In the first study of its kind towards the design and synthesis of easy-to-handle aluminium precursors that decompose at temperatures <200 °C: informed ligand choice and structural design of the compounds has caused inbuilt fluxionality leading to a markable decrease in the onset of decomposition temperatures. Eight thiourea ligands [L1H-L8H] were chosen with the steric bulk on the N atoms of these ligands varied systematically [L1-4H: RN(H)CS(NMe2) and L5-8H: RN(H)CS(NEt2); R=Me (L1H and L5H), Et (L2H and L6H), iPr (L3H and L7H) and Ph (L4H and L8H). Three families of aluminium compounds were synthesised by the reaction of these thiourea ligands with trimethylamine alane [Al(Lx)3 (1-7), trimethylaluminium [MeAl(Lx)2] (8-11) and triethylaluminium [EtAl(Lx)2] (12-14) respectively. The three most spatially encumbered compounds (Al(L3)3 (2), Al(L6)3 (5) and Al(L7)3 (6) are highly fluxional in solution and displayed lengthening of the Al-N bond as compared to the other compounds. Both factors directly affect the activation temperature of these compounds. The remaining compounds were not shown to display any of these behaviours and consequently have higher thermal decomposition temperatures. SCXRD, 1H and 13C{1H} NMR, variable temperature 1H NMR, MS and EA have been used to study the structure and solution dynamics of 1-14. This has directly been linked to the decomposition profiles of the compounds to assess their viability as precursors, evidencing that what we see in the solution state is present in the solid state too. Density functional theory calculations have been carried out to elucidate the various bonding modes observed for compounds 1-7. Tandem MS has been employed to better understand the breakdown of the molecules.

We built a custom device to subject an antibody fragment A33 Fab to controlled stress conditions that combined pH, temperature, agitation, and LED-based light exposure in polypropylene microplates; to simulate the real-world challenges it may encounter during storage and transportation and to evaluate the key degradation routes in Fab formulations. We also explored the addition of Tween 80 as a surfactant and the impact of plate surface siliconisation. Monomer loss and fragmentation was monitored by size-exclusion chromatography, aggregate formation determined by changes in hydrodynamic radius in DLS, and chemical modifications identified through intact mass analysis by LC-MS, and N-terminal sequencing. The findings indicated that the light exposure conditions often interacted with other factors. In particular, light exposure in the UV to blue range led to chemical degradations that led to greater susceptibility to aggregation, particularly at elevated temperatures. Interestingly, while Tween 80 provided stabilization, particularly within siliconized plates, the presence of Tween 80 also promoted losses due to light exposure, consistent with previous findings that Tween 80 could act as a photosensitizer. Exposure of A33 Fab to light led to sequential losses of amino acids selectively from only the heavy chain C-terminus, indicating a photosensitive hotspot in that region of the Fab structure. These also suggested that photoinduced clipping of the heavy chain C-terminus increased the susceptibility of A33 Fab to fragmentation into heavy and light chains and aggregation, consistent with previous work in which flexibility-suppressing mutations of the hinge region decreased the aggregation kinetics.

The hinge region of antibody fragments plays a crucial role in their stability and aggregation properties. In this study, we investigated the effects of hinge truncations on the thermal stability and aggregation propensity of the A33 Fab antibody fragment. Eight Fab variants were engineered by introducing stop codons to truncate 1-8 residues at the hinge region (heavy chain residues 221-228). These variants were then expressed, purified, and characterized in terms of stability and aggregation propensity using SDS-PAGE, SEC-HPLC, LC-MS, and thermal stability assays. Our findings demonstrate that truncating the hinge region can enhance the thermal stability and reduce the aggregation of Fab fragments, and that progressive truncations identified an optimal hinge length for stability. Notably, the 227TGA variant exhibited a significant 14.5% reduction in aggregation rate compared to the wild type, without compromising thermal stability. By contrast, 221TGA removed all of the hinge and reduced the aggregation rate by 13%, but also decreased the thermal stability. These results suggest that hinge truncation is a promising strategy for improving the developability of therapeutic antibody Fab fragments by mitigating some of the stability issues associated with aggregation.

Non-thermal plasma has been an emerging technology for water treatment for decades. In this study, we have designed and fabricated a bubbling plasma batch reactor using an atmospheric pressure dielectric barrier discharge with a hydrophobic porous membrane. The reactor performance is assessed for purifying synthetic contaminated water samples containing chemical contaminant sulfamethoxazole (SMX), a widely used antibiotic, and biological contaminant E. coli K12. The SMX decontamination tests indicate that the degradation process is not first-order and the reaction rate dwindle with increasing initial concentration. The yield at 50% removal achieves its highest value of 8.12 g/kWh for 50 mg/L SMX sample. For inactivation of E. coli K12 tests, the inactivation process is also not first-order, and the pathogen is completely inactivated for 102 CFU/mL and 104 CFU/mL cases after 10 min and 45 min of plasma treatment, respectively. For the 108 CFU/mL sample, a 5-log reduction is achieved after 60 min of treatment. The developed plasma reactor can achieve fast deployment in point of use, low cost for manufacturing, and simple for maintenance. Moreover, it can be used for in-situ water purification in future long duration crewed space missions as well as tackling with water pollution issues on our planet.

While the use of antibiotics has been reported as extensive in the rearing of agricultural animals, insufficient information is available on the antibiotic residues in animal products and the adverse impact that consistent low-level exposure to antibiotics might have on the human body and its microbiome. The aim of this study was to estimate the antibiotic concentrations that humans are exposed to via their diet using the concentration of antibiotics in animal food products and water and an online survey on dietary intake. A total of 131 participants completed the dietary intake survey, with the majority belonging to the omnivorous diet group (76.3%). Distinct dietary trends were observed in the omnivorous and unknown groups eating animal products, with specific food types dominating each meal: pork (e.g., ham) and dairy products (e.g., milk, yoghurt) during breakfast, beef (e.g., burgers) and chicken (e.g., chicken breast) products during lunch, and fish (e.g., salmon fillet) during dinner. In total, 34 different animal-based food and drink products were tested for the presence of ten different antibiotics. Of all the products tested, over 35% exceeded the acceptable daily antibiotic intake for amoxicillin, ampicillin, and enrofloxacin.

Phytosterols are lipophilic compounds found in plants with structural similarity to mammalian cholesterol. They cannot be endogenously produced by mammals and therefore always originate from diet. There has been increased interest in dietary phytosterols over the last few decades due to their association with a variety of beneficial health effects including low-density lipoprotein cholesterol lowering, anti-inflammatory and anti-cancerous effects. They are proposed as potential moderators for diseases associated with the central nervous system where cholesterol homeostasis is found to be imperative (multiple sclerosis, dementia, etc.) due to their ability to reach the brain. Here we utilised an enzyme-assisted derivatisation for sterol analysis (EADSA) in combination with a liquid chromatography tandem mass spectrometry (LC-MSn) to characterise phytosterol content in human serum. As little as 100 fg of plant sterol was injected on a reversed phase LC column. The method allows semi-quantitative measurements of phytosterols and their derivatives simultaneously with measurement of cholesterol metabolites. The identification of phytosterols in human serum was based on comparison of their LC retention times and MS2, MS3 spectra with a library of authentic standards. Free campesterol serum concentration was in the range from 0.30 - 4.10µg/mL, β-sitosterol 0.16 - 3.37µg/mL and fucosterol was at lowest concentration range from 0.05 - 0.38µg/mL in ten individuals. This analytical methodology could be applied to the analysis of other biological fluids and tissues.

Staphylococcus aureus is a leading cause of nosocomial implant-associated infections, causing significant morbidity and mortality, underscoring the need for rapid, non-invasive, and cost-effective diagnostics. Here, we optimise the synthesis of renal-clearable gold nanoclusters (AuNCs) for enhanced catalytic activity with the aim of developing a sensitive colourimetric diagnostic for bacterial infection. All-atom molecular dynamics (MD) simulations confirm the stability of glutathione-coated AuNCs and surface access for peroxidase-like activity in complex physiological environments. We subsequently develop a biosensor by encapsulating these optimised AuNCs in bacterial toxin-responsive liposomes, which is extensively studied by various single-particle techniques. Upon exposure to S. aureus toxins, the liposomes rupture, releasing AuNCs that generate a colourimetric signal after kidney-mimetic filtration. The biosensor is further validated in vitro and in vivo using a hyaluronic acid (HA) hydrogel implant infection model. Urine samples collected from mice with bacteria-infected HA hydrogel implants turn blue upon substrate addition, confirming the suitability of the sensor for non-invasive detection of implant-associated infections. This platform has significant potential as a versatile, cost-effective diagnostic tool. Implant-associated infections with Staphylococcus aureus pose serious clinical challenges. Here, the authors develop a biosensor based on toxin-responsive liposomes encapsulating gold nanoclusters, providing a non-invasive, colourimetric diagnostic tool for bacterial infection detection with urinary readout.

Cyclopropane fatty acid synthases (CFAS) are a class of S-adenosylmethionine (SAM) dependent methyltransferase enzymes able to catalyse the cyclopropanation of unsaturated phospholipids. Since CFAS enzymes employ SAM as a methylene source to cyclopropanate alkene substrates, they have the potential to be mild and more sustainable biocatalysts for cyclopropanation transformations than current carbene-based approaches. This work describes the characterisation of E. coli CFAS (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with up to 58 % conversion and 73 % ee and the absolute configuration (9S,10R) was established. Substrate tolerance of ecCFAS was found to be correlated with the electronic properties of phospholipid headgroups and for the first time ecCFAS was found to catalyse cyclopropanation of both phospholipid chains to form dicyclopropanated products. In addition, mutagenesis and in silico experiments were carried out to identify the enzyme residues with key roles in catalysis and to provide structural insights into the lipid substrate preference of ecCFAS. Finally, the biocatalytic synthesis of methyl dihydrosterculate 1 and its deuterated analogue was also accomplished combining recombinant ecCFAS with the SAM regenerating AtHMT enzyme in the presence of CH3I and CD3I respectively.

Improving the range of substrates accepted by enzymes with high catalytic activity remains an important goal for the industrialisation of biocatalysis. Many enzymes catalyse two-substrate reactions which increases the complexity in engineering them for the synthesis of alternative products. Often mutations are found independently that can improve the acceptance of alternatives to each of the two substrates. Ideally, we would be able to combine mutations identified for each of the two alternative substrates, and so reprogramme new enzyme variants that synthesise specific products from their respective two-substrate combinations. However, as we have previously observed for E. coli transketolase, the mutations that improved activity towards aromatic acceptor aldehydes, did not successfully recombine with mutations that switched the donor substrate to pyruvate. This likely results from several active site residues having multiple roles that can affect both of the substrates, as well as structural interactions between the mutations themselves. Here, we have designed small libraries, including both natural and non-natural amino acids, based on the previous mutational sites that impact on acceptance of the two substrates, to achieve up to 630× increases in k cat for the reaction with 3-formylbenzoic acid (3-FBA) and pyruvate. Computational docking was able to determine how the mutations shaped the active site to improve the proximity of the 3-FBA substrate relative to the enamine-TPP intermediate, formed after the initial reaction with pyruvate. This work opens the way for small libraries to rapidly reprogramme enzyme active sites in a plug and play approach to catalyse new combinations of two-substrate reactions.

While the use of a wide range of antibiotics has been reported as extensive in the rearing of agricultural animals, extremely limited information is available on the antibiotic residues in animal products and the adverse impact consistent low-level exposure to antibiotics might have on the human body as well as its microbiome. The aim of this study was to estimate the possible antibiotic concentrations humans are exposed to via their diet using the concentration of antibiotics in animal food products and water, and an online survey on dietary habits. A total of 131 participants completed the dietary habits survey, the majority belonging to the omnivorous diet habit. Distinct dietary trends were observed into omnivorous and unknown groups eating food-producing animal products, with specific food types dominating each meal: pork (e.g. ham) and dairy products (e.g. milk, yoghurt) during breakfast, beef (e.g. burger) and chicken (e.g. chicken breast) products during lunch, and fish (e.g. salmon fillet) during dinner. 34 different animal-based food and drink products were tested for the presence of ten different antibiotics. Low levels of nine of the ten antibiotics were detected across the samples tested with amoxicillin and trimethoprim being the most frequently detected antibiotics from all samples with concentrations ranging from 216.7-6866.9 μg/kg and 55.2-461.7 μg/kg, respectively. Of all products tested, over 35% exceeded the acceptable daily intake antibiotic concentration for amoxicillin, ampicillin, and enrofloxacin. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Committee of the University College London gave ethical approval for this work, project number [19139/001]. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

Herein we report on photodecarboxylations of various substituted maleimides, resulting in an elimination reaction. Furthermore, we establish facile wavelength tunability through modulation of the maleimide double bond substituents. We envisage that these versatile reagents, which are readily constructed and diversified by nucleophilic substitution reactions on bromomaleimides, will offer new opportunities for triggered photorelease.

Electron-poor aryl nitriles are promising reagents for bioconjugation due to their high electrophilicity and selectivity for reaction with thiols, albeit generally in a reversible manner. A transient species has previously been observed in such reactions, involving the addition of two thiols to the nitrile functional group, forming a tetrahedral amino dithioacetal (ADTA). In this work, the reaction of heteroaryl nitriles with bis-thiols is explored in an attempt to generate stable ADTAs, which could facilitate new bioconjugation protocols. By use of a 1,2-dithiol, or the incorporation of an electrophilic trap into the aryl nitrile design, the formation of stable products is achieved. The resultant "nitrile bis-thiol" (NBT) reaction is then explored in the context of protein modification, specifically to carry out antibody conjugation. By addition of these nitriles to the reduced disulfide bond of an antibody fragment, it is shown that, depending on the reagent design, cysteine-to-lysine transfer or disulfide bridged NBT products can be generated. Both represent site-selective conjugates and are shown to be stable when challenged with glutathione under physiological conditions and upon incubation in serum. Furthermore, the NBT reaction is tested in the more challenging context of a full antibody, and all four disulfide bonds are effectively modified by these new one-carbon bridging reagents. Overall, this reaction of heteroaryl-nitriles with bis-thiols is shown to be highly efficient and versatile, of tunable reversibility, and offers enticing prospects as a new addition to the toolbox of biocompatible "click"-type reactions.

Herein we report a fundamental discovery on the use of tris(dialkylamino)phosphine reagents for peptide and protein modification. We discovered that C-terminal thiophosphonium species, which are uniquely stable, could be selectively and rapidly generated from their disulfide counterparts. In sharp and direct contrast, internal thiophosphonium species rapidly degrade to dehydroalanine. We demonstrate this remarkable chemoselectivity on a bis-cysteine model peptide, and the formation of a stable C-terminal-thiophosphonium adduct on an antibody fragment, as well as characterise the species in various small molecule/peptide studies.

ZAG is a multifunctional glycoprotein with a class I MHC-like protein fold and an alpha 1-alpha 2 lipid-binding groove. The intrinsic ZAG ligand is unknown. Our previous studies showed that ZAG binds the dansylated C-11 fatty acid, DAUDA, differently to the boron dipyrromethane C-16 fatty acid, C-16-BODIPY. Here, the molecular basis for this difference was elucidated. Multi-wavelength analytical ultracentrifugation confirmed that DAUDA and C-16-BODIPY individually bind to ZAG and compete for the same binding site. Molecular docking of lipid-binding in the structurally related Cluster of differentiation 1 proteins predicted nine conserved ligand contact residues in ZAG. Twelve mutants were accordingly created by alanine scanning site directed mutagenesis for characterisation. Mutation of Y12 caused ZAG to misfold. Mutation of K147, R157 and A158 abrogated C-16-BODIPY but not DAUDA binding. L69 and T169 increased the fluorescence emission intensity of C-16-BODIPY but not of DAUDA compared to wild-type ZAG and showed that C-16-BODIPY binds close to T169 and L69. Distance measurements of the crystal structure revealed K147 forms a salt bridge with D83. A range of bioactive bulky lipids including phospholipids and sphingolipids displaced DAUDA from the ZAG binding site but unexpectedly did not displace C-16-BODIPY. We conclude that the ZAG alpha 1-alpha 2 groove contains separate but overlapping sites for DAUDA and C-16-BODIPY and is involved in binding to a bulkier and wider repertoire of lipids than previously reported. This work suggested that the in vivo activity of ZAG may be dictated by its lipid ligand.

Natural substances are increasingly being developed for use in health-related applications. Honey has attracted significant interest, not only for its physical and chemical properties, but also for its antibacterial activity. For the first time, suspensions of Black Forest honeydew honey and manuka honey UMF 20+ were examined for their antibacterial properties against Escherichia coli and Staphylococcus epidermidis using flow cytometry. The inhibitory effect of honey on bacterial growth was evident at concentrations of 10, 20 and 30 v/v%. The minimum inhibitory effects of both honey types against each bacterium were also investigated and reported. Electrospray ionisation (ESI) mass spectrometry was performed on both Black Forest honeydew honey and manuka honey UMF 20+. Manuka honey had a gluconic concentration of 2519 mg/kg, whilst Black Forest honeydew honey had a concentration of 2195 mg/kg. Manuka honey demonstrated the strongest potency when compared to Black Forest honeydew honey; therefore, it was incorporated into nanofiber scaffolds using pressurised gyration and 10, 20 and 30 v/v% manuka honey-polycaprolactone solutions. Composite fibres were analysed for their morphology and topography using scanning electron microscopy. The average fibre diameter of the manuka honey-polycaprolactone scaffolds was found to range from 437 to 815 nm. The antibacterial activity of the 30 v/v% scaffolds was studied using S. epidermidis. Strong antibacterial activity was observed with a bacterial reduction rate of over 90%. The results show that honey composite fibres formed using pressurised gyration can be considered a natural therapeutic agent for various medicinal purposes, including wound-healing applications.

Correction for 'Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis' by Maurício Morais et al., Org. Biomol. Chem., 2017, 15, 2947-2952, DOI: .

Given detrimental impacts induced by estrogens at trace level, determination of them is significant but challenging due to their low content in environmental samples and inherent weak ionisation. A modified derivatisation-based methodology was applied for the first time to detect estrogen in free and conjugated forms including some isomers simultaneously using liquid chromatography tandem mass spectrometry (LC-MSn). Derivatisation reaction with previously used 1,2-dimethyl-1H-imidazole-5-sulphonyl chloride allowed significant increase of mass spectrometric signal of analytes and also provided distinctive fragmentation for their confirmation even in complicated matrix. Then satisfactory recovery (>75%) for the majority of analytes was achieved following optimisation of solid phase extraction (SPE) factors. The linearity was validated over a wide concentration with the correlation coefficient around 0.995. The repeatability of this methodology was also confirmed via the intra-day and inter-day precision and was less than 11.73%. Validation of method quantification limits (MQLs) for all chosen estrogens was conducted using 1000 mL surface water, ranging from 7.0 to 132.3 pg L-1. The established methodology was applied to profile presence of targeted estrogens in natural surface water samples. Out of the ten compounds of interest, three free estrogens (E1, E2, E3) and two sulphate estrogens (E1-3S and E2-3S) were found over their MQLs, being in the range of 0.05-0.32 ng L-1.