular assembly and PEGylation (attachment of a polyethylene glycol polymer chain) of peptides can be an effective strategy to develop antimicrobial peptides with increased stability, antimicrobial efficacy and hemocompatibility. However, how the self-assembly properties and PEGylation affect their lipid membrane interaction is still an unanswered question. In this work, we use state-of-the-art small angle X-ray and neutron scattering (SAXS/SANS) together with neutron reflectometry (NR) to study the membrane interaction of a series of multidomain peptides, with and without PEGylation, known to self-assemble into nanofibers. Our approach allows us to study both how the structure of the peptide and the membrane are affected by the peptide–lipid interactions. When comparing self-assembled peptides with monomeric peptides that are not able to undergo assembly due to shorter chain length, we found that the nanofibers interact more strongly with the membrane. They were found to insert into the core of the membrane as well as to absorb as intact fibres on the surface. Based on the presented results, PEGylation of the multidomain peptides leads to a slight net decrease in the membrane interaction, while the distribution of the peptide at the interface is similar to the non-PEGylated peptides. Based on the structural information, we showed that nanofibers were partially disrupted upon interaction with phospholipid membranes. This is in contrast with the considerable physical stability of the peptide in solution, which is desirable for an extended in vivo circulation time.

The naturally occurring peptide indolicidin from bovine neutrophils exhibits strong biological activity against a broad spectrum of microorganisms. This is believed to arise from selective interactions with the negatively charged cytoplasmic lipid membrane found in bacteria. We have investigated the peptide interaction with supported lipid model membranes using a combination of complementary surface sensitive techniques: neutron reflectometry (NR), atomic force microscopy (AFM), and quartz crystal microbalance with dissipation monitoring (QCM-D). The data are compared with small-angle X-ray scattering (SAXS) results obtained with lipid vesicle/peptide solutions. The peptide membrane interaction is shown to be significantly concentration dependent. At low concentrations, the peptide inserts at the outer leaflet in the interface between the headgroup and tail core. Insertion of the peptide results in a slight decrease in the lipid packing order of the bilayer, although not sufficient to cause membrane thinning. By increasing the indolicidin concentration well above the physiologically relevant conditions, a deeper penetration of the peptide into the bilayer and subsequent lipid removal take place, resulting in a slight membrane thinning. The results suggest that indolicidin induces lipid removal and that mixed indolicidin-lipid patches form on top of the supported lipid bilayers. Based on the work presented using model membranes, indolicidin seems to act through the interfacial activity model rather than through the formation of stable pores.

Sialic acid binding immunoglobulin-type lectin (Siglec) family are inhibitory receptors with diverse roles in the immune system. Siglec family contains 14 members in human and 9 in murine. Differentially expressed on various white blood cells. Here in this review we are focusing on CD22, also known as Sialic Acid-Binding Ig-Like Lectin 2 (Siglec-2). CD22 gene is located on 19q13.12 and is encoding a 140 kD type I transmembrane glycoprotein on the surface of B cells and is part of the immunoglobulin (Ig) superfamily and has been found only on B cells. CD22 has been shown to play a major role in establishing a baseline level of B-cell inhibition, and thus is a critical determinant of homeostasis in humoral immunity.

På huden lever en sammansättning av olika mikroorganismer såsom bakterier, svampar och virus. Dessa mikroorganismer kallas hudens mikrobiom. Sammansättningen av en individs mikrobiom kan ge mycket information om en individens hälsa. För att undersöka sammansättningen av bakterier på hudytan med exempelvis qPCR, behöver bakterier samlas in och DNA extraheras. Bakteriekoncentrationen på hudens torrare områden som exempelvis armar har normalt en relativt låg bakteriekoncentration vid 10²-10⁴ bakterier per cm². Huden koloniseras till stor del av grampositiva bakterier. Grampositiva bakterier är i regel svårare att lysera än andra bakterier och kräver därför hårdare lysering. En bra extraktionsmetod ska erhålla mycket DNA utan att påverka dess kvalité. I detta arbete utvärderades initialt fem olika extraktionsmetoder på bakteriesuspension med Staphylococcus aureus (S. aureus), både direkt på bakteriesuspension men också från svabb. Utvärdering gjordes på PureLink Microbiome DNA Purification Kit, QlAamp PowerFecal Pro, QlAamp DNA Mini Kit och KOH-EDTA. Metoden med QlAamp DNA Mini Kit testades med två olika protokoll och räknades som två separata metoder. Metoderna som gav bäst resultat vid initial utvärdering var PureLink Microbiome och KOH-EDTA. Därefter utvärderades dessa två metoderna på prov insamlat med svabb från huden på 10 frivilliga deltagare. 

The use of neutrons as a scattering probe to investigate biological membranes has steadily grown in the past three decades, shedding light on the structure and behaviour of this ubiquitous and fundamental biological barrier. Meanwhile, the rise of antibiotic resistance has catalysed a renewed interest in understanding the mechanisms underlying the dynamics of antibiotics interaction with the bacterial cell envelope. It is widely recognised that the key reason behind the remarkable success of Gram-negative pathogens in developing antibiotic resistance lies in the effectiveness of their outer membrane (OM) in defending the cell from antibacterial compounds. Critical to its function, the highly asymmetric lipid distribution between the inner and outer bilayer leaflets of the OM, adds an extra level of complexity to the study of this crucial defence barrier. Here we review the opportunities offered by neutron scattering techniques, in particular reflectometry, to provide structural information on the interactions of antimicrobials with in vitro models of the OM. The differential sensitivity of neutrons towards hydrogen and deuterium makes them a unique probe to study the structure and behaviour of asymmetric membranes. Molecular-level understanding of the interactions between antimicrobials and the Gram-negative OM provides valuable insights that can aid drug development and broaden our knowledge of this critically important biological barrier.

The stability of biologically produced pharmaceuticals is the limiting factor to various applications, which can be improved by formulation in solid-state forms, mostly via lyophilization. Knowledge about the protein structure at the molecular level in the solid state and its transition upon rehydration is however scarce, and yet it most likely affects the physical and chemical stability of the biological drug. In this work, synchrotron small- and wide-angle X-ray scattering (SWAXS) are used to characterize the structure of a model protein, lysozyme, in the solid state and its structural transition upon rehydration to the liquid state. The results show that the protein undergoes distortion upon drying to adopt structures that can continuously fill the space to remove the protein-air interface that may be formed upon dehydration. Above a hydration threshold of 35 wt %, the native structure of the protein is recovered. The evolution of SWAXS peaks as a function of water content in a broad range of concentrations is discussed in relation to the structural changes in the protein. The findings presented here can be used for the design and optimization of solid-state formulations of proteins with improved stability.

In recent years, many papers have reported a catalytic “acceleration” of enzymes when immobilized on gold nanoparticles. Concordantly, gold nanoparticles are often considered as an inert and safe nanomaterial, and are widely used for various purposes, e.g., experiments with humans are being conducted in vivo. In this work we have carried out an in-detail study of catalytic properties of citrate-capped gold nanoparticles and gold nanoparticle-protein conjugates using three model proteins – enzymes glucose oxidase and catalase, and catalytically inactive protein bovine serum albumin. Catalytic properties were studied at different protein-nanoparticle ratios. UV–Vis, DLS, AFM and ζ potential measurements confirmed protein-nanoparticle conjugate formation. Catalytic activity measurements were conducted using oxygen electrode and the data were analyzed by modeling the activity of conjugates. The designed experiments demonstrated that gold nanoparticles form stable conjugates with all the investigated proteins, yet they do not increase catalytic activity of the investigated enzymes – in certain conditions gold nanoparticles mimic enzymatic reactions, which may be misattributed to accelerated enzymatic catalysis. Additionally, we present specific key points demonstrating why it may be difficult to differentiate between enzyme- and gold nanoparticle-catalyzed reactions, as well as suggest specific measurements enabling better differentiation. We do not claim that enzymes cannot be accelerated on nanoparticles in general, but rather emphasize, that experimental results demonstrating atypical catalytic performance of enzymes on nanoparticles should be interpreted with additional care, and a widely propagated view of “inert gold nanoparticles” should probably be reconsidered. 

This thesis investigates responses to external cues in oral bacteria on a molecular level. Paper I maps Ser/Thr/Tyr phosphorylated proteins in relation to the general proteome in an oral commensal streptococcus (Streptococcus gordonii DL1). The identified phosphoproteins were involved in various bacterial processes, several associated to dysbiosis and development of biofilm-induced disease. Comparison against phosphoproteomes of other bacteria showed many similarities. This is of interest for the identification of shared phosphorylation profiles. 

Paper II studies differences between the S. gordonii DL1 general proteomes in planktonic and biofilm growth phases, and the regulatory effects of salivary mucin MUC5B on protein expression in the biofilm cells. Regulations in protein expression between the different growth conditions provides insights in bacterial mechanisms for adaptation to the biofilm lifestyle. 

Paper III examines the regulatory roles of salivary MUC5B on biofilm attachment and metabolic output in two clinical isolates of oral commensals, S. gordonii CW and Actinomyces naeslundii CW. S. gordonii facilitated adhesion of A. naeslundii to MUC5B during early attachment. Both bacteria were also able to utilize MUC5B as a sole nutrient source during early biofilm formation, individually and synergistically in a dual species biofilm. The specific responses elicited by MUC5B in paper II-III seem to promote commensal colonization while down-regulating dysbiosis-related biofilm activities. 

Microbiological studies are often focused on dysbiosis and development of disease. However, mechanisms that promote eubiosis are equally important to understand how health can be maintained. Findings associated with responses to external cues in oral bacteria may contribute to future development of novel preventative strategies and identification of predictive biomarkers for oral health. 

The field of anion recognition in aqueous media is dominated by multiple positive charges and/or metal ions containing synthetic receptors. Here, we report the synthesis of a highly water-compatible oxyanion-imprinted neutral receptor by cross-linking the polymerizable diary! urea host monomer and phenyl phosphonic acid (PPA) complex in the presence of a large number of comonomers containing hydroxyl or methyl ester groups. We have demonstrated that the affinity of the imprinted receptor for anions is easily tuned by decorating the hydrophilic and hydrophobic end groups around the binding pocket of the imprinted receptor. The binding characteristics of the synthesized artificial binders were evaluated in aqueous and nonaqueous environments. We have demonstrated practical applicability of the PPA-imprinted receptor by packing in the solid-phase extraction cartridge which was used to separate the ubiquitous pesticide glyphosate from water. Also, the developed receptor can selectively bind to phosphorylated tyrosine amino acids and phosphorylated tyrosine octapeptides in aqueous buffered media than to other phosphorylated and nonphosphorylated analogues. The approach utilized here to use an inexpensive dummy template can be utilized for the synthesis of imprinted receptors for anions with phosphate motifs.

Lipid nanoparticles (LNPs) are promising drug and gene carriers. Upon intravenous administration, LNPs' experience different degree of cellular uptake depending on their formulation. Currently, in vitro and in vivo studies are the gold standard for assessing the fate of nano carriers once administered, but they are time consuming and expensive. In this work, we propose a time and cost-effective method to screen a wide range of LNP formulations and select the most promising candidates for in vitro and in vivo studies. Two different approaches were explored to investigate the binding affinity between LNPs and serum proteins using sensor functionalisation with either protein specific antibody or PEG specific antibody. The first approach allowed to identify the presence of a specific protein in the protein corona of lipid particles (reconstituted and native high-density lipoproteins (rHDL and HDL), and low-density lipoproteins LDL); while the second one provided a versatile platform for the immobilisation of pegylated-particles in order to follow the interaction with serum proteins and hence predict the composition of LNP protein corona. Sensing was done using Quartz Crystal Microbalance with Dissipation (QCM-D) but the approach is extendable to other surface sensing techniques such as Surface Plasmon Resonance (SPR) or ellipsometry.

The use of polymerizable hosts in anion imprinting has led to powerful receptors with high oxyanion affinity and specificity in both aqueous and non-aqueous environments. As demonstrated in previous reports, a carefully tuned combination of orthogonally interacting binding groups, for example, positively charged and neutral hydrogen bonding monomers, allows receptors to be constructed for use in either organic or aqueous environments, in spite of the polymer being prepared in non-competitive solvent systems. We here report on a detailed experimental design of phenylphosphonic and benzoic acid-imprinted polymer libraries prepared using either urea-or thiourea-based host monomers in the presence or absence of cationic comonomers for charge-assisted anion recognition. A comparison of hydrophobic and hydrophilic crosslinking monomers allowed optimum conditions to be identified for oxyanion binding in non-aqueous, fully aqueous, or high-salt media. This showed that recognition improved with the water content for thiourea-based molecularly imprinted polymers (MIPs) based on hydrophobic EGDMA with an opposite behavior shown by the polymers prepared using the more hydrophilic crosslinker PETA. While the affinity of thiourea-based MIPs increased with the water content, the opposite was observed for the oxourea counterparts. Binding to the latter could however be enhanced by raising the pH or by the introduction of cationic amine-or Na+-complexing crown ether-based comonomers. Use of high-salt media as expected suppressed the amine-based charge assistance, whereas it enhanced the effect of the crown ether function. Use of the optimized receptors for removing the ubiquitous pesticide glyphosate from urine finally demonstrated their practical utility.

This paper reports results of a reexamination of some poorly understood peculiarities of laccases, an enzyme family which has been extensively studied in our laboratories as well as by others for some years. The issue that is reconsidered here is the previously proposed existence of “active” and “resting” forms of laccases. The presence of fungal laccases with partly reduced active sites is demonstrated. Of further interest is that an aggregated state in solution, not to our knowledge previously noted for laccase, has been found by using small-angle X-ray scattering as well as thorough analysis of the results of several biochemical experiments. Under some conditions, this aggregated state may correlate with the resting form of the laccases, although this resting form could have a broader significance. It was shown that Trametes ochracea laccase had some anomalous characteristics, which could be correlated with the high concentration of the “resting” enzyme. The mechanism of formation of resting laccase is suggested. Knowledge of the resting state is of importance for in vitro studies. Additionally, a suggestion about the possible regulatory role of this form in vivo is mentioned.

The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as Selectins, Galectins, and Siglecs are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human HEK293 cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knock-in of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galβ1-3GalNAcα1-O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3O-sulfotransferase 2 (GAL3ST2) in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3O-sulfotransferase 4 (GAL3ST4) resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step towards expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.

Periodontitis is an inflammatory oral disease that affects a large part of the adult population, causing significant costs and suffering. The key pathogen, Porphyromonas gingivalis, secretes gingipains, which are highly destructive proteases and the most important virulence factors in the pathogenesis of the disease. Currently, periodontitis is diagnosed mainly by mechanical manual probing and radiography, often when the disease has already progressed significantly. The possibilities of detecting gingipain activity in gingival fluid could enable early-stage diagnosis and facilitate treatment. Here, we describe a sensitive nanoparticle-based nanoplasmonic biosensor for the detection of the proteolytic activity of gingipains. Gold nanoparticles (AuNPs) were self-assembled as a submonolayer in multiwell plates and further modified with casein or IgG. The proteolytic degradation of the protein coating was tracked by monitoring the shift in the localized surface plasmon resonance (LSPR) peak position. The sensor performance was investigated using model systems with trypsin and purified gingipains (subtypes Kgp and RgpB) and further validated using supernatants from cultures of P. gingivalis. Proteolytic degradation by proteases in buffer results in a concentration- and time-dependent blueshift of the LSPR band of about 1-2 nm when using casein as a substrate. In bacterial supernatants, the degradation of the protein coating resulted in unspecific binding of proteins present in the complex sample matrix to the nanoparticles, which instead triggered a redshift of about 2 nm of the LSPR band. A significant LSPR shift was seen only in samples with gingipain activity. The sensor showed a limit of detection < 0.1 mu g/mL (4.3 nM), which is well below gingipain concentrations detected in severe chronic periodontitis cases (similar to 50 mu g/mL). This work shows the possibility of developing cost-effective nanoparticle-based biosensors for rapid detection of protease activity for chair-side periodontal diagnostics.

Atherosclerosis arises from build-up of plaque in the blood, can result in cardiovascular disease and is the largest killer in the west. Low- and high-density lipoproteins are involved in the disease development by depositing and removing lipids to and from artery walls. These processes are complex and not fully understood however, therefore determining the specific roles of the components involved is of fundamental importance in the treatment of the disease.

The work presented in this thesis investigates the production of recombinant tailor-deuterated cholesterol, the structure of cholesterol-containing model membranes and interactions of both native and reconstituted lipoproteins with model membranes. Deuteration is commonly used in neutron scattering for biological samples to provide highly important contrast and the complexity of the native lipoproteins leads to the use of more simple model systems where the compositions can be altered and investigated systematically.

A protocol was developed to produce matchout-deuterated cholesterol for use in neutron scattering studies, as cholesterol is a hugely important component in membranes. The verification of the matchpoint of cholesterol was determined by small-angle neutron scattering and the localisation of cholesterol in model membranes was determined through the use of neutron reflectometry. The interactions of the native and reconstituted lipoproteins with model membranes were also followed by neutron reflectometry, while the structural characterisation of the reconstituted lipoproteins was carried out by small-angle scattering.

Lipoproteins play a central role in the development of atherosclerosis. High and low-density lipoproteins (HDL and LDL), known as 'good' and 'bad' cholesterol, respectively, remove and/or deposit lipids into the artery wall. Hence, insight into lipid exchange processes between lipoproteins and cell membranes is of particular importance in understanding the onset and development of cardiovascular disease. In order to elucidate the impact of phospholipid tail saturation and the presence of cholesterol in cell membranes on these processes, neutron reflection was employed in the present investigation to follow lipid exchange with both HDL and LDL against model membranes. Mirroring clinical risk factors for the development of atherosclerosis, lower exchange was observed in the presence of cholesterol, as well as for an unsaturated phospholipid, compared to faster exchange when using a fully saturated phospholipid. These results highlight the importance of membrane composition on the interaction with lipoproteins, chiefly the saturation level of the lipids and presence of cholesterol, and provide novel insight into factors of importance for build-up and reversibility of atherosclerotic plaque. In addition, the correlation between the results and well-established clinical risk factors suggests that the approach taken can be employed also for understanding a broader set of risk factors including, e.g., effects of triglycerides and oxidative stress, as well as local effects of drugs on atherosclerotic plaque formation.

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body.

Panther Fusion® system is a fully automated in vitro diagnostic system that has a high flow of sample-to-results and allows the performance of in vitro diagnostics tests using real-time Polymerase Chain Reaction (real-time PCR). The purpose of this project is to validate whether it is possible to use a kit from Amplidiag on Panther fusion open access to diagnose vancomycin resistant enterococcus (VRE). In addition, this project aims to evaluate and analyze E-swab samples directly instead of enrichment broth. It is important to be able to detect VanA and VanB genes in samples because VRE harbors these genes. Sensitivity and efficiency of Panther fusion open access were tested by analyzing the samples simultaneously with Bio-Rad CFX384/96 which is the routine method for VRE diagnostic at Clinical Microbiology in Lund. Specificity of a previously published PCR-mix (PCR-mix) that can detect VanA and VanB genes in samples was compared with Amplidiag assay mix 2 used in the routine method. In order to evaluate the difference in specificity and sensitivity between E-swab and enrichment broth, the analysis was performed on both E-swab and enrichment broth with both Panther fusion open access and Bio-Rad CFX384/96-PCR. MgCl2 concentration was optimized to 3,0 mM. Both sensitivity and efficiency of Panther fusion open access were higher with PCR-mix than those with Amplidiag assay mix 2 and it showed optimal linearity. Specificity test of PCR-mix detected only VanA and VanB genes. The results of inoculated the negative VRE-patient samples (E-swab) indicated that they were unreasonable because some of the samples and controls gave results of both genes. Replacement of the VRE-diagnostic method on Bio-Rad CFX384/96 with Panther fusion open access requires performing additional experiments directly on E-swab and vancomycin variable enterococcus (VVE) samples using Panther fusion open access.