This study aimed to evaluate the suitability of sea buckthorn by-products, including pomace without seeds (SBPW), pomace with seeds (SBPS), and leaves (SBL) as potential livestock feed ingredients. Proximate composition, amino acids, fatty acids, minerals, vitamins, and metabolizable energy (ME) were assessed and compared to the daily requirements of livestock, assuming an inclusion rate of 2.5% of dry matter intake in different animal species. SBPW exhibited the highest metabolizable energy (13.7–17 MJ/kg DM across species), exceeding 2.5% of daily requirements in most animal species, suggesting it is a good energy source, whereas SBL contributed the least (8.1–13.6 MJ/kg DM across species). SBL was mineral rich, with notably elevated Fe, Ca, Mg, and Mn concentrations, supporting its use as a mineral supplement, whereas both pomaces contained low mineral levels. SBL and especially SBPS revealed promising amino acid profiles, showing potential as protein source, meeting 2.5% daily amino acid requirements across most categories, especially gilts and pregnant sows. SBPS contained high levels of arginine (31.4 g/kg DM) and lysine (11.3 g/kg DM), while SBL also supplied substantial amounts of essential amino acids (e.g., lysine 10.2 g/kg DM). Sulfur-containing amino acids were limiting in all residues, with methionine especially low (e.g., SBPW 0.9 g/kg DM). SBPW provided insufficient essential amino acids for most categories, suggesting it would have to be combined with protein feeds to achieve a balanced ration. SBPS was rich in polyunsaturated (notably alpha-linolenic: 1.39 g/kg DM) fatty acids, while SBPW contained more monounsaturated and SBL more saturated fats. Both pomaces provided similar linoleic acid (≈2.5 g/kg DM), exceeding pig but not poultry requirements at 2.5% dry matter intake. All residues were rich in vitamin E, particularly SBPW (720 mg/kg DM), and pomaces contained substantial β-carotene (SBPW 258 mg/kg DM, SBPS 151 mg/kg DM), with potential benefits for gestating animals and those experiencing high oxidative stress. Overall, this study provides an initial assessment of sea buckthorn residues as alternative feed resources and offers guidance for their strategic inclusion in animal diets supporting more sustainable, circular feeding systems.

Continued research into structurally and functionally diverse biosurfactants is crucial to identify new biosurfactants that are cost-competitive and suitable for diverse industrial niches. Herein, we report the successful application of wood waste torrefied at 225 °C (T225) and food waste (FWS) as media components in three different formulationsT225, T225-FWS, and FWSfor the concomitant production of rhamnolipids and biopolymers by Burkholderia thailandensis E264. The rhamnolipids (carbon chain length C8-C16) exhibited media-dependent congener differences, and the biopolymers were identified to be glycolipopeptides with molecular weights of 4.4 × 105-5.5 × 105 Da. T225-FWS exhibited the highest thermostability and retained ∼30% of its residual mass at 400 °C, suggesting potential high-temperature applications, including oil recovery and industrial cleaning. Rhamnose was the dominant sugar in T225 and T225-FWS glycolipopeptides, whereas in FWS, galactose was dominant. FAME analysis by GC-MS revealed lipid chains ranging from C12 to C18 in the biopolymers. All three biopolymers were anionic and possessed β-hemolytic, emulsification, and drop-collapse activities against crude oil and engine oil. Moreover, T225, T225-FWS, and FWS biopolymers could reduce the surface tension of water up to 36.8, 35.4, and 48.5 mN/m, respectively. This study could lead to resource-efficient synthesis of structurally and, hence, functionally tunable biosurfactants from low-cost, completely waste-derived media in biorefinery operations.

Moisture content influences the chemical and physical properties of various natural products. It affects the stability, mechanical properties, and biodegradability of biologically originated polymers. Lignin, a major component of plant biomass, is a renewable resource with potential applications in sustainable materials and energy production. Accurate determination of water or moisture content in lignin is critical for assessing its quality, processing behavior, and application potential, and a fast and convenient method for this is therefore desirable. In this study, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy combined with partial least squares regression (PLS-R) was developed as a unified method capable of reproducing results from all three conventional water/moisture determination techniques: vaporization-based coulometric Karl Fischer titration (vap-C-KFT), oven drying (7 h and 48 h), and freeze drying, covering four parameters in total. Spectral data from reference lignin samples were used to calibrate four chemometric models using OPUS software, with each model corresponding to one of the conventional water or moisture determination methods. The models demonstrated good predictive performance and robustness, with root mean square error of cross-validation (RMSECV) values ranging from 0.53% to 0.66%, and root mean square errors of prediction (RMSEP) values of 0.63% for vap-C-KFT, 0.70% for freeze drying, 0.33% for oven drying 48 h, and 1.0% for oven drying 7 h. These results demonstrate that ATR-FTIR-PLS-R provides a rapid, simple, and cost-effective alternative for accurately predicting the water and moisture content of lignin across multiple established reference methods.

N-Hydroxysuccinimidyl (NHS) esters are widely employed in peptide coupling; however, their behavior in derivatization for high-performance liquid chromatography (HPLC) analysis differs from the synthesis point of view. This work evaluated the performance of NHS derivatization reagents for amino compounds using liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI–MS/MS); later, their E/Z photoisomerization was studied. These reagents were azobenzene-based carbamates (AzoA and AzoB), or esters (AzoC and AzoD), fluorescent NHS-esters incorporating Coumarin and Pyrene moieties. It was observed that NHS-esters are susceptible to different side reactions (alkaline hydrolysis, alcoholysis, and transesterification), which later affect their performance, especially if the goal is to analyze β-Alanine (β-Ala). To highlight this potential pitfall for analytical chemists, the extent of these side reactions was studied in different conditions: e.g., the base used, aqueous borate buffer or tertiary amines if the reaction is carried out at room temperature or 50 °C, and with or without alcohol. It was found that, in contrast to NHS-esters, NHS-carbamates are free from β-Ala interfering (confirmed with first-time synthesized carbamate AzoA). Furthermore, the study explored replacing the NHS group in AzoD ester (commercially known as Dabcyl-OSu, Dabcyl-SE or Dabcyl-NHS) with 1-hydroxybenzotriazolyl leaving group, creating AzoD-OBt. The LC–ESI–MS/MS study shows that the AzoD-OBt reagent can overcome the side reaction of NHS-ester, which would benefit the derivatization yield toward the analytes.

Vervain (Verbena officinalis L., Verbenaceae family) is a perennial plant which grows widely in Europe. It is rich in iridoids, phenolic acids, phenylpropanoid glycosides, flavonoids and terpenoids. Verbena has traditionally been used in folk medicine to calm the nervous system, but there is a lack of scientific data about it. The aim of this study was to explore and characterise the chemical profile and neurotropic effects of V. officinalis dry extracts and their amino acid-based preparations. We determined a total of eight main phenolic compounds and 17 amino acids in the V. officinalis dry extracts. To evaluate the neurotropic effects of the verbena extracts, the following behavioural pharmacology tests were used: Open Field Test, Elevated Plus Maze, Black-and-White Box Test and Tail Suspension Test. The dry aqueous–ethanolic extract (extractant 70% ethanol) demonstrated strong anxiolytic and antidepressant effects, while its dry modified extracts with valine and arginine consistently exhibited pronounced sedative activity across all studies. For example, the Tail Suspension Test demonstrated that the total immobility time in animals receiving the dry aqueous–ethanolic extract was the lowest, being 1.22-fold (p < 0.05) lower than in control animals and 2.25-fold (p < 0.05) lower than in the animals treated with the reference drug preparation (“Sedaphyton”). A novel aqueous-based gel formulation feasible for semi-solid extrusion (SSE) 3D printing was designed. This printing gel enables the fabrication of new oral dosage forms for V. officinalis dry extracts. The effects of pharmaceutical preparations on the human central nervous system require clinical studies.

Aquatic dissolved organic matter (DOM) is an ultracomplex mixture of compounds covering a wide range of masses and with an unknown extent of isomeric complexity, making its structural elucidation and quantification highly challenging. Electrospray ionization high-resolution mass spectrometry (ESI-HRMS) has advanced DOM analysis, but accurate concentration determination remains limited by the lack of a response factor correction. Here, we address this limitation by introducing novel deuterated compounds as internal standards that mimic DOM structures. Using a d5-labeled compound free of isobaric interferences in DOM, we assessed ionization suppression in various aquatic sample extracts and improved concentration-based linearity in a coastal DOM reference material. Our results show that deuterated carboxylic acid-rich standards enable "pseudoquantification" by correcting for ionization suppression and instrument drift. Applying this approach, we estimate that DOM consists of 20-30% acids in river, coastal, and deep-ocean reference samples using an Orbitrap system. The same samples were estimated to contain 11-24% acids using 15T FT-ICR-MS, highlighting platform differences. Additionally, we establish a ∼1 ng L-1 feature detection limit for DOM compounds in seawater via a standard LC-MS gradient method. These findings demonstrate that deuterated standards provide a simple, practical way to improve DOM pseudoquantification, enhancing our understanding of its chemical composition in environmental studies.

This work focused on understanding the influence of dyeing methods and dye origin on the fluorescence spectra of wool yarns dyed with bloodred webcap (Cortinarius sanguineus), and exploring the potential and limitations of non-invasive fluorescence spectrometry in differentiating natural dyes on wool textiles. Wool fibers dyed with bloodred webcap specimens from five different geographic origins or with emodin standards, and using nine different mordanting procedures, were investigated using three-dimensional (3D) fiber optic fluorescence spectrometry, resulting in a dataset of 20 excitation-emission matrices (EEMs). In addition, the effect of self-absorption on EEMs of this dataset was investigated. The ability of the fluorescence-based method to discriminate dyes from different taxonomic families was also assessed by combining the bloodred webcap data with EEMs from a more diverse dataset which includes 18 different species. The dyeing method had some effect on the collected EEMs, potentially complicating the identification of a dye source in a historical textile if the mordanting procedure is unknown. The geographic origin of the fungi samples in this dataset showed minimal impact on the fluorescence spectra. Principal component analysis of a more diverse set of EEMs was helpful in differentiating dye taxonomic families, indicating utility of fluorescence spectrometry in qualitative determinations of some natural dye sources. While fluorescence alone may be insufficient for the unequivocal identification of natural dyes, this non-invasive, non-destructive, and rapid technique can provide useful additional information for identification when used in combination with other analytical techniques.

In recent years, lignin analysis utilizing quantitative nuclear magnetic resonance (qNMR) has attracted considerable interest and has been the subject of numerous studies. However, evaluating the measurement uncertainty of qNMR results of lignin remains a challenge. Specifically, uncertainty originating from lignin sampling or subsampling has been overlooked in a large majority of articles. Although lignin is a reasonably homogeneous substance, it is nevertheless a solid, and individual samples collected from the same bulk may have somewhat different compositions depending on mixing and the amount of sample taken. The objective of this study was to evaluate the influence of sampling uncertainty on qNMR analysis of lignin-based analysis as a case study, with an exclusive focus on the relative quantification method. The results from this study demonstrate that sample-to-sample variations can contribute to approximately half of the variability in actual qNMR measurements. The relative standard deviation (RSD) of sample-to-sample variability was 2.4%. In contrast, the other sources of variability related to qNMR, including measurement, baseline irregularities, and partial peak overlap, caused an RSD of 4.4%. The total variability RSD was 5.0%. In this article, two calculation approaches were presented for evaluating the uncertainty due to sampling from replicate measurement data of different samples, which may be helpful for practitioners in the field.

Elevated concentrations of pharmaceutically active compounds (PhACs) in the water bodies are posing a serious threat to the aquatic microbiota and other organisms. In this context, anaerobic ammonium oxidizing (anammox) bacteria carry a great potential to degrade PhACs through their innate metabolic pathways. This study investigates the influence of short-term exposure to lower and higher concentrations (0.8 mg L-1, 0.06 mg L-1, respectively) of antibiotics on the anammox process under distinct operational conditions (starvation/non-starvation) in moving bed biofilm reactor (MBBR). During batch operations that lasted for up to 6 h, the total nitrogen removal efficiency (TNRE) and total nitrogen conversion rate (TNCR) reached a maximum of 93 ± 5 % and 6.97 ± 1.30 mg N g-1 TSS d-1, respectively. Evidently, at higher PhAC levels, the anammox process was active, and up to 75 % PhAC removal efficiency was obtained within 6 h of the batch cycle. Most importantly, the anammox biofilm effectively eliminated the PhACs compounds, i.e., ciprofloxacin (CIP), ofloxacin (OFL), and norfloxacin (NOR) present at higher (0.8 mg L-1) and lower (0.06 mg L-1) total PhACs (sum of CIP, NOR, OFL) concentrations. Furthermore, 16S rRNA sequencing analyses showed a mixture of nitrifying, denitrifying, and anammox bacterial commodities enriched on the carriers' surface with a high relative abundance of Candidatus Brocadia, primarily responsible for catalyzing the anammox process. This study showed the intricate relationship between PhAC concentrations, TNCR, and antibiotic elimination in the wastewater treatment, and the results obtained set up a new breakthrough in wastewater treatment. Future research should investigate the mechanisms that underlie the anammox biofilms' resistance to various types of PhACs and investigate the long-term stability and scalability of this process with real wastewater influents.

Antibiotic resistance refers to the ability of microbes to grow in the presence of an antibiotic that would have originally killed or inhibited the growth of these microorganisms. Microorganisms resistant to antibiotics exist in humans, animals and in the environment. Resistant microbes can spread from animals to humans and vice versa either through direct contact or through the environment. Resistant bacteria survive in the body during a course of antibiotics and continue to multiply. Treatment of antibiotic-resistant infections takes more time, costs more, and sometimes may prove impossible. The aim of the AMR-RITA project was to develop recommendations based on scientific evidence including the "One Health" principle for the formulation of policy on antibiotic resistance. In order to achieve the goal, the role of human behaviour, human and animal medicine, and the environment was implicated in the development of antibiotic resistance. The evaluation of the resistance spread routes, risks and levels, and the possible measures to control the spread of antibiotic resistance were identified. Topics related to antibiotic resistance were analysed in medicine, veterinary medicine and environment subsections. Existing data were combined with new data to assess the transmission routes and mechanisms of antibiotic resistance. For this purpose, samples were collected from people, animals, food, and the environment. The analysis of the samples focused on the main resistent organisms, resistance genes and antibiotic residues. As a result of the study, we conclude that the use of antibiotics in Estonia is generally low compared to other European countries. However, there are bottlenecks that concern both human and veterinary medicine. In both cases, we admit that for some diagnoses there were no treatment guidelines and antibiotics were used for the wrong indications. The lack of specialists of clinical microbiology is a problem in Estonain hospitals. For example, many hospitals lack an infection control specialist. The major worrying trends are the unwarranted use of broad-spectrum antibiotics in humans and the high use of antibiotics critical for human medicine (cephalosporins, quinolones) in the teratment of animals. If more antibiotics are being used, resistance will also spread. We found that those cattle farms that use more cephalosporins also have higher levels of resistance (ESBL-mediated resistance). It also turned out that genetically close clusters of bacteria are often shared by humans and animals. This is evidence of a transfer of resistance between species. However, such transfer occurs slowly, and we did not detect any transfer events in the recent years. Antibiotic residues, just like other drug residues, can reach the environment. The use of slurry and composted sewage sludge as fertilizer are the main pathways. We detected fluroquinolones and tetracyclines in comparable concentrations in slurry and uncomposted sewage sludge. Composting reduces the content of drug residues, and the efficiency of the process depends on the technology used. In addition to antibiotic residues, we also determined some other drug residues accumulating in the environment. High levels of diclofenac and carbamazepine in surface water are a special concern. These are medicines for human use only, so they reach the environment through sewage treatment plants. Based on the results obtained during the research, we propose a series of evidence-based recommendations to the state for the formulation of antimicrobial resistance policy. We propose that Estonia needs sustainable AMR surveillance institution, which (1) continuously collects and analyses data on the use of antimicrobials and antimicrobial resistance and provides regular feedback to relevant institutions (state, health and research institutions), (2) assesses the reliability of the data and ensures carrying out additional and confirming studies, (3) coordinates the activities of national and international research and monitoring networks and projects. We recommend creation of a competence centre that would deal with the topic of AMR across all fields. This should also include funding for research.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThe study was commissioned and funded by the Estonian Research Agency with support from the European Regional Development Fund of the program "Strengthening of sectoral research and development" (RITA) activity 1 "Strategic TA supporting activity". The study was completed in the period July 1, 2019 - June 31, 2022 to implement the objectives of the Ministry of Rural Affairs, the Ministry of Social Affairs the Ministry of the Environment. The research budget was 947,370 euros.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:University of Tartu Ethics CommitteeI 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.YesI 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).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Global deficit of fertilizers is evident and as a result of the war in Ukraine the situation is worsening. At the same time, not all the possible major resources of nutrients have found appropriate application in agriculture, as for example sewage and its sludge, which are among the most promising sources of P, N and organic matter. Sewage sludge (SS) usage as an agricultural fertilizer has a number of limitations. Amongst them persistent organic pollutants (POPs) have a special role due to their ability to affect living organisms. Ongoing work directed to eliminating POPs present in SS is widely reported, but due to the complexity of these compounds no universal success has been achieved. In the case of several undesired residual compounds promising results have been gained using SS composting with different bulking agents. The current study was concentrated on the degradation of some widely used persistent POPs that are present in SS, namely carbamazepine (CBZ) and triclosan (TCS). Peat alone as a bulking agent widely used in the mixture with sewage sludge in Estonia is not suitable for initiating the process of composting. Additions of carbon-rich matter, as for example draff, might provide the relevant conditions needed for launching this process.

Recuperating bioactive compounds and nutrients from agricultural by-products for livestock feed is a potential solution for their reuse. Three of the agricultural by-products whose potential needs to be further explored are hempseed (Cannabis sativa L.) hull and sea buckthorn (Hippophae rhamnoides) pomace and leaves. Free amino compounds content has not yet been explored for these samples and they were compared to oat (Avena sativa) bran and apple (Malus domestica Borkh.) pomace, since they have already been employed as livestock feed. For the analysis of amino compounds, the samples were derivatized with diethyl ethoxymethylenemalonate (DEEMM) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, in an approach called derivatization-targeted analysis. Overall, 46 free amino compounds were quantified, and a correlation matrix revealed that amino compounds’ profile of sea buckthorn leaves resembles that of oat bran, indicating that it is the most suitable agricultural by-product to be employed as feed supplement.

Amino compounds are of significant interest in dietary, clinical, and quality control fields. Efficient extraction is crucial for comprehensive metabolomics, especially for amino acids and biogenic amines, but traditional solid-phase extraction (SPE) methods are costly and require large solvent volumes. Miniaturized SPE techniques, like pipette-tip micro-solid-phase extraction (PT-µ-SPE), offer promising alternatives by improving throughput and reducing solvent and sorbent usage. This study presents PT-µ-SPE for the screening and quantification of amino compounds in bee products, particularly honey. The method involves derivatization with diethyl ethoxymethylenemalonate (DEEMM) and analysis using liquid chromatography-triple quadrupole mass spectrometry. Silica-based SCX sorbents were effective for a broad range of amino compounds, while WCX sorbents were better for aliphatic amines. The method's extraction efficiency was assessed across sample loading, washing, and elution solution, with recovery rates of 70 - 120% in oat bran, sea buckthorn leaves, and honey extracts. Matrix effects were observed for four amino compounds in honey. Limits of detection (LoD) and quantification (LoQ) ranged from 0.37 to 57 µg L⁻¹ and 1.13 to 174 µg L⁻¹, respectively. Covering 48 amino compounds under different PT-µ-SPE conditions, this method has been applied to several samples, demonstrating accuracy, environmental sustainability, cost-effectiveness, portability, and versatility in amino compound analysis.

Apple pomace represents an underexploited source of bioactive compounds. This study examines the optimization of total phenolic content (TPC) and antioxidant extraction yield of apple pomace (variety: Belorusskoje malinovoje) using response surface methodology. The green extraction technique used was ultrasound-assisted extraction, and it was compared with conventional solvent extraction. The impact of extraction time and amplitude of ultrasound-assisted extraction on the yield of polyphenols and antioxidants has been evaluated. Total phenolic content was determined using an established TPC assay. The antioxidant activity of the apple pomace was determined using established assays 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+). Furthermore, the potential of apple pomace as a feed material was explored by assessing its nutritional composition, vitamins, minerals, fatty acids, and amino acid content. The extraction of antioxidants and phenolic compounds was efficiently optimized using RSM. The optimal conditions for TPC and DPPH• analyses were achieved with an extraction time of 17.5 min and an ultrasound-assisted extraction amplitude of 20%. Optimal conditions for ABTS•+ were 5 min extraction time and 20% amplitude. Conventional and ultrasound-assisted extraction methods yielded comparable results. Moreover, apple pomace exhibits potential as a feed ingredient despite its modest protein content. This study contributes to the utilization of apple pomace by providing additional information on its antioxidant content and nutritional composition, thus contributing to its sustainable utilization in various industries, especially the livestock feed sector.

Aims C‐reactive protein (CRP) is used to determine the effect of antibiotic treatment on sepsis in neonates/infants. We aimed to develop pharmacokinetic–pharmacodynamic (PKPD) model of meropenem and CRP in neonates/infants and evaluate its predictive performance of CRP dynamics. Methods Data from neonates/infants treated with meropenem in 3 previous studies were analysed. To the previously developed meropenem PK models, the addition of turnover, transit or effect compartment, delay differential equation PD models of CRP as a function of meropenem concentration or its cumulative area under the curve (AUC) were evaluated. The percentage of neonates/infants (P 0.1 , P 0.2 ) in whom the ratio of the fifth day CRP to its peak value was predicted with an error of <0.1 (<0.2) was calculated. Results A total of 60 meropenem treatment episodes (median [range] gestational age 27.6 [22.6–40.9] weeks, postnatal age 13 [2–89] days) with a total of 351 CRP concentrations (maximum value 65.5 [13–358.4] mg/L) were included. Turnover model of CRP as a function of meropenem cumulative AUC provided the best fit and included CRP at the start of treatment, use of prior antibiotics, study and causative agent Staphylococcus aureus or enterococci as covariates. Using meropenem population predictions and data available at 0, 24, 48, 72 h after the start of treatment, P 0.1 (P 0.2 ) was 36.4, 36.4, 60.6 and 66.7% (70.0, 66.7, 72.7 and 78.7%), respectively. Conclusion The developed PKPD model of meropenem and CRP as a function of meropenem cumulative AUC incorporating several patient characteristics predicts CRP dynamics with an error of <0.2 in most neonates/infants.

Glacial groundwater can mobilize deep-seated methane from beneath glaciers and permafrost in the Arctic, leading to atmospheric emissions of this greenhouse gas. We present a temporal, hydro-chemical dataset of methane-rich groundwater collected during two melt seasons from a high Arctic glacial forefield to explore the seasonal dynamics of methane emissions. We use methane and ion concentrations and the isotopic composition of water and methane to investigate the sources of groundwater and the origin of the methane that the groundwater transports to the surface. Our results suggest two sources of groundwater, one shallow and one deep, which mix, and moderate methane dynamics. During summer, deep methane-rich groundwater is diluted by shallow oxygenated groundwater, leading to some microbial methane oxidation prior to its emergence at the surface. Characterization of the microbial compositions in the groundwater shows that microbial activity is an important seasonal methane sink along this flow-path. In the groundwater pool studied, we found that potential methane emissions were reduced by an average of 29% (±14%) throughout the summer due to microbial oxidation. During winter, deep groundwater remains active while many shallow systems shut down due to freezing, reducing subsurface methane oxidation, and potentially permitting larger methane emissions. Our results suggest that ratios of the different groundwater sources will change in the future as aquifer capacities and recharge volumes increase in a warming climate.

Rhodotorula toruloides is a non-conventional, oleaginous yeast able to naturally accumulate high amounts of microbial lipids. Constraint-based modeling of R. toruloides has been mainly focused on the comparison of experimentally measured and model predicted growth rates, while the intracellular flux patterns have been analyzed on a rather general level. Hence, the intrinsic metabolic properties of R. toruloides that make lipid synthesis possible are not thoroughly understood. At the same time, the lack of diverse physiological data sets has often been the bottleneck to predict accurate fluxes. In this study, we collected detailed physiology data sets of R. toruloides while growing on glucose, xylose, and acetate as the sole carbon source in chemically defined medium. Regardless of the carbon source, the growth was divided into two phases from which proteomic and lipidomic data were collected. Complemental physiological parameters were collected in these two phases and altogether implemented into metabolic models. Simulated intracellular flux patterns demonstrated the role of phosphoketolase in the generation of acetyl-CoA, one of the main precursors during lipid biosynthesis, while the role of ATP citrate lyase was not confirmed. Metabolic modeling on xylose as a carbon substrate was greatly improved by the detection of chirality of D-arabinitol, which together with D-ribulose were involved in an alternative xylose assimilation pathway. Further, flux patterns pointed to metabolic trade-offs associated with NADPH allocation between nitrogen assimilation and lipid biosynthetic pathways, which was linked to large-scale differences in protein and lipid content. This work includes the first extensive multi-condition analysis of R. toruloides using enzyme-constrained models and quantitative proteomics. Further, more precise kcat values should extend the application of the newly developed enzyme-constrained models that are publicly available for future studies.

The conjugates of an adenosine mimetic and oligo-l-arginine or oligo-d-arginine (ARCs) were initially designed in our research group as inhibitors and photoluminescent probes targeting basophilic protein kinases. Here, we explored a panel of ARCs and their fluorescent derivatives in biochemical assays with members of the protein arginine methyltransferase (PRMT) family, focusing specifically on PRMT1. In the binding/displacement assay with detection of fluorescence anisotropy, we found that ARCs and arginine-rich peptides could serve as high-affinity ligands for PRMT1, whereas the equilibrium dissociation constant values depended dramatically on the number of arginine residues within the compounds. The fluorescently labeled probe ARC-1081 was displaced from its complex with PRMT1 by both S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH), indicating binding of the adenosine mimetic of ARCs to the SAM/SAH-binding site within PRMT1. The ARCs that had previously shown microsecond-lifetime photoluminescence in complex with protein kinases did not feature such property in complex with PRMT1, demonstrating the selectivity of the time-resolved readout format. When tested against a panel of PRMT family members in single-dose inhibition experiments, a micromolar concentration of ARC-902 was required for the inhibition of PRMT1 and PRMT7. Overall, our results suggest that the compounds containing multiple arginine residues (including the well-known cell-penetrating peptides) are likely to inhibit PRMT and thus interfere with the epigenetic modification status in complex biological systems, which should be taken into consideration during interpretation of the experimental data.

The oleaginous yeast Rhodotorula toruloides is a promising host for sustainable bioproduction due to its capacity to naturally utilize xylose present in lignocellulosic biomass, an abundant and renewable resource. However, its xylose consumption pathway is still not completely understood. To better understand the potential limitations in xylose utilization in R. toruloides , heterologous xylose reductase from Scheffersomyces stipitis , together with the native and heterologous xylulokinases from three different microorganisms ( Scheffersomyces stipitis, Candida intermedia , and Escherichia coli ) were overexpressed solely and in combination. The overexpression of xylulokinases showed more significant improvements in terms of xylose consumption rate compared to the single overexpression of xylose reductase. When the heterologous xylulokinase from Escherichia coli was overexpressed, the specific xylose consumption rate was improved by 66% and the maximum specific growth rate by 30% compared to the parental strain. The xylose specific consumption rate increased by 146% and the maximum specific growth rate increased by 118% when heterologous genes for xylose reductase and xylulokinase from E. coli were overexpressed together. These results suggest that the low expression of xylulokinase in R. toruloides , which has been reported previously, could limit its sugar consumption, while supporting higher lipid accumulation in this yeast.### Competing Interest StatementThe authors have declared no competing interest.

Multidimensional fluorescence spectroscopy was assessed as a non-invasive and non-destructive method for the analysis of components in natural textile dyes. Results demonstrate that components in the natural dyes fluoresce and wool's intrinsic fluorescence is, in many cases, not a considerable analytical interferent. In the case of some self-dyed reference yarns, like those dyed with northern and lady's bedstraws, wood horsetail, safflower, salted shield lichen, dyer's madder and cochineal, the fluorescence excitation-emission matrices (EEMs) are sufficiently characteristic for using them as a primary means of identification (or assignment to a family of dyes). With most of the studied yellow and green dyes (heather, silver birch, some bloodred webcap treatments, alkanet), however, the spectra can be used as additional information for identification. This study reports multidimensional fluorescence data for a collection of wools dyed with natural dyes (31 dyed wool yarn samples that were self-dyed with 18 different natural dyes) that were used as references in a case study of two historical textiles for which liquid chromatography-mass spectrometry was used as a confirmatory technique. Given its utility as a rapid and non-destructive/non-invasive method with information-rich multidimensional EEM output, the front-face fluorescence spectroscopy of surfaces using a fiber optic probe is a promising technique for the analysis of dyes on cultural heritage textiles.