Tentative Identification Research Articles

Untargeted analysis and tentative identification of unknown substances in human tears by ultra-high performance liquid chromatography-high resolution mass spectrometry: Pilot study

In this work, a new approach for the identification of unknown compounds in human tears has been developed and validated using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) linked to an intelligent data acquisition mode (AcquireX DS-dd-MS2) coupled to an automated data processing software (Compound Discoverer™ 3.2). As a pilot research study, four human tear samples from volunteers were analyzed. Data were acquired in both positive and negative ionization modes and exact mass, isotope pattern, and MS2 spectra match were used for the tentative identification. Following this approach, 58 substances were identified, 47 in positive mode and 11 in negative mode, with an estimated concentration ranging from 0.1 to 9000 ng mL−1. Most of them were amino acids, hormones, metabolites, and pharmaceuticals. In order to validate the proposed method, the system suitability was evaluated and 29 commercial analytical standards of the tentatively identified substances were analyzed, of which 28 were confirmed obtaining a high identification accuracy (96.6 %). These results confirm that the screening tool presented in this work can facilitate the discovery of new metabolites, novel potential biomarkers, and substances to which the person is exposed, such as pollutants.

Opacities of singly and doubly ionized neodymium and uranium for kilonova emission modeling

ABSTRACT Even though the electromagnetic counterpart AT2017gfo to the binary neutron star merger GW170817 is powered by the radioactive decay of r-process nuclei, only few tentative identifications of light r-process elements have been made so far. One of the major limitations for the identification of heavy nuclei is incomplete or missing atomic data. While substantial progress has been made on lanthanide atomic data over the last few years, for actinides there has been less emphasis, with the first complete set of opacity data only recently published. We perform atomic structure calculations of neodymium (Z = 60) as well as the corresponding actinide uranium (Z = 92). Using two different codes [flexible atomic code (fac) and hartree–fock-relativistic (hfr)] for the calculation of the atomic data, we investigate the accuracy of the calculated data (energy levels and electric dipole transitions) and their effect on kilonova opacities. For the fac calculations, we optimize the local central potential and the number of included configurations and use a dedicated calibration technique to improve the agreement between theoretical and available experimental atomic energy levels (AELs). For ions with vast amounts of experimental data available, the presented opacities agree quite well with previous estimations. On the other hand, the optimization and calibration method cannot be used for ions with only few available AELs. For these cases, where no experimental nor benchmarked calculations are available, a large spread in the opacities estimated from the atomic data obtained with the various atomic structure codes is observed. We find that the opacity of uranium is almost double the neodymium opacity.

Dietary Olive Leaf Extract Differentially Modulates Antioxidant Defense of Normal and Aeromonas hydrophila-Infected Common Carp (Cyprinus carpio) via Keap1/Nrf2 Pathway Signaling: A Phytochemical and Biological Link.

Olive leaves are an immense source of antioxidant and antimicrobial bioactive constituents. This study investigated the effects of dietary incorporation of olive leaf extract (OLE) on the growth performance, hematobiochemical parameters, immune response, antioxidant defense, histopathological changes, and some growth- and immune-related genes in the common carp (Cyprinus carpio). A total of 180 fish were allocated into four groups with triplicate each. The control group received the basal diet without OLE, while the other three groups were fed a basal diet with the OLE at 0.1, 0.2, and 0.3%, respectively. The feeding study lasted for 8 weeks, then fish were challenged with Aeromonas hydrophila. The results revealed that the group supplied with the 0.1% OLE significantly exhibited a higher final body weight (FBW), weight gain (WG%), and specific growth rate (SGR) with a decreased feed conversion ratio (FCR) compared to the other groups (p < 0.05). An increase in immune response was also observed in the fish from this group, with higher lysosome activity, immunoglobulin (IgM), and respiratory burst than nonsupplemented fish, both before and after the A. hydrophila challenge (p < 0.05). Similarly, the supplementation of the 0.1% OLE also promoted the C. carpio's digestive capacity pre- and post-challenge, presenting the highest activity of protease and alkaline phosphatase (p < 0.05). In addition, this dose of the OLE enhanced fish antioxidant capacity through an increase in the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and decreased hepatic lipid peroxidation end products (malondialdehyde-MDA), when compared to the control group, both pre- and post-infection (p < 0.05). Concomitantly with the superior immune response and antioxidant capacity, the fish fed the 0.1% OLE revealed the highest survival rate after the challenge with A. hydrophila (p < 0.05). A significant remarkable upregulation of the hepatic sod, nrf2, and protein kinase C transcription levels was detected as a vital approach for the prevention of both oxidative stress and inflammation compared to the infected unsupplied control group (p < 0.05). Interestingly, HPLC and UPLC-ESI-MS/MS analyses recognized that oleuropein is the main constituent (20.4%) with other 45 compounds in addition to tentative identification of two new compounds, namely oleuroside-10-carboxylic acid (I) and demethyl oleuroside-10-carboxylic acid (II). These constituents may be responsible for the OLE exerted potential effects. To conclude, the OLE at a dose range of 0.66-0.83 g/kg w/w can be included in the C. carpio diet to improve the growth, antioxidant capacity, and immune response under normal health conditions along with regulating the infection-associated pro-inflammatory gene expressions, thus enhancing resistance against A. hydrophila.

Unraveling the secrets of plant roots: Simplified method for large scale root exudate sampling and analysis in Arabidopsis thaliana.

Background: Plants exude a plethora of compounds to communicate with their environment. Although much is known about above-ground plant communication, we are only beginning to fathom the complexities of below-ground chemical communication channels. Studying root-exuded compounds and their role in plant communication has been difficult due to the lack of standardized methodologies. Here, we develop an interdisciplinary workflow to explore the natural variation in root exudate chemical composition of the model plant Arabidopsis thaliana. We highlight key challenges associated with sampling strategies and develop a framework for analyzing both narrow- and broad-scale patterns of root exudate composition in a large set of natural A. thaliana accessions. Methods: Our method involves cultivating individual seedlings in vitro inside a plastic mesh, followed by a short hydroponic sampling period in small quantities of ultrapure water. The mesh makes it easy to handle plants of different sizes and allows for large-scale characterization of individual plant root exudates under axenic conditions. This setup can also be easily extended for prolonged temporal exudate collection experiments. Furthermore, the short sampling time minimizes the duration of the experiment while still providing sufficient signal even with small volume of the sampling solution. We used ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) for untargeted metabolic profiling, followed by tentative compound identification using MZmine3 and SIRIUS 5 software, to capture a broad overview of root exudate composition in A. thaliana accessions. Results: Based on 28 replicates of the Columbia genotype (Col-0) compared with 10 random controls, MZmine3 identified 354 metabolites to be present only in Col-0 by negative ionization. Of these, 254 compounds could be annotated by SIRIUS 5 software. Conclusions: The methodology developed in this study can be used to broadly investigate the role of root exudates as chemical signals in plant belowground interactions.

First report of Klebsiella Leaf Streak on Sorghum Caused by Klebsiella variicola in Haryana, India.

Sorghum (Sorghum bicolor [L.] Moench) is one of the top ten cereal crops in the world and is grown for fodder and seed purposes. During the fall of 2019 to 2022, a disease causing small to long streaks on leaves was observed in sorghum fields of Hisar (29° 9' 6.6996'' N, 75° 43' 16.0428'' E), Rohtak (28° 53' 43.8540'' N, 76° 36' 23.8068'' E) and Mohindergarh (28° 16' 6.0492'' N, 76° 9' 3.3552'' E) regions of Haryana between July and October. The reddish brown streaks were observed in the interveinal spaces of upper and lower leaves. The disease incidence reached 20-30% of plants in affected fields. The diseased leaf tissues were disinfected with 70% alcohol and placed in a tube with sterile water. After 30 minutes, 100 µl of the suspension was inoculated onto nutrient agar medium, incubated at 28 ± 2°C for three days, and a pure culture was obtained by restreaking on nutrient agar (Janse, 2005). The rod-shaped gram-negative bacterium with round, cream to white colonies was positive for methyl red, citrate utilization, urease activity, and glucose, lactose, sorbitol, rhamnose and sucrose fermentation tests. The genomic DNA of the bacterial suspension was extracted and 16S rDNA was amplified using universal 27F/1492R primers (Marchesi et al. 1998), resulting in tentative identification as Klebsiella sp. It was further confirmed with PCR amplification of Klebsiella specific primers (F:5'-CGCGTACTATACGCCATGAACGTA-3'; R:5'-ACCGTTGATCACTTCGGTCAGG-3') for gyrA gene (Brisse and Verhoef 2001). Discrete PCR amplicons of 1,500 (16S rDNA) and 300 bp (gyrA) were observed in a 1% (w/v) agarose gel. Forward and reverse DNA sequencing of both amplicons of the Hisar isolate (VMKV101) was carried out using a BDT v3.1 Cycle sequencing kit and consensus sequences were generated by using the program SeqMan Ultra (DNASTAR Lasergene). Sequences of the PCR products were deposited in GenBank with accession numbers MZ569433 (16S rDNA) and OP390080 (gyrA). The 16S rDNA sequence was 97.32% similar to K. variicola strain 13450 (CP026013; 1,450/1,490 bp) and the gyrA sequence had 99.66% similarity to K. variicola strain FH-1 (CP054254; 297/298 bp). A 16S RNA-based phylogenetic tree done by MEGA11 (Tamura et al. 2021) using the Maximum Likelihood method showed that strain VMKV101 clustered with K. variicola type strain F2R9. The complete bacterial genome (GCA025629215), sequenced by the Ion GeneStudio S5 system using Ion 530 chips (Thermo Fisher Scientific), was 99.03% identical by average nucleotide identity (ANI) to the type genome (CP045783) of Klebsiella variicola, with 87.8% genome coverage. For pathogenicity testing, a bacterial suspension (10 ml, 1×107 colony forming units/ml) was injected into the whole whorl after mechanical injury on 15-20 days old seedlings of the susceptible genotype HC-171, then plants were incubated at 35 ± 2°C, >80% relative humidity. Control plants were injected with sterile distilled water. Initial symptoms were observed on leaves of inoculated plants after 5 to 7 days as narrow, small longitudinal reddish brown streaks. As the disease progressed, the streaks on the leaf blade increased in number and size maintaining the reddish brown color. These streaks had slightly wavy margins and were surrounded by bright yellow halos. After 15 to 20 days, the streaks were 0.5 to 2.0 mm wide and 1.0 to 5.0 cm long, occasionally up to 10.0 cm long on both side of the leaves. Over time, neighboring streaks coalesced to form large necrotic areas. All inoculated plants exhibited identical symptoms. No symptoms were observed on control leaves. The reisolated bacterium from diseased sorghum leaves showed exactly the same morphological, biochemical and 16S RNA and gyrA molecular characteristics. To our knowledge, this is the first report of K. variicola causing a leaf streak disease on sorghum. Klebsiella species primarily cause diseases in humans and animals, but K. variicola has been found to incite banana soft rot (Fan et al. 2015) and K. aerogenes to cause stem rot in pearl millet (Malik et al. 2022). Differences of prevalence, spread and control between K. variicola and two other bacteria (Xanthomonas vasicola pv. holcicola causing Bacterial leaf streak; Paraburkholderia andropogonis causing Bacterial leaf stripe) causing leaf streak diseases on sorghum need to be determined. The identification of Klebsiella leaf streak disease lays the groundwork for future investigations into epidemiology and management of K. variicola on sorghum.

Searching for pharmaceutically active products and metabolites in environmental waters of Peru by HRMS-based screening: Proposal for future monitoring and environmental risk assessment

The presence of pharmaceutical active products (PhACs) in the aquatic environment is a matter of current concern, and there is an increasing trend to include these compounds in water quality monitoring programs and environmental risk assessments. Several studies have reported the presence of PhACs in environmental waters worldwide, but only a few studies have focused on Latin American countries. Thus, available information on the occurrence of parent pharmaceuticals, especially their metabolites, is very scarce. Peru is one of the less monitored countries in terms of contaminants of emerging concern (CECs) in waters, and only one study has been found, which was focused on the quantification of selected PhACs in urban wastewater and surface water. The aim of this work is to complement the previous data reported on PhACs in the aquatic environment by application of a wide-scope high-resolution (HRMS)-based screening, making use of target and suspect approaches. In the present work, 30 pharmaceuticals, drugs or other compounds (sweeteners, UV filters, etc.) and 21 metabolites have been identified, with antibiotics (and metabolites) being the most prevalent compounds. The use of liquid chromatography (LC) coupled to ion mobility-HRMS allowed the tentative identification of parent compounds and metabolites, for which the analytical reference standard was not available, with a high level of confidence in their identification. Based on the results obtained, a strategy for the monitoring of PhACs and relevant metabolites in environmental waters from Peru and for subsequent risk assessment is proposed. Our data will also help to focus future studies to evaluate the removal efficiency of wastewater treatment plants and the impact of treated water in receiving water bodies.

Human metabolism and basic pharmacokinetic evaluation of AP-238: A recently emerged acylpiperazine opioid.

As a consequence of recently implemented legal restrictions on fentanyl analogs, a new generation of acylpiperazine opioids appeared on the illicit drug market. AP-238 was the latest opioid in this series to be notified by the European Early Warning System in 2020 and was involved in an increasing number of acute intoxications. AP-238 metabolism was investigated to provide useful markers of consumption. For the tentative identification of the main phase I metabolites, a pooled human liver microsome assay was performed. Further, four whole blood and two urine samples collected during post-mortem examinations and samples from a controlled oral self-administration study were screened for anticipated metabolites. In total, 12 AP-238 phase I metabolites were identified through liquid chromatography-quadrupole time-of-flight mass spectrometry in the in vitro assay. All of these were confirmed in vivo, and additionally, 15 phase I and five phase II metabolites were detected in the human urine samples, adding up to a total of 32 metabolites. Most of these metabolites were also detected in the blood samples, although mostly with lower abundances. The main in vivo metabolites were built by hydroxylation combined with further metabolic reactions such as O-methylation or N-deacylation. The controlled oral self-administration allowed us to confirm the usefulness of these metabolites as proof of intake in abstinence control. The detection of metabolites is often crucial to documenting consumption, especially when small traces of the parent drug can be found in real samples. The in vitro assay proved to be suitable for the prediction of valid biomarkers of novel synthetic opioid intake.

Organic contaminants in bio-based fertilizer treated soil: Target and suspect screening approaches

Using bio-based fertilizer (BBF) in agricultural soil can reduce the dependency on chemical fertilizer and increase sustainability by recycling nutrient-rich side-streams. However, organic contaminants in BBFs may lead to residues in the treated soil. This study assessed the presence of organic contaminants in BBF treated soils, which is essential for evaluating sustainability/risks of BBF use. Soil samples from two field studies amended with 15 BBFs from various sources (agricultural, poultry, veterinary, and sludge) were analyzed. A combination of QuEChERS-based extraction, liquid chromatography quadrupole time of flight mass spectrometry-based (LC-QTOF-MS) quantitative analysis, and an advanced, automated data interpretation workflow was optimized to extract and analyze organic contaminants in BBF-treated agricultural soil. The comprehensive screening of organic contaminants was performed using target analysis and suspect screening. Of the 35 target contaminants, only three contaminants were detected in the BBF-treated soil with concentrations ranging from 0.4 ng g−1 to 28.7 ng g−1; out of these three detected contaminants, two were also present in the control soil sample. Suspect screening using patRoon (an R-based open-source software platform) workflows and the NORMAN Priority List resulted in tentative identification of 20 compounds (at level 2 and level 3 confidence level), primarily pharmaceuticals and industrial chemicals, with only one overlapping compound in two experimental sites. The contamination profiles of the soil treated with BBFs sourced from veterinary and sludge were similar, with common pharmaceutical features identified. The suspect screening results suggest that the contaminants found in BBF-treated soil might come from alternative sources other than BBFs.

Ovicidal Effect on Haemonchus contortus of Extract Partitions Shrubby Plants of the Tropical Dry Forest and Potentially Active Compounds Identification by UHPLC-Q/Orbitrap/MS/MS

The in vitro anthelmintic effect of the extracts on Haemonchus contortus (H. contortus) of three forage species in the tropical dry forest is known; however, there is no information about the effects of the extract partitions, nor their chemical composition. The objectives of this study were to evaluate the in vitro ovicidal activity of H. contortus in extract partitions of the species Gliricidia sepium, Leucaena leucocephala, and Pithecellobium dulce, and to identify the compounds present in the extract partitions with the highest activity by employing ultra HPLC Quadrupole orbitrap mass spectrometry. Four extract partitions, hexane, dichloromethane, ethyl acetate, and hydroethanolic from the three forage species were assessed in an inhibition of egg hatching (IEH) assay. The extract partitions with the highest anthelmintic activity (AA) were subjected to analysis, from which the tentative identification of the compounds was established. The extract partitions, including dichloromethane from Gliricidia sepium, ethyl acetate from Leucaena leucocephala, and hydroethanolic from Pithecellobium dulce showed a greater anthelmintic effect, with IC50 values of 0.39, 0.86, and 0.27 mg/mL for the IEH, respectively. Metabolites with in vitro AA potential included flavonoids, fatty acid esters, hydroxycinnamic acids, organic oxygenated compounds of the benzene class and substituted derivatives, phenolic glycosides, and phenols.

Preparative Fractionation of Phenolic Compounds and Isolation of an Enriched Flavonol Fraction from Winemaking Industry By-Products by High-Performance Counter-Current Chromatography.

High-performance counter-current chromatography (HPCCC) was used as a tool for the isolation and fractionation of phenolic compounds (PCs) in extracts from wine lees (WL) and grape pomace (GP). The biphasic solvent systems applied for HPCCC separation were n-butanol:methyl tert-butyl ether:acetonitrile:water (3:1:1:5) with 0.1% trifluoroacetic acid (TFA) and n-hexane:ethyl acetate:methanol:water (1:5:1:5). After refining the ethanol:water extracts of GP and WL by-products by ethyl acetate extraction, the latter system yielded an enriched fraction of the minor family of flavonols. Recoveries of 112.9 and 105.9 mg of purified flavonols (myricetin, quercetin, isorhamnetin, and kaempferol) in GP and WL, respectively, from 500 mg of ethyl acetate extract (equivalent to 10 g of by-product) were obtained. The HPCCC fractionation and concentration capabilities were also exploited for the characterization and tentative identification of constitutive PCs by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). In addition to the isolation of the enriched flavonol fraction, a total of 57 PCs in both matrixes were identified, 12 of which were reported for the first time in WL and/or GP. The application of HPCCC to GP and WL extracts may be a powerful approach to isolate large amounts of minor PCs. The composition of the isolated fraction demonstrated quantitative differences in the individual compound composition of GP and WL, supporting the potential exploitation of these matrixes as sources of specific flavonols for technological applications.

Classifying micropollutants by environmental risk in groundwater using screening analysis associated to a hybrid multicriteria method combining (Q)SAR tools, fuzzy AHP and ELECTRE

This study aimed to evaluate and monitor pesticides in groundwater of the Serra Geral aquifer, located in the Paraná Basin 3 (southern Brazil), using Liquid Chromatography coupled with a Quadrupole-Time-of-Flight Mass Spectrometer (LC-QTOF MS). A total of 117 samples, collected in three different moments, were analyzed over 36 months. Groundwater samples from 35 wells and four surface water points were monitored in each sampling campaign. A pesticide screening methodology was proposed with the tentative identification of 1607 pesticides and pesticide metabolites. The application of the proposed methodology resulted in the verification of 29 pesticides and pesticide metabolites, 7 as confirmed analytes and 22 as suspect compounds. (Q)SAR in silico predictions and GUS index calculation provided data on the potential environmental risk of the identified compounds, with eight endpoints considered. After in silico predictions, an alternative hybrid multicriteria method was applied, combining the weighting of endpoints of fuzzy AHP and micropollutants classification by environmental risk using ELECTRE. The fuzzy AHP results indicated the greatest importance of mutagenicity among the eight evaluated indicators, while the scarce influence of the physicochemical properties on the environmental risk suggested their exclusion from the model. Accordingly, the ELECTRE results highlighted the prevalence of thiamethoxam and carbendazim as the most dangerous for the environment. The application of the proposed method enabled the selection of the compounds that must be monitored, considering mutagenicity and toxicity predictions for environmental risk analysis.

Organophosphate esters (OPEs) in plastic food packaging: non-target recognition, and migration behavior assessment

Organophosphate esters (OPEs) are widely used as plasticizers in plastic food packaging; however, the migration of OPEs from plastic to food is largely unstudied. We do not even know the specific number of OPEs that exist in the plastic food packaging. Herein, an integrated target, suspect, and nontarget strategy for screening OPEs was optimized using ultrahigh-performance liquid chromatography–high-resolution mass spectrometry (UHPLC–HRMS). The strategy was used to analyze 106 samples of plastic food packaging collected in Nanjing city, China, in 2020. HRMS allowed full or tentative identification of 42 OPEs, of which seven were reported for the first time. Further, oxidation products of bis(2,4-di-tert-butylphenyl) pentaerythritol diphosphite (AO626) in plastics were identified, implying that the oxidation of organophosphite antioxidants (OPAs) could be an important indirect source of OPEs in plastics. The migration of OPEs was examined with four simulated foods. Twenty-six out of 42 OPEs were detected in at least one of the four simulants, particularly isooctane, in which diverse OPEs were detected at elevated concentrations. Overall, the study supplements the list of OPEs that humans could ingest as well as provides essential information regarding the migration of OPEs from plastic food packaging to food.

TOWARDS A RADIOCARBON-BASED CHRONOLOGY OF URBAN NORTHERN MESOPOTAMIA IN THE EARLY TO MID-SECOND MILLENNIUM BC: INITIAL RESULTS FROM KURD QABURSTAN

ABSTRACTRadiocarbon (14C) data for 2nd millennium BC urban sites in northern Mesopotamia have been lacking until recently. This article presents a preliminary dataset and Bayesian model addressing the Middle and early Late Bronze Age (Old Babylonian and pre/early Mittani) strata of Kurd Qaburstan—one of the largest archaeological sites on the Erbil plain of Iraqi Kurdistan. The results place the large, densely occupied and fortified Middle Bronze Age city in the first part of the 18th century BC, an outcome consistent with the site’s tentative identification as ancient Qabra. A long occupation gap (up to two centuries) probably ensued, before a smaller town confined to the high mound and part of the northeastern lower town resumed in the late 16th and early 15th centuries BC, possibly before this region became part of the Late Bronze Age kingdom of Mittani.

Tentative Identification of Compounds, Antioxidant, and Antimicrobial Activity of the Edible Part of Benincasa hispida L. fruit (Cucurbitaceae)

The edible part of Benicasa hispida (Thunb.) Cogn. fruit is traditionally used in Southeast Sulawesi to treat high blood pressure, typhoid fever, and body cooling. The present study evaluated the chemical compounds present in the 80% ethanol of the edible part of the plant using phytochemical screening and an LC-MS analysis, antioxidant activity based on assays on total phenolics content (TPC), total flavonoids content (TFC), and DPPH, and antimicrobial activity towards Salmonella typhi, Escherichia coli, Staphylococcus aureus, and Candida albicans. Phytochemical screening revealed the presence of tannins, flavonoids, terpenoids, steroids, and saponins in the extract. As many as eighteen compounds (1-18) were tentatively identified in the extract, including sugars, a simple phenolic, a tricarboxylic acid, a peptide, flavonoids, quinic acid derivatives, phytosterols, triterpenoids, and saponins. The extract exhibited remarkable antioxidant activity with an SC50 value of 23.4 µg/mL, although its TPC (1.1±0.1 mg GAE/g extract) and TFC (1.0±0.1 mg QE/g extract) values were considered in low amounts. The extract was found inactive to inhibit the microbial growths of all tested microbes. However, raffinose (3) present in the extract might be beneficial as a prebiotic to promote a healthy human gut. The study concludes that the 80% ethanol extract of the edible part of B. hispida fruit could be used to develop natural antioxidant agents and nutraceuticals.

Heartworms in Halichoerus grypus: first records of Acanthocheilonema spirocauda (Onchocercidae; Filarioidea) in 2 grey seals from the North Sea.

The assumed definitive host of the heartworm Acanthocheilonema spirocauda (Onchocerdidae; Filarioidea) is the harbour seal (Phoca vitulina). This filaroid nematode parasitizing in cardiac ventricles and blood vessel lumina of harbour seals (P. vitulina) has a low prevalence and seldom causes severe health impacts. The seal louse (Echinophthirius horridus) is the assumed intermediate host for transmission of A. spirocauda filariae between seals, comprising a unique parasite assembly conveyed from the terrestrial ancestors of pinnipeds. Although grey seals (Halichoerus grypus) are infected by seal lice, heartworm infection was not verified. Analysing a longterm dataset compiled over decades (1996–2021) of health monitoring seals along the German coasts comprising post mortem investigations and archived parasites, 2 cases of A. spirocauda infected male grey seals were detected. Tentative morphological identification was confirmed with molecular tools by sequencing a section of mtDNA COI and comparing nucleotide data with available heartworm sequence. This is the first record of heartworm individuals collected from the heart of grey seals at necropsy. It remains puzzling why heartworm infection occur much less frequently in grey than in harbour seals, although both species use the same habitat, share mixed haul-outs and consume similar prey species. If transmission occurs directly via seal louse vectors on haul-outs, increasing seal populations in the North- and Baltic Sea could have density dependent effects on prevalence of heartworm and seal louse infections. It remains to be shown how species-specificity of filarial nematodes as well as immune system traits of grey seals influence infection patterns of A. spirocauda.

Valorization of Avocado Seed Wastes for Antioxidant Phenolics and Carbohydrates Recovery Using Deep Eutectic Solvents (DES).

Avocado seeds represent the chief waste produced in avocado processing, leading not only to environmental problems regarding its elimination but to a loss of economic profitability. In fact, avocado seeds are known as interesting sources of bioactive compounds and carbohydrates, so their utilization may reduce the negative effect produced during the industrial manufacture of avocado-related products. In this sense, deep eutectic solvents (DES) are a novel greener alternative than organic solvents to extract bioactive polyphenols and carbohydrates. The study was based on a Box-Behnken experimental design to study the effect of three factors, temperature (40, 50, 60 °C), time (60, 120, 180 min) and water content (10, 30, 50% v/v) on the responses of total phenolic (TPC) and flavonoid content (TFC), antioxidant capacity (measured as ABTS and FRAP) and xylose content in the extract. The DES Choline chloride:glycerol (1:1) was used as solvent on avocado seed. Under optimal conditions, TPC: 19.71 mg GAE/g, TFC: 33.41 mg RE/g, ABTS: 20.91 mg TE/g, FRAP: 15.59 mg TE/g and xylose: 5.47 g/L were obtained. The tentative identification of eight phenolic compounds was assayed via HPLC-ESI. The carbohydrate content of the solid residue was also evaluated, and that solid was subjected to two different processing (delignification with DES and microwave-assisted autohydrolysis) to increase the glucan susceptibility to enzymes, and was also assayed reaching almost quantitative glucose yields. These results, added to the non-toxic, eco-friendly, and economic nature of DES, demonstrate that these solvents are an efficient alternative to organic solvents to recover phenolics and carbohydrates from food wastes.

Integrating data dependent and data independent non-target screening methods for monitoring emerging contaminants in the Pearl River of Guangdong Province, China

The occurrence of Contaminants of Emerging Concern (CECs) in the Pearl River of Guandong province, China, was characterized using a nontarget screening (NTS) strategy combining both data dependent and data independent acquisition techniques. Our analysis identified 620 unique compounds, including pharmaceuticals (137), pesticides (124), industrial materials (68), personal care products (32), veterinary drugs (27), plasticizers or flame retardants (11), etc. Out of these compounds, 40 CECs were found with a detection frequency of over 60 %, including diazepam, a well-known drug to treat anxiety, insomnia, convulsion, etc., which had the highest detection rate at 98 %. Risk quotients (RQs) were calculated for CECs identified with high confidence (Level 1, confirmed with authentic standards), and it was found that 12 CECs had RQs > 1, with notable concern for pretilachlor (detection frequency: 48 %; 0.8–19.0 ng/L), bensulfuron-methyl (86 %, 3.1–56.2 ng/L), imidacloprid (80 %, 5.3–62.8 ng/L) and thiamethoxam (86 %, 9.1–99.9 ng/L), which exhibited RQs exceeding the threshold of concern (RQ > 1) at 46–80 % of sampling sites. Additionally, tentative identification of potential structurally related compounds provided valuable insight into the parent-product relationships in complex samples. This study highlights the importance and urgency of using NTS for CECs in the environment and presents a novel data sharing approach, which facilitates other scientists to assess, investigate further, and perform retrospective analyses.

UPLC-QToF-ESI-MS identification and anthelmintic activity of Mitragyna inermis (Willd.) Kuntze (Rubiaceae)

Medicinal plants attract the attention of many researchers to find natural and safe remedies for various resistant diseases. Leaves of Mitragyna inermis are widely used in traditional veterinary medicine for the treatment of gastrointestinal strongyles of small ruminants. The aim of the current study is to estimate the antioxidant, anthelmintic and the larval toxicity of the aqueous and hydroethanolic extracts of this plant in addition to the hexane, dichloromethane and ethanol fractions of the hydroethanolic extract. Investigation of the most active extract using Ultra Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight Electrospray Ionization Mass Spectrometry (UPLC-QToF-ESI-MS). Both plant extracts showed good antioxidant activity by scavenging the 2,2′-diphényl-1-picrylhydrazyl (DPPH) radical and reducing the ferric ion. Similarly, they were no-toxic to Artemia salina larvae (CL50 > 0.1 μg/mL). Also, they significantly reduced larval migration and motility of Haemonchus contortus adult worms (p < 0.001). The hexane, dichloromethane and ethanolic fractions of the hydroethanolic extract showed low activity compared to crude extracts except for the hexane fraction on H. contortus adult worms (p < 0.001) while it showed a poor result on larvae. It thus appears that the anthelmintic activity of the extract may be linked to the synergistic action of these compounds. The UPLC-QToF-ESI-MS analysis revealed the tentative identification of 15 compounds including 7 alkaloids. The results of the present study confirm the anthelmintic activity of M. inermis in traditional veterinary medicine.

Chemical profiling and cytotoxic potential of the n-butanol fraction of Tamarix nilotica flowers

BackgroundCancer represents one of the biggest healthcare issues confronting humans and one of the big challenges for scientists in trials to dig into our nature for new remedies or to develop old ones with fewer side effects. Halophytes are widely distributed worldwide in areas of harsh conditions in dunes, and inland deserts, where, to cope with those conditions they synthesize important secondary metabolites highly valued in the medical field. Several Tamarix species are halophytic including T.nilotica which is native to Egypt, with a long history in its tradition, found in its papyri and in folk medicine to treat various ailments.MethodsLC–LTQ–MS–MS analysis and 1H-NMR were used to identify the main phytoconstituents in the n- butanol fraction of T.nilotica flowers. The extract was tested in vitro for its cytotoxic effect against breast (MCF-7) and liver cell carcinoma (Huh-7) using SRB assay.ResultsT.nilotica n-butanol fraction of the flowers was found to be rich in phenolic content, where, LC–LTQ–MS–MS allowed the tentative identification of thirty-nine metabolites, based on the exact mass, the observed spectra fragmentation patterns, and the literature data, varying between tannins, phenolic acids, and flavonoids. 1H-NMR confirmed the classes tentatively identified. The in-vitro evaluation of the n-butanol fraction showed lower activity on MCF-7 cell lines with IC50 > 100 µg/mL, while the higher promising effect was against Huh-7 cell lines with an IC50= 37 µg/mL.ConclusionOur study suggested that T.nilotica flowers' n-butanol fraction is representing a promising cytotoxic candidate against liver cell carcinoma having potential phytoconstituents with variable targets and signaling pathways.

Chenopodium murale Juice Shows Anti-Fungal Efficacy in Experimental Oral Candidiasis in Immunosuppressed Rats in Relation to Its Chemical Profile.

Chenopodium murale (Syn. Chenopodiastrum murale) (amaranthaceae) is used in the rural Egypt to treat oral ulcers in newborn children. The current study aimed to discover new natural products suitable for treating candidiasis disease with minimal side effects. Characterization of bioactive compounds by LC-QTOF-HR-MS/MS from Chenopodium murale fresh leaves' juice (CMJ) was carried out in order to elucidate their potential anti-fungal and immunomodulatory effects in oral candidiasis in immunosuppressed rats. An oral ulcer candidiasis model was created in three stages: (i) immunosuppression by drinking dexamethasone (0.5 mg/L) for two weeks; (ii) Candida albicans infection (3.00 × 106 viable cell/mL) for one week; and (iii) treatment with CMJ (0.5 and 1.0 g/kg orally) or nystatin (1,000,000 U/L orally) for one week. Two doses of CMJ exhibited antifungal effects, for example, through a significant reduction in CFU/Petri (236.67 ± 37.86 and 4.33 ± 0.58 CFU/Petri), compared to the Candida control (5.86 × 104 ± 1.21 CFU/Petri), p ≤ 0.001. In addition, CMJ significantly induced neutrophil production (32.92% ± 1.29 and 35.68% ± 1.77) compared to the Candida control level of 26.50% ± 2.44. An immunomodulatory effect of CMJ at two doses appeared, with a considerable elevation in INF-γ (103.88 and 115.91%), IL-2 (143.50, 182.33%), and IL-17 (83.97 and 141.95% Pg/mL) compared with the Candida group. LC-MS/MS analysis operated in negative mode was used for tentative identification of secondary (SM) metabolites based on their retention times and fragment ions. A total of 42 phytoconstituents were tentatively identified. Finally, CMJ exhibited a potent antifungal effect. CMJ fought Candida through four strategies: (i) promotion of classical phagocytosis of neutrophils; (ii) activation of T cells that activate IFN-γ, IL-2, and IL-17; (iii) increasing the production of cytotoxic NO and H2O2 that can kill Candida; and (iv) activation of SOD, which converts superoxide to antimicrobial materials. These activities could be due to its active constituents, which are documented as anti-fungal, or due to its richness in flavonoids, especially the active compounds of kaempferol glycosides and aglycone, which have been documented as antifungal. After repetition on another type of small experimental animal, their offspring, and an experimental large animal, this study may lead to clinical trials.