Optimization of skin sampling based on tape stripping for lipidome analysis by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry.

In this study, skin sampling by tape stripping for lipid analysis was optimized by examining the lipid profiles of the stratum corneum (SC), focusing on the composition and levels of ceramides (Cer), diacylglycerols (DG), and triacylglycerols (TG), using nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry. Significant variations in the number and composition of the identified lipids, particularly Cer and neutral lipid species, were observed across different skin locations, including the forearm, forehead, cheek, and neck. Analysis of the layer-to-layer lipid profiles of the seven consecutive layers revealed a gradual decrease in DG and TG levels from the outermost to the innermost layers, with certain Cer subclasses showing increases in the second to fourth layers and subsequent decreases. Comparative analysis of lipid profiles from adjacent spots demonstrated statistical consistency and persistent differences between spots. Pooling layers were evaluated as an alternative method for representing SC layers, and their efficiencies were assessed by varying the number of pooled layers. We found that pooling five consecutive layers was effective in terms of the number and levels of identified lipids. Additionally, investigations into the matrix effect and extraction efficiency upon pooling layers indicated that pooling up to five layers did not significantly affect ionization suppression or reduce extraction recovery.

Integrated GC-MS and UPLC-ESI-QTOF-MS based untargeted metabolomics analysis of in vitro raised tissues of Digitalis purpurea L.

Digitalis purpurea L. is one of the important plant species of Nilgiris, Kashmir and Darjeeling regions of India, belonging to the family Plantaginaceae, with well-known pharmacological applications. In the present investigation, an in vitro culture technique of indirect shoot organogenesis of D. purpurea is being explored; the biochemical attributes, the antioxidant activities and the metabolomic analyses were made by utilizing untargeted Gas Chromatography-Mass Spectrometry (GC-MS) and Ultra Performance Liquid Chromatography coupled with electronspray ionization/quadrupole-time-of-flight-mass spectrometry (UPLC-ESI-QTOF-MS) approaches. Initially, the leaf explants were used for callus induction and proliferation and maximum callusing frequency (94.44%) and fresh biomass (4.9 g) were obtained on MS, fortified with 8.8 µM BAP (6-benzyl amino purine) + 0.9 µM 2,4-D (2,4-dichlorophenoxyacetic acid), subsequently shoot formation (indirect organogenesis) was noted on the same MS medium with a shoot induction frequency of 83.33%. Later on, the biochemical and antioxidant potential of in vivo-, in vitro grown leaf and leaf derived callus were assessed. Significantly higher total phenol, flavonoid, DPPH (2,2-diphenyl-1-picrylhydrazyl), POD (peroxidase) and SOD (superoxide dismutase) activities were noticed in in vitro grown callus and leaf tissues compared with field grown leaf. The GC-MS analysis of each methanolic extract (in vivo-, in vitro derived leaf and leaf derived callus) displayed the presence of more than 75 bioactive compounds viz loliolide, stigmasterin, alpha-tocopherol, squalene, palmitic acid, linoleic acid, beta-amyrin, campesterol etc. possessing immense therapeutic importance. The UPLC-MS based metabolite fingerprinting of each methanolic extracts were conducted in both positive and negative ionization mode. The obtained results revealed variation in phytochemical composition in field - and laboratory grown tissues, indicating the impact of in vitro culture conditions on plant tissues. The detected phytocompounds belongs to various classes such as flavonoids, steroids, terpenoids, carbohydrates, tannins, lignans etc. The medicinally important metabolites identified were 20, 22-dihydrodigoxigenin, digoxigenin monodigitoxoside, apigenin, luteolin, kaempferide, rosmarinic acid, nepitrin and others. The results of the present study suggest that in vitro culture of D. purpurea could successfully be utilized for the novel drug discovery by producing such important phytocompounds of commercial interest in shorter duration without harming the plants' natural population.

Valorization of grape pomace extracts against cranberry, elderberry, rose hip berry, goji berry and raisin extracts: Phytochemical profile and in vitro biological activity.

The circular economy is gaining attention around the world as a sustainable approach to tackling environmental problems, promoting more responsible management of resources. The aim of this work is the valorization of grape pomace as a waste product of agrifood chain. We prepared decoction (DC), ultrasound-assisted and microwave-assisted extracts (UAE and MAE respectively) of grape pomace, determining their phytochemical profile (using HPLC-ESI-Q-TOF-MS), antioxidant activity and enzyme inhibitory effects. Then, the results were compared with those of raisins and several edible berries already present in the market. Grape pomace extracts presented the highest total phenolic content (62-68 mg gallic acid equivalents/g; mg GAE/g), whereas the concentrations in the other berries were 4-43 mg GAE/g. These results were in agreement with the higher antioxidant activity and tyrosinase inhibition observed in grape pomace compared with the other berries, except for the metal chelating activity. The main compounds in grape pomace extracts were flavonoids (particularly quercetin glycosides), followed by organic acids (citric, isocitric and gallic acids). These results open new perspectives in the development of food supplements and nutraceuticals based on grape pomace extracts.

UHPLC-ESI-ms/ms-based characterisation of phenolics and flavonoids in hydroalcoholic extract of Clitoria ternatea seeds and their cytotoxic potential against breast and pancreatic cancer.

Fourteen known phenolics (1-14) and ten known flavonoids (15-24) were identified in the hydroalcoholic extract of Clitoria ternatea seeds. Phenolics such as gentisic acid (3), protocatechuic acid (5), o-coumaric acid (12) and p-coumaric acid (13) and among flavonoids, myricetin (16) were detected as the major compounds. The compounds were determined simultaneously in a multiple reaction monitoring (MRM) mode using the Acquity UPLC-H class coupled with TQD-MS/MS with an ESI source. The total phenolic and flavonoid contents were also determined. The hydroalcoholic extract evaluated for its cytotoxic effect against breast (MDA-MB-231) and pancreatic (PANC-1) cancer cells showed significant (p < 0.05) cytotoxicity with IC50 values of 45.4 and 96.5 µg/mL respectively. In addition, the cancer cells treated with the crude extract also showed elevated reactive oxygen species (ROS), upregulated caspase -8/-9/-3 activities and apoptosis of cells treated with C. ternatea extracts compared to control suggesting the pharmacological importance of this herb.

Hydrophilic interaction liquid chromatography: An efficient tool for assessing thorium interaction with phosphorylated biomimetic peptides.

In the field of nuclear toxicology, the knowledge of the interaction of actinides (An) with biomolecules is of prime concern in order to elucidate their toxicity mechanism and to further develop selective decorporating agents. In this work, we demonstrated the great potential of hydrophilic interaction liquid chromatography (HILIC) to separate polar thorium (Th) biomimetic peptide complexes, as a key starting point to tackle these challenges. Th4+ was used as plutonium (Pu4+) analogue and pS16 and pS1368 as synthetic di- and tetra-phosphorylated peptides capable of mimicking the interaction sites of these An in osteopontin (OPN), a hyperphosphorylated protein. The objective was to determine the relative affinity of pS16 and pS1368 towards Th4+, and to evaluate the pS1368 selectivity when Th4+ was in competition complexation reaction with UO22+ at physiological pH. To meet these aims, HILIC was simultaneously coupled to electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS), which allowed to identify online the molecular structure of the separated complexes and quantify them, in a single step. Dedicated HILIC conditions were firstly set up to separate the new dimeric Th2(peptide)2 complexes with good separation resolution (peptide = pS16 or pS1368). By adding pS16 and pS1368 in different proportions relatively to Th4+, we found that lower or equal proportions of pS16 with respect to pS1368 were not sufficient to displace pS1368 from Th2pS13682 and pS16 proportion higher than pS1368 led to the formation of a predominant ternary complex Th2(pS16)(pS1368), demonstrating preferential Th4+ binding to the tetra-phosphorylated peptide. Finally, online identification and quantification of the formed complexes when Th4+ and UO22+ were mixed in equimolar ratio relatively to pS1368 showed that in spite of pS1368 has been specifically designed to coordinate UO22+, pS1368 is also Th4+-selective and exhibits stronger affinity for this latter than for UO22+. Hence, the results gathered through this approach highlight the impact of Th4+ coordination chemistry on its interaction with pS1368 and more widely to its affinity for biomolecules.

High levels of the health-relevant antioxidant selenoneine identified in the edible mushroom Boletus edulis.

BACKGROUND: Selenoneine, the selenium analogue of the sulfur antioxidant ergothioneine, has been ascribed a multitude of beneficial health effects. Natural nutritional sources for this selenium species are, hence, of high interest. So far marine fish is the only significant selenoneine source consumed by larger parts of the population worldwide. METHODS: As selenoneine and ergothioneine share their biosynthetic pathways and transport mechanisms and the popular edible porcini mushroom Boletus edulis is rich in ergothioneine and selenium, we conducted a preliminary study investigating a composite sample of two specimens of B. edulis for their selenoneine content by HPLC coupled to elemental and molecular mass spectrometry after aqueous extraction. RESULTS: Selenium speciation analysis by HPLC-ICPMS revealed that ca. 860 µg Se kg-1 wet mass (81 % of the total Se) co-eluted with a selenoneine standard and a minor selenium species with Se-methylselenoneine. The presence of selenoneine was rigorously proven by HPLC-ESI-Orbitrap MS. The selenoneine content of the investigated specimens of B. edulis was higher than that of commonly consumed muscle of marine fish species, like tuna or mackerel. CONCLUSION: This is the first report of a terrestrial food source containing significant selenoneine levels. Our results suggest that B. edulis might represent a complementary natural supply with this health-relevant selenium species for humans.

A cost-effectiveness analysis of intraosseous basivertebral nerve ablation for the treatment of chronic low back pain.

BACKGROUND CONTEXT: Randomized trials have demonstrated the superiority of intraosseous basivertebral nerve ablation (BVNA) compared with sham and standard care in terms of improvements in pain, disability, and health-related quality of life in patients with vertebrogenic chronic low back pain (cLBP). PURPOSE: To assess the cost effectiveness of BVNA in patients with vertebrogenic cLBP compared to standard care alone. STUDY DESIGN/SETTING: A model-based economic analysis. PATIENT SAMPLE: Base case analysis used INTRACEPT, a randomized trial comparing BVNA with standard care in 140 patients with vertebrogenic cLBP, recruited from 23 sites across the United States, with a follow-up, up to 5 years. Scenario analyses compared data from the Surgical Multi-center Assessment of Radiofrequency Ablation for the Treatment of Vertebrogenic Back Pain (SMART) randomized trial against a sham control, and a single-arm study. OUTCOME MEASURES: Costs and quality-adjusted life years (QALYs) were calculated to determine the incremental cost-effectiveness ratio (ICER). METHODS: A cost-effectiveness model was built in Microsoft Excel® to evaluate the costs and health outcomes of patients undergoing BVNA using the Intracept Procedure (Relievant Medsystems) to treat vertebrogenic cLBP from a US payor perspective. Alternative scenario sensitivity analyses and probabilistic sensitivity analyses were conducted to assess the robustness of the model results. QALYs were discounted at 3.0% per year. RESULTS: Base case analysis showed that BVNA relative to standard care alone was a cost-effective strategy for the management of patients with vertebrogenic cLBP, with an ICER of US$11,376 per QALY at a 5-year time horizon from introduction of the procedure. Modeling demonstrated a >99% probability that this was cost effective in the US, based on a willingness-to-pay threshold of US$100,000 to US$150,000. Various sensitivity and scenario analyses produced ICERs that all remained below this threshold. CONCLUSIONS: BVNA with the Intracept Procedure offers patients with vertebrogenic cLBP, clinicians, and healthcare systems a cost-effective treatment compared to standard care alone.

Aligning Post-Column ESI-MS, MALDI-MS, and Coagulation Bioassay Data of Naja spp., Ophiophagus hannah, and Pseudonaja textillis Venoms Chromatographically to Assess MALDI-MS and ESI-MS Complementarity with Correlation of Bioactive Toxins to Mass Spectrometric Data.

Snakebite is a serious health issue in tropical and subtropical areas of the world and results in various pathologies, such as hemotoxicity, neurotoxicity, and local swelling, blistering, and tissue necrosis around the bite site. These pathologies may ultimately lead to permanent morbidity and may even be fatal. Understanding the chemical and biological properties of individual snake venom toxins is of great importance when developing a newer generation of safer and more effective snakebite treatments. Two main approaches to ionizing toxins prior to mass spectrometry (MS) analysis are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). In the present study, we investigated the use of both ESI-MS and MALDI-MS as complementary techniques for toxin characterization in venom research. We applied nanofractionation analytics to separate crude elapid venoms using reversed-phase liquid chromatography (RPLC) and high-resolution fractionation of the eluting toxins into 384-well plates, followed by online LC-ESI-MS measurements. To acquire clear comparisons between the two ionization approaches, offline MALDI-MS measurements were performed on the nanofractionated toxins. For comparison to the LC-ESI-MS data, we created so-called MALDI-MS chromatograms of each toxin. We also applied plasma coagulation assaying on 384-well plates with nanofractionated toxins to demonstrate parallel biochemical profiling within the workflow. The plotting of post-column acquired MALDI-MS data as so-called plotted MALDI-MS chromatograms to directly align the MALDI-MS data with ESI-MS extracted ion chromatograms allows the efficient correlation of intact mass toxin results from the two MS-based soft ionization approaches with coagulation bioassay chromatograms. This facilitates the efficient correlation of chromatographic bioassay peaks with the MS data. The correlated toxin masses from ESI-MS and/or MALDI-MS were all around 6-8 or 13-14 kDa, with one mass around 20 kDa. Between 24 and 67% of the toxins were observed with good intensity from both ionization methods, depending on the venom analyzed. All Naja venoms analyzed presented anticoagulation activity, whereas pro-coagulation was only observed for the Pseudonaja textillis venom. The data of MALDI-MS can provide complementary identification and characterization power for toxin research on elapid venoms next to ESI-MS.

Phytochemical, biological, and computational investigations of Ephedra alata Decne. growing in salinity conditions of Arabian Peninsula.

Ephedra alata Decne is a medicinal plant widely used in traditional medicine for the management of bronchial asthma and cancer. Phytochemical analysis and biological activities, including antioxidant and anticancer effects, were investigated in the current work as new findings for the plant E. alata, a species growing wildly in the marsh and saline environments of the central area of Saudi Arabia. The Ultra Pressure Liquid Chromatography coupled with Electron spray ionization-Quadropole-Time of flight (UPLC-ESI-Q-TOF) system was used for the phytochemical analysis of the plant constituents. In addition, Polyphenolic profiling including the total phenolic (TPC) and flavonoid (TFC) contents of the plant extracts were measured. Phenolic acids were found at the highest relative percentages among all the identified compounds and were measured at 66.07 mg GAE (Gallic acid equivalent). The UPLC analysis of the E. alata extract indicated the presence of chlorogenic acid, syringic acid, caffeic acid, vanillic acid, rosmarinic acid, umbelliferone, isorhoifolin, and apigenin at the highest relative percentages. Mineral analysis indicated that the microelement content of E. alata was relatively low, except for magnesium (Mg). In vitro antioxidant assays revealed the ability of the plant to scavenge DPPH free radicals, reduced molybdenum ions, and ferrous at levels of 14.63, 19.97, and 27.78 mg Trolox equivalents, respectively. The extract induced transition metal chelation at 31.36 mg EDTA equivalents. The extract induced cytotoxic effects against MDA-231 and A549 cell lines at IC50 levels of 25.31 and 39.81 µg/mL, respectively. The plant extract inhibited the colonization and migration of cancer cells as part of its potential anticancer effects. In addition, major E. alata constituents like isorhoifolin, chlorogenic acid, apigenin, and rosmarinic acid exhibited the lowest binding energy to the CAIX enzyme at - 8.41, - 6.64, - 6.32, and - 6.26 kcal/mol, respectively, compared to the binding energy (- 7.72 kcal/mol) of the co-crystallized ligand (Y0R). The docking results further supported the selection of the CAIX enzyme as a standard predictive therapeutic target, since it exhibited significant binding interactions with the major constituents of the plant.

Changes in Plasma Concentration of Free Proteinogenic and Non-Proteinogenic Amino Acids in High-Performance Sprinters over a 6-Month Training Cycle.

Background/Objectives: Free amino acids substantially contribute to energy metabolism. Also, their profile may identify (over)training status and effectiveness. The long-term effects of speed-power training on plasma free amino acid (PFAA) profiles are not known. We aimed to observe variations in PFAA levels in high-performance sprinters in a six-month training cycle. Methods: Ten male athletes (24.6 ± 3.3 years) were examined during four training phases: transition (1 month), general preparation (2 months), specific preparation (1 month), and pre-competition/competition (2 months). Venous blood was collected at rest, after exhaustive exercise, and recovery. Forty-two PFAAs were analyzed by the LC-ESI-MS/MS method. Results: Significant decreases in resting concentrations were observed between the transition and competition phases for glutamine (762 ± 117 vs. 623 ± 53 µmol∙L-1; p < 0.001, η2 = 0.47) and histidine (89 ± 15 vs. 75 ± 10 µmol∙L-1; p = 0.010, η2 = 0.27), whereas ß-alanine (30 ± 7 vs. 41 ± 9 µmol∙L-1; p = 0.024, η2 = 016) and sarcosine (3.6 ± 0.4 vs. 4.8 ± 0.6 µmol∙L-1; p = 0.006, η2 = 0.188) levels increased. Between the specific and competition phases, significant decreases in the resting levels of 1-methylhistidine (22.1 ± 19.4 vs. 9.6 ± 8.8 µmol∙L-1; p = 0.14, η2 = 0.19), 3-methylhistidine (7.1 ± 1.5 vs. 6.5 ± 1.6 µmol∙L-1; p = 0.009, η2 = 0.18), citrulline (40 ± 10 vs. 29 ± 4 µmol∙L-1; p = 0.05, η2 = 0.29), and ornithine (74 ± 15 vs. 56 ± 10 µmol∙L-1; p = 0.015, η2 = 185) were noticed. Also, for ß-alanine and sarcosine, the pattern of response to exercise strongly changed between the training phases. Blood ammonia levels at exhaustion decreased between the transition and competition phases (32 ± 4 vs. 23 ± 5 µmol∙L-1; p < 0.001, η2 = 0.67), while lactate, the phenylalanine-tyrosine ratio, the glutamine-glutamate ratio, hematological parameters, and cardiorespiratory indices remained at similar levels. Conclusions: Speed-power training seems to affect PFAAs involved in skeletal muscle metabolic pathways responsible for neutralizing toxic ammonia (glutamine, arginine, citrulline, ornithine), attenuating the deleterious effects of H+ ions (histidine, ß-alanine), and reducing exercise-induced protein breakdown (1- and 3-methylhistidine). Our findings suggest that sprint-oriented training supports metabolic pathways that are responsible for the removal of harmful metabolites produced during exercise.

Phytochemical Investigations, Antioxidant and Insecticidal Properties of Essential Oil and Extracts from the Aerial Parts of Pelargonium graveolens from Morocco.

The essential oil and the aqueous and ethanolic extracts obtained from the aerial parts of Pelargonium graveolens cultivated in Morocco were studied for their antioxidant and insecticidal activity against rice weevils (Sitophylus oryzae). The total phenolic content of the extracts was determined by a spectrophotometric method and the phenolic compounds were extensively characterized by HPLC-PDA/ESI-MS. To evaluate antioxidant potential, three in vitro assays were used. In the DPPH test, the ethanolic extract was the most active, followed by the aqueous extract and the essential oil. In the reducing power assay, excellent activity was highlighted for both extracts, while in the Fe2+ chelating activity assay, weak activity was observed for both the essential oil and the ethanolic extract and no activity for the aqueous extract. Concerning insecticide activity, the toxicity of the essential oil and the extracts was tested against rice weevils; the lethal concentrations LC50 and LC99 were determined, as well as the lethal time required for the death of 50% (LT50) and 99% (LT99) of the weevils. The essential oil had the highest activity; 100% mortality of S. oryzae was observed around 5, 9, and 8 days for the essential oil and the aqueous and ethanolic extracts, respectively.

A Comparative Study of LC-MS and FIA-(ESI)MS for Quantitation of S-Allyl-L-Cysteine in Aged Garlic Supplements.

The increasing consumption of food supplements demands the development of improved analytical methodologies to ensure their quality and authenticity. In this paper, two new approaches, liquid chromatography coupled to mass spectrometry (LC-MS) and flow injection analysis-(electrospray ionization) mass spectrometry (FIA-(ESI)MS), were optimized and validated for their application in the quantitative analysis of bioactive S-allyl-L-cysteine (SAC) in commercial aged garlic supplements (AGS). Although both methodologies were found to be useful for the sensitive and precise quantitation of SAC, the LC-MS approach allowed the differential determination of SAC and its bioactive diastereoisomer, S-1-propenyl-L-cysteine (S1PC), together with the identification of a number of organosulfur compounds typical of garlic. Mass fingerprints by FIA-(ESI)MS were proposed as an advantageous alternative to LC-MS analysis when the fast (4 min/sample) screening of AGS for their SAC content is intended, as in applications aimed at high-throughput quality control or standardization. Finally, the results gathered by the application of these two methodologies evidenced the highly variable composition of commercial AGS, as well as the identification of a number of potential composition frauds affecting their genuineness and benefits on health.

Study on Selenium Assimilation and Transformation in Radish Sprouts Cultivated Using Maillard Reaction Products.

The organic selenium (Se), particularly in the form of selenoamino acids, in non-edible sections or by-products of Se-enriched plants, has the potential to generate Maillard reaction products (MRPs) during thermal treatment or fermentation. To elucidate the recycling process of organic selenium in foods and improve the utilization rate of Se, the biotransformation of organic selenium was studied by the cultivation of edible radish sprouts with Se-MPRs. Maillard reactions were simulated using selenoamino acids (SeAAs; selenomethionine and methylselenocysteine) and reducing sugars (glucose and fructose) for preparing Se-MRPs. The structures of the possible dehydrated Se-MRPs were analyzed using a HPLC-ESI-MS/MS system based on their fragmentation patterns and Se isotopic characteristics. Se absorption by the radish sprouts cultivated using Se-MRPs was estimated by the corresponding Se in the SeAAs and the total Se contents. The capabilities of SeAA transformation and total Se assimilation by the sprouts were related to the substrate composition during the Se-Maillard reaction. A particular Se-MRP (selenomethionine + fructose) increased SeAAs transformation by 33.8% compared to selenomethionine. However, glucose and fructose seemed to inhibit the transformation of the Se-MRPs to SeAAs by 10.0 to 59.1% compared to purified Se-MRPs. These results provide key references for the efficient utilization of organic Se in the cultivation of Se-enriched sprouts.

Does Practice Match Protocol? A Comparison of Triage-to-Provider Time Among More vs. Less Acute Emergency Department Patients.

Introduction The Emergency Severity Index (ESI) stratifies emergency department (ED) patients for triage, from "most acute" (level 1) to "least acute" (level 5). Many EDs have a split flow model where less acute (ESI 4 and 5) are seen in a fast track, while more acute (ESI 1, 2, and 3) are seen in the acute care area. A core principle of emergency medicine (EM) is to attend to more acute patients first. Deliberately designating an area for less acute patients to be initially assessed quickly by a first provider might result in them being seen before more acute patients. This study aims to determine the percentage of less acute patients seen by a provider sooner after triage than more acute patients who arrived within 10 minutes of one another. Additionally, this study compares the fast track and acute care areas to see if location affects triage-to-provider time. Methods A random convenience sample of 252 ED patients aged ≥18 was taken. Patients were included if their ESI was available for the provider during sign-up. Patients were excluded if they were directly sent to the ED psychiatric area or attended to by the author. We collected data on the ESI level, time stamps for triage and first provider sign-up, and the location to which the patient was triaged (fast track vs. acute care). Paired patients' ESI levels, locations, and triage and first provider sign-up times were compared. Results  The study included 126 pairs of patients. There was a statistically significant difference in triage-to-provider times for paired ESI 2 vs. 3 patients (60.5 vs. 35.5 minutes, p = 0.0007) and overall paired high- vs. low-acuity patients (55 vs. 39.5 minutes, p = 0.004). However, in 34.8% of paired ESI 2 vs. 3 patients, the ESI 3 patient was seen prior to the paired ESI 2 patient, and in 39.4% of overall paired high vs. low acuity patients, the less acute patient was seen before the more acute patient. Additionally, patients in the acute care area had significantly shorter median triage-to-provider times (~40 minutes) compared to those in the fast track area for ESI 2 (acute care) vs. ESI 3 (fast track) and overall high acuity (acute care) vs. low acuity (fast track). Nonetheless, approximately one-third of ESI 3 patients triaged to fast track were seen before ESI 2 patients triaged to the acute care area. Conclusion The split flow model reduces overall ED length of stay, improving flow volume, revenue, and patient satisfaction. However, it comes at the expense of the fundamental ethos of EM and potentially subverts the intended triage process. Although most more acute patients are seen by a provider sooner after triage than less acute patients, a substantial number are seen later, which could delay urgent medical needs and negatively impact patients' outcomes. Furthermore, patients triaged to acute care are, in general, seen sooner post-triage than identical-ESI-level fast track patients, suggesting fast track might not function as intended (for low-acuity patients to be quickly assessed and initiate diagnostic and treatment plans). We intend to follow this exploratory study with a more comprehensive, multivariate analysis that will consider confounding variables such as initial vital signs, how busy a provider was that day, etc. The future study will also examine patient outcomes to determine the impact on more acute patients of the split flow model and, in particular, on less acute patients being seen sooner by a first provider.

Metabolic Pathway of Osilodrostat in Equine Urine Established through High-resolution Mass Spectrometric Characterization for Doping Control.

OBJECTIVE: Osilodrostat, used to treat Cushing's disease, exhibits an anabolic effect, leading to its classification as a prohibited substance in horseracing and equestrian sports. This study reports the characterization of osilodrostat metabolites in horse urine and elucidates its metabolic pathways for the first time for doping control purposes. METHODS: Osilodrostat was administered nasoesophageally to four thoroughbreds (one gelding and three mares) at a dose of 50 mg each. Potential metabolites were extensively screened via our developed generic approach employing differential analysis to identify metabolites. Specifically, high-resolution mass spectral data were compared between pre- and post-administration samples on the basis of criteria of fold-changes of peak areas and their P values. Potential metabolite candidates were further identified through mass spectral interpretations using product ion scan data. RESULTS: A total of 37 metabolites were identified after comprehensive analysis. Osilodrostat was predominantly metabolized into a mono-hydroxylated form M1c (~40%) alongside osilodrostat glucuronide M2 (~17%). Given their longest detection time (2 weeks after administration) and the identification of several conjugates of osilodrostat and M1c, including a novel conjugate of riburonic acid, we recommend monitoring both osilodrostat and M1c after hydrolysis during the screening stage. However, only osilodrostat can be used for confirmation because of the availability of a reference material. CONCLUSION: It is advisable to screen for both osilodrostat and its mono-hydroxylated metabolite M1c to effectively monitor horse urine for the potential misuse or abuse of osilodrostat. For suspicious samples, confirmation of osilodrostat using its reference material is required.

Quantification of osilodrostat in horse urine using LC/ESI-HRMS to establish an elimination profile for doping control.

Aim: The use of osilodrostat, developed as a medication for Cushing's disease but categorized as an anabolic agent, is banned in horses by both the International Federation of Horseracing Authorities and the Fédération Equestre Internationale. For doping control purposes, elimination profiles of hydrolyzed osilodrostat in horse urine were established and the detectability of free forms of osilodrostat and its major metabolite, mono-hydroxylated osilodrostat (M1c), was investigated.Materials & methods: Post-administration urine samples obtained from a gelding and three mares were analyzed to establish the elimination profiles of osilodrostat using a validated method involving efficient enzymatic hydrolysis followed by LC/ESI-HRMS analysis.Results: Applying the validated quantification method with an LLOQ of 0.05 ng/ml, hydrolyzed osilodrostat could be quantified in post-administration urine samples from 48 to 72 h post-administration; by contrast, both hydrolyzed osilodrostat and M1c were detected up to 2 weeks. In addition, confirmatory analysis identified the presence of hydrolyzed osilodrostat for up to 72 h post-administration.Conclusion: For doping control purposes, we recommend monitoring both hydrolyzed M1c and osilodrostat because of the greater detectability of M1c and the availability of a reference material of osilodrostat, which is essential for confirmatory analysis.

[Box: see text].

Phenolic Compound Profiles, Antioxidant and Cytoprotective Activity of Elaeagnus conferta Roxb. Fruit.

In this paper, three varieties of Elaeagnus conferta Roxb fruits prepared by ultrasonic-assisted extraction from a subtropical region southwest of China were utilized as raw materials to investigate their phenolic profiles, antioxidant activities, and protective effects on injured human umbilical vein endothelial cells (HUVECs). The ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) findings revealed that fifteen substances, including seven phenolic acids, seven flavonoids, and one gallic acid derivative, were discovered. The dihydromyricetin, ellagic acid, gallic acid were the predominant phenolic compounds in all E.conferta fruits. These E.conferta fruits extracts shown excellent antioxidant activity varied from 2.258 ± 0.03 ~ 7.844 ± 0.39 µM Trolox/g and protective effect on HUVECs injured by H2O2 through decrease the level of ROS, MDA, LDH and enhance the SOD level. These finding indicate that E.conferta is a valuable source of high-capacity antioxidants that might be used as an alternative material for food industries.

In Vivo Glucose Transporter-2 Regulation of Dorsomedial Versus Ventrolateral VMN Astrocyte Metabolic Sensor and Glycogen Metabolic Enzyme Gene Expression in Female Rat.

Astrocyte glycogenolysis shapes ventromedial hypothalamic nucleus (VMN) regulation of glucostasis in vivo. Glucose transporter-2 (GLUT2), a plasma membrane glucose sensor, controls hypothalamic primary astrocyte culture glycogen metabolism in vitro. In vivo gene silencing tools and single-cell laser-catapult-microdissection/multiplex qPCR techniques were used here to examine whether GLUT2 governs dorsomedial (VMNdm) and/or ventrolateral (VMNvl) VMN astrocyte metabolic sensor and glycogen metabolic enzyme gene profiles. GLUT2 gene knockdown diminished astrocyte GLUT2 mRNA in both VMN divisions. Hypoglycemia caused GLUT2 siRNA-reversible up-regulation of this gene profile in the VMNdm, but down-regulated VMNvl astrocyte GLUT2 transcription. GLUT2 augmented baseline VMNdm and VMNvl astrocyte glucokinase (GCK) gene expression, but increased (VMNdm) or reduced (VMNvl) GCK transcription during hypoglycemia. GLUT2 imposed opposite control, namely stimulation versus inhibition of VMNdm or VMNvl astrocyte 5'-AMP-activated protein kinase-alpha 1 and -alpha 2 gene expression, respectively. GLUT2 stimulated astrocyte glycogen synthase (GS) gene expression in each VMN division. GLUT2 inhibited transcription of the AMP-sensitive glycogen phosphorylase (GP) isoform GP-brain type (GPbb) in each site, yet diminished (VMNdm) or augmented (VMNvl) astrocyte GP-muscle type (GPmm) mRNA. GLUT2 enhanced VMNdm and VMNvl glycogen accumulation during euglycemia, and curbed hypoglycemia-associated VMNdm glycogen depletion. Results show that VMN astrocytes exhibit opposite, division-specific GLUT2 transcriptional responsiveness to hypoglycemia. Data document divergent GLUT2 control of GCK, AMPK catalytic subunit, and GPmm gene profiles in VMNdm versus VMNvl astrocytes. Ongoing studies seek to determine how differential GLUT2 regulation of glucose and energy sensor function and glycogenolysis in each VMN location may affect local neuron responses to hypoglycemia.

An interactive R-based custom quantification program for semi-quantitative analysis of triacylglycerols in bovine milk.

The R programming language, RStudio, and open-source software solutions for analysis of liquid chromatography-mass spectrometry (LC-MS) data have been used with user-written R-based custom quantification programs (CQP) for semi-quantification of triacylglycerols (TAGs) in bovine milk lipid extracts. Using the peak-finding capabilities of the package "xcms" in RStudio, peaks were integrated, and retention times aligned, normalized, and then used for semi-quantitative analysis of a custom set of four extraction internal standards (EISs) and 29 TAG regioisomers using the choice of four analytical internal standards (AISs). Alternating stereospecific numbering (sn) 1,3 TAG regioisomers (standards 1, 3, and 5 of six calibration standards) and sn-1,2 TAG regioisomers (standards 2, 4, and 6 of six standards) were used to make a set of six calibration standards, which were used for quantification using a linear fit model, polynomial fit model, power fit model, level-bracketed linear fit, replicate-bracketed polynomial fit, replicate-bracketed power fit, and replicate- and level-bracketed linear fit and response factors. For example, the linear fit for EIS1 gave an unacceptable coefficient of determination (CoD), r2 = 0.9616, whereas the polynomial fit gave r2 = 0.9908 and the power fit gave r2 = 0.9928, while the double-bracketed linear fit gave CoDs of r2 = 0.9960, 0.9848, and 0.9781 for the three brackets, yet gave the least % difference to known calibration concentrations. For unparalleled transparency, the CQP produced webpages that allowed every step in the data processing and quantification sequence to be verified and reproduced, and contained interactive figures. The data are publicly available using a digital object identifier (DOI). The R code can be downloaded and used with the downloadable data to reproduce the results, to modify the code and further customize the results, or to copy and paste and adapt the code to other quantification applications.

In-line RNA-based microreactor direct mass spectrometry for ultrasensitive and rapid assay of terminal deoxynucleotidyl transferase activity.

Terminal deoxynucleotidyl transferase (TdT), a unique template-independent DNA polymerase, plays a crucial role in the human adaptive immune system and is considered a promising biomarker for the diagnosis of various forms of acute or chronic leukemia. The accurate and sensitive detection of trace TdT is of pivotal importance to fulfill the significant medical interest in understanding its pathological functions and diagnosing TdT-related diseases. We hereby present an in-line RNA-based microreactor direct mass spectrometry (MS) method and its application for ultrasensitive, accurate, and rapid analysis of trace TdT activity in leukemic cell samples. A specially designed RNA-based microreactor is fabricated by immobilizing short RNA sequence via covalent Au-S bond on the inner surface of a capillary pre-modified with three-dimensional porous layer (PL) and Au nanoparticles (AuNPs). Utilizing this PL@Au@RNA microreactor, the signal of target TdT is conversed into reporter molecules (adenine), which exhibit a strong MS response. This conversion process enables efficient signal amplification and enhances detection sensitivity. The outlet end of the PL@Au@RNA microreactor is deliberately crafted into a porous tip, serving as an electrospray ionization (ESI) interface to directly couple to ESI-MS in-line. This design facilitates the direct transmission of the generated signaling molecules into the MS system, eliminating the need for laborious sample treatment procedures. By implementing this RNA-based microreactor in direct MS analysis, we have achieved remarkable sensitivity in detecting TdT activity with the limit-of-detection of 4 × 10-9 U, surpassing other reported methods in literature by three to four orders of magnitude. Furthermore, each assay requires a minimal sample volume of merely 10 nL. This method has successfully demonstrated its application in accurately and efficiently detecting TdT activity in leukemia cells, and its detection results are consistent with those obtained by ELISA kits.