Development of Viral-Vectored Vaccines and Virus Replicon Particle-Based Neutralisation Assay against Mayaro Virus

Mayaro virus (MAYV) is an emerging alphavirus causing acute febrile illness associated with chronic polyarthralgia. Although MAYV is currently restricted to tropical regions in South America around the Amazon basin, it has the potential to spread globally by Aedes species mosquitoes. In addition, there are currently no specific therapeutics or licenced vaccines against MAYV infection. We have previously shown that an adenovirus based Mayaro vaccine (ChAdOx1 May) was able to provide full protection against MAYV challenge in vaccinated A129 mice and induced high neutralising antibody titres. In this study, we have constructed a replication deficient simian adenovirus (ChAdOx2) and a Modified Ankara Virus (MVA) based vaccine expressing the MAYV structural cassette (sMAYV) similar to ChAdOx1 May, and characterised recombinant MAYV E2 glycoprotein expressed in a mammalian system for immune monitoring. We demonstrate that ChAdOx2 May was able to induce high antibody titres similar to ChAdOx1 May, and MVA May was shown to be an effective boosting strategy following prime vaccination with ChAdOx1 or ChAdOx2 May. In order to measure MAYV neutralising ability, we have developed a virus replicon particle-based neutralisation assay which effectively detected neutralising antibodies against MAYV. In summary, our study indicates the potential for further clinical development of the viral vectored MAYV vaccines against MAYV infections.     

Regulation of CD4+ and CD8+ T Cell Biology by Short-Chain Fatty Acids and Its Relevance for Autoimmune Pathology

The gut microbiota encodes a broad range of enzymes capable of synthetizing various metabolites, some of which are still uncharacterized. One well-known class of microbiota-derived metabolites are the short-chain fatty acids (SCFAs) such as acetate, propionate, butyrate and valerate. SCFAs have long been considered a mere waste product of bacterial metabolism. Novel results have challenged this long-held dogma, revealing a central role for microbe-derived SCFAs in gut microbiota-host interaction. SCFAs are bacterial signaling molecules that act directly on host T lymphocytes by reprogramming their metabolic activity and epigenetic status. They have an essential biological role in promoting differentiation of (intestinal) regulatory T cells and in production of the anti-inflammatory cytokine interleukin-10 (IL-10). These small molecules can also reach the circulation and modulate immune cell function in remote tissues. In experimental models of autoimmune and inflammatory diseases, such as inflammatory bowel disease, multiple sclerosis or diabetes, a strong therapeutic potential of SCFAs through the modulation of effector T cell function was observed. In this review, we discuss current research activities toward understanding a relevance of microbial SCFA for treating autoimmune and inflammatory pathologies from in vitro to human studies.     

Design of CK2β-Mimicking Peptides as Tools To Study the CK2α/CK2β Interaction in Cancer Cells

The ubiquitously expressed Ser/Thr kinase CK2 is a key regulator in a variety of key processes in normal and malignant cells. Due to its distinctive anti-apoptotic and tumor-driving properties, elevated levels of CK2 have frequently been found in tumors of different origin. In recent years, development of CK2 inhibitors has largely been focused on ATP-competitive compounds; however, targeting the CK2α/CK2β interface has emerged as a further concept that might avoid selectivity issues. To address the CK2 subunit interaction site, we have synthesized halogenated CK2β-mimicking cyclic peptides modified with the cell-penetrating peptide sC18 to mediate cellular uptake. We investigated the binding of the resulting chimeric peptides to recombinant human CK2α using a recently developed fluorescence anisotropy assay. The iodinated peptide sC18-I-Pc was identified as a potent CK2α ligand (K_i=0.622 μm ). It was internalized in cells to a high extent and exhibited significant cytotoxicity toward cancerous HeLa cells (IC₅₀=37 μm ) in contrast to non-cancerous HEK-293 cells. The attractive features and functionalities of sC18-I-Pc offer the opportunity for further improvement.     

A Sensitive and Robust Method for Direct Determination of Lipolytic Activity in Natural Milk Environment

Microbial lipases may be produced during milk storage and processing. This can lead to organoleptic changes in the corresponding dairy products. Thus, monitoring of lipase activity in milk is desirable. Turbidity of milk prevents a direct photometric measurement of lipase activity using chromophore- or fluorophore-based assays. Laborious pretreatments or alternative analytical methods normally have to be used. With the method for the determination of lipolytic activity (MeDeLi) proposed here, it is possible to measure lipase activity directly in the natural milk utilizing tailored fluorometric substrates. Only a defatting step is carried out initially for the MeDeLi. Then, the conversion of added lipase substrate is carried out in the unmodified milk without addition of any solutions or any enzyme extraction procedure which may influence the enzyme activity. Thereafter, the milk sample is treated with two solutions to remove the turbidity of milk by dissolution. A valid and sensitive fluorometric measurement is then possible. The applicability of the MeDeLi was demonstrated in comparison with tests published previously: The limit of detection for lipolytic activity measured by MeDeLi was the lowest, with 41 pkat/mL. Raw milk, milk products, and spoiled milk samples were also investigated with the MeDeLi.     

Sulfur speciation in soil by S K-Edge XANES spectroscopy: comparison of spectral deconvolution and linear combination fitting

Defined, quartz-diluted mixtures of sulfur (S) compounds with different oxidation state (OS) were analyzed by K-edge XANES spectroscopy using linear combination fitting (LCF) and spectrum deconvolution by fitting several Gaussian and arctangent functions (GCF). Additionally, for different soils the S speciation as calculated by both methods was compared with results of a wet-chemical S speciation. For mixtures of FeS, L-cysteine, and Na2SO4, the S speciation was recovered with satisfactory accuracy and precision by both methods at the 2 and 0.2 mg S g(-1) level. For GCF, white-line peaks must be normalized with respect to their OS-specific absorption cross-section. LCF must be conducted with dilute reference compounds to avoid self-absorption effects. For mixtures of FeS, FeS2, S°, and L-cysteine, both procedures showed poor accuracy. For the soils, similar percentages of reduced inorganic S, organic S, and sulfate were calculated by LCF, GCF, and wet chemical S speciation. GCF allows a fair estimation of S species groups with different OS (inorganic reduced S, organic reduced S, organic intermediate S, oxidized S) in soils without standards. If dilute standards of all S compounds assumed to be present in a sample are available, LCF is more objective and allows a more detailed S speciation.     

Novel copolymers showing interactions of amidinium-carboxylate groups in water

Four hydrophobic amidine containing monomers N-(pyridin-2-yl)methacrylamide (3), 5-(pyridin-2-ylamino)pentyl methacrylate (7), S-5-(isopropenylcarbonyloxy)pentyl-N,N-diphenylthiourea (9) and methyl-5-guanidino-2-methacrylamidopentanoate (12) were synthesized and copolymerized with a water soluble co-monomer N,N-dimethylacrylamide (13) or N-isopropylacrylamide (22). The second component was a copolymer containing methacrylic acid (19) and 13/22. By combination of these two components the formation of amidinium-carboxylate interactions was determined by dynamic light scattering (DLS), ¹H-NMR spectroscopy and rheology measurements.     

The microbial diversity of water kefir

The microbial diversity of water kefir, made from a mixture of water, dried figs, a slice of lemon and sucrose was studied. The microbial consortia residing in the granules of three water kefirs of different origins were analyzed. A collection of 453 bacterial isolates was obtained on different selective/differential media. Bacterial isolates were grouped with randomly amplified polymorphic DNA (RAPD)-PCR analyses. One representative of each RAPD genotype was identified by comparative 16S rDNA gene sequencing. The predominant genus in water kefirs I and II was Lactobacillus, which accounted for 82.1% in water kefir I and 72.1% in water kefir II of the bacterial isolates. The most abundant species in water kefirs I and II were Lactobacillus hordei and Lb. nagelii followed by considerably lower numbers of Lb. casei. Other lactic acid bacteria (LAB) were identified as Leuconostoc mesenteroides and Lc. citreum in all three water kefirs. The most abundant species in water kefir III was Lc. mesenteroides (28%) and Lc. citreum (24.3%). A total of 57 LAB belonging to the species of Lb. casei, Lb. hordei, Lb. nagelii, Lb. hilgardii and Lc. mesenteroides were able to produce exopolysacchrides from sucrose. Non LABs were identified as Acetobacter fabarum and Ac. orientalis. The Acetobacter species were more prevalent in consortium III. Cluster analyses of RAPD-PCR patterns revealed an interspecies diversity among the Lactobacillus and Acetobacter strains. Aditionally, Saccharomyces cerevisiae, Lachancea fermentati, Hanseniaospora valbyensis and Zygotorulaspora florentina were isolated and identified by comparison of partial 26S rDNA sequences and FTIR spectroscopy.     

Concentrated Raw Fibers Enhance the Fiber-Degrading Capacity of a Synthetic Human Gut Microbiome

The consumption of prebiotic fibers to modulate the human gut microbiome is a promising strategy to positively impact health. Nevertheless, given the compositional complexity of the microbiome and its inter-individual variances, generalized recommendations on the source or amount of fiber supplements remain vague. This problem is further compounded by availability of tractable in vitro and in vivo models to validate certain fibers. We employed a gnotobiotic mouse model containing a 14-member synthetic human gut microbiome (SM) in vivo, characterized a priori for their ability to metabolize a collection of fibers in vitro. This SM contains 14 different strains belonging to five distinct phyla. Since soluble purified fibers have been a common subject of studies, we specifically investigated the effects of dietary concentrated raw fibers (CRFs)—containing fibers from pea, oat, psyllium, wheat and apple—on the compositional and functional alterations in the SM. We demonstrate that, compared to a fiber-free diet, CRF supplementation increased the abundance of fiber-degraders, namely Eubacterium rectale, Roseburia intestinalis and Bacteroides ovatus and decreased the abundance of the mucin-degrader Akkermansia muciniphila. These results were corroborated by a general increase of bacterial fiber-degrading α-glucosidase enzyme activity. Overall, our results highlight the ability of CRFs to enhance the microbial fiber-degrading capacity.     

Cytotoxicity of Ultrasmall Gold Nanoparticles on Planktonic and Biofilm Encapsulated Gram‐Positive Staphylococci

The emergence of multidrug resistant bacteria, especially biofilm‐associated Staphylococci, urgently requires novel antimicrobial agents. The antibacterial activity of ultrasmall gold nanoparticles (AuNPs) is tested against two gram positive: S. aureus and S. epidermidis and two gram negative: Escherichia coli and Pseudomonas aeruginosa strains. Ultrasmall AuNPs with core diameters of 0.8 and 1.4 nm and a triphenylphosphine‐monosulfonate shell (Au0.8MS and Au1.4MS) both have minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 25 × 10^?6m [Au]. Disc agar diffusion test demonstrates greater bactericidal activity of the Au0.8MS nanoparticles over Au1.4MS. In contrast, thiol‐stabilized AuNPs with a diameter of 1.9 nm (AuroVist) cause no significant toxicity in any of the bacterial strains. Ultrasmall AuNPs cause a near 5 log bacterial growth reduction in the first 5 h of exposure, and incomplete recovery after 21 h. Bacteria show marked membrane blebbing and lysis in biofilm‐associated bacteria treated with ultrasmall AuNP. Importantly, a twofold MIC dosage of Au0.8MS and Au1.4MS each cause around 80%–90% reduction in the viability of Staphylococci enveloped in biofilms. Altogether, this study demonstrates potential therapeutic activity of ultrasmall AuNPs as an effective treatment option against staphylococcal infections.     

Temperature‐Induced Switchable Adhesion using Nickel–Titanium–Polydimethylsiloxane Hybrid Surfaces

A switchable dry adhesive based on a nickel–titanium (NiTi) shape‐memory alloy with an adhesive silicone rubber surface has been developed. Although several studies investigate micropatterned, bioinspired adhesive surfaces, very few focus on reversible adhesion. The system here is based on the indentation‐induced two‐way shape‐memory effect in NiTi alloys. NiTi is trained by mechanical deformation through indentation and grinding to elicit a temperature‐induced switchable topography with protrusions at high temperature and a flat surface at low temperature. The trained surfaces are coated with either a smooth or a patterned adhesive polydimethylsiloxane (PDMS) layer, resulting in a temperature‐induced switchable surface, used for dry adhesion. Adhesion tests show that the temperature‐induced topographical change of the NiTi influences the adhesive performance of the hybrid system. For samples with a smooth PDMS layer the transition from flat to structured state reduces adhesion by 56%, and for samples with a micropatterned PDMS layer adhesion is switchable by nearly 100%. Both hybrid systems reveal strong reversibility related to the NiTi martensitic phase transformation, allowing repeated switching between an adhesive and a nonadhesive state. These effects have been discussed in terms of reversible changes in contact area and varying tilt angles of the pillars with respect to the substrate surface.