Conformations of a highly expressed Z19 α-zein studied with AlphaFold2 and MD simulations.

α-zeins are amphiphilic maize seed storage proteins with material properties suitable for a multitude of applications e.g., in renewable plastics, foods, therapeutics and additive manufacturing (3D-printing). To exploit their full potential, molecular-level insights are essential. The difficulties in experimental atomic-resolution characterization of α-zeins have resulted in a diversity of published molecular models. However, deep-learning α-zein models are largely unexplored. Therefore, this work studies an AlphaFold2 (AF2) model of a highly expressed α-zein using molecular dynamics (MD) simulations. The sequence of the α-zein cZ19C2 gave a loosely packed AF2 model with 7 α-helical segments connected by turns/loops. Compact tertiary structure was limited to a C-terminal bundle of three α-helices, each showing notable agreement with a published consensus sequence. Aiming to chart possible α-zein conformations in practically relevant solvents, rather than the native solid-state, the AF2 model was subjected to MD simulations in water/ethanol mixtures with varying ethanol concentrations. Despite giving structurally diverse endpoints, the simulations showed several patterns: In water and low ethanol concentrations, the model rapidly formed compact globular structures, largely preserving the C-terminal bundle. At ≥ 50 mol% ethanol, extended conformations prevailed, consistent with previous SAXS studies. Tertiary structure was partially stabilized in water and low ethanol concentrations, but was disrupted in ≥ 50 mol% ethanol. Aggregated results indicated minor increases in helicity with ethanol concentration. ß-sheet content was consistently low (∼1%) across all conditions. Beyond structural dynamics, the rapid formation of branched α-zein aggregates in aqueous environments was highlighted. Furthermore, aqueous simulations revealed favorable interactions between the protein and the crosslinking agent glycidyl methacrylate (GMA). The proximity of GMA epoxide carbons and side chain hydroxyl oxygens simultaneously suggested accessible reactive sites in compact α-zein conformations and pre-reaction geometries for methacrylation. The findings may assist in expanding the applications of these technologically significant proteins, e.g., by guiding chemical modifications.

Fast collective motions of backbone in transmembrane α helices are critical to water transfer of aquaporin.

Fast collective motions are widely present in biomolecules, but their functional relevance remains unclear. Herein, we reveal that fast collective motions of backbone are critical to the water transfer of aquaporin Z (AqpZ) by using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. A total of 212 residue site-specific dipolar order parameters and 158 15N spin relaxation rates of the backbone are measured by combining the 13C- and 1H-detected multidimensional ssNMR spectra. Analysis of these experimental data by theoretic models suggests that the small-amplitude (~10°) collective motions of the transmembrane α helices on the nanosecond-to-microsecond timescales are dominant for the dynamics of AqpZ. The MD simulations demonstrate that these collective motions are critical to the water transfer efficiency of AqpZ by facilitating the opening of the channel and accelerating the water-residue hydrogen bonds renewing in the selectivity filter region.

Effect of water glass treatment for zirconia and silane coupling on bond strength of resin cement.

OBJECTIVE: To evaluate the ability of the water glass treatment to penetrate zirconia and improve the bond strength of resin cement. MATERIAL AND METHODS: Water glass was applied to zirconia specimens, which were then sintered. The specimens were divided into water-glass-treated and untreated zirconia (control) groups. The surface properties of the water-glass-treated specimens were evaluated using surface roughness and electron probe micro-analyser (EPMA) analysis. A resin cement was used to evaluate the tensile bond strength, with2 and without a silane-containing primer. After 24 h in water storage at 37 °C and thermal cycling, the bond strengths were statistically evaluated with t-test, and the fracture surfaces were observed using SEM. RESULTS: The water glass treatment slightly increased the surface roughness of the zirconia specimens, and the EPMA analysis detected the water glass penetration to be 50 µm below the zirconia surface. The application of primer improved the tensile bond strength in all groups. After 24 h, the water-glass-treated zirconia exhibited a tensile strength of 24.8 ± 5.5 MPa, which was significantly higher than that of the control zirconia (17.6 ± 3.5 MPa) (p < 0.05). After thermal cycling, the water-glass-treated zirconia showed significantly higher tensile strength than the control zirconia. The fracture surface morphology was mainly an adhesive pattern, whereas resin cement residue was occasionally detected on the water-glass-treated zirconia surfaces. CONCLUSION: The water glass treatment resulted in the formation of a stable silica phase on the zirconia surface. This process enabled silane coupling to the zirconia and improved the adhesion of the resin cement.

An eco-friendly and facile method for oil-water separation using the bio-Zn oxide-based superhydrophobic membrane.

This manuscript presents a novel approach for developing an environmentally friendly and effective oil-water separation membrane. Achieving a superhydrophobic (SH) coating on textile fabric (TF) involved a two-step process. Initially, the surface roughness was enhanced by applying bio-zinc oxide (ZnO) nanoparticles obtained from Thymbra spicata L. Subsequently, the roughened surface was modified with stearic acid, a material known for its low surface energy. The bio-ZnO nanoparticles exhibit a circular morphology with an average size of 21 nm. The coating demonstrated remarkable mechanical stability, maintaining SH properties even after an abrasion length of 300 mm. Chemical stability studies revealed that the prepared membrane retained SH properties within a pH range of 5-11, which ensures robust performance. Absorption capacity measurements showcased different capacities for n-hexane (Hex), corn oil (C.O), and silicone oil (S.O), with consistent performance over 10 absorption-desorption cycles. High oil-water separation efficiencies were achieved for hexane, C.O, and S.O, emphasizing the coating's versatility. Flux rate measurements demonstrated that oil passed through the membrane efficiently, with the highest flux observed for Hex. The prepared SH membrane has superior mechanical and chemical stability and high separation efficiencies, which positions it as a promising candidate for diverse industrial applications.

Formulation and Characterization of Soybean Oil-in-Water Emulsions Stabilized Using Gelatinized Starch Dispersions from Plant Sources.

Consumers are concerned about employing green processing technologies and natural ingredients in different manufacturing sectors to achieve a "clean label" standard for products and minimize the hazardous impact of chemical ingredients on human health and the environment. In this study, we investigated the effects of gelatinized starch dispersions (GSDs) prepared from six plant sources (indica and japonica rice, wheat, corn, potatoes, and sweet potatoes) on the formulation and stability of oil-in-water (O/W) emulsions. The effect of gelatinization temperature and time conditions of 85-90 °C for 20 min on the interfacial tension of the two phases was observed. Emulsification was performed using a primary homogenization condition of 10,000 rpm for 5 min, followed by high-pressure homogenization at 100 MPa for five cycles. The effects of higher oil weight fractions (15-25% w/w) and storage stability at different temperatures for four weeks were also evaluated. The interfacial tension of all starch GSDs with soybean oil decreased compared with the interfacial tension between soybean oil and water as a control. The largest interfacial tension reduction was observed for the GSD from indica rice. Microstructural analysis indicated that the GSDs stabilized the O/W emulsion by coating oil droplets. Emulsions formulated using a GSD from indica rice were stable during four weeks of storage with a volume mean diameter (d4,3) of ~1 µm, minimal viscosity change, and a negative ζ-potential.

Removal of Azo Dyes from Water Using Natural Luffa cylindrica as a Non-Conventional Adsorbent.

Reducing high concentrations of pollutants such as heavy metals, pesticides, drugs, and dyes from water is an emerging necessity. We evaluated the use of Luffa cylindrica (Lc) as a natural non-conventional adsorbent to remove azo dye mixture (ADM) from water. The capacity of Lc at three different doses (2.5, 5.0, and 10.0 g/L) was evaluated using three concentrations of azo dyes (0.125, 0.250, and 0.500 g/L). The removal percent (R%), maximum adsorption capacity (Qm), isotherm and kinetics adsorption models, and pH influence were evaluated, and Fourier-transform infrared spectroscopy and scanning electron microscopy were performed. The maximum R% was 70.8% for 10.0 g L-1Lc and 0.125 g L-1 ADM. The Qm of Lc was 161.29 mg g-1. Adsorption by Lc obeys a Langmuir isotherm and occurs through the pseudo-second-order kinetic model. Statistical analysis showed that the adsorbent dose, the azo dye concentration, and contact time significantly influenced R% and the adsorption capacity. These findings indicate that Lc could be used as a natural non-conventional adsorbent to reduce ADM in water, and it has a potential application in the pretreatment of wastewaters.

Design and Synthesis of Dimethylaminomethyl-Substituted Curcumin Derivatives: Potent Anti-Inflammatory, Anti-Oxidant, and Radioprotection Activity, Improved Aqueous Solubility Compared with Curcumin.

Although the wide variety of bioactivities of curcumin has been reported by researchers, the clinical application of curcumin is still limited due to its poor aqueous solubility. In view of this, a series of dimethylaminomethyl-substituted curcumin derivatives were designed and synthesized (compounds 1-15). Acetate of these derivatives were prepared (compounds 1a-15a). The Mannich reaction and aldol condensation reaction are the main reactions involved in this study. Compounds 6, 10, 12, 3a, 5a, 6a, 7a, 8a, 10a, 11a, 12a, 13a, 14a, and 15a exhibited better in vitro anti-inflammatory activity compared to curcumin in the RAW264.7 cell line. Compounds 5, 1a, 5a, 8a, and 12a exhibited better in vitro antioxidant activity compared to curcumin in the PC 12 cell line. Compounds 11, 13, 5a, 7a, and 13a exhibited better in vitro radiation protection compared to curcumin in the PC 12 cell line. The aqueous solubilities of all the curcumin derivative acetates were greatly improved compared to curcumin.

Microencapsulation of Essential Oils Using Faba Bean Protein and Chia Seed Polysaccharides via Complex Coacervation Method.

The aim of this study was to develop microcapsules containing juniper or black pepper essential oils, using a combination of faba bean protein and chia seed polysaccharides (in ratios of 1:1, 1:2, 2:1). By synergizing these two polymers, our goal was to enhance the efficiency of essential oil microencapsulation, opening up various applications in the food industry. Additionally, we aimed to investigate the influence of different polymer mixing ratios on the properties of the resulting microcapsules and the course of the complex coacervation process. To dissolve the essential oils and limit their evaporation, soybean and rapeseed oils were used. The powders resulting from the freeze-drying of coacervates underwent testing to assess microencapsulation efficiency (65.64-87.85%), density, flowability, water content, solubility, and hygroscopicity. Additionally, FT-IR and DSC analyses were conducted. FT-IR analysis confirmed the interactions between the components of the microcapsules, and these interactions were reflected in their high thermal resistance, especially at a protein-to-polysaccharide ratio of 2:1 (177.2 °C). The water content in the obtained powders was low (3.72-7.65%), but it contributed to their hygroscopicity (40.40-76.98%).

Theoretical Study of Cyanidin-Resveratrol Copigmentation by the Functional Density Theory.

Anthocyanins are colored water-soluble plant pigments. Upon consumption, anthocyanins are quickly absorbed and can penetrate the blood-brain barrier (BBB). Research based on population studies suggests that including anthocyanin-rich sources in the diet lowers the risk of neurodegenerative diseases. The copigmentation caused by copigments is considered an effective way to stabilize anthocyanins against adverse environmental conditions. This is attributed to the covalent and noncovalent interactions between colored forms of anthocyanins (flavylium ions and quinoidal bases) and colorless or pale-yellow organic molecules (copigments). The present work carried out a theoretical study of the copigmentation process between cyanidin and resveratrol (CINRES). We used three levels of density functional theory: M06-2x/6-31g+(d,p) (d3bj); ωB97X-D/6-31+(d,p); APFD/6-31+(d,p), implemented in the Gaussian16W package. In a vacuum, the CINRES was found at a copigmentation distance of 3.54 Å between cyanidin and resveratrol. In water, a binding free energy ∆G was calculated, rendering -3.31, -1.68, and -6.91 kcal/mol, at M06-2x/6-31g+(d,p) (d3bj), ωB97X-D/6-31+(d,p), and APFD/6-31+(d,p) levels of theory, respectively. A time-dependent density functional theory (TD-DFT) was used to calculate the UV spectra of the complexes and then compared to its parent molecules, resulting in a lower energy gap at forming complexes. Excited states' properties were analyzed with the ωB97X-D functional. Finally, Shannon aromaticity indices were calculated and isosurfaces of non-covalent interactions were evaluated.

Quantification of resistant starch content in rice after hydrothermal treatments using terahertz spectroscopy.

Since hydrothermal treatments can enhance resistant starch (RS) content in rice and provide health benefits when consumed, a less laborious and non-destructive method to determine RS content is needed. Terahertz (THz) spectroscopy is hypothesized as a suitable method to quantify RS content in rice after hydrothermal treatment with its sensitivity for the intermolecular forces increase in the formation of RS. In this study, we first used the traditional in vitro hydrolysis method to determine the content of RS in rice. Then, the potential of starch absorbance peaks to quantify RS content after three commonly used hydrothermal methods, soaking, mild heat-moisture treatment, and parboiling, was investigated. The second derivative intensities of the peak at 9.0, 10.5, 12.1, and 13.1 THz were confirmed as being correlated with RS content and showed the high accuracy to predict RS content in samples (R2 > 0.96). Our results indicate the RS content of hydrothermally treated rice can be accurately quantified using these peaks.

Near-infrared spectroscopy to quantify overall thermal process intensity during high-moisture extrusion of soy protein concentrate.

High-moisture extrusion (HME) is widely used to produce meat analogues. During HME the plant-based materials experience thermal and mechanical stresses. It is complicated to separate their effects on the final products because these effects are interrelated. In this study we hypothesize that the intensity of the thermal treatment can explain a large part of the physicochemical changes that occur during extrusion. For this reason, near-infrared (NIR) spectroscopy was used as a novel method to quantify the thermal process intensity during HME. High-temperature shear cell (HTSC) processing was used to create a partial least squares (PLS) regression curve for processing temperature under controlled processing conditions (root mean standard error of cross-validation (RMSECV) = 4.00 °C, coefficient of determination of cross-validation (R2CV) = 0.97). This PLS regression model was then applied to HME extrudates produced at different screw speeds (200-1200 rpm) and barrel temperatures (100-160 °C) with two different screw profiles to calculate the equivalent shear cell temperature as a measure for thermal process intensity. This equivalent shear cell temperature reflects the effects of changes in local temperature conditions, residence time and thermal stresses. Furthermore, it can be related to the degree of texturization of the extrudates. This information can be used to gain new insights into the effect of various process parameters during HME on the thermal process intensity and extrudate quality.

Water-in-oil Pickering emulsion using ergosterol as an emulsifier solely.

As a crucial component of the fungal cell membranes, ergosterol has been demonstrated to possess surface activity attributed to its hydrophobic region and polar group. However, further investigation is required to explore its emulsification behavior upon migration to the oil-water interface. Therefore, this study was conducted to analyze the interface properties of ergosterol as a stabilizer for water in oil (W/O) emulsion. Moreover, the emulsion prepared under the optimal conditions was utilized to load the water-soluble bioactive substance with the chlorogenic acid as the model molecules. Our results showed that the contact angle of ergosterol was 117.017°, and its dynamic interfacial tension was obviously lower than that of a pure water-oil system. When the ratio of water to oil was 4: 6, and the content of ergosterol was 3.5 % (ergosterol/oil phase, w/w), the W/O emulsion had smaller particle size (438 nm), higher apparent viscosity, and better stability. Meanwhile, the stability of loaded chlorogenic acid was improved under unfavorable conditions (pH 1.2, 90 °C, ultraviolet irradiation, and oxidation), which were 73.87 %, 59.53 %, 62.53 %, and 69.73 %, respectively. Additionally, the bioaccessibility of chlorogenic acid (38.75 %) and ergosterol (33.69 %), and the scavenging rates of the emulsion on DPPH radicals (81.00 %) and hydroxyl radicals (82.30 %) were also enhanced. Therefore, a novel W/O Pickering emulsion was prepared in this work using ergosterol as an emulsifier solely, which has great potential for application in oil-based food and nutraceutical formulations.

Enhancement of soil aggregation and physical properties through fungal amendments under varying moisture conditions.

Soil structure and aggregation are crucial for soil functionality, particularly under drought conditions. Saprobic soil fungi, known for their resilience in low moisture conditions, are recognized for their influence on soil aggregate dynamics. In this study, we explored the potential of fungal amendments to enhance soil aggregation and hydrological properties across different moisture regimes. We used a selection of 29 fungal isolates, recovered from soils treated under drought conditions and varying in colony density and growth rate, for single-strain inoculation into sterilized soil microcosms under either low or high moisture (≤-0.96 and -0.03 MPa, respectively). After 8 weeks, we assessed soil aggregate formation and stability, along with soil properties such as soil water content, water hydrophobicity, sorptivity, total fungal biomass and water potential. Our findings indicate that fungal inoculation altered soil hydrological properties and improved soil aggregation, with effects varying based on the fungal strains and soil moisture levels. We found a positive correlation between fungal biomass and enhanced soil aggregate formation and stabilization, achieved by connecting soil particles via hyphae and modifying soil aggregate sorptivity. The improvement in soil water potential was observed only when the initial moisture level was not critical for fungal activity. Overall, our results highlight the potential of using fungal inoculation to improve the structure of agricultural soil under drought conditions, thereby introducing new possibilities for soil management in the context of climate change.

Ionic Liquid Gating Induces Anomalous Permeation through Membrane Channel Proteins.

Membrane channel proteins (MCPs) play key roles in matter transport through cell membranes and act as major targets for vaccines and drugs. For emerging ionic liquid (IL) drugs, a rational understanding of how ILs affect the structure and transport function of MCP is crucial to their design. In this work, GPU-accelerated microsecond-long molecular dynamics simulations were employed to investigate the modulating mechanism of ILs on MCP. Interestingly, ILs prefer to insert into the lipid bilayer and channel of aquaporin-2 (AQP2) but adsorb on the entrance of voltage-gated sodium channels (Nav). Molecular trajectory and free energy analysis reflect that ILs have a minimal impact on the structure of MCPs but significantly influence MCP functions. It demonstrates that ILs can decrease the overall energy barrier for water through AQP2 by 1.88 kcal/mol, whereas that for Na+ through Nav is increased by 1.70 kcal/mol. Consequently, the permeation rates of water and Na+ can be enhanced and reduced by at least 1 order of magnitude, respectively. Furthermore, an abnormal IL gating mechanism was proposed by combining the hydrophobic nature of MCP and confined water/ion coordination effects. More importantly, we performed experiments to confirm the influence of ILs on AQP2 in human cells and found that treatment with ILs significantly accelerated the changes in cell volume in response to altered external osmotic pressure. Overall, these quantitative results will not only deepen the understanding of IL-cell interactions but may also shed light on the rational design of drugs and disease diagnosis.

Machine learning-aided evaluation of oxidative strength of cold atmospheric plasma-treated water.

Plasma medicine is gaining attraction in the medical field, particularly the use of cold atmospheric plasma (CAP) in biomedicine. The chemistry of the plasma is complex, and the reactive oxygen species (ROS) within it are the basis for the biological effect of CAP on the target. Understanding how the oxidative power of ROS responds to diverse plasma parameters is vital for standardizing the effective application of CAP. The proven applicability of machine learning (ML) in the field of medicine is encouraging, as it can also be applied in the field of plasma medicine to correlate the oxidative strength of plasma-treated water (PTW) according to different parameters. In this study, plasma-treated water was mixed with potassium iodide-starch reagent for color formation that could be linked to the oxidative capacity of PTW. Corresponding images were captured resulting from the exposure of the color-forming agent to water treated with plasma for different time points. Several ML models were trained to distinguish the color changes sourced by the oxidative strength of ROS. The AdaBoost Classifier (ABC) algorithm demonstrated better performance among the classification models used by extracting color-based features from the images. Our results, with a test accuracy of 63.5%, might carry a potential for future standardization in the field of plasma medicine with an automated system that can be created to interpret the oxidative properties of ROS in different plasma treatment parameters via ML.

Impact of an Ionic Liquid Solution on Horseradish Peroxidase Activity.

Horseradish peroxidase (HRP) is an enzyme that oxidizes pollutants from wastewater. A previous report indicated that peroxidases can have an enhancement in initial enzymatic activity in an aqueous solution of 0.26 M 1-ethyl-3-methylimidazolium ethyl sulfate ([EMIm][EtSO4]) at neutral pH. However, the atomistic details remain elusive. In the enzymatic landscape of HRP, compound II (Cpd II) plays a key role and involves a histidine (H42) residue. Cpd II exists as oxoferryl (2a) or hydroxoferryl (2b(FeIV)) forms, where 2a is the predominantly observed form in experimental studies. Intriguingly, the ferric 2b(FeIII) form seen in synthetic complexes has not been observed in HRP. Here, we have investigated the structure and dynamics of HRP in pure water and aqueous [EMIm][EtSO4] (0.26 M), as well as the reaction mechanism of 2a to 2b conversion using polarizable molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations. When HRP is solvated in aq [EMIm][EtSO4], the catalytic water displaces, and H42 directly orients over the ferryl moiety, allowing a direct proton transfer (PT) with a significant energy barrier reduction. Conversely, in neat water, the reaction of 2a to 2b follows the previously reported mechanism. We further investigated the deprotonated form of H42. Analysis of the electric fields at the active site indicates that the aq [EMIm][EtSO4] medium facilitates the reaction by providing a more favorable environment compared with the system solvated in neat water. Overall, the atomic level supports the previous experimental observations and underscores the importance of favorable electric fields in the active site to promote catalysis.

Intermolecular hydrogen bonding behavior of amino acid radical cations.

Amino acid and peptide radicals are of broad interest due to their roles in biochemical oxidative damage, pathogenesis and protein radical catalysis, among others. Using density functional theory (DFT) calculations at the ωB97X-D/def2-QZVPPD//ωB97X-D/def2-TZVPP level of theory, we systematically investigated the hydrogen bonding between water and fourteen α-amino acids (Ala, Asn, Cys, Gln, Gly, His, Met, Phe, Pro, Sel, Ser, Thr, Trp, and Tyr) in both neutral and radical cation forms. For all amino acids surveyed, stronger hydrogen-bonding interactions with water were observed upon single-electron oxidation, with the greatest increases in hydrogen-bonding strength occurring in Gly, Ala and His. We demonstrate that the side chain has a significant impact on the most favorable hydrogen-bonding modes experienced by amino acid radical cations. Our computations also explored the fragmentation of amino acid radical cations through the loss of a COOH radical facilitated by hydrogen bonding. The most favorable pathways provided stabilization of the resulting cationic fragments through hydrogen bonding, resulting in more favorable thermodynamics for the fragmentation process. These results indicate that non-covalent interactions with the environment have a profound impact on the structure and chemical fate of oxidized amino acids.

Coprecipitation of Fe/Cr Hydroxides at Organic-Water Interfaces: Functional Group Richness and (De)protonation Control Amounts and Compositions of Coprecipitates.

Iron/chromium hydroxide coprecipitation controls the fate and transport of toxic chromium (Cr) in many natural and engineered systems. Organic coatings on soil and engineered surfaces are ubiquitous; however, mechanistic controls of these organic coatings over Fe/Cr hydroxide coprecipitation are poorly understood. Here, Fe/Cr hydroxide coprecipitation was conducted on model organic coatings of humic acid (HA), sodium alginate (SA), and bovine serum albumin (BSA). The organics bonded with SiO2 through ligand exchange with carboxyl (-COOH), and the adsorbed amounts and pKa values of -COOH controlled surface charges of coatings. The adsorbed organic films also had different complexation capacities with Fe/Cr ions and Fe/Cr hydroxide particles, resulting in significant differences in both the amount (on HA > SA(-COOH) ≫ BSA(-NH2)) and composition (Cr/Fe molar ratio: on BSA(-NH2) ≫ HA > SA(-COOH)) of heterogeneous precipitates. Negatively charged -COOH attracted more Fe ions and oligomers of hydrolyzed Fe/Cr species and subsequently promoted heterogeneous precipitation of Fe/Cr hydroxide nanoparticles. Organic coatings containing -NH2 were positively charged at acidic pH because of the high pKa value of the functional group, limiting cation adsorption and formation of coprecipitates. Meanwhile, the higher local pH near the -NH2 coatings promoted the formation of Cr(OH)3. This study advances fundamental understanding of heterogeneous Fe/Cr hydroxide coprecipitation on organics, which is essential for successful Cr remediation and removal in both natural and engineered settings, as well as the synthesis of Cr-doped iron (oxy)hydroxides for material applications.

Impact of four different extraction methods and three different reconstitution solvents on the untargeted metabolomics analysis of human and rat urine samples.

Unsuitable sample preparation may result in loss of important analytes and consequently affect the outcome of untargeted metabolomics. Due to species differences, different sample preparations may be required within the same biological matrix. The study aimed to compare the in-house sample preparation method for urine with methods from literature and to investigate the transferability of sample preparation from human urine to rat urine. A total of 12 different conditions for protein precipitation were tested, combining four different extraction solvents and three different reconstitution solvents using an untargeted liquid-chromatography high resolution mass spectrometry (LC-HRMS) metabolomics analysis. Evaluation was done based on the impact on feature count, their detectability, as well as the reproducibility of selected compounds. Results showed that a combination of methanol as extraction and acetonitrile/water (75/25) as reconstitution solvent provided improved results at least regarding the total feature count. Additionally, it was found that a higher amount of methanol was most suitable for extraction of rat urine among the tested conditions. In comparison, human urine requires significantly less volume of extraction solvent. Overall, it is recommended to systematically optimize both, the extraction method, and the reconstitution solvent for the used biofluid and the individual analytical settings.

Comparative near Infrared (NIR) spectroscopy calibrations performance of dried and undried forage on dry and wet matter bases.

The application of Near Infrared (NIR) spectroscopy for analyzing wet feed directly on farms is increasingly recognized for its role in supporting harvest-time decisions and refining the precision of animal feeding practices. This study aims to evaluate the accuracy of NIR spectroscopy calibrations for both undried, unprocessed samples and dried, ground samples. Additionally, it investigates the influence of the bases of reference data (wet vs. dry basis) on the predictive capabilities of the NIR analysis. The study utilized 492 Corn Whole Plant (CWP) and 405 High Moisture Corn (HMC) samples, sourced from various farms across Italy. Spectral data were acquired from both undried, unground and dried, ground samples using laboratory bench NIR instruments, covering a spectral range of 1100 to 2498 nm. The reference chemical composition of these samples was analyzed and presented in two formats: on a wet matter basis and on a dry matter basis. The study revealed that calibrations based on undried samples generally exhibited lower predictive accuracy for most traits, with the exception of Dry Matter (DM). Notably, the decline in predictive performance was more pronounced in highly moist products like CWP, where the average error increased by 60-70%. Conversely, this reduction in accuracy was relatively contained (10-15%) in drier samples such as HMC. The Standard Error of Cross-Validation (SECV) values for DMres, Ash, CP, and EE were notably low, at 0.39, 0.30, 0.29, 0.21% for CWP and 0.49, 0.14, 0.25, 0.14% for HMC, respectively. These results align with previous studies, indicating the reliability of NIR spectroscopy in diverse moisture contexts. The study attributes this variance to the interference caused by water in 'as is' samples, where the spectral features predominantly reflect water content, thereby obscuring the spectral signatures of other nutrients. In terms of calibration development strategies, the study concludes that there is no significant difference in predictive performance between undried calibrations based on either 'dry matter' or 'as is' basis. This finding emphasizes the potential of NIR spectroscopy in diverse moisture contexts, although with varying degrees of accuracy contingent upon the moisture content of the analyzed samples. Overall, this research provides valuable insights into the calibration strategies of NIR spectroscopy and its practical applications in agricultural settings, particularly for on-farm forage analysis.