Biocatalytic N-Alkylation of Amines Using Either Primary Alcohols or Carboxylic Acids via Reductive Aminase Cascades.

The alkylation of amines with either alcohols or carboxylic acids represents a mild and safe alternative to the use of genotoxic alkyl halides and sulfonate esters. Here we report two complementary one-pot systems in which the reductive aminase (RedAm) from Aspergillus oryzae is combined with either (i) a 1° alcohol/alcohol oxidase (AO) or (ii) carboxylic acid/carboxylic acid reductase (CAR) to affect N-alkylation reactions. The application of both approaches has been exemplified with respect to substrate scope and also preparative scale synthesis. These new biocatalytic methods address issues facing alternative traditional synthetic protocols such as harsh conditions, overalkylation and complicated workup procedures.

Citrullinated isomer of myelin basic protein can induce inflammatory responses in astrocytes.

Purpose: During the course of demyelinating inflammatory diseases, myelin-derived proteins, including myelin basic protein(MBP), are secreted into extracellular space. MBP shows extensive post-translational modifications, including deimination/citrullination. Deiminated MBP is structurally less ordered, susceptible to proteolytic attack, and more immunogenic than unmodified MBP. This study investigated the effect of the deiminated/citrullinated isomer of MBP(C8) and the unmodified isomer of MBP(C1) on cultured primary astrocytes. Methods: MBP charge isomers were isolated/purified from bovine brain. Primary astrocyte cultures were prepared from the 2-day-old Wistar rats. For evaluation of glutamate release/uptake a Fluorimetric glutamate assay was used. Expression of peroxisome proliferator-activated receptor-gamma(PPAR-γ), excitatory amino acid transporter 2(EAAT2), the inhibitor of the nuclear factor kappa-B(ikB) and high mobility group-B1(HMGB1) protein were assayed by Western blot analysis. IL-17A expression was determined in cell medium by ELISA. Results: We found that MBP(C8) and MBP(C1) acted differently on the uptake/release of glutamate in astrocytes: C1 increased glutamate uptake and did not change its release, whereas C8 decreased glutamate release but did not change its uptake. Both isomers increased the expression of PPAR-γ and EAAT2 to the same degree. Western blots of cell lysates revealed decreased expression of ikB and increased expression of HMGB1 proteins after treatment of astrocytes by C8. Moreover, C8-treated cells released more nitric oxide and proinflammatory IL-17A than C1-treated cells. Conclusions: These data suggest that the most immunogenic deiminated isomer C8, in parallel to the decreases in glutamate release, elicits an inflammatory response and enhances the secretion of proinflammatory molecules via activation of nuclear factor kappa B(NF-kB). Summary statement: The most modified-citrullinated myelin basic protein charge isomer decreases glutamate release, elicits an inflammatory response and enhances the secretion of proinflammatory molecules via activation of nuclear factor kappa B in astrocytes.

Optical anisotropy of flagellin layers: in situ and label-free measurement of adsorbed protein orientation using OWLS.

The surface adsorption of the protein flagellin was followed in situ using optical waveguide lightmode spectroscopy (OWLS). Flagellin did not show significant adsorption on a hydrophilic waveguide, but very rapidly formed a dense monolayer on a hydrophobic (silanized) surface. The homogeneous and isotropic optical layer model, which has hitherto been generally applied in OWLS data interpretation for adsorbed protein films, failed to characterize the flagellin layer, but it could be successfully modeled as an uniaxial thin film. This anisotropic modeling revealed a significant positive birefringence in the layer, suggesting oriented protein adsorption. The adsorbed flagellin orientation was further evidenced by monitoring the surface adsorption of truncated flagellin variants, in which the terminal protein regions or the central (D3) domain were removed. Without the terminal regions the protein adsorption was much slower and the resulting films were significantly less birefringent, implying that intact flagellin adsorbs on the hydrophobic surface via its terminal regions.

Adhesive properties and inflammatory potential of citrullinated myelin basic protein peptide 45-89.

Deimination of arginyl residue of myelin basic protein (MBP) reduces cationicity of MBP and impedes the normal myelin membrane assembly. Less ordered structure of MBP is more susceptible to proteolytic attack that may lead to the release of highly immunogenic deiminated peptides into extracellular milieu. We have studied the association of peptides 45-89 derived from citrullinated MBP (C8 isomer) and phosphorylated MBP (C3 isomer) with the myelin lipids in a model membrane system using optical waveguide lightmode spectrometry. The analysis of association/dissociation kinetics to planar lipids under controlled hydrodynamic conditions has shown that MBP 45-89 peptide from citrullinated C8 isomer is less effectively adsorbed on the lipid membrane, than peptide from phosphorylated C3 isomer and packing densities for phosphorylated 45-89 MBP peptide is higher than for citrullinated forms. On the other hand, our results shown that continuous (24 h) exposure of mixed oligodendrocyte/microglial cells to peptides 45-89 from MBP-C8 induces apoptosis via mitochondrial pathway. In addition, peptides 45-89 stimulated the secretion of nitric oxide from microglial cells via induction of iNOS and decreased the level of the inhibitory protein IkB, indicating involvement of the transcription factor NF-kB in these processes. Our results suggest that some citrullinated peptides, initially released from oligodendrocytes, might activate microglia, which produces reactive nitrogen species and generates in turn fatal feedbacks that kill oligodendrocytes.

The NutriChip project--translating technology into nutritional knowledge.

Advances in food transformation have dramatically increased the diversity of products on the market and, consequently, exposed consumers to a complex spectrum of bioactive nutrients whose potential risks and benefits have mostly not been confidently demonstrated. Therefore, tools are needed to efficiently screen products for selected physiological properties before they enter the market. NutriChip is an interdisciplinary modular project funded by the Swiss programme Nano-Tera, which groups scientists from several areas of research with the aim of developing analytical strategies that will enable functional screening of foods. The project focuses on postprandial inflammatory stress, which potentially contributes to the development of chronic inflammatory diseases. The first module of the NutriChip project is composed of three in vitro biochemical steps that mimic the digestion process, intestinal absorption, and subsequent modulation of immune cells by the bioavailable nutrients. The second module is a miniaturised form of the first module (gut-on-a-chip) that integrates a microfluidic-based cell co-culture system and super-resolution imaging technologies to provide a physiologically relevant fluid flow environment and allows sensitive real-time analysis of the products screened in vitro. The third module aims at validating the in vitro screening model by assessing the nutritional properties of selected food products in humans. Because of the immunomodulatory properties of milk as well as its amenability to technological transformation, dairy products have been selected as model foods. The NutriChip project reflects the opening of food and nutrition sciences to state-of-the-art technologies, a key step in the translation of transdisciplinary knowledge into nutritional advice.

Sodium nitroprusside, a nitric oxide donor, fails to bypass the block of neuronal differentiation in PC12 cells imposed by a dominant negative Ras protein.

Nitric oxide (NO) is a mediator of a diverse array of inter- and intracellular signal transduction processes. The aim of the present study was to analyze its possible role as a second messenger in the process of neuronal differentiation of PC12 pheochromocytoma cells. Upon NGF treatment wildtype PC12 cells stop dividing and develop neurites. In contrast, a PC12 subclone (designated M-M17-26) expressing a dominant-negative mutant Ras protein keeps proliferating and fails to grow neurites after NGF treatment. Sodium nitroprusside (SNP), an NO donor, was found to induce the p53 protein and to inhibit proliferation of both PC12 and M-M17-26 cells, but failed to induce neuronal differentiation in these cell lines. Key signaling pathways (the ERK and Akt pathways) were also not affected by SNP treatment, and the phosphorylation of CREB transcription factor was only slightly stimulated. It is thus concluded from the results presented in this paper that NO is unable to activate signaling proteins acting downstream or independent of Ras that are required for neuronal differentiation.

Review of Label-Free Monitoring of Bacteria: From Challenging Practical Applications to Basic Research Perspectives.

Novel biosensors already provide a fast way to detect the adhesion of whole bacteria (or parts of them), biofilm formation, and the effect of antibiotics. Moreover, the detection sensitivities of recent sensor technologies are large enough to investigate molecular-scale biological processes. Usually, these measurements can be performed in real time without using labeling. Despite these excellent capabilities summarized in the present work, the application of novel, label-free sensor technologies in basic biological research is still rare; the literature is dominated by heuristic work, mostly monitoring the presence and amount of a given analyte. The aims of this review are (i) to give an overview of the present status of label-free biosensors in bacteria monitoring, and (ii) to summarize potential novel directions with biological relevancies to initiate future development. Optical, mechanical, and electrical sensing technologies are all discussed with their detailed capabilities in bacteria monitoring. In order to review potential future applications of the outlined techniques in bacteria research, we summarize the most important kinetic processes relevant to the adhesion and survival of bacterial cells. These processes are potential targets of kinetic investigations employing modern label-free technologies in order to reveal new fundamental aspects. Resistance to antibacterials and to other antimicrobial agents, the most important biological mechanisms in bacterial adhesion and strategies to control adhesion, as well as bacteria-mammalian host cell interactions are all discussed with key relevancies to the future development and applications of biosensors.

Rapid inactivation of SARS-CoV-2 by titanium dioxide surface coating.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission occurs via airborne droplets and surface contamination. Titanium dioxide (TiO 2) coating of surfaces is a promising infection control measure, though to date has not been tested against SARS-CoV-2. Methods: Virus stability was evaluated on TiO 2- and TiO 2-Ag (Ti:Ag atomic ratio 1:0.04)-coated 45 x 45 mm ceramic tiles. After coating the tiles were stored for 2-4 months before use. We tested the stability of both SARS-CoV-2 Spike pseudotyped virions based on a lentiviral system, as well as fully infectious SARS-CoV-2 virus. For the former, tile surfaces were inoculated with SARS-CoV-2 spike pseudotyped HIV-1 luciferase virus. At intervals virus was recovered from surfaces and target cells infected. For live virus,  after illuminating tiles for 0-300 min virus was recovered from surfaces followed by infection of Vero E6 cells. % of infected cells was determined by flow cytometry detecting SARS-CoV-2 nucleocapsid protein 24 h post-infection. Results: After 1 h illumination the pseudotyped viral titre was decreased by four orders of magnitude. There was no significant difference between the TiO 2 and TiO 2-Ag coatings. Light alone had no significant effect on viral viability. For live SARS-CoV-2, virus was already significantly inactivated on the TiO 2 surfaces after 20 min illumination. After 5 h no detectable active virus remained. Significantly, SARS-CoV-2 on the untreated surface was still fully infectious at 5 h post-addition of virus. Overall, tiles coated with TiO 2 120 days previously were able to inactivate SARS-CoV-2 under ambient indoor lighting with 87% reduction in titres at 1h and complete loss by 5h exposure. Conclusions: In the context of emerging viral variants with increased transmissibility, TiO 2 coatings could be an important tool in containing SARS-CoV-2, particularly in health care facilities where nosocomial infection rates are high.

Data evaluation for surface-sensitive label-free methods to obtain real-time kinetic and structural information of thin films: A practical review with related software packages.

Interfacial layers are important in a wide range of applications in biomedicine, biosensing, analytical chemistry and the maritime industries. Given the growing number of applications, analysis of such layers and understanding their behavior is becoming crucial. Label-free surface sensitive methods are excellent for monitoring the formation kinetics, structure and its evolution of thin layers, even at the nanoscale. In this paper, we review existing and commercially available label-free techniques and demonstrate how the experimentally obtained data can be utilized to extract kinetic and structural information during and after formation, and any subsequent adsorption/desorption processes. We outline techniques, some traditional and some novel, based on the principles of optical and mechanical transduction. Our special focus is the current possibilities of combining label-free methods, which is a powerful approach to extend the range of detected and deduced parameters. We summarize the most important theoretical considerations for obtaining reliable information from measurements taking place in liquid environments and, hence, with layers in a hydrated state. A thorough treamtmaent of the various kinetic and structural quantities obtained from evaluation of the raw label-free data are provided. Such quantities include layer thickness, refractive index, optical anisotropy (and molecular orientation derived therefrom), degree of hydration, viscoelasticity, as well as association and dissociation rate constants and occupied area of subsequently adsorbed species. To demonstrate the effect of variations in model conditions on the observed data, simulations of kinetic curves at various model settings are also included. Based on our own extensive experience with optical waveguide lightmode spectroscopy (OWLS) and the quartz crystal microbalance (QCM), we have developed dedicated software packages for data analysis, which are made available to the scientific community alongside this paper.

Natural Compounds as Target Biomolecules in Cellular Adhesion and Migration: From Biomolecular Stimulation to Label-Free Discovery and Bioactivity-Based Isolation.

Plants and fungi can be used for medical applications because of their accumulation of special bioactive metabolites. These substances might be beneficial to human health, exerting also anti-inflammatory and anticancer (antiproliferative) effects. We propose that they are mediated by influencing cellular adhesion and migration via various signaling pathways and by directly inactivating key cell adhesion surface receptor sites. The evidence for this proposition is reviewed (by summarizing the natural metabolites and their effects influencing cellular adhesion and migration), along with the classical measuring techniques used to gain such evidence. We systematize existing knowledge concerning the mechanisms of how natural metabolites affect adhesion and movement, and their role in gene expression as well. We conclude by highlighting the possibilities to screen natural compounds faster and more easily by applying new label-free methods, which also enable a far greater degree of quantification than the conventional methods used hitherto. We have systematically classified recent studies regarding the effects of natural compounds on cellular adhesion and movement, characterizing the active substances according to their organismal origin (plants, animals or fungi). Finally, we also summarize the results of recent studies and experiments on SARS-CoV-2 treatments by natural extracts affecting mainly the adhesion and entry of the virus.

Is it time for biocatalysis in fragment-based drug discovery?

The use of biocatalysts for fragment-based drug discovery has yet to be fully investigated, despite the promise enzymes hold for the synthesis of poly-functional, non-protected small molecules. Here we analyze products of the biocatalysis literature to demonstrate the potential for not only fragment generation, but also the enzyme-mediated elaboration of these fragments. Our analysis demonstrates that biocatalytic products can readily populate 3D chemical space, offering diverse catalytic approaches to help generate new, bioactive molecules.

Asymmetric synthesis of primary amines catalyzed by thermotolerant fungal reductive aminases.

Chiral primary amines are important intermediates in the synthesis of pharmaceutical compounds. Fungal reductive aminases (RedAms) are NADPH-dependent dehydrogenases that catalyse reductive amination of a range of ketones with short-chain primary amines supplied in an equimolar ratio to give corresponding secondary amines. Herein we describe structural and biochemical characterisation as well as synthetic applications of two RedAms from Neosartorya spp. (NfRedAm and NfisRedAm) that display a distinctive activity amongst fungal RedAms, namely a superior ability to use ammonia as the amine partner. Using these enzymes, we demonstrate the synthesis of a broad range of primary amines, with conversions up to >97% and excellent enantiomeric excess. Temperature dependent studies showed that these homologues also possess greater thermal stability compared to other enzymes within this family. Their synthetic applicability is further demonstrated by the production of several primary and secondary amines with turnover numbers (TN) up to 14 000 as well as continous flow reactions, obtaining chiral amines such as (R)-2-aminohexane in space time yields up to 8.1 g L-1 h-1. The remarkable features of NfRedAm and NfisRedAm highlight their potential for wider synthetic application as well as expanding the biocatalytic toolbox available for chiral amine synthesis.

Natural heterogeneous catalysis with immobilised oxidase biocatalysts.

The generation of immobilised oxidase biocatalysts allowing multifunctional oxidation of valuable chemicals using molecular oxygen is described. Engineered galactose oxidase (GOase) variants M1 and M3-5, an engineered choline oxidase (AcCO6) and monoamine oxidase (MAO-N D9) displayed long-term stability and reusability over several weeks when covalently attached on a solid support, outperforming their free counterparts in terms of stability (more than 20 fold), resistance to heat at 60 °C, and tolerance to neat organic solvents such as hexane and toluene. These robust heterogenous oxidation catalysts can be recovered after each reaction and be reused multiple times for the oxidation of different substrates.

Optical biosensors for cell adhesion.

Planar optical waveguides offer an ideal substratum for cells on which to reside. The materials from which the waveguides are made--high refractive index transparent dielectrics--correspond to the coatings of medical implants (e.g., the oxides of niobium, tantalum, and titanium) or the high molecular weight polymers used for culture flasks (e.g., polystyrene). The waveguides can furthermore be modified both chemically and morphologically while retaining their full capability for generating an evanescent optical field that has its greatest strength at the interface between the solid substratum and the liquid phase with which it is invariably in contact (i.e., the culture medium bathing the cells), decaying exponentially perpendicular to the interface at a rate controllable by varying the material parameters of the waveguide. Analysis of the perturbation of the evanescent field by the presence of living cells within it enables their size, number density, shape, refractive index (linked to their constitution) and so forth to be determined, the number of parameters depending on the number of waveguide lightmodes analyzed. No labeling of any kind is necessary, and convenient measurement setups are fully compatible with maintaining the cells in their usual environment. If the temporal evolution of the perturbation is analyzed, even more information can be obtained, such as the amount of material (microexudate) secreted by the cell while residing on the surface. Separation of parallel effects simultaneously contributing to the perturbation of the evanescent field can be accomplished by analysis of coupling peak shape when a grating coupler is used to measure the propagation constants of the waveguide lightmodes.

Optical monitoring of stem cell-substratum interactions.

Modulation of the coupling of light into a waveguide via a grating, together with a novel approach to analyzing the data, is used to investigate the attachment of human embryonal carcinoma stem cells to three substrata: silica-titania (representative of artificial implants); poly-lysine (a commonly used laboratory cell culture substrate); and mucin (the coating of the mucosae). By considering both in-coupling peak width and position, the secretion of microexudate by the cells, the formation of filopodia, and the overall change in their shape (spreading) can be distinguished. This cannot be achieved by the conventional microscopic imaging approach. Moreover, we obtain the kinetics of these processes with excellent time resolution.

Self-assembly of rodlike receptors from bulk solution.

Optical waveguide lightmode spectroscopy has been used to observe the deposition of bacterial flagellar filaments of mean length 350 nm from bulk solution onto a smooth planar substratum, chemically modified to covalently bind the flagellar filaments on contact. At the highest practicable bulk concentration, the filaments follow the theoretically predicted kinetics of random sequential addition of highly elongated rigid rods to the substratum, but addition terminates with the rods almost perpendicular to the substratum. Rod-rod correlations in the bulk anomalously accelerate the rate of arrival of the filaments at the surface of the substratum, relative to spheres. At lower concentrations, this effect is absent, and the rods have time to order themselves on the substratum, forming a two-dimensional array.

Staphylococcus aureus resists UVA at low irradiance but succumbs in the presence of TiO2 photocatalytic coatings.

The aim of this study was to evaluate the bactericidal effect of reactive oxygen species (ROS) generated upon irradiation of photocatalytic TiO2 surface coatings using low levels of UVA and the consequent killing of Staphylococcus aureus. The role of intracellular enzymes catalase and superoxide dismutase in protecting the bacteria was investigated using mutant strains. Differences were observed in the intracellular oxidative stress response and viability of S. aureus upon exposure to UVA; these were found to be dependent on the level of irradiance and not the total UVA dose. The wild type bacteria were able to survive almost indefinitely in the absence of the coatings at low UVA irradiance (LI, 1 mW/cm2), whereas in the presence of TiO2 coatings, no viable bacteria were measurable after 24 h of exposure. At LI, the lethality of the photocatalytic effect due to the TiO2 surface coatings was correlated with high intracellular oxidative stress levels. The wild type strain was found to be more resistant to UVA at HI compared with an identical dose at LI in the presence of the TiO2 coatings. The UVA-irradiated titania operates by a "stealth" mechanism at low UVA irradiance, generating low levels of extracellular lethal ROS against which the bacteria are defenceless because the low light level fails to induce the oxidative stress defence mechanism of the bacteria. These results are encouraging for the deployment of antibacterial titania surface coatings wherever it is desirable to reduce the environmental bacterial burden under typical indoor lighting conditions.

Structural hysteresis and hierarchy in adsorbed glycoproteins.

The adsorption and desorption of the giant heavily glycosylated protein mucin from solutions of different bulk concentrations have been followed at the nanometer scale using high resolution molecular microscopy based on optical waveguide lightmode spectroscopy. Modeling the layer as a uniaxial thin film allowed the in situ determination of adsorbed mass, mean layer thickness, and structural anisotropy. These parameters manifest highly significant adsorption-desorption hysteresis, indicating at least two dominant glycoprotein conformational types (i.e., molecular states, structurally and kinetically distinguishable). One of them is proposed to be a conformationally extended state that engenders uniaxial symmetry and dominates layers generated from low bulk concentrations. The revealed structure and the mechanism by which it is formed are postulated to be a general feature of the self-assembly of large glycoproteins. We expect that, inter alia, this knowledge will be relevant for understanding the extraordinary effectiveness of mucin thin films as boundary lubricants.

How Does a Photocatalytic Antimicrobial Coating Affect Environmental Bioburden in Hospitals?

BACKGROUND The healthcare environment is recognized as a source for healthcare-acquired infection. Because cleaning practices are often erratic and always intermittent, we hypothesize that continuously antimicrobial surfaces offer superior control of surface bioburden. OBJECTIVE To evaluate the impact of a photocatalytic antimicrobial coating at near-patient, high-touch sites in a hospital ward. SETTING The study took place in 2 acute-care wards in a large acute-care hospital. METHODS A titanium dioxide-based photocatalytic coating was sprayed onto 6 surfaces in a 4-bed bay in a ward and compared under normal illumination against the same surfaces in an untreated ward: right and left bed rails, bed control, bedside locker, overbed table, and bed footboard. Using standardized methods, the overall microbial burden and presence of an indicator pathogen (Staphylococcus aureus) were assessed biweekly for 12 weeks. RESULTS Treated surfaces demonstrated significantly lower microbial burden than control sites, and the difference increased between treated and untreated surfaces during the study. Hygiene failures (>2.5 colony-forming units [CFU]/cm2) increased 2.6% per day for control surfaces (odds ratio [OR], 1.026; 95% confidence interval [CI], 1.009-1.043; P=.003) but declined 2.5% per day for treated surfaces (OR, 0.95; 95% CI, 0.925-0.977; P<.001). We detected no significant difference between coated and control surfaces regarding S. aureus contamination. CONCLUSION Photocatalytic coatings reduced the bioburden of high-risk surfaces in the healthcare environment. Treated surfaces became steadily cleaner, while untreated surfaces accumulated bioburden. This evaluation encourages a larger-scale investigation to ascertain whether the observed environmental amelioration has an effect on healthcare-acquired infection. Infect Control Hosp Epidemiol 2018;39:398-404.

Imine Reductases, Reductive Aminases, and Amine Oxidases for the Synthesis of Chiral Amines: Discovery, Characterization, and Synthetic Applications.

Synthesis of the chiral amine moiety is a key challenge for synthetic organic chemistry due to its prevalence in many biologically active molecules. Imine reductase and amine oxidase enzymes have enabled the biocatalytic synthesis of a host of chiral amine compounds. In this chapter, procedures for the synthesis of chiral amines using imine reductases (IREDs), the recently discovered IRED homologues reductive aminases, and amine oxidases (AOs) are described. Amine oxidases have been the subject of mutagenesis approaches for improvement of substrate scope. The high-throughput screening method for determining active variants in amine oxidase libraries is illustrated. Finally, in an approach which takes inspiration from nature, many enzymes can be combined with each other in cascade reactions. The incorporation of imine reductase and monoamine oxidase biocatalysts into several cascade reactions, both in vitro and in vivo (where the approach moves toward synthetic biology), is reported.