Agilent Technologies (United Kingdom)

Fluid-preserved specimens are central to the scientific and cultural value of natural history collections, yet their conservation is challenged by chemical and physical instabilities of both the specimens and their preservation media. Here we report the application of handheld spatially offset Raman spectroscopy (SORS) to noninvasively characterize historical specimens from their conservation status perspective. This goes beyond previously reported basic determination of the major constituents of preservation fluids by providing detailed chemical information on minor dissolved components, such as lipids, protein fragments, and residual fixation products, as well as organic deposits on container walls. This provides insight into fluid degradation, leakage, and specimen-fluid interactions of sealed wet collection items. Furthermore, we demonstrate the capability of directly probing specimen composition. All measurements were performed in situ without opening containers, demonstrating the robustness and versatility of SORS for comprehensive monitoring wet collection status under museum conditions and offering curators actionable insights into degradation processes and long-term collection integrity.

Decentralised manufacture (DM) of personalised medicines via 3D printing (3DP) is now viable in the United Kingdom under new legislation with regulatory agencies across the globe working to follow suit. Quality control (QC) and ensuring dose accuracy for small-batch production of extended-release (ER) printlets (3DP tablets) remains a challenge. We demonstrate, for the first time, a non-destructive, volumetric method for drug quantification using Transmission Raman spectroscopy (TRS) combined with partial least squares regression (PLSR), suitable for high-throughput QC of individual ER printlets in DM settings. A simple theophylline-based pharma-ink was developed for direct powder extrusion (DPE) 3DP of cylindrical ER printlets in 3 sizes, maintaining a constant surface area-to-volume (SA/V) ratio to ensure comparable release profiles. In vitro dissolution confirmed equivalent ER profiles across all sizes, meeting pharmacopeial requirements. A PLSR model calibrated with only the smallest and largest printlet TRS spectra accurately predicted drug content in all 3 printlet sizes (R² = 0.9948, RMSEP = 0.5611% w/w) with no statistical differences compared to the reference method. These findings establish TRS as a rapid, non-destructive drug content QC tool for personalised, dose-flexible 3DP medicines and support its implementation in decentralised pharmaceutical manufacturing workflows.

Abstract Maintaining cold-chain integrity is vital for vaccines to ensure they remained within the recommended temperature limits to ensure stability and avoid degradation as storage temperature is one of the key factors contributing to rendering products substandard or ‘out of specification’. Heat-exposed vaccines closely resemble the chemical composition of the stable product making them very difficult to detect and testing as such is not routinely carried out at various points in the supply chain due to the lack of tools to identify effects of heat exposure in the field, a particular issue in countries with high-temperature climates. Here, we propose rapid and low-cost tests based on simple glucose assays to detect heat-exposed degraded sucrose-containing vaccines through its inherent gradual conversion to glucose at elevated temperatures. Bioluminescent and colorimetric assays and a clinical biochemical analyser for urine samples could successfully determine effects of heat exposure by detecting a significant increase in glucose levels. We show that this increase in glucose also correlates with the loss of vaccine potency. When vaccines were incubated at 37 and 45°C, the bioluminescent assay was able to detect an increase in glucose levels from 12 hours of heat exposure. The biochemical analyser could successfully detect if a COVID-19 vaccine had been exposed to 37 and 45°C. Most importantly, the colorimetric assay has the advantage of noticing a colour change by eye upon simply mixing the vaccine with a reagent without the need for a plate reader or any other sophisticated devices. To our knowledge, this is the first device-free test of its kind to determine the heat-exposed vaccines, making it an ideal test for deploying at various points in the supply chain in low- and middle-income countries to ensure the integrity of vaccine cold-chain. Although this test does not replace the more definitive potency assays, it could initially be used as a rapid and low-cost test to identify substandard sucrose-containing vaccines within supply chains, in support of WHO’s Prevent, Detect, and Respond strategy.

The conservation and characterization of preservation fluids are crucial for maintaining specimen integrity in natural history fluid collections. However, characterizing these fluids analytically poses significant challenges, especially as noninvasive methods are preferred to avoid opening jars and reduce the risk of compromising specimens. This proof-of-concept study investigates the feasibility of using a hand-held spatially offset Raman spectroscopy (SORS) instrument to determine the chemical composition of preservation fluids through their original glass containers. Results demonstrate that SORS can noninvasively verify the chemical identity of dominant excipients in these fluids measured through a historic glass jar. Additionally, multivariate analysis combined with SORS measurements successfully differentiated several types of typical preservation fluids prepared as mixtures of different alcohols in water, such as glycerol, ethanol, methanol, and formaldehyde. The proposed noninvasive approach was also able to differentiate between different concentration points of components in water within the same type of preservation fluid.

Fluorescent insulin is commonly used for a range of detection and imaging purposes. Achieving site-selective insulin labeling affords superior labeling yield while retaining its biological activity. Insulin labeling is usually achieved using commercial kits with minimal emphasis on the site and degree of labeling. To bridge this gap, this work highlights the essential parameters concerning the development of fluorescent insulin and reflects them on the biological activity of insulin in vivo. To this end, monolabeled insulin at the N-terminal of A chain (GlyA1-N-FITC-insulin) was prepared using the minimal equivalents of fluorescein isothiocyanate (FITC) dye. In our hands, temperature and pH control were the main parameters affecting the reaction yield, with no dilabeled insulin being attained. To label the N-terminal of the B chain (PheB1-N-FITC-insulin), di-tert-butyl decarbonate, known as Boc anhydride, was used before FITC labeling. The attained insulin conjugates, namely, GlyA1-N-FITC-insulin and PheB1-N-FITC-insulin, were characterized using protein mass spectroscopy and peptide analysis. A third fluorescent conjugate was prepared using α-haloacetyl-based chemistry. This chemistry's advantage is maintaining the chain A N-terminal amine basicity, which was essential for its activity. Using α-haloacetyl-based chemistry, azide group-functionalized insulin was prepared, which was further clicked with fluorescent dye affording GlyA1-N-Cy5-insulin. According to the in vivo efficacy study of the three insulin conjugates, both fluorescent GlyA1-N-FITC-insulin and GlyA1-N-Cy5-insulin retained the insulin biological activity, suggesting no structural alteration upon the conjugation conditions. Hence, both GlyA1-N-FITC-insulin and GlyA1-N-Cy5-insulin are effective in labeling and, more importantly, maintaining the in vivo activity of insulin. Lastly, in vitro binding of GlyA1-N-FITC-insulin was successful when it was assayed in NIH/3T3 fibroblast cells. This work has provided facile conjugation approaches for site-specific insulin labeling with dyes or clickable chemistry in conjunction with insulin's in vivo biological activity.

Tidal energy, with its potential to provide a consistent energy output and reduce carbon emissions, has garnered significant interest. This study, which evaluates the performance of tidal turbine blades in seawater conditions and with sand particles, presents a novel approach. A slurry rig was developed to examine composite materials, and a glass fibre-reinforcement polymeric material was tested over a range of particle sizes, velocities, and impact angles. In addition, this paper used a new test protocol with 14 days (336 h) and 91 days (2184 h) of pre-exposure time of materials before testing. The results, which show significant changes in the erosive mechanisms of GFRP in short- and long-term pre-exposure time as a function of these variables, have profound implications for the design and performance of tidal turbine blades. The study also utilised scanning electron microscopy (SEM), depth profiling analysis, and erosion mapping techniques to compare the erosion behaviours of GFRP. These tools can be used to optimise such materials in tidal turbine conditions.

We adapted the SureSelect XT HS2 DNA protocol to prepare libraries using as input material samples after single-cell Whole Genome Amplification (scWGA), instead of genomic DNA samples, for library preparation for Illumina single-cell Whole Genome Sequencing (scWGS). This protocol can be employed either manually (Section 2: Option A) or in combination with automation using the Bravo Automated Liquid Handling Platform (Section 2: Option B).

Metabolomic analyses in alkaptonuria (AKU) have recently revealed alternative pathways in phenylalanine-tyrosine (phe-tyr) metabolism from biotransformation of homogentisic acid (HGA), the active molecule in this disease. The aim of this research was to study the phe-tyr metabolic pathway and whether the metabolites upstream of HGA, increased in nitisinone-treated patients, also undergo phase 1 and 2 biotransformation reactions. Metabolomic analyses were performed on serum and urine from patients partaking in the SONIA 2 phase 3 international randomised-controlled trial of nitisinone in AKU (EudraCT no. 2013-001633-41). Serum and urine samples were taken from the same patients at baseline (pre-nitisinone) then at 24 and 48 months on nitisinone treatment (patients N = 47 serum; 53 urine) or no treatment (patients N = 45 serum; 50 urine). Targeted feature extraction was performed to specifically mine data for the entire complement of theoretically predicted phase 1 and 2 biotransformation products derived from phenylalanine, tyrosine, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid, in addition to phenylalanine-derived metabolites with known increases in phenylketonuria. In total, we observed 13 phase 1 and 2 biotransformation products from phenylalanine through to HGA. Each of these products were observed in urine and two were detected in serum. The derivatives of the metabolites upstream of HGA were markedly increased in urine of nitisinone-treated patients (fold change 1.2–16.2) and increases in 12 of these compounds were directly proportional to the degree of nitisinone-induced hypertyrosinaemia (correlation coefficient with serum tyrosine = 0.2–0.7). Increases in the urinary phenylalanine metabolites were also observed across consecutive visits in the treated group. Nitisinone treatment results in marked increases in a wider network of phe-tyr metabolites than shown before. This network comprises alternative biotransformation products from the major metabolites of this pathway, produced by reactions including hydration (phase 1) and bioconjugation (phase 2) of acetyl, methyl, acetylcysteine, glucuronide, glycine and sulfate groups. We propose that these alternative routes of phe-tyr metabolism, predominantly in urine, minimise tyrosinaemia as well as phenylalanaemia.