Charge site manipulation to enhance top-down fragmentation efficiency.

In recent years, top-down mass spectrometry has become a widely used approach to study proteoforms; however, improving sequence coverage remains an important goal. Here, two different proteins, α-synuclein and bovine carbonic anhydrase, were subjected to top-down collision-induced dissociation (CID) after electrospray ionisation. Two high-boiling solvents, DMSO and propylene carbonate, were added to the protein solution in low concentration (2%) and the effects on the top-down fragmentation patterns of the proteins were systematically investigated. Each sample was measured in triplicate, which revealed highly reproducible differences in the top-down CID fragmentation patterns in the presence of a solution additive, even if the same precursor charge state was isolated in the quadrupole of the instrument. Further investigation supports the solution condition-dependent selective formation of different protonation site isomers as the underlying cause of these differences. Higher sequence coverage was often observed in the presence of additives, and the benefits of this approach became even more evident when datasets from different solution conditions were combined, as increases up to 35% in cleavage coverage were obtained. Overall, this approach therefore represents a promising opportunity to increase top-down fragmentation efficiency.

Reinforcing the Evidence of Mitochondrial Dysfunction in Long COVID Patients Using a Multiplatform Mass Spectrometry-Based Metabolomics Approach.

Despite the recent and increasing knowledge surrounding COVID-19 infection, the underlying mechanisms of the persistence of symptoms for a long time after the acute infection are still not completely understood. Here, a multiplatform mass spectrometry-based approach was used for metabolomic and lipidomic profiling of human plasma samples from Long COVID patients (n = 40) to reveal mitochondrial dysfunction when compared with individuals fully recovered from acute mild COVID-19 (n = 40). Untargeted metabolomic analysis using CE-ESI(+/-)-TOF-MS and GC-Q-MS was performed. Additionally, a lipidomic analysis using LC-ESI(+/-)-QTOF-MS based on an in-house library revealed 447 lipid species identified with a high confidence annotation level. The integration of complementary analytical platforms has allowed a comprehensive metabolic and lipidomic characterization of plasma alterations in Long COVID disease that found 46 relevant metabolites which allowed to discriminate between Long COVID and fully recovered patients. We report specific metabolites altered in Long COVID, mainly related to a decrease in the amino acid metabolism and ceramide plasma levels and an increase in the tricarboxylic acid (TCA) cycle, reinforcing the evidence of an impaired mitochondrial function. The most relevant alterations shown in this study will help to better understand the insights of Long COVID syndrome by providing a deeper knowledge of the metabolomic basis of the pathology.

Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

Thirty years of fruitful collaborations between a physician and mass spectrometrists in diabetes field.

The nonenzymatic protein glycation and the subsequent formation of advanced glycation end products is a process involved in the long-term complications of diabetes. In this context the collaboration, in the last 30 years, between my research group, operating in the DPT of Medicine of Padua University, and the mass spectrometric group, operating in CNR of Padua, are described and discussed. The development of new mass spectrometric techniques has allowed investigation more indepth, starting from the applications on small molecules responsible for the browning observed in the interactions between sugars and proteins, and growing up to intact proteins as albumin, immunoglobulin, hemoglobin, and so forth, with the determination of their glycation levels as well as their glycation sites. This study has helped to clarify the role of advanced glycation end products in the pathogenesis of the chronic complications of diabetes. In particular the results obtained in diabetic nephropathy, diabetic cardiovascular disease and in placenta samples of patients affected by gestational diabetes are described in this review.

Suppressing the background of LC-ESI-MS analysis of permethylated glycans using the active background ion reduction device.

Mass spectrometry (MS) has revolutionized analytical chemistry, enabling precise identification and quantification of chemical species, which is pivotal for biomarker discovery and understanding complex biological systems. Despite its versatility, the presence of background ions in MS analysis hinders the sensitive detection of low-abundance analytes. Therefore, studies aimed at lowering background ion levels have become increasingly important. Here, we utilized the commercially available Active Background Ion Reduction Device (ABIRD) to suppress background ions and assess its effect on the liquid chromatography-electrospray ionization (LC-ESI)-MS analyses of N-glycans on the Q Exactive HF mass spectrometer. We also investigated the effect of different solvent vapors in the ESI source on N-glycan analysis by MS. ABIRD generally had no effect on high-mannose and neutral structures but reduced the intensity of some structures that contained sialic acid, fucose, or both when methanol vapor filled the ESI source. Based on our findings on the highest number of identified N-glycans from human serum, methanol vapor in the ion source compartment may enhance N-glycan LC-ESI-MS analyses by improving the desolvation of droplets formed during the ESI process due to its high volatility. This protocol may be further validated and extended to advanced bottom-up proteomic/glycoproteomic studies for the analysis of peptide/glycopeptide ions by MS.

Mn(II) Promoted Divergent-Convergent Domino Reaction Giving Dinuclear Tetrasubstituted Pyrrole Complex.

Domino reaction of benzo[d]thiazole-2-methylamine (S1) has been developed in the presence of MnCl2 ⋅ 4H2O, leading to tetrasubstituted pyrrole coordinated dinuclear Mn(II) complex 1 ([MnClP]2, P-=2,3,4,5-tetrakis(benzo[d]thiazol-2-yl)pyrrol-1-ide). The reaction process has been studied by assigning a series of intermediates based on time-dependent mass spectrometry, control experiments, crystallography, and density functional theory (DFT) theoretical calculation. A plausible mechanism involving an unprecedented divergent-convergent domino sequence has been proposed. Compound S1 could be activated by MnCl2 ⋅ 4H2O via coordination, which divergently produces two intermediates imine II (1-(benzo[d]thiazol-2-yl)-N-(benzo[d]thiazol-2-ylmethyl)methanimine) and alkene C (1,2-bis(benzo[d]thiazol-2-yl)ethene) through oxidative self-condensation and free radical coupling followed by elimination, respectively. They could then react with each other convergently via formal [3+2] cycloaddition to give deprotonated tetrasubstituted pyrrole coordinated intermediate [MnClP] after aromatization. Dimerization of [MnClP] produces the final product 1. Three C-C bonds and one C-N bond are formed through this six-step domino sequence. The corresponding organic skeleton (HP: 2,2',2'',2'''-(1H-pyrrole-2,3,4,5-tetrayl)tetrakis(benzo[d]thiazole)) has been obtained from 1 and shows a higher fluorescent quantum yield (52 %) than the reported 3,4-diphenyl substituted analogue 2,2'-(3,4-diphenyl-1H-pyrrole-2,5-diyl)bis(benzo[d]thiazole) (DPB) (42 %).

Protein-Protein Stabilization in VIVO/8-Hydroxyquinoline-Lysozyme adduct: A Spectroscopic, Crystallographic, and Computational Study.

The binding of the potential drug [VIVO(8-HQ)2], where 8-HQ is 8-hydroxyquinolinato, with hen egg white lysozyme (HEWL) was evaluated through spectroscopic (electron paramagnetic resonance, EPR, and UV-visible), spectrometric (electrospray ionization-mass spectrometry, ESI-MS), crystallographic (X-ray diffraction, XRD), and computational (DFT and docking) studies. ESI-MS indicates the interaction of [VIVO(8-HQ)(H2O)]+ and [VIVO(8-HQ)2(H2O)] species with HEWL. Room temperature EPR spectra suggest both covalent and non-covalent binding of the two different V-containing fragments. XRD analyses confirm these findings, showing that [VIVO(8-HQ)(H2O)]+ interacts covalently with the solvent exposed Asp119, while cis-[VIVO(8-HQ)2(H2O)] non-covalently with Arg128 and Lys96 from a symmetry mate. The covalent binding of [VIVO(8-HQ)(H2O)]+ to Asp119 is favored by a π-π contact with Trp62 and a H-bond with Asn103 of a symmetry-related molecule. Additionally, the covalent binding of VVO2+ to Asp48 and non-covalent binding of other V-containing fragments to Arg5, Cys6, and Glu7 is revealed. Molecular docking indicates that, in the absence of the interactions occurring at the protein-protein interface close to Asp119, the binding to Glu35 or Asp52 should be preferred. Such a protein-protein stabilization could be more common than what believed up today, at least in the solid state, and should be considered in the characterization of metal-protein adducts.

Extraction of naturally occurring peptides versus the tryptic digestion of proteins from fetal versus adult bovine serum for LC-ESI-MS/MS.

The naturally occurring peptides and digested proteins of fetal versus adult bovine serum were compared by LC-ESI-MS/MS after correction against noise from blank injections and random MS/MS spectra as statistical controls. Serum peptides were extracted by differential precipitation with mixtures of acetonitrile and water. Serum proteins were separated by partition chromatography over quaternary amine resin followed by tryptic digestion. The rigorous X!TANDEM goodness of fit algorithm that has a low error rate as demonstrated by low FDR q-values (q ≤ 0.01) showed qualitative and quantitative agreement with the SEQUEST cross correlation algorithm on 12,052 protein gene symbols. Tryptic digestion provided a quantitative identification of the serum proteins where observation frequency reflected known high abundance. In contrast, the naturally occurring peptides reflected the cleavage of common serum proteins such as C4A, C3, FGB, HPX, A2M but also proteins in lower concentration such as F13A1, IK, collagens and protocadherins. Proteins associated with cellular growth and development such as actins (ACT), ribosomal proteins like Ribosomal protein S6 (RPS6), synthetic enzymes and extracellular matrix factors were enriched in fetal calf serum. In contrast to the large literature from cord blood, IgG light chains were absent from fetal serum as observed by LC-ESI-MS/MS and confirmed by ELISA.

Sheet Models for Methylaluminoxane (MAO) Activators? A Theoretical Case Study involving rac-Me2Si(η5-C9H6)2Zr (SBIZr) Complexes.

Activation of SBIZrMe2 or SBIZrMeCl and a sheet model for an active component of hydrolytic MAO, (MeAlO)16(Me3Al)6, (16,6) has been studied by DFT. Contact ion-pair formation occurs through the intermediacy of SBIZrMe(Cl) or SBIZrMe2 reacting with sheet 16,6 to furnish SBIZrMe-µ-X(MeAlO)16(Me3Al)6 (2, X=Me, Cl). Contact ion-pairs 2 would be in equilibrium with heterodinuclear catalyst precursors [SBIZrMe2AlMe2][(MeAlO)16(Me3Al)6X] (3 (X=Me, Cl) through reversible binding of Me3Al at higher Al : Zr ratios. Calculations show that formation of ion-pairs 3 from contact ion-pairs 2 is more favourable for the SBIZr compared with the parent Cp2Zr complexes. TD-DFT calculations were conducted on relevant SBIZr complexes to relate the results to earlier spectroscopic studies of catalyst activation using UV-Vis spectroscopy. Finally, propene insertion into ion-pairs 2, SBIZrMe-µ-MeB(C6F5)3 (6) and [SBIZrMe][B(C6F5)4] (7) was studied at M06-2X/TZVP level of theory. These studies suggest that contact ion-pairs 2 are significantly less reactive towards insertion than 6 or 7, in disagreement with experiment.

Derivatization procedure of estradiol with a combination of MPDNP-F and 4-dimethylaminopyridine to generate product ion containing estradiol-skeleton for reliable determination of its serum/plasma concentrations by LC/ESI-MS/MS.

The quantification of serum/plasma estradiol (E2) is useful for the diagnosis, pathological analysis, and monitoring of the therapeutic efficacy of estrogen-dependent diseases. In this study, an improved derivatization method using 1-(2,4-dinitro-5-fluorophenyl)-4,4-dimethylpiperazinium iodide (MPDNP-F) was developed and combined with liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) for the sensitive and specific quantification of the serum/plasma E2. In the new method, the reaction time was reduced to 15 min from 90 min (two-step reaction in the previous method) by the direct reaction of MPDNP-F with E2 at 60°C in the presence of 4-dimethylaminopyridine (DMAP). DMAP served as the organic catalyst and had a less negative effect on the LC/ESI-MS/MS instrument compared to the non-volatile inorganic salt (NaHCO3), which was used in the previous method. The collision-induced dissociation of the molecular cation ([M]+) of the resulting derivative provided a product ion containing the E2-skeleton ([M-NO2-H]+), which significantly enhanced the assay sensitivity and specificity; compared to the dansyl chloride derivatization, which is the currently most-used derivatization procedure for the LC/ESI-MS/MS assays of E2, the MPDNP-F derivatization had significantly fewer interfering peaks and a clear and flat baseline in the serum sample analysis. The MPDNP-F derivatization-LC/ESI-MS/MS method enabled the precise and accurate quantification of E2 even at a 5.0 pg/mL concentration (lower limit of quantification) with a small sample volume (100 µL of serum/plasma) and had a tolerance for the matrix effect. This method was also proven to serve as a more sensitive and specific alternative to the clinically used chemiluminescence enzyme immunoassay.

Arsenobetaine amide: a novel arsenic species detected in several mushroom species.

The total arsenic mass fraction as well as the arsenic speciation were studied in four different mushroom species with inductively coupled plasma mass spectrometry and high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry, respectively. Arsenic mass fractions detected in the mushrooms were covering a range from 0.3 to 22 mg As kg-1 dry mass. For the arsenic speciation, species like arsenobetaine, inorganic arsenic, or dimethylarsinic acid were found, which are commonly detected in mushrooms, but it was also proven that the recently discovered novel compound homoarsenocholine is present in Amanita muscaria and Ramaria sanguinea. Moreover, a previously unidentified arsenic species was isolated from Ramaria sanguinea and identified as trimethylarsonioacetamide, or in short: arsenobetaine amide. This new arsenical was synthesized and verified by spiking experiments to be present in all investigated mushroom samples. Arsenobetaine amide could be an important intermediate to further elucidate the biotransformation pathways of arsenic in the environment.

Study of metalation of thioredoxin by gold(I) therapeutic compounds using combined liquid chromatography/capillary electrophoresis with inductively coupled plasma/electrospray MS/MS detection.

The reactivity of thioredoxin (Trx1) with the Au(I) drug auranofin (AF) and two therapeutic N-heterocyclic carbene (NHC)2-Au(I) complexes (bis [1-methyl-3-acridineimidazolin-2-ylidene]gold(I) tetrafluoroborate (Au3BC) and [1,3-diethyl-4,5-bis(4methoxyphenyl)imidazol-2-ylidene]gold(I) (Au4BC)) was investigated. Direct infusion (DI) electrospray ionization (ESI) mass spectrometry (MS) allowed information on the structure, stoichiometry, and kinetics of formation of Trx-Au adducts. The fragmentation of the formed adducts in the gas phase gave insights into the exact Au binding site within the protein, demonstrating the preference for Trx1 Cys32 or Cys35 of AF or the (NHC)2-Au(I) complex Au3BC, respectively. Reversed-phase HPLC suffered from the difficulty of elution of gold compounds, did not preserve the formed metal-protein adducts, and favored the loss of ligands (phosphine or NHC) from Au(I). These limitations were eliminated by capillary electrophoresis (CE) which enabled the separation of the gold compounds, Trx1, and the formed adducts. The ICP-MS/MS detection allowed the simultaneous quantitative monitoring of the gold and sulfur isotopes and the determination of the metallation extent of the protein. The hyphenation of the mentioned techniques was used for the analysis of Trx1-Au adducts for the first time.

Micro simulated moving bed chromatography-mass spectrometry as a continuous on-line process analytical tool.

Continuous manufacturing is becoming increasingly important in the (bio-)pharmaceutical industry, as more product can be produced in less time and at lower costs. In this context, there is a need for powerful continuous analytical tools. Many established off-line analytical methods, such as mass spectrometry (MS), are hardly considered for process analytical technology (PAT) applications in biopharmaceutical processes, as they are limited to at-line analysis due to the required sample preparation and the associated complexity, although they would provide a suitable technique for the assessment of a wide range of quality attributes. In this study, we investigated the applicability of a recently developed micro simulated moving bed chromatography system (µSMB) for continuous on-line sample preparation for MS. As a test case, we demonstrate the continuous on-line MS measurement of a protein solution (myoglobin) containing Tris buffer, which interferes with ESI-MS measurements, by continuously exchanging this buffer with a volatile ammonium acetate buffer suitable for MS measurements. The integration of the µSMB significantly increases MS sensitivity by removing over 98% of the buffer substances. Thus, this study demonstrates the feasibility of on-line µSMB-MS, providing a versatile PAT tool by combining the detection power of MS for various product attributes with all the advantages of continuous on-line analytics.

Development of an analytical method to quantify N-acyl-homoserine lactones in bacterial cultures, river water, and treated wastewater.

N-Acyl-homoserine lactones (AHL) play a major role in the communication of Gram-negative bacteria. They influence processes such as biofilm formation, swarming motility, and bioluminescence in the aquatic environment. A comprehensive analytical method was developed to elucidate the "chemical communication" in pure bacterial cultures as well as in the aquatic environment and engineered environments with biofilms. Due to the high diversity of AHLs and their low concentrations in water, a sensitive and selective LC-ESI-MS/MS method combined with solid-phase extraction was developed for 34 AHLs, optimized and validated to quantify AHLs in bacterial conditioned medium, river water, and treated wastewater. Furthermore, the developed method was optimized in terms of enrichment volume, internal standards, limits of detection, and limits of quantification in several matrices. An unanticipated variety of AHLs was detected in the culture media of Pseudomonas aeruginosa (in total 8 AHLs), Phaeobacter gallaeciensis (in total 6 AHLs), and Methylobacterium mesophilicum (in total 15 AHLs), which to our knowledge have not been described for these bacterial cultures so far. Furthermore, AHLs were detected in river water (in total 5 AHLs) and treated wastewater (in total 3 AHLs). Several detected AHLs were quantified (in total 24) using a standard addition method up to 7.3±1.0 µg/L 3-Oxo-C12-AHL (culture media of P. aeruginosa).

Development and validation of stability indicating assay method for mitapivat: Identification of novel hydrolytic, photolytic, and oxidative forced degradation products employing quadrupole-time of flight mass spectrometry.

Mitapivat is a novel, first-in-class orally active pyruvate kinase activator approved by the US Food and Drug Administration in 2022 for the treatment of hemolytic anemia. There is no literature available regarding the identification of degradation impurities of mitapivat. The present study deals with the degradation behavior of mitapivat under various stress conditions such as hydrolytic, photolytic, thermal, and oxidative stress. The multivariate analysis found that the independent variables, that is, molarity, temperature, and time, are interacting with each other to affect the degradation of mitapivat. A specific, accurate, and precise high-performance liquid chromatographic (HPLC) method was developed to separate mitapivat from its degradation products. The separation was achieved on the C-18 column (250 mm × 4.6 mm × 5 µm) using the combination of 0.1% formic acid buffer and acetonitrile in gradient elution profile. The method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use Q2(R2) guideline. LC-electrospray ionization-Quadrupole-time of flight was employed to identify degradation products. A total of seven novel degradation products of mitapivat were identified based on tandem mass spectrometry and accurate mass measurement. In-silico toxicity of mitapivat and its degradation products was qualitatively evaluated by the DEREK toxicity prediction tool.

Microbiome of seventh-century old Parsurameswara stone monument of India and role of desiccation-tolerant cyanobacterium Lyngbya corticicola on its biodeterioration.

The Parsurameswara stone monument, built in the seventh century, is one of the oldest stone monuments in Odisha, India. Metagenomic analysis of the biological crust samples collected from the stone monument revealed 17 phyla in the microbiome, with Proteobacteria being the most dominant phylum, followed by cyanobacteria. Eight cyanobacteria were isolated. Lyngbya corticicola was the dominant cyanobacterium in all crust samples and could tolerate six months of desiccation in vitro. With six months of desiccation, chlorophyll-a decreased; however, carotenoid and cellular carbohydrate contents of this organism increased in the desiccated state. Resistance to desiccation, high carotenoid content, and effective trehalose biosynthesis in this cyanobacterium provide a distinct advantage over other microbiomes. Comparative metabolic profiles of the biological crust and L. corticicola show strongly corrosive organic acids such as dichloroacetic acid, which might be responsible for the biocorrosion of stone monuments.

Peptide-Based Mass Spectrometry for the Investigation of Protein Complexes.

In the last two decades, biological mass spectrometry has become the gold standard for the identification of proteins in biological samples. The technological advancement of mass spectrometers and the development of methods for ionization, gas phase transfer, peptide fragmentation as well as for acquisition of high-resolution mass spectrometric data marked the success of the technique. This chapter introduces peptide-based mass spectrometry as a tool for the investigation of protein complexes. It provides an overview of the main steps for sample preparation starting from protein fractionation, reduction, alkylation and focus on the final step of protein digestion. The basic concepts of biological mass spectrometry as well as details about instrumental analysis and data acquisition are described. Finally, the most common methods for data analysis and sequence determination are summarized with an emphasis on its application to protein-protein complexes.

Influence of Nutrient Deprivation on the Antioxidant Capacity and Chemical Profile of Two Diatoms from Genus Chaetoceros.

The limited availability of phosphate, nitrogen and silicon in the growth media affects the growth, cellular processes, and metabolism of diatoms. Silicon deficiency primarily affects diatom morphology, while phosphate deficiency reduces the production of nucleic acids and phospholipids. Differences in pigment and protein composition are mainly due to nitrogen deficiency. In this study, Chaetoceros socialis and Chaetoceros costatus were cultured under phosphate, nitrogen, and silicon deprivation conditions. The diatom biomass was collected during the stationary growth phase and extracted with 70% ethanol under ultrasonication. The chemical profiles of the extracts were analyzed by high-performance liquid chromatography with high-resolution mass spectrometry with electrospray ionisation (UHPLC-ESI-HRMS), while the antioxidant capacity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and oxygen radical absorbance capacity (ORAC) assays. Pigments, fatty acids, sterols, and derivatives were detected in both species. The total phenolic content in the extracts ranged from 46.25 ± 1.08 to 89.38 ± 6.21 mg of gallic acid equivalent (GAE)/L and from 29.58 ± 1.08 to 54.17 ± 1.18 mg GAE/L. for C. costatus and C. socialis, respectively. Antioxidant activity was higher in C. costatus extracts, especially those obtained from nitrogen-deprived media. The results of this study contribute to the existing knowledge and the ongoing efforts to overcome application and commercialization barriers of microalgae for wide-ranging potential in different industries.

Improvement of Alginate Extraction from Brown Seaweed (Laminaria digitata L.) and Valorization of Its Remaining Ethanolic Fraction.

This study aimed to improve the conventional procedure of alginate isolation from the brown seaweed (Laminaria digitata L.) biomass and investigate the possibility of further valorization of the ethanolic fraction representing the byproduct after the degreasing and depigmentation of biomass. The acid treatment of biomass supported by ultrasound was modeled and optimized regarding the alginate yield using a response surface methodology based on the Box-Behnken design. A treatment time of 30 min, a liquid-to-solid ratio of 30 mL/g, and a treatment temperature of 47 °C were proposed as optimal conditions under which the alginate yield related to the mass of dry biomass was 30.9%. The use of ultrasonic radiation significantly reduced the time required for the acid treatment of biomass by about 4 to 24 times compared to other available conventional procedures. The isolated alginate had an M/G ratio of 1.08, which indicates a greater presence of M-blocks in its structure and the possibility of forming a soft and elastic hydrogel with its use. The chemical composition of the ethanolic fraction including total antioxidant content (293 mg gallic acid equivalent/g dry weight), total flavonoid content (14.9 mg rutin equivalent/g dry weight), contents of macroelements (the highest content of sodium, 106.59 mg/g dry weight), and microelement content (the highest content of boron, 198.84 mg/g dry weight) was determined, and the identification of bioactive compounds was carried out. The results of ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis confirmed the presence of 48 compounds, of which 41 compounds were identified as sugar alcohol, phenolic compounds, and lipids. According to the 2,2-diphenyl-1-picrylhydrazyl assay, the radical scavenging activity of the ethanolic fraction (the half-maximal inhibitory concentration of 42.84 ± 0.81 µg/mL) indicated its strong activity, which was almost the same as in the case of the positive control, synthetic antioxidant butylhydroxytoluene (the half-maximal inhibitory concentration of 36.61 ± 0.79 µg/mL). Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus) were more sensitive to the ethanolic fraction compared to Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei). The obtained results indicated the possibility of the further use of the ethanolic fraction as a fertilizer for plant growth in different species and antifouling agents, applicable in aquaculture.

Safety outcomes and improvement in pain scores after radiologist-performed fluoroscopy-guided interlaminar cervical epidural steroid injection.

INTRODUCTION: Image-guided spine injections are an important tool in the management of patients with a variety of spinal pathologies. Our practice offers radiologist-performed fluoroscopy-guided interlaminar cervical epidural steroid injection (ESI) routinely performed in the outpatient setting. The purpose of this study was to evaluate clinical outcomes and improvement in pain scores after radiologist-performed cervical ESI. METHODS: An institutional database was used to retrospectively identify cervical injections performed between October 2016 and October 2022. All injections were performed at the C7-T1 level utilizing 1.0 mL of 10 mg/mL dexamethasone without epidural anesthetic. The Numerical Rating Scale (NRS) was used to assess pain improvement. Cervical MRI was reviewed to assess pre-injection cervical disease severity. Patient charts were reviewed for any post-injection complications. RESULTS: A total of 251 cervical injections in 186 patients met our inclusion criteria with mean clinical follow up of 28.5 months (range 0.2 - 73.0 months). No patients experienced any major complications after injection. Post-injection pain scores were available for 218 of 251 injections (86.9%) with mean follow-up of 11.8 days (range 6 - 57 days). There was a significant improvement in pain scores from a mean pre-injection NRS score of 5.2/10 to 3.0/10 (p < .0001). 117 patients (53.7%) reported ≥ 50% improvement after injection. Patients who had prior injection were more likely to report ≥ 50% pain improvement after subsequent injection (p = .012). CONCLUSION: Radiologist-performed fluoroscopy-guided interlaminar cervical ESI at the C7-T1 level is a safe and effective tool in the management of patients with cervical pathology.