To keep their functional properties, it is crucial that protein aggregates maintain their structure after spray drying and that the powders can be fully rehydrated. In this study, microgels and fractal aggregates were prepared by heating a mixture of milk serum protein concentrate and lactose (40/60; %, w/w) at 85 °C for 15 min by varying the pH. Various fractions of native proteins were added to the systems prior to spray drying. This study showed that microgels and fractal aggregates kept their structure after spray drying and reconstitution. The particle morphology could be correlated to the stiffness of the interface of the feed droplet. The forced imbibition rate showed a negative correlation with increasing amount of aggregated proteins in the powders that seems to be a result of denatured/aggregated proteins present at the surface. These findings are of importance for the formulation of spray-dried powders with improved rehydration characteristics. © 2020 The Author(s)

The LignoJet project aimed to achieve an intermediate lignin-oil product miscible with fossil feedstock and with a significantly reduced oxygen content. A technical concept for production has been studied that involves combined catalysed depolymerisation and hydrodeoxygenation, so called hydrogenolytic depolymerisation, of kraft lignin. Kraft lignin was separated through membrane ultrafiltration from softwood and eucalyptus black liquor followed by precipitation through LignoBoost technology. A difference in lignin properties was observed between ultrafiltration of softwood and eucalyptus black liquor through 15 and 150kDa ceramic membranes. Lignin-oils with similar oxygen content were produced regardless of origin and fractionation technique. A lignin-oil with favourable properties as precursor for refinery integration for jet fuel production as produced in small-scale batch experiments using nickel-based catalyst. Stable pumpable oils with melting point of less than 25-50 deg C and with 20-30% lower oxygen content and aromatic content were obtained that would be suitable as jet fuel precursors. The estimated production cost was found to be competitive with that of other liquid biofuels, while additional revenues could potentially be achieved by also producing chemical and materials from suitable fractions of the lignin-oil.

Metal oxide nanoparticles are widely used in both consumer products and medical applications, but the knowledge regarding exposure-related health effects is limited. However, it is challenging to investigate nanoparticle interaction processes with biological systems. The overall aim of this project was to improve the possibility to predict exposure-related health effects of metal oxide nanoparticles through interdisciplinary collaboration by combining workflows from the pharmaceutical industry, nanomaterial sciences, and occupational medicine. Specific aims were to investigate nanoparticle-protein interactions and possible adverse immune reactions. Four different metal oxide nanoparticles; CeOx nanocrystals with 5% or 14% Gd, Co3O4, and Fe2O3, were characterized by dynamic light scattering and high-resolution transmission electron microscopy. Nanoparticle-binding proteins were identified and screened for HLA-binding peptides in silico. Monocyte interaction with nanoparticle–protein complexes was assessed in vitro. Herein, for the first time, immunogenic properties of nanoparticle-binding proteins have been characterized. The present study indicates that especially Co3O4-protein complexes can induce both ‘danger signals’, verified by the production of inflammatory cytokines and simultaneously bind autologous proteins, which can be presented as immunogenic epitopes by MHC class II. The clinical relevance of these findings should be further evaluated to investigate the role of metal oxide nanoparticles in the development of autoimmune disease. The general workflow identified experimental difficulties, such as nanoparticle aggregate formation and a lack of protein-free buffers suitable for particle characterization, protein analyses, as well as for cell studies. This confirms the importance of future interdisciplinary collaborations. © 2021 The Author(s). 

Freeze-drying is the preferred method to manufacture proteins in their solid state thus the understanding of the relationship between cycle parameters and cake properties remains of great interest. The present study aims to investigate the influence of the freezing conditions in the material properties at different layers throughout the dried structure, in the presence and absence of a protein. Placebo and protein formulations were dried applying different cooling rates: slow, fast and fast cooling with annealing. Non-uniform visual cake appearance, different pore sizes and endothermic events for release of structural water were observed throughout the cake at different freezing rates indicating heterogeneous properties of the dried material likely due to heating gradients during freezing. However, annealing increased the crystallinity and eliminated material inhomogeneities across the cake. The crystalline phase was mainly comprised of δ and hemihydrate mannitol (MHH) distributed at different ratios and influenced by the presence of the protein. The undesired formation of MHH is associated to currently used freezing temperatures or amorphous to crystalline material ratios. Thus, the correlation between the freezing step parameters and resulting material structure is a step forward to provide a better understanding of the freeze-dried cake formation and product quality improvement. 

Objectives: Novel therapeutics are urgently required for the treatment of carbapenem-resistant Acinetobacter baumannii (CRAB) causing critical infections with high mortality. Here we assessed the therapeutic potential of the clinical-stage drug candidate EBL-1003 (crystalline free base of apramycin) in the treatment of CRAB lung infections. Methods: The genotypic and phenotypic susceptibility of CRAB clinical isolates to aminoglycosides and colistin was assessed by database mining and broth microdilution. The therapeutic potential was assessed by target attainment simulations on the basis of time–kill kinetics, a murine lung infection model, comparative pharmacokinetic analysis in plasma, epithelial lining fluid (ELF) and lung tissue, and pharmacokinetic/pharmacodynamic (PKPD) modelling. Results: Resistance gene annotations of 5451 CRAB genomes deposited in the National Database of Antibiotic Resistant Organisms (NDARO) suggested >99.9% of genotypic susceptibility to apramycin. Low susceptibility to standard-of-care aminoglycosides and high susceptibility to EBL-1003 were confirmed by antimicrobial susceptibility testing of 100 A. baumannii isolates. Time–kill experiments and a mouse lung infection model with the extremely drug-resistant CRAB strain AR Bank #0282 resulted in rapid 4-log CFU reduction both in vitro and in vivo. A single dose of 125 mg/kg EBL-1003 in CRAB-infected mice resulted in an AUC of 339 h × μg/mL in plasma and 299 h × μg/mL in ELF, suggesting a lung penetration of 88%. PKPD simulations suggested a previously predicted dose of 30 mg/kg in patients (creatinine clearance (CLCr) = 80 mL/min) to result in >99% probability of –2 log target attainment for MICs up to 16 μg/mL. Conclusions: This study provides proof of concept for the efficacy of EBL-1003 in the treatment of CRAB lung infections. Broad in vitro coverage, rapid killing, potent in vivo efficacy, and a high probability of target attainment render EBL-1003 a strong therapeutic candidate for a priority pathogen for which treatment options are very limited. © 2020 The Author(s)

Background: The clinical-stage drug candidate EBL-1003 (apramycin) represents a distinct new subclass of aminoglycoside antibiotics for the treatment of drug-resistant infections. It has demonstrated best-in-class coverage of resistant isolates, and preclinical efficacy in lung infection models. However, preclinical evidence for its utility in other disease indications has yet to be provided. Here we studied the therapeutic potential of EBL-1003 in the treatment of complicated urinary tract infection and acute pyelonephritis (cUTI/AP). Methods: A combination of data-base mining, antimicrobial susceptibility testing, time-kill experiments, and four murine infection models was used in a comprehensive assessment of the microbiological coverage and efficacy of EBL-1003 against Gram-negative uropathogens. The pharmacokinetics and renal toxicology of EBL-1003 in rats was studied to assess the therapeutic window of EBL-1003 in the treatment of cUTI/AP. Findings: EBL-1003 demonstrated broad-spectrum activity and rapid multi-log CFU reduction against a phenotypic variety of bacterial uropathogens including aminoglycoside-resistant clinical isolates. The basicity of amines in the apramycin molecule suggested a higher increase in positive charge at urinary pH when compared to gentamicin or amikacin, resulting in sustained drug uptake and bactericidal activity, and consequently in potent efficacy in mouse infection models. Renal pharmacokinetics, biomarkers for toxicity, and kidney histopathology in adult rats all indicated a significantly lower nephrotoxicity of EBL-1003 than of gentamicin. Interpretation: This study provides preclinical proof-of-concept for the efficacy of EBL-1003 in cUTI/AP. Similar efficacy but lower nephrotoxicity of EBL-1003 in comparison to gentamicin may thus translate into a higher safety margin and a wider therapeutic window in the treatment of cUTI/API. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section. © 2021 The Author(s)

Liquefaction of lignocellulosic biomass through fast pyrolysis, to yield fast pyrolysis bio-oil (FPBO), is a technique that has been extensively researched in the quest for finding alternatives to fossil feedstocks to produce fuels, chemicals, etc. Properties such as high oxygen content, acidity, and poor storage stability greatly limit the direct use of this bio-oil. Furthermore, high coking tendencies make upgrading of the FPBO by hydrodeoxygenation in fixed-bed bed hydrotreaters challenging due to plugging and catalyst deactivation. This study investigates a novel two-step hydroprocessing concept; a continuous slurry-based process using a dispersed NiMo-catalyst, followed by a fixed bed process using a supported NiMo-catalyst. The oil product from the slurry-process, having a reduced oxygen content (15 wt%) compared to the FPBO and a comparatively low coking tendency (TGA residue of 1.4 wt%), was successfully processed in the downstream fixed bed process for 58 h without any noticeable decrease in catalyst activity, or increase in pressure drop. The overall process resulted in a 29 wt% yield of deoxygenated oil product (0.5 wt% oxygen) from FPBO with an overall carbon recovery of 68%.

The freeze-drying of proteins, along with excipients, offers a solution for increasing the shelf-life of protein pharmaceuticals. Using differential scanning calorimetry, thermogravimetric analysis, sorption calorimetry, and synchrotron small-angle X-ray scattering (SAXS), we have characterized the properties at low (re)hydration levels of the protein lysozyme, which was freeze-dried together with the excipient sucrose. We observe that the residual moisture content in these samples increases with the addition of lysozyme. This results from an increase in equilibrium water content with lysozyme concentration at constant water activity. Furthermore, we also observed an increase in the glass transition temperature (Tg) of the mixtures with increasing lysozyme concentration. Analysis of the heat capacity step of the mixtures indicates that lysozyme does not participate in the glass transition of the sucrose matrix; as a result, the observed increase in the Tg of the mixtures is the consequence of the confinement of the amorphous sucrose domains in the interstitial space between the lysozyme molecules. Sorption calorimetry experiments demonstrate that the hydration behavior of this formulation is similar to that of the pure amorphous sucrose, while the presence of lysozyme only shifts the sucrose transitions. SAXS analysis of amorphous lysozyme-sucrose mixtures and unfolding of lysozyme in this environment show that prior to unfolding, the size and shape of lysozyme in a solid sucrose matrix are consistent with its native state in an aqueous solution. The results obtained from our study will provide a better understanding of the low hydration behavior of protein-excipient mixtures and support the improved formulation of biologics. © 2023 The Authors. 

The mechanisms of glass transitions and the behavior of small solute molecules in a glassy matrix are some of the most important topics of modern thermodynamics. Water plays an important role in the physical and chemical stability of lyophilized biologics formulations, in which glassy carbohydrates act as cryoprotectants and stabilizers. In this study, sorption calorimetry was used for simultaneous measurements of water activity and the enthalpy of water sorption by amorphous sucrose, trehalose and maltodextrins. Moreover, the heat capacity of these carbohydrates in mixtures with water was measured by DSC in a broad range of water contents. The hydration enthalpies of glassy sucrose, trehalose and maltodextrins are exothermic, and the enthalpy change of water-induced isothermal glass transitions is higher for small molecules. The partial molar enthalpy of mixing of water in slow experiments is about -18 kJ mol-1, but less exothermic in the case of small molecules at fast hydration scan rates. By measuring the heat capacities of disaccharides and maltodextrins as a function of water content, we separated the contributions of carbohydrates and water to the total heat capacities of the mixtures. The combination of these data allowed testing of thermodynamic models describing the hydration-induced glass transitions. The heat capacity changes calculated by the fitting of the hydration enthalpy data for disaccharides are in good agreement with the heat capacity data obtained by DSC, while for maltodextrins, the effect of sub-Tg transitions should be taken into account. Combining the data obtained by different techniques, we found a distinct difference in the behavior of water in glassy polymers compared to that in glassy disaccharides. By understanding the behavior of water in glassy carbohydrates, these results can be used to improve the design of freeze-dried formulations of proteins and probiotics.

The ice crystallization and melting in systems where the equilibrium state is difficult to reach is one of the growing areas in pharmaceutical freeze-drying research. The quality of the final freeze-dried product depends on the parameters of the cooling step, which affect the ice nucleation and growth. In this paper, we present a DSC study of ice crystallization and melting in a sucrose-water system. Using two different types of thermal cycles, we examine the influence of cooling and heating rates on the thermal behavior of sucrose-water solutions with water contents between 50 and 100 wt%. The DSC results show that low cooling rates provide crystallization at higher temperatures and lead to lower amount of non-freezing water. Consequently, the glass transition and ice melting properties observed upon heating depend on the cooling conditions in the preceding step. Based on the experimental results, we investigate the reasons for the existence of the two steps on DSC heating curves in sucrose-water systems: the glass transition step and the onset of ice melting. We show that diffusion of water can be the limiting factor for ice growth and melting in the sucrose-water system when the amorphous phase is in a liquid state. In particular, when the diffusion coefficient drops below 10−14 m2/sec, the ice crystals growth or melting becomes strongly suppressed even above the glass transition temperature. Understanding the diffusion limitations in the sucrose-water system can be used for the optimization of the freeze-drying protocols for proteins and probiotics. © 2022 The Authors

In this paper, we demonstrate the coupling of synchrotron small angle X-ray scattering (SAXS) to asymmetrical flow-field flow fractionation (AF4) for protein characterization. To the best of our knowledge, this is the first time AF4 is successfully coupled to a synchrotron for on-line measurements on proteins. This coupling has potentially high impact, as it opens the possibility to characterize individual constituents of sensitive and/or complex samples, not suited for separation using other techniques, and for low electron density samples where high X-ray flux is required, e.g., biomolecules and biologics. AF4 fractionates complex samples in native or close to native environment, with low shear forces and system surface area. Many orders of magnitude in size can be fractionated in one measurement, without having to reconfigure the experimental setup. We report AF4 fractionations with correlated UV and statistically adequate SAXS data of bovine serum albumin and a monoclonal antibody and evaluate SAXS data recorded for the two protein systems. Graphical Abstract: [Figure not available: see fulltext.] © 2023, The Author(s).

A well-known Squaramide-Cinchonine organocatalyst was immobilized in a controlled way onto three types of commercial porous glass beads EziGTM (EziG OPAL, EziG Amber and EziG Coral) and applied in asymmetric Michael reactions. The performance of the immobilized catalysts was evaluated under batch and continuous flow conditions showing promising results in both approaches. In batch reactions, 0.8 and 1.6 mol% of the immobilized cinchonine-squaramide provided the products with excellent yields (up to 99%) and enantioselectivities (up to 99% ee). These excellent results were also verified in the case of continuous flow reactions, where also 0.8 and 1.6 mol% of the catalyst immobilized onto the glass beads afforded the product with extraordinary yields (up to 99%) and very high enantioselectivities (up to 97% ee). The immobilized catalysts could be recycled (up to 7 cycles) using both approaches. 

Transition-metal-catalyzed asymmetric reactions have been a powerful tool in organic synthesis for many years. The design of chiral ligands with the right configuration is fundamental to induce high regio- and stereoselectivity to catalytic reactions and to achieve high turnover numbers and high yields. A challenge is the control of prochiral centers with similar electronic properties in a similar steric environment within the same molecule. Over the last 10 years, a range of novel rigid C-stereogenic chiral phosphine ligands has been developed and successfully applied in various types of asymmetric transformations. Many of these ligands are of a di-, tri-, or multidentate nature. The purpose of this Perspective is to highlight recent synthetic achievements (since 2010) with spiro-phosphines and other rigid phosphines and discuss some mechanistic aspects of the catalytic reactions. 

This article evaluates the current gaps around the impact of post-manufacturing processes on the product qualities of protein-based biologics, with a focus on user centricity. It includes the evaluation of the regulatory guidance available, describes a collection of scientific literature and case studies to showcase the impact of post-manufacturing stresses on product and dosing solution quality. It also outlines the complexity of clinical handling and the need for communication, and alignment between drug providers, healthcare professionals, users, and patients. Regulatory agencies provide clear expectations for drug manufacturing processes, however, guidance supporting post-product manufacturing handling is less defined and often misaligned. This is problematic as the pharmaceutical products experience numerous stresses and processes which can potentially impact drug quality, safety and efficacy. This article aims to stimulate discussion amongst pharmaceutical developers, health care providers, device manufacturers, and public researchers to improve these processes. Patients and caregivers’ awareness can be achieved by providing relevant educational material on pharmaceutical product handling. 

Deep eutectic solvents (DESs) are a mixture of two or more components, and at a particular composition, they become liquids at room temperature. When the compounds that constitute the DESs are primary metabolites namely, amino acids, organic acids, sugars, or choline derivatives, the DESs are called natural deep eutectic solvents (NADESs). NADESs fully represent green chemistry principles. These solvents are highly welcome, as they are obtained from renewable resources, and gratifyingly are biodegradable and biocompatible. They are an alternative to room-temperature ionic liquids (RTILs). From the pharmaceutical industry’s point of view, they are highly desirable, but they unfortunately have been rarely used despite their enormous potential. In this review, we look at their impact on the asymmetric catalytic synthesis of key target molecules via metal-based catalysis, biocatalysis, and organocatalysis. In many cases, the NADESs that have been used are chiral and can even promote enantioselective reactions; this crucial and very exciting aspect is also discussed and analyzed. © 2024 by the authors.

This article is a history of an industrial-academic partnership that started almost two decades ago and details the evolution of a relationship between a small academic research group and a spin-out company located in Portugal. Their activities have ranged from the development of new metal-based catalytic systems for asymmetric epoxidations, allylic alkylations, and arylations to the development of novel cinchona-based organocatalysts for asymmetric hydrosilylations and Michael additions. Current common interests are centered on the development of novel chiral Natural Deep Eutectic Solvent systems, which they are investigating in different types of reaction systems.

Photocatalytic nanoparticles offer antimicrobial effects under illumination due to the formation of reactive oxygen species (ROS), capable of degrading bacterial membranes. ROS may, however, also degrade human cell membranes and trigger toxicity. Since antimicrobial peptides (AMPs) may display excellent selectivity between human cells and bacteria, these may offer opportunities to effectively “target” nanoparticles to bacterial membranes for increased selectivity. Investigating this, photocatalytic TiO2 nanoparticles (NPs) are coated with the AMP LL-37, and ROS generation is found by C11-BODIPY to be essentially unaffected after AMP coating. Furthermore, peptide-coated TiO2 NPs retain their positive ζ-potential also after 1–2 h of UV illumination, showing peptide degradation to be sufficiently limited to allow peptide-mediated targeting. In line with this, quartz crystal microbalance measurements show peptide coating to promote membrane binding of TiO2 NPs, particularly so for bacteria-like anionic and cholesterol-void membranes. As a result, membrane degradation during illumination is strongly promoted for such membranes, but not so for mammalian-like membranes. The mechanisms of these effects are elucidated by neutron reflectometry. Analogously, LL-37 coating promoted membrane rupture by TiO2 NPs for Gram-negative and Gram-positive bacteria, but not for human monocytes. These findings demonstrate that AMP coating may selectively boost the antimicrobial effects of photocatalytic NPs. 

The choriogenin H – EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17β-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts. 

Protein-based drugs often require targeted drug delivery for optimal therapy. A successful strategy to increase the circulation time of the protein in the blood is to link the therapeutic protein with an albumin-binding domain. In this work, we characterized such a protein-based drug, GA-Z. Using asymmetrical flow field-flow fractionation coupled with multi-angle light scattering (AF4-MALS) we investigated the GA-Z monomer-dimer equilibrium as well as the molar binding ratio of GA-Z to HSA. Using small angle X-ray scattering, we studied the structure of GA-Z as well as the complex between GA-Z and HSA. The results show that GA-Z is predominantly dimeric in solution at pH 7 and that it binds to monomeric as well as dimeric HSA. Furthermore, GA-Z binds to HSA both as a monomer and a dimer, and thus, it can be expected to stay bound also upon dilution following injection in the blood stream. The results from SAXS and binding studies indicate that the GA-Z dimer is formed between two target domains (Z-domains). The results also indicate that the binding of GA-Z to HSA does not affect the ratio between HSA dimers and monomers, and that no higher order oligomers of the complex are seen other than those containing dimers of GA-Z and dimers of HSA.

There is a strong need for innovative and efficient drug delivery systems for ocular therapy development. However, testing intravitreal drug delivery systems without using live animals is challenging. Ex vivo animal models offer an interesting alternative. We analyzed the potential of using fresh porcine eyes obtained from the local slaughterhouse as a model for testing the intravitreal biodistribution and retention of liposomes with or without polyethylene glycol (PEG) conjugation and with different surface charges. The histology of the eyes was analyzed to localize the liposomes, and it was found that liposomes with PEG absorbed rapidly on the retina (within 1 h), with positively charged and PEG-coated liposomes being retained for at least 24 h. In parallel, fluorophotometry was employed on intact eyes, to determine the pharmacokinetics of the fluorophore calcein, as a substitute for a small hydrophilic therapeutic compound. We found a 4.5-fold increase in the vitreous half-life of calcein loaded in liposomes, compared with the free solution. Retinal toxicity was addressed using murine-derived retinal explant cultures. Liposomes were non-toxic up to 500 µg/mL. Toxicity was observed at 5 mg/mL for anionic and cationic liposomes, with 2-fold and 2.5-fold increased photoreceptor cell death, respectively. Overall, we could show that important ocular drug delivery considerations such as pharmacokinetics and biodistribution can be estimated in ex vivo porcine eyes, and may guide subsequent in vivo experiments.

Despite several promising candidates, there is a paucity of drug treatments available for patients suffering from retinal diseases. An important reason for this is the lack of suitable delivery systems that can achieve sufficiently high drug uptake in the retina and its photoreceptors. A promising and versatile method for drug delivery to specific cell types involves transporter-targeted liposomes, i.e., liposomes surface-coated with substrates for transporter proteins highly expressed on the target cell. We identified strong lactate transporter (monocarboxylate transporter, MCT) expression on photoreceptors as a potential target for drug delivery vehicles. To evaluate MCT suitability for drug targeting, we used PEG-coated liposomes and conjugated these with different monocarboxylates, including lactate, pyruvate, and cysteine. Monocarboxylate-conjugated and dye-loaded liposomes were tested on both human-derived cell-lines and murine retinal explant cultures. We found that liposomes conjugated with pyruvate consistently displayed higher cell uptake than unconjugated liposomes or liposomes conjugated with lactate or cysteine. Pharmacological inhibition of MCT1 and MCT2 reduced internalization, suggesting an MCT-dependent uptake mechanism. Notably, pyruvate-conjugated liposomes loaded with the drug candidate CN04 reduced photoreceptor cell death in the murine rd1 retinal degeneration model while free drug solutions could not achieve the same therapeutic effect. Our study thus highlights pyruvate-conjugated liposomes as a promising system for drug delivery to retinal photoreceptors, as well as other neuronal cell types displaying high expression of MCT-type proteins. © 2023 The Authors

Recently, cGMP analogues have been investigated for the treatment of inherited retinal degenerations (IRD) using intravitreal injections. However, higher vitreous elimination rates limit the possibility to treat the retina with small molecule drugs. Here, we investigated the potential of lipid nanocapsules (LNCs) as vehicles to reduce clearance and prolong the delivery of cGMP analogue, CN03 to the retinal photoreceptors. Initially LNCs were investigated for both topical/periocular and intravitreal administration routes. While LNC-mediated drug permeation through the cornea proved to be too low for clinical applications, intravitreal application showed significant promise. Intravitreally administered LNCs containing fluorescent tracer in ex vivo porcine eyes showed complete intravitreal dispersal within 24 h. Ocular bio-distribution on histological sections showed that around 10 % of the LNCs had reached the retina, and 40 % accumulated in the ciliary body. For comparison, we used fluorescently labeled liposomes and these showed a different intraocular distribution with 48 % accumulated in the retina, and almost none were in the ciliary body. LNCs were then tested in retinal explants prepared from wild-type (WT) and rd1 mouse. In WT retina LNCs showed no significant toxic effects up to a concentration of 5 mg/mL. In rd1 retina, the LNC/CN03 formulation protected rd1 photoreceptors with similar efficacy to that of free CN03, demonstrating the usefulness of LNC/CN03 formulation in the treatment of IRD. Overall, our results indicate the suitability of LNCs for intraocular administration and drug delivery to both the retina and the ciliary body. © 2023 The Author(s)

Calcium carbonate, particularly in the form of calcite, is an abundant mineral widely used in both human-made products and biological systems. The calcite surface possesses a high surface energy, making it susceptible to the adsorption of organic contaminants. Moreover, the surface is also reactive towards a range of chemicals, including water. Consequently, studying and maintaining a clean and stable calcite surface is only possible under ultrahigh vacuum conditions and for limited amounts of time. When exposed to air or solution, the calcite surface undergoes rapid transformations, demanding a comprehensive understanding of the properties of calcite surfaces in different environments. Similarly, attention must also be directed towards the kinetics of changes, whether induced by fluctuating environments or at constant condition. All these aspects are encompassed in the expression “dynamic nature”, and are of crucial importance in the context of the diverse applications of calcite. In many instances, the calcite surface is modified by adsorption of fatty acids to impart a desired nonpolar character. Although the binding between carboxylic acid groups and calcite surfaces is strong, the fatty acid layer used for surface modification undergoes significant alterations when exposed to water vapour and liquid water droplets. Therefore, it is also crucial to understand the dynamic nature of the adsorbed layer. This review article provides a comprehensive overview of the current understanding of both the dynamics of the calcite surface as well as when modified by fatty acid surface treatments. Calcium carbonate, particularly in the form of calcite and surface modified calcite, is an abundant mineral widely used in both human-made and biological systems.

A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis, Enterococcus faecium and multidrug resistant (MDR) Staphylococcus aureus strains [best compound minimal inhibitory concentrations (MICs): range, <0.03125-0.25 μg/mL] and against the Gram-negatives Acinetobacter baumannii and Klebsiella pneumoniae (best compound MICs: range, 1-4 μg/mL). Lead compound 7a was identified with favorable solubility and plasma protein binding, good metabolic stability, selectivity for bacterial topoisomerases, and no toxicity issues. The crystal structure of 7a in complex with Pseudomonas aeruginosa GyrB24 revealed its binding mode at the ATP-binding site. Expanded profiling of 7a and 7h showed potent antibacterial activity against over 100 MDR and non-MDR strains of A. baumannii and several other Gram-positive and Gram-negative strains. Ultimately, in vivo efficacy of 7a in a mouse model of vancomycin-intermediate S. aureus thigh infection was also demonstrated. © 2023 The Authors. 

To find means of controlling the size and density of particles intended for inhalation the relationship between droplet and particle size during spray drying was investigated. Lactose solutions were atomized with a two-fluid nozzle and dried in a laboratory spray drier. The effects of nozzle orifice diameter, atomization airflow and feed concentration on droplet and particle size were examined. Mass median diameter of both droplets and particles were analyzed with laser diffraction. In addition, scanning electron microscopy and transmission electron microscopy were used for studies of particle shape and morphology. It was demonstrated that nozzle orifice diameter and airflow, but not feed concentration controlled the droplet size during atomization. Increasing droplet size increased particle size but the effect was also influenced by feed concentration. Particles from solutions of a low concentration (1% w/w) were smaller than those from higher concentrations (5-20% w/w). This may be partly explained by lower yields at higher feed concentrations, but may also be related to differences in drying rate. Spray-dried lactose solutions formed hollow particles, and it was suggested that the shell thickness of the particles increased with increasing feed concentration

X-ray photoelectron spectroscopy (XPS) was employed to quantify adsorption of polyelectrolytes from aqueous solutions of low ionic strength onto mica, glass, and silica. Silica surfaces were conditioned in base or in acid media as last pre-treatment step (silica-base last or silica-acid last, respectively). Consistency in the determined adsorbed amount, Γ, was obtained independent of the choice of XPS mode and with the two quantification approaches used in the data evaluation. Under the same adsorption conditions, the adsorbed amount, Γ, varied as Γmica &gt; Γsilica-base last ≈ Γglass &gt; Γsilica-acid last. In addition, the adsorbed amount increased with decreasing polyelectrolyte charge density (100% to 1% of segments being charged) for all substrates. Large adsorbed amount was measured for low-charge density polyelectrolytes, but the number of charged segments per square nanometer was low due to steric repulsion between polyelectrolyte chains that limited the adsorption. The adsorbed amount of highly charged polyelectrolytes was controlled by electrostatic interactions and thus limited to that needed to neutralize the substrate surface charge density. For silica, the adsorbed amount depended on the cleaning method, suggesting that this process influenced surface concentration and fraction of different silanol groups. Our results demonstrate that for silica, a higher density and/or more acidic silanol groups are formed using base, rather than acid, treatment in the last step. © 2022 The Authors.

The introduction of the Green Chemistry Principles in the late 1990s formed the basis for a transition to a greener environment. These Principles have become an integral part in the work on designing chemical processes, especially for large-scale manufacture. The ultimate target is the achievement of a sustainable production method allowing hundreds of tons of valuable materials to be prepared. For this purpose, a holistic view must be applied to the elements constituting a fullyfledged process encompassing layout of the synthetic route, defining starting materials and their origin, output of product and quality features, quantity of effluent streams and waste, recovery and recycling of chemicals involved, and energy consumption. These parameters form a complex matrix where the individual components are in a complicated relationshipwith each other. This short review addresses these issues and the benefits of life-cycle assessment and metrics commonly used to measure the performance of chemical manufacturing all from a pharmaceutical industry perspective as experienced by the author. 

What factors will play a role in the decision process when looking at the reasons for certain reactions being adopted for large-scale industrial applications? An examination reveals that there are several parameters that have the power to be influential in this regard, either on their own or in combination, all dependent on the circumstances. Seen from a pharmaceutical manufacturing point of view, only certain structural motifs are of interest for in-depth investigations and, hence, are in demand of synthetic methodologies allowing the preparation of quantities beyond what sensibly is achieved by ordinary laboratory procedures. This inevitably leads to many reactions and even reaction types to fall by the wayside. A further criterion to fulfill is that a given synthesis must perform in a robust and consistent manner, irrespective of scale. Many methods used in the laboratory are demanding but can be handled and controlled by scientists well-versed in the art of organic synthesis. On scale-up, such reactions could behave in a capricious way, for example under the influence of issues with heat transfer and stirring efficiency, sensitivity to trace amounts of water, or being exposed to a non-inert atmosphere, which could lead to various levels of problems or even complete failure. Another significant aspect is that a key performance indicator in medicinal chemistry is the capability to make novel chemical compounds amenable to patent protection. This “race” into the unknown will inevitably demand the discovery and development of unprecedented synthetic methodologies, which eventually will have to prove their capabilities on a larger scale. Finally, in times where green chemistry and sustainability are top-rated criteria for any chemical process, there is bound to be a major reluctance to apply procedures that cannot guarantee the highest level of environmental concern for both ethical and legal reasons. © 2023 The Author(s)

Enzymes have been exploited by humans for thousands of years in brewing and baking, but it is only recently that biocatalysis has become a mainstream technology for synthesis. Today, enzymes are used extensively in the manufacturing of pharmaceuticals, food, fine chemicals, flavors, fragrances and other products. Enzyme immobilization technology has also developed in parallel as a means of increasing enzyme performance and reducing process costs. The aim of this review is to present and discuss some of the more recent promising technical developments in enzyme immobilization, including the supports used, methods of fabrication, and their application in synthesis. The review highlights new support technologies such as the use of well-established polysaccharides in novel ways, the use of magnetic particles, DNA, renewable materials and hybrid organic–inorganic supports. The review also addresses how immobilization is being integrated into developing biocatalytic technology, for example in flow biocatalysis, the use of 3D printing and multi-enzymatic cascade reactions. © 2021 by the authors. 

The catalyst cinchonidine-squaramide was immobilized within three different deep eutectic solvents (DES): (Betaine: D-Sorbitol: Water), (Betaine: D-Xylitol: Water) and (Betaine: D-Mannitol: Water) and evaluated in a well-known asymmetric Michael addition. These reactions provided excellent yields (up to 99%) and enantioselectivities (up to 98%) using only 1 mol% of catalyst. It was also possible to achieve 9 cycles in reactions with DES (Betaine: D-Sorbitol: Water), proving the high recyclability of this system. In the reactions realized with only 0.5 mol% of catalyst, it was possible to achieve 5 cycles and the products were obtained with high yields (up to 95%) and excellent enantioselectivities (up to 94%), using DES (Betaine: D-Sorbitol: Water)..

In this work, a waxy barley starch-PEG aqueous two-phase system (ATPS) phase diagram was constructed, and starch microsphere preparation was explored at different phase diagram positions. The aim was to investigate starch-PEG ATPS phase behaviour and relate this to starch crystallisation and microsphere formation. The hypothesis was that phase diagram position would influence the starch microsphere preparation and the properties of the microspheres. The microsphere formation process was investigated with regard to microsphere development and starch crystallisation kinetics. Microsphere physicochemical properties and their development during different stages of the preparation were studied by examining freshly produced, freeze-dried, and redispersed microspheres. Enzymatic hydrolysis of redispersed microspheres was also investigated. It was possible to produce microspheres from different positions in the phase diagram using 24 h incubation at 25 °C. However, the operational area for the used production conditions was relatively small compared to the biphasic region of the phase diagram. The main findings were that the starch-PEG ATPS phase behaviour can affect the rate of microsphere formation and particle size, but the additional properties of the dried and redispersed microspheres did not differ to a considerable extent. Thus, we have identified a robust production space where production parameters such as time to obtain microspheres can be considerably influenced by the ATPS system phase diagram position.

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase–cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a ‘folate trap’. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism. © 2023, The Author(s).

The use of DEHP (diethylhexyl phthalate) is now banned for most applications in Europe; the exception is for blood bags, where its toxicity is overshadowed by its ability to extend the storage life of red blood cells. Another plasticiser, BTHC (butanoyl trihexyl citrate), is used in paediatric blood bags but does not stabilise blood cells as effectively. Interactions between plasticisers and lipids are investigated with a phospholipid, DMPC, to understand the increased stability of blood cells in the presence of DEHP as well as bioaccumulation and identify differences with BTHC. Mixed monolayers of DMPC and DEHP or BTHC were studied on Langmuir troughs where surface pressure/area isotherms can be measured. Neutron reflection measurements were made to determine the composition and structure of these mixed layers. A large amount of plasticiser can be incorporated into a DMPC monolayer but once an upper limit is reached, plasticiser is selectively removed from the interface at high surface pressures. The upper limit is found to occur between 40-60 mol% for DEHP and 20-40 mol% for BTHC. The areas per molecule are also different with DEHP being in the range of 50-100 Å2 and BTHC being 65-120 Å2. Results indicate that BTHC does not fit as well as DEHP in DMPC monolayers which could help explain the differences observed with regards to the stability of blood cells.

Purpose: Our purpose was to identify human ovarian extracellular matrix (ECM) components that would support in vitro culture of human ovarian tissue and be compatible with possible future clinical applications. We characterized ovarian expression of laminins and selected three laminin tripeptides for culture experiments to be compared with Matrigel, an undefined and animal-based mixture of ECM components. Methods: Expression of the 12 laminin genes was determined on transcript and protein levels using cortical tissue samples (n = 6), commercial ovary RNA (n = 1), follicular fluid granulosa cells (n = 20), and single-cell RNA-sequencing data. Laminin 221 (LN221), LN521, LN511, and their mixture were chosen for a 7-day culture experiment along with Matrigel using tissue from 17 patients. At the end of the culture, follicles were evaluated by scoring and counting from serial tissue sections, apoptosis measured using in situ TUNEL assay, proliferation by Ki67 staining, and endocrine function by quantifying steroids in culture media using UPLC-MS/MS. Results: Approximately half of the cells in ovarian cortex expressed at least one laminin gene. The overall most expressed laminin α-chains were LAMA2 and LAMA5, β-chains LAMB1 and LAMB2, and γ-chain LAMC1. In culture experiments, LN221 enhanced follicular survival compared with Matrigel (p < 0.001), whereas tissue cultured on LN521 had higher proportion of secondary follicles (p < 0.001). LN511 and mixture of laminins did not support the cultures leading to lower follicle densities and higher apoptosis. All cultures produced steroids and contained proliferating cells. Conclusions: LN221 and LN521 show promise in providing xeno-free growth substrates for human ovarian tissue cultures, which may help in further development of folliculogenesis in vitro for clinical practices. The system could also be used for identification of adverse effects of chemicals in ovaries.

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

In the reperfusion era, a new paradigm of treating patients with endovascular treatment (EVT) and neuroprotective drugs is emerging as a promising therapeutic option for patients with acute ischemic stroke (AIS). In this context, ApTOLL, a Toll-like receptor 4 (TLR4) antagonist with proven neuroprotective effect in preclinical models of stroke and a very good pharmacokinetic and safety profile in healthy volunteers, is a promising first-in-class aptamer with the potential to address this huge unmet need. This protocol establishes the clinical trial procedures to conduct a Phase Ib/IIa clinical study (APRIL) to assess ApTOLL tolerability, safety, pharmacokinetics, and biological effect in patients with AIS who are eligible for EVT. This will be a multicenter, double-blind, randomized, placebo-controlled, Phase Ib/IIa clinical study to evaluate the administration of ApTOLL together with EVT in patients with AIS. The study population will be composed of men and non-pregnant women with confirmed AIS with a <6h window from symptoms onset to ApTOLL/placebo administration. The trial is currently being conducted and is divided into two parts: Phase Ib and Phase IIa. In Phase Ib, 32 patients will be allocated to four dose ascending levels to select, based on safety criteria, the best two doses to be administered in the following Phase IIa in which 119 patients will be randomized to three arms of treatment (dose A, dose B, and placebo).

Importance ApTOLL is a TLR4 antagonist with proven preclinical neuroprotective effect and a safe profile in healthy volunteers. Objective To assess the safety and efficacy of ApTOLL in combination with endovascular treatment (EVT) for patients with ischemic stroke. Design, Setting, and Participants This phase 1b/2a, double-blind, randomized, placebo-controlled study was conducted at 15 sites in Spain and France from 2020 to 2022. Participants included patients aged 18 to 90 years who had ischemic stroke due to large vessel occlusion and were seen within 6 hours after stroke onset; other criteria were an Alberta Stroke Program Early CT Score of 6 to 10, estimated infarct core volume on baseline computed tomography perfusion of 5 to 70 mL, and the intention to undergo EVT. During the study period, 4174 patients underwent EVT. Interventions In phase 1b, 0.025, 0.05, 0.1, or 0.2 mg/kg of ApTOLL or placebo; in phase 2a, 0.05 or 0.2 mg/kg of ApTOLL or placebo; and in both phases, treatment with EVT and intravenous thrombolysis if indicated. Main Outcomes and Measures The primary end point was the safety of ApTOLL based on death, symptomatic intracranial hemorrhage (sICH), malignant stroke, and recurrent stroke. Secondary efficacy end points included final infarct volume (via MRI at 72 hours), NIHSS score at 72 hours, and disability at 90 days (modified Rankin Scale [mRS] score). Results In phase Ib, 32 patients were allocated evenly to the 4 dose groups. After phase 1b was completed with no safety concerns, 2 doses were selected for phase 2a; these 119 patients were randomized to receive ApTOLL, 0.05 mg/kg (n = 36); ApTOLL, 0.2 mg/kg (n = 36), or placebo (n = 47) in a 1:1:√2 ratio. The pooled population of 139 patients had a mean (SD) age of 70 (12) years, 81 patients (58%) were male, and 58 (42%) were female. The primary end point occurred in 16 of 55 patients (29%) receiving placebo (10 deaths [18.2%], 4 sICH [7.3%], 4 malignant strokes [7.3%], and 2 recurrent strokes [3.6%]); in 15 of 42 patients (36%) receiving ApTOLL, 0.05 mg/kg (11 deaths [26.2%], 3 sICH [7.2%], 2 malignant strokes [4.8%], and 2 recurrent strokes [4.8%]); and in 6 of 42 patients (14%) receiving ApTOLL, 0.2 mg/kg (2 deaths [4.8%], 2 sICH [4.8%], and 3 recurrent strokes [7.1%]). ApTOLL, 0.2 mg/kg, was associated with lower NIHSS score at 72 hours (mean difference log-transformed vs placebo, −45%; 95% CI, −67% to −10%), smaller final infarct volume (mean difference log-transformed vs placebo, −42%; 95% CI, −66% to 1%), and lower degrees of disability at 90 days (common odds ratio for a better outcome vs placebo, 2.44; 95% CI, 1.76 to 5.00). Conclusions and Relevance In acute ischemic stroke, 0.2 mg/kg of ApTOLL administered within 6 hours of onset in combination with EVT was safe and associated with a potential meaningful clinical effect, reducing mortality and disability at 90 days compared with placebo. These preliminary findings await confirmation from larger pivotal trials.

Oligonucleotide (ON) conjugates are increasingly important tools for various molecular diagnostics, nanotechnological applications, and for the development of nucleic acid-based therapies. Multiple labeling of ONs can further equip ON-conjugates and provide improved or additional tailored properties. Typically, the preparation of ON multiconjugates involves additional synthetic steps and/or manipulations in post-ON assembly. This report describes the simplified methodology allowing for multiple labeling of ONs on a solid support and is compatible with phosphodiester as well as phosphorothioate (PS) ONs. The current approach utilizes two novel alkyne- A nd amino-functionalized linker phosphoramidites that can be readily synthesized from a common aminodiol intermediate in three steps. The combination of new linkers provides orthogonal functionalities, which allow for multiple attachments of similar or varied moieties. The linkers are incorporated into ONs during automated solid-phase ON synthesis, and the conjugation with functional entities is achieved by either amide bond formation or by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The versatility of the approach is demonstrated by the synthesis of 5′-site ON multiconjugates with small molecules, peptides, and fatty acids as well as in the preparation of an internal peptide-ON conjugate. 

In this study, we developed a novel solid lipid nanoparticle (SLN) formulation for drug delivery of small hydrophilic cargos to the retina. The new formulation, based on a gel core and composite shell, allowed up to two-fold increase in the encapsulation efficiency. The type of hydrophobic polyester used in the composite shell mixture affected the particle surface charge, colloidal stability, and cell internalization profile. We validated SLNs as a drug delivery system by performing the encapsulation of a hydrophilic neuroprotective cyclic guanosine monophosphate analog, previously demonstrated to hold retinoprotective properties, and the best formulation resulted in particles with a size of ±250 nm, anionic charge > −20 mV, and an encapsulation efficiency of ±60%, criteria that are suitable for retinal delivery. In vitro studies using the ARPE-19 and 661W retinal cell lines revealed the relatively low toxicity of SLNs, even when a high particle concentration was used. More importantly, SLN could be taken up by the cells and the release of the hydrophilic cargo in the cytoplasm was visually demonstrated. These findings suggest that the newly developed SLN with a gel core and composite polymer/lipid shell holds all the characteristics suitable for the drug delivery of small hydrophilic active molecules into retinal cells.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has unmasked mankind’s vulnerability to biological threats. Although higher age is a major risk factor for disease severity in COVID-19, several predisposing risk factors for mortality are related to low cardiorespiratory and metabolic fitness, including obesity, cardiovascular disease, diabetes, and hypertension. Reaching physical activity (PA) guideline goals contribute to protect against numerous immune and inflammatory disorders, in addition to multi-morbidities and mortality. Elevated levels of cardiorespiratory fitness, being non-obese, and regular PA improves immunological function, mitigating sustained low-grade systemic inflammation and age-related deterioration of the immune system, or immunosenescence. Regular PA and being non-obese also improve the antibody response to vaccination. In this review, we highlight potential physiological, cellular, and molecular mechanisms that are affected by regular PA, increase the host antiviral defense, and may determine the course and outcome of COVID-19. Not only are the immune system and regular PA in relation to COVID-19 discussed, but also the cardiovascular, respiratory, renal, and hormonal systems, as well as skeletal muscle, epigenetics, and mitochondrial function. © 2021 by the authors. 

The chemistry of silepins and related seven-membered heterocycles containing group 14 elements is reviewed. 

In several countries forest-based biofuels are being developed and to some extent also deployed. Fast pyrolysis bio-oil produced from, for example, sawdust, has now been coprocessed in fluid catalytic cracking refinery units in a number of commercial trials. However, this application is limited to about 10% of the total feed, and coprocessing in conventional fixed bed hydrotreaters is necessary to reach the high potential with this feedstock. Feeding and upgrading of fast pyrolysis bio-oil in a fixed bed reactor configuration is still problematic due to the inherent bio-oil properties. Stabilization of reactive compounds in fast pyrolysis bio-oil and mild hydrotreatment in a separate refining unit prior to refinery integration has therefore been developed the past decade. Another approach, presented here, involves complete dewatering of fast pyrolysis bio-oil by azeotropic distillation using mesityl oxide as the solvent, followed by conversion of the abundant hydroxyl compounds via mixed anhydride esterification methodology using an external source of mixed carboxylic acids of different chain lengths originating from renewable tall oil fatty acids, providing a lipophilic feed component. Dewatering and derivatizing were carried out in reactors up to 50 dm3 with a mass ratio of fast pyrolysis bio-oil to tall oil fatty acid of 10:13. The produced lipophilic oils were miscible with a petroleum light gas oil fraction and exhibited superior stability even after accelerated aging at elevated temperature (80 °C). The derivatized oils were thus mixed with light gas oil, with a proportion of 30 wt % derivatized oil in final blends and hydrotreated continuously in pilot fixed bed reactors for 14 days at 4 operating conditions without plugging or excessive exotherms. The test conditions were varied; the reactor pressure was either 55 or 80 bar, temperature 380 or 400 °C, and liquid hourly space velocity either 1 or 2 h-1 during the hydrotreatment. Successful hydrodeoxygenation and desulfurization were accomplished, whereas an increasing nitrogen concentration could be observed in the liquid products with the particular catalyst and reaction conditions employed. The observed hydrogen consumption (15-20 g/kg feed) was compared with the stoichiometric consumption for direct deoxygenation and with typical consumptions for industrial hydrotreated vegetable oil processing. The measured biogenic carbon content in hydrotreated liquid products (26.7%) agreed extremely well with the calculated biogenic carbon content in the hydrotreating feed (26.6%) that consisted of the blend of derivatized oil and petroleum light gas oil. The overall results are very promising since simple unit operations can be used to produce derivatized fast pyrolysis bio-oils that do not need additional standalone hydrotreating units but can be coprocessed in existing ones

The nitroaldol reaction is demonstrated as an efficient dynamic covalent reaction in phosphate buffers at neutral pH. Rapid equilibration was recorded with pyridine-based aldehydes, and dynamic oligomerization could be achieved, leading to nitroaldol dynamers of up to 17 repeating units. The dynamers were applied in a coherent stimuli-responsive molecular system in which larger dynamers transiently existed out-of-equilibrium in a neutral aqueous system rich in formaldehyde, controlled by nitromethane.

Two novel bicyclo[6.1.0]nonyne (BCN) linker derivatives, which can be directly incorporated into oligonucleotide sequences during standard automated solid-phase synthesis, are reported. Stabilities of BCN-carbinol and two BCN-oligonucleotides are evaluated under acidic conditions. In addition, derivatized BCN linkers (non-acidic and acid treated) are evaluated for strain-promoted alkyne-azide cycloaddition (SPAAC). 

2′-O-(N-(Aminoethyl)carbamoyl)methyl-modified 5-methyluridine (AECM-MeU) and 5-methylcytidine (AECM-MeC) phosphoramidites are reported for the first time and prepared in multigram quantities. The syntheses of AECM-MeU and AECM-MeC nucleosides are designed for larger scales (approx. 20 g up until phosphoramidite preparation steps) using low-cost reagents and minimizing chromatographic purifications. Several steps were screened for best conditions, focusing on the most crucial steps such as N3 and/or 2′-OH alkylations, which were improved for larger scale synthesis using phase transfer catalysis (PTC). Moreover, the need of chromatographic purifications was substantially reduced by employing one-pot synthesis and improved work-up strategies. © 2021 by the authors. 

T cell-driven diseases account for considerable morbidity and disability globally and there is an urgent need for new targeted therapies. Both cancer cells and activated T cells have an altered redox balance, and up-regulate the DNA repair protein MTH1 that sanitizes the oxidized nucleotide pool to avoid DNA damage and cell death. Herein we suggest that the up-regulation of MTH1 in activated T cells correlates with their redox status, but occurs before the ROS levels increase, challenging the established conception of MTH1 increasing as a direct response to an increased ROS status. We also propose a heterogeneity in MTH1 levels among activated T cells, where a smaller subset of activated T cells does not up-regulate MTH1 despite activation and proliferation. The study suggests that the vast majority of activated T cells have high MTH1 levels and are sensitive to the MTH1 inhibitor TH1579 (Karonudib) via induction of DNA damage and cell cycle arrest. TH1579 further drives the surviving cells to the MTH1low phenotype with altered redox status. TH1579 does not affect resting T cells, as opposed to the established immunosuppressor Azathioprine, and no sensitivity among other major immune cell types regarding their function can be observed. Finally, we demonstrate a therapeutic effect in a murine model of experimental autoimmune encephalomyelitis. In conclusion, we show proof of concept of the existence of MTH1high and MTH1low activated T cells, and that MTH1 inhibition by TH1579 selectively suppresses pro-inflammatory activated T cells. Thus, MTH1 inhibition by TH1579 may serve as a novel treatment option against autoreactive T cells in autoimmune diseases, such as multiple sclerosis.

The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.

Modifications of mineral surfaces were performed in order to gain insight into what surface properties are decisive of the accumulation of dental plaque. A non-charged, hydrophilic surface was made by two consecutive plasma polymerizations, firstly with allyl alcohol, secondly with acrylic acid, followed by adsorption of a poly(ethylene glycol)-poly(ethylene imine) adduct. A strongly hydrophobic surface was obtained by plasma polymerization of hexamethyldisiloxane. Ellipsometry was used to monitor protein interaction with the surfaces. The hydrophilic surface gave very little adsorption of both a model protein, IgG, and of saliva proteins. The hydrophobic surface, on the other hand, adsorbed high amounts of both types of proteins. In vitro adhesion of an oral bacterium, S. mutans, as well as in uivo studies, gave the opposite result, the hydrophobic surface giving less adhesion and less plaque accumulation than the hydrophilic surface. A tentative explanation of this behaviour is that the saliva proteins that bind to the hydrophobic surface adsorb in an unnatural conformation which does not favour bacteria adherence.

Competetive adsorption from a ternary mixture of human serum albumin (HSA), human IgG, and human fibrinogen (FGN) at concentrations corresponding to blood plasma diluted 1/100 was investigated with the combination of Total Internal Reflection Fluorescence spectroscopy (TIRF) and ellipsometry. As substrates, three different plasma polymer surfaces, representing different surface charge and surface energy, were prepared from hexamethyldisiloxane (PP-HMDSO), acrylic acid (PP-AA), and 1,2-diaminocyclohexane (PP-DACH). In addition, adsorption from single and binary protein systems was investigated with ellipsometry. At the hydrophobic PP-HMDSO little or no displacement of any of the proteins was observed. The adsorbed layer was dominated by HSA and IgG, although Fgn was also present to a smaller extent. On PP-DACH and PP-AA, representing positively and negatively charged hydrophilic surfaces, respectively, Fgn completely dominated the adsorbed layer while HSA was almost absent and IgG was present only at a very low level.