An untargeted metabolomics approach to study changes of the medium during human cornea culture.

INTRODUCTION: Two main approaches (organ culture and hypothermia) for the preservation and storage of human donor corneas are globally adopted for corneal preservation before the transplant. Hypothermia is a hypothermic storage which slows down cellular metabolism while organ culture, a corneal culture performed at 28-37 °C, maintains an active corneal metabolism. Researchers, till now, have just studied the impact of organ culture on human cornea after manipulating and disrupting tissues. OBJECTIVES: The aim of the current work was to optimize an analytical procedure which can be useful for discovering biomarkers capable of predicting tissue health status. For the first time, this research proposed a preliminary metabolomics study on medium for organ culture without manipulating and disrupting the valuable human tissues which could be still used for transplantation. METHODS: In particular, the present research proposed a method for investigating changes in the medium, over a storage period of 20 days, in presence and absence of a human donor cornea. An untargeted metabolomics approach using UHPLC-QTOF was developed to deeply investigate the differences on metabolites and metabolic pathways and the influence of the presence of the cornea inside the medium. RESULTS: Differences in the expression of some compounds emerged from this preliminary metabolomics approach, in particular in medium maintained for 10 and 20 days in presence but also in the absence of cornea. A total of 173 metabolites have been annotated and 36 pathways were enriched by pathway analysis. CONCLUSION: The results revealed a valuable untargeted metabolomics approach which can be applied in organ culture metabolomics.

Influence of extraction solvents on the chemical constituents and biological activities of Astragalus aduncus from Turkey flora: In vitro and in silico insights.

The n-hexane, ethyl acetate, ethanol, ethanol/water (70% ethanol), and water extracts of Astragalus aduncus aerial parts were investigated for their antioxidant potential, enzyme inhibition activity (anti-acetylcholinesterase [AChE], anti-butyrylcholinesterase [BChE], antityrosinase, antiamylase, and antiglucosidase) and antiproliferative effect (against colon adenocarcinoma cell line [HT-29], gastric cancer cell line [HGC-27], prostate carcinoma cell line [DU-145], breast adenocarcinoma cell line [MDA-MB-231], and cervix adenocarcinoma cell line [HeLa]). In addition, the phytochemical profile of the extracts was evaluated using validated spectrophotometric and high-pressure liquid chromatography-electrospray ionization/tandem mass spectroscopy methods. Generally, the 70% ethanol extract demonstrated the strongest antioxidant properties, and it was the richest source of total phenolic constituents. Our findings indicated that the ethyl acetate extract was the most potent BChE inhibitor (11.44 mg galantamine equivalents [GALAE]/g) followed by the ethanol extract (8.51 mg GALAE/g), while the ethanol extract was the most promising AChE inhibitor (3.42 mg GALAE/g) followed by the ethanol/water extract (3.17 mg GALAE/g). Excellent tyrosinase inhibitory activity (66.25 mg kojic acid equivalent/g) was observed in ethanol/water extracts of the aerial part of A. aduncus. Тhese results showed that the most cytotoxic effects were exhibited by the ethyl acetate extract against HGC-27 cells (IC50: 36.76 µg/mL), the ethanol extract against HT-29 cells (IC50: 30.79 µg/mL), and the water extract against DU-145 cells (IC50: 37.01 µg/mL). A strong correlation was observed between the highest total flavonoid content and the highest content of individual compounds in the ethanol extract, including rutin, hyperoside, isoquercitrin, delphinidin-3,5-diglucoside (delphinidin-3,5-O-diglucoside), and kaempferol-3-glucoside (kaempferol-3-O-glucoside). In the present study, the A. aduncus plant was considered a new source of antioxidants, enzyme inhibitors, and anticancer agents and could be used as a future health-benefit natural product.

Establishing a link between the chemical composition and biological activities of Gladiolus italicus Mill. from the Turkish flora utilizing in vitro, in silico and network pharmacological methodologies.

OBJECTIVES: Five solvent extracts (n-hexane, ethyl acetate, ethanol, ethanol/water (70%), and water) of Gladiolus italicus Mill. from Turkey were evaluated for chemical and biological properties. METHODS: Antioxidant activities, inhibitory properties against key enzymes involved in the etiology of chronic diseases were tested, as well as cytotoxic effects on different cell lines. Chemical characterization was also carried out to determine the most abundant compounds of each extract. RESULTS: The highest total phenolic content (TPC) was observed in the water extract while highest TFC in ethanol/water extract. The most abundant compounds in the extracts were hyperoside (69041.06 mg kg-1), isoquercitrin (46239.49 mg kg-1), delphindin-3,5-diglucoside (42043.81 mg kg-1), myricetin (21486.61 mg kg-1), and kaempferol-3-glucoside (21199.76 mg kg-1). Molecular dynamic (MD) simulations confirmed the structural stability and dynamic conformational integrity of these complexes over a period of 100 ns. In network pharmacology, A total of 657 unique target genes were screened: 52 associated with programmed cell death-1 (PD-1), 85 with vascular endothelial growth factor receptor-2 (VEGFR2), and 130 with fibroblast growth factor receptor-2 (FGFR2), identifying crucial gene interactions for these proteins. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted, revealing significant interactions and pathways such as the advanced glycation end products (AGE) and their receptors (RAGE) signaling pathway in diabetic complications and T- helper 17 (Th17) cell differentiation, among others. This elucidation of complex networks involving key genes like AKT Serine/Threonine Kinase 1 (AKT1), MYC proto-oncogene (MYC), tumor protein 53 (TP53), Interleukin 6 (IL6), and tumor necrosis factor (TNF) provides a promising foundation for the development of targeted therapies in the treatment of non-communicable diseases. CONCLUSION: These results show that G. italicus could be a natural source of potent antioxidants and enzyme inhibitors which need to be further explored for the development of biopharmaceuticals.

Dual Anta-Inhibitors Targeting Protein Kinase CK1δ and A2A Adenosine Receptor Useful in Neurodegenerative Disorders.

Currently, the number of patients with neurodegenerative pathologies is estimated at over one million, with consequences also on the economic level. Several factors contribute to their development, including overexpression of A2A adenosine receptors (A2AAR) in microglial cells and up-regulation and post-translational alterations of some casein kinases (CK), among them, CK-1δ. The aim of the work was to study the activity of A2AAR and CK1δ in neurodegeneration using in-house synthesized A2A/CK1δ dual anta-inhibitors and to evaluate their intestinal absorption. Experiments were performed on N13 microglial cells, which were treated with a proinflammatory CK cocktail to simulate an inflammatory state typical of neurodegenerative diseases. Results showed that the dual anta-inhibitors have the ability to counteract the inflammatory state, even if compound 2 is more active than compound 1. In addition, compound 2 displayed an important antioxidant effect similar to the reference compound ZM241385. Since many known kinase inhibitors are very often unable to cross lipid bilayer membranes, the ability of A2A/CK1δ double anta-inhibitors to cross the intestinal barrier was investigated by an everted gut sac assay. HPLC analysis revealed that both compounds are able to cross the intestinal barrier, making them promising candidates for oral therapy.

Development of Innovative Vitamin D Enrichment Designs for Two Typical Italian Fresh Cheeses: Burrata and Giuncata.

The aim of this research was to develop innovative cheeses fortified with vitamin D3 (VD3). Formulation studies and analyses of textural properties and chemicals were carried out for these developments. Two traditional Italian varieties of cheese (giuncata and burrata) were studied. For giuncata, the fortification of milk for cheese production provided a VD3 retention level of 43.9 ± 0.6% in the food matrix. For burrata, the VD3 ingredient was incorporated into the creamy inner part after mixing, maintaining the textural quality of the product (adhesiveness 4.3 ± 0.4 J × 10-3; firmness 0.7 ± 0.0 N; and cohesiveness 0.8 ± 0.2). The optimized enrichment designs allowed to obtain homogenous contents of VD3 during the production of giuncata (0.48 ± 0.01 µg/g) and burrata cheeses (0.32 ± 0.02 µg/g). Moreover, analyses revealed the high stability of VD3 during the storage of the two fortified cheese types (2 weeks, 4 °C). These fortification designs could be implemented at an industrial scale to obtain new cheese types enriched in VD3 and thus contribute to the reduction in VD deficiency prevalence.

Fiber-Sample Distance, An Important Parameter To Be Considered in Headspace Solid-Phase Microextraction Applications.

To define and control the parameters which impact headspace solid-phase microextraction (HS-SPME), it is important to reach the highest level of reproducibility. The present study aims to assess, for the first time, the effect of fiber-sample distance during HS-SPME in pre-equilibrium conditions. Analyses were primarily performed on mixtures of standard volatiles compounds (alkanes, alcohols, organic acids) designed in our lab and then on various food matrices (wine, chicken, cheese, tea), repeating already published experiments. Extractions were performed varying fiber penetration depths (10-60 mm) at different times (10-60 min) and temperatures of extraction (30-80 °C). The study revealed that variation of the distance between the fiber and the sample into the vial clearly impacts the results obtained during HS-SPME when conditions are such that no equilibrium is reached in HS. For example, in wine analysis, the percentage of octanoic acid at 80 °C was higher at 40 mm (7.5 ± 0.2%) than that at 20 mm (4.4 ± 0.3%). Moreover, regardless of the extraction temperature, the lower the time of extraction, the stronger the dependence on the fiber-sample distance. Indeed, at 60 °C, the obtained response factors for octadecane at 20 and 40 mm of fiber penetration were 21.8 and 44.5, respectively, after 10 min of extraction, 54.1 and 71.0 after 30 min, and 79.4 and 82.4 after 60 min of extraction. The analyses have been here corroborated by a theoretical model based on the diffusion equation. Therefore, to improve the method robustness during HS-SPME studies, we suggest specifying the fiber penetration depth or the fiber-sample distance with the other parameters of extraction.

New sensible method to quantize the intestinal absorption of receptor ligands.

In recent years, special attention has been paid to the A3 adenosine receptor (A3AR) as a possible pharmacological target to treat intestinal inflammation. In this work, it was set up a novel method to quantify the concentration of a promising anti-inflammatory agent inside and outside of intestinal barrier using the everted gut sac technique. The compound chosen for the present study is one of the most potent and selective A3AR agonist reported so far, named AR 170 (N6-methyl-2-phenylethynyl-5'-N-methylcarboxamidoadenosine). In order to evaluate the intestinal absorption of AR 170 the radioligand binding assay in comparison with HPLC-DAD was used. Results showed that the compound is absorbed via passive diffusion by paracellular pathway. The concentrations determined in the serosal (inside the sac) fluid by radioligand binding assay are in good agreement with those obtained through the widely used HPLC/MS protocol, demonstrating the reliability of the method. It is worthwhile to note that the radioligand binding assay allows detecting very low concentrations of analyte, thus offering an excellent tool to measure the intestinal absorption of receptor ligands. Moreover, the AR 170 quantity outside the gut sac and the interaction with A3AR could presuppose good topical anti-inflammatory effects of this compound.

Fecal Proteomic Analysis in Healthy Dogs and in Dogs Suffering from Food Responsive Diarrhea.

Different laboratory markers are routinely used in the diagnosis and management of gastrointestinal (GI) disease in dogs. In the present study, starting from feces from both healthy dogs and dogs suffering from food responsive diarrhea (FRD), we tried to find proteins differently expressed in the two groups of dogs, by using a proteomic approach. Interestingly, we found that the immunoglobulin J-chain isoform 1 (species: Canis lupus familiaris) was identified only in diseased dogs (not in healthy). J-chain combines especially IgA monomers to IgA dimers and plays a crucial role for their secretions into mucosal interface. Being the first study of that kind in the dog, it is only possible to hypothesize that their presence could be likely due to an increased activation of the immune system or to a mucosal damage or both in FRD patients. Similarly, it is still impossible to assess whether this protein could be used as diagnostic/prognostic marker of GI disease; however, this study represents a promising first step toward fecal proteomics in canine GI disorders.

Studies on the Interaction between Poly-Phosphane Gold(I) Complexes and Dihydrofolate Reductase: An Interplay with Nicotinamide Adenine Dinucleotide Cofactor.

A class of gold(I) phosphane complexes have been identified as inhibitors of dihydrofolate reductase (DHFR) from E. coli, an enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), using NADPH as a coenzyme. In this work, to comprehend the nature of the interaction at the basis of these inhibitory effects, the binding properties of bis- and tris-phosphane gold(I) chloride compounds in regards to DHFR have been studied by emission spectroscopy and spectrophotometric assays. The lack of cysteine and seleno-cysteine residues in the enzyme active site, the most favorable sites of attack of Au(I) moieties, makes this work noteworthy. The interaction with the gold compounds results into the quenching of the DHFR tryptophan's emissions and in an enhancement of their intrinsic emission intensities. Moreover, a modulating action of NADPH is highlighted by means of an increase of the gold compound affinity toward the enzyme; in fact, the dissociation constants calculated for the interactions between DHFR and each gold compound in the presence of saturating NADPH were lower than the ones observed for the apo-enzyme. The fluorimetric data afforded to Kd values ranged from 2.22 ± 0.25 µM for (PPh3)2AuCl in the presence of NADPH to 21.4 ± 3.85 µM for 4L3AuTf in the absence of NADPH. By elucidating the energetic aspects of the binding events, we have attempted to dissect the role played by the gold phosphane/protein interactions in the inhibitory activity, resulting in an exothermic enthalpy change and a positive entropic contribution (ΔH° = -5.04 ± 0.08 kcal/mol and ΔS° = 7.34 ± 0.005 cal/mol·K).

Analysis of 17 polyphenolic compounds in organic and conventional legumes by high-performance liquid chromatography-diode array detection (HPLC-DAD) and evaluation of their antioxidant activity.

Consumption of legumes has become popular among health-conscious consumers due to the high levels of nutrients such as proteins, dietary fibres, minerals, vitamins and micronutrients like polyphenols. Ten legumes cultivars organically and conventionally grown were compared regarding their chemical profiles in terms of total phenolic content (TPC), total anthocyanin content (TAC), total flavonoid content (TFC) and their antioxidant capacity. The chemical profiles (i.e. free phenolics and anthocyanins) were determined with aid of new high-performance liquid chromatography-diode array detection (HPLC-DAD) method able to identify two individual anthocyanins and 15 polyphenols. Results showed that phenolic acids, namely gallic acid, caffeic acid, syringic acid and ferulic acid, are mainly significantly higher in the organic samples with respect to the conventional ones. All organic legumes showed a higher antioxidant capacity with respect to conventional samples with the exception of red and yellow dehulled lentils.

Efficacy of the Volatile Oil from Water Celery (Helosciadium nodiflorum, Apiaceae) against the Filariasis Vector Culex quinquefasciatus, the Housefly Musca domestica, and the African Cotton Leafworm Spodoptera littoralis.

Water celery (Helosciadium nodiflorum, Apiaceae) is a common and invasive hydrophyte occurring in rivers and slow waters of Europe. In the past, it was consumed as a vegetable and spices, but currently it has mostly been abandoned. As a result, this plant produces an unexploited biomass occluding rivers and slow waters. With the aim to valorise water celery as an important biomass source for the development of botanical insecticides, in this work, we investigated the inhibitory effects of the essential oil hydrodistilled from its aerial parts against larvae of the filariasis vector Culex quinquefasciatus, adults of the housefly Musca domestica, and larvae of the African cotton leafworm Spodoptera littoralis. The essential oil, which was characterized by the insecticidal limonene (39.8%) and myristicin (35.3%), exerted important toxic effects on 3rd instar larvae of C. quinquefasciatus and S. littoralis and on adults of M. domestica, with LD50 of 50.4 µl l-1 , 116.7 µg larva-1 , and 45.1 (male) and 70.1 (female) µg adult-1 , respectively. The oil did not show any noticeable inhibitory activity on the acetylcholinesterase enzyme. Our findings add scientific evidences highlighting water celery as an important biomass source to be used for the control of insect pests and vectors of high economic importance.

Nutritional composition, bioactive compounds and volatile profile of cocoa beans from different regions of Cameroon.

Analysis of the complex composition of cocoa beans provides fundamental information for evaluating the quality and nutritional aspects of cocoa-based food products, nutraceuticals and supplements. Cameroon, the world's fourth largest producer of cocoa, has been defined as "Africa in miniature" because of the variety it habitats. In order to evaluate the nutritional characteristics of cocoa beans from five different regions of Cameroon, we studied their polyphenolic content, volatile compounds and fatty acids composition. The High Performance Thin Layer Chromatography (HPTLC) analysis showed that the Mbalmayo sample had the highest content of theobromine (11.6 mg/g) and caffeic acid (2.1 mg/g), while the Sanchou sample had the highest level of (-)-epicatechin (142.9 mg/g). Concerning fatty acids, the lowest level of stearic acid was found in the Mbalmayo sample while the Bertoua sample showed the highest content of oleic acid. Thus, we confirmed that geographical origin influences the quality and nutritional characteristics of cocoa from these regions of Cameroon.

Quantification of two derivatives of malic acid first-time discovered in coffee: Influence of postharvest processing method.

This study quantified, for the first time, 2-isopropylmalic and 3-isopropylmalic acids, in green, roasted and espresso coffee by UHPLC-MS/MS. Moreover, it reports the influence of postharvest processing methods (natural, washed and honey) on their content. New extraction techniques were developed and validated from three coffee matrices (green, roasted and espresso). Honey coffee exhibited levels substantially higher of 2-isopropylmalic acid than those processed by natural and washed methods (p < 0.05). Specifically, 2-isopropylmalic acid levels in honey green, roasted and espresso coffee samples were 48.24 ± 7.31 ng/g, 168.8 ± 10.88 ng/g and 177.5 ± 9.49 ng/g, respectively. This research highlights the significant impact of processing methods on the chemical profile of coffee and introduces 2-isopropylmalic and 3-isopropylmalic acids as potential quality indicators. Moreover, it suggests that the fermentation stage during processing may play a crucial role in their formation, laying the foundation for optimizing coffee processing to enhance quality.

Comprehensive investigation of coffee acidity on eight different brewing methods through chemical analyses, sensory evaluation and statistical elaboration.

Even if the acids composition and their role in coffee still need to be clarified, acidity is one of the main sought-after features in coffee and it is becoming one of the main quality markers. Hence, the aim of this paper was to evaluate the main parameters influencing coffee acidity with a focus on carboxylic acids. To the best of our knowledge, this is the first study regarding filter coffee prepared from specialty and mainstream coffee, differently roasted and through eight diverse extraction methods. Coffee cup chemical composition in terms of organic and chlorogenic acids, caffein and physicochemical parameters were correlated with perceived sourness and mouthfeel to better understand the influence of extracted compounds on the final beverage acidity. Statistical tools revealed that a major impact of chlorogenic acids emerged in pH and titratable acidity, while the sensorial sourness appeared more correlated with organic acids concentration. Thus, these findings suggests that organic acids could be potential predictors of beverage perceived acidity.

Assessing the Chemical Profile and Biological Potentials of Tamarix aphylla (L.) H.Karst. and Tamarix senegalensis DC. by In Vitro, In Silico, and Network Methodologies.

The present study aimed to investigate the chemical profile, antioxidant, and enzyme inhibition properties of extracts from fruits and aerial parts (leaves and twigs) of Tamarix aphylla and T. senegalensis. Hexane, dichloromethane, ethyl acetate (EtOAc), and methanol extracts were prepared sequentially by maceration. Results revealed that EtOAc extracts of T. senegalensis and T. aphylla fruits contained the highest total phenolic content (113.74 and 111.21 mg GAE/g) while that of T. senegalensis (38.47 mg RE/g) recorded the highest total flavonoids content. Among the quantified compounds; ellagic, gallic, 3-hydroxybenzoic, caffeic, syringic, p-coumaric acids, isorhamnetin, procyanidin B2, and kaempferol were the most abundant compounds in the two species. EtOAc extracts of the two organs of T. senegalensis in addition to MeOH extract of T. aphylla aerial parts displayed the highest chelating power (21.00-21.30 mg EDTAE/g, p > 0.05). The highest anti-AChE (3.11 mg GALAE/g) and anti-BChE (3.62 mg GALAE/g) activities were recorded from the hexane and EtOAc extracts of T. senegalensis aerial parts and fruits, respectively. EtOAc extracts of the fruits of the two species exerted the highest anti-tyrosinase (anti-Tyr) activity (99.44 and 98.65 mg KAE/g, p > 0.05). Also, the EtOAc extracts of the both organs of the two species exhibited highest anti-glucosidase activity (0.88-0.90 mmol ACAE/g, p > 0.05) while the best anti-α-amylase activity was recorded from the dichloromethane extract of T. senegalensis fruits (0.74 mmol ACAE/g). In this study, network pharmacology was employed to examine the connection between compounds from Tamarix and their potential effectiveness against Alzheimer's disease. The compounds demonstrated potential interactions with pivotal genes including APP, GSK3B, and CDK5, indicating a therapeutic potential. Molecular docking was carried out to understand the binding mode and interaction of the compounds with the target enzymes. Key interactions observed, such as H-bonds, promoted the binding, and weaker ones, such as van der Waals attractions, reinforced it. These findings suggest that these two Tamarix species possess bioactive properties with health-promoting effects.

Microwave-assisted hydrodiffusion and gravity extraction: An efficient method to produce pomegranate juice.

Green technologies based on microwaves have been developed by the food industry to produce organoleptically acceptable fruit juices without preliminary processing. Microwave irradiation coupled with hydrodiffusion and gravity (MHG) combines microwave heating with the earth's gravity, allowing the collection of hydrophilic substances released from the irradiated matrix. To the best of our knowledge, MHG extraction has never been experimented to produce pomegranate juice. In this work, we have evaluated it as a potential alternative to the conventional squeezing. A central composite design study (CCD) allowed the selection of the best extractive conditions (irradiation power and extraction time) to obtain a pomegranate juice with higher yield, polyphenol (e.g., catechin and delphinidin-3,5-glucoside) content, and related bioactivities (antioxidant and antidiabetic) than the one obtained by squeezing while maintaining the chemical-physical properties. Thus, this technique appears to be a functional alternative to producing high value pomegranate juice.

Central nervous system activities of Hypericum origanifolium extract via GABAergic and opioidergic mechanisms.

Pharmacological effects of hydroalcoholic extract prepared from Hypericum origanifolium Willd. (Guttiferae) on behavioral parameters and pain perceptions of mice were investigated in this study. Depression, anxiety, spontaneous locomotor activity, and motor coordination parameters of mice were assessed by modified forced swimming, hole board, activity cage, and rota-rod tests, respectively. In addition, antinociceptive effect was evaluated by performing hot-plate, tail-clip, and formalin tests. Reboxetine (20 mg/kg), diazepam (1 mg/kg), and morphine (10 mg/kg) were used as reference antidepressant, anxiolytic, and analgesic drugs, respectively. Phytochemical analyses exhibited that chlorogenic acid (2317.12 ppm) and rutin (2108.79 ppm) were the main phenolic compounds in the H. origanifolium extract. The extract (50, 100, and 250 mg/kg) induced significant antidepressant, anxiolytic, and antinociceptive activities following the acute administrations. Anxiolytic effect was antagonized by flumazenil (a benzodiazepine receptor antagonist, 2.5 mg/kg, i.p.) pre-treatment, which indicated the participation of GABA(A)-benzodiazepine receptor complex in the activity. Moreover, centrally and peripherally mediated antinociception reversed by naloxone (a non-selective opioid receptor antagonist, 5 mg/kg, i.p.) pre-treatment, indicating the involvement of opioid system in the pharmacological action. These findings are the first to indicate the pharmacological effects of the H. origanifolium extract on the emotional state and pain perceptions of mice.

Biogenic amines as freshness index of meat wrapped in a new active packaging system formulated with essential oils of Rosmarinus officinalis.

Biogenic amines (BAs) are considered as an important indicator of freshness and quality of food. In this work, a new active packaging (AP) system for meat that, incorporating essential oil of Rosmarinus officinalis at 4% (w/w), inhibits the increase of BAs and the bacteria involved into their production was developed. BAs were analyzed by a SPE-HPLC-DAD method during the storage time of meat (0-7 d, 4 °C). Results showed that, in each monitored day, Biogenic Amine Index (BAI) expressed in mg kg(-1) is lower in meat wrapped in AP with respect to that packed in polycoupled packaging (PP) (from 19% to 62%). A strong correlation was found between the inhibition of increase of putrescine, cadaverine, histamine and their bacteria producers such as Enterobacteriaceae, Pseudomonas spp. and Brocothrix thermospacta. By exploiting antimicrobial and antioxidant action of essential oil of R. officinalis, the new APs contribute to increase the shelf life of fresh meat and to preserve its important nutrients.

Fecal Protein Profile in Eight Dogs Suffering from Acute Uncomplicated Diarrhea before and after Treatment.

Acute diarrhea is a very frequent condition affecting dogs; nevertheless, little is known about what happens in the GI tract during such conditions. Proteomics allows the study of proteins present in a specific biologic substrate, and fecal proteomic investigations have been recently implemented to study GI diseases in dogs. In the present study, the fecal protein profiles of eight dogs suffering from acute uncomplicated diarrhea at the time of inclusion was investigated for the first time, and then the same patients were followed, replicating two further evaluations at two subsequent time points (after 2 and 14 days from the first presentation), with the aim of gaining possible new insights regarding the pathologic changes in the gastrointestinal environment during such conditions. Two-dimensional gel electrophoresis (2-DE) was performed, followed by mass spectrometry. Nine spots, corresponding to four (groups of) proteins (i.e., albumin, alkaline phosphatase, chymotrypsin-C-like, and some immunoglobulins), showed significant differences at two or more of the three time points investigated, almost all behaving similarly and decreasing at T1 (2 days after the onset of the condition) and significantly increasing at T2 (14 days after the onset), mainly evidencing a reaction of the organism. Further studies including a greater number of patients and possibly different techniques are needed to confirm the present findings.

Acmella oleracea extracts as green pesticides against eight arthropods attacking stored products.

Developing sustainable control tools for managing noxious pests attacking stored foodstuffs is a timely research challenge. Acmella oleracea (L.) R. K. Jansen is a crop widely cultivated for its multiple usages on an industrial level. In this study, the extracts prepared with A. oleracea aerial parts were applied on wheat kernels for the management of eight important arthropod pests attacking stored products, i.e., Cryptolestes ferrugineus, Tenebrio molitor, Oryzaephilus surinamensis, Trogoderma granarium, Tribolium castaneum, Tribolium confusum, Alphitobius diaperinus (adults/larvae), and Acarus siro (adults/nymphs). Extraction of A. oleracea was optimized on the base of the yield and content of spilanthol and other N-alkylamides which were analysed by HPLC-DAD-MS. Two concentrations of n-hexane or methanol extracts (500 ppm and 1000 ppm), obtained through Soxhlet extraction, were tested to acquire mortality data on the above-mentioned pests after 4, 8, and 16 h and 1 to 7 days of exposure. Both extracts achieved complete mortality (100.0%) of C. ferrugineus adults. In the case of A. diaperinus adults, mortalities were very low at any concentrations of both extracts. In general, the n-hexane extract was more efficient than methanol extract against almost all species and stages. Considering both extracts, the susceptibility order, from most to least susceptible species/stage, was C. ferrugineus adults > A. diaperinus larvae > C. ferrugineus larvae > T. granarium adults > T. molitor larvae > O. surinamensis adults > O. surinamensis larvae > T. confusum larvae > T. castaneum larvae > A. siro adults > T. molitor adults > A. siro nymphs > T. granarium larvae > T. castaneum adults > T. confusum adults > A. diaperinus adults. Our research provides useful knowledge on the efficacy of N-alkylamides-rich A. oleracea extracts as grain protectants, pointing out the importance of targeting the most susceptible species/ developmental stages.