High-performance Liquid Chromatography Fractions Research Articles

Turning a 3D Printer into a HPLC Fraction Collector: A Tool for Compound-Specific Stable Isotope Measurements

Compound-specific isotope analysis (CSIA) can provide unique insights into the cycling of elements including carbon and nitrogen. One approach for CSIA is the use of high-performance liquid chromatography (HPLC) to separate compounds of interest, followed by analysis of these compounds using an elemental analyser coupled to an isotope ratio mass spectrometer. A key component of this technique is the fraction collector, which automatically collects compounds as they are separated by HPLC. Here, we present a fraction collector that is a simple adaptation of a 3D printer, and, thus, can be easily adopted by any laboratory already equipped for HPLC. In addition to the much lower cost compared to commercial alternatives, this adaptation has the advantage for CSIA that the 3D printer is able to heat the collected fractions, which is not true for many commercial fraction collectors. Heating allows faster evaporation of the solvent, so that the dried compounds can be measured by EA–IRMS immediately. The procedure can be repeated consecutively so that diluted solutions can have the compounds concentrated for analysis. Any computer-controlled HPLC can be integrated to the fraction collector used here by means of AutoIt.

Bile acids as putative social signals in Mozambique tilapia (Oreochromis mossambicus)

Chemical cues provide potential mates with information about reproductive status and resource-holding potential. In the Mozambique tilapia (Oreochromis mossambicus), males can distinguish female reproductive status through chemical cues, and accessibility of males to females depends on their position in the hierarchy, determined in part by chemical cues. Here, we hypothesized that tilapia faecal cues are attractive to conspecifics once released into the water. C18 solid-phase extracts of faeces from dominant males and pre-ovulatory females evoked stronger olfactory epithelium electrical responses (EOG) than, respectively, subordinate males and post-spawning females. Mass spectrometry of the reverse-phase C18 high-performance liquid chromatography fractions of these extracts with highest EOG, identified by amino acids and bile acids. Faeces from pre-ovulatory females contain significantly higher concentrations of cholic acid (CA) and taurocholic acid (TCH) than both post-spawning females and males. A pool of amino acids had no effect on aggression or attraction in males. However, males were attracted to the scent of pre-ovulatory female faeces, as well as CA and TCH, when applied separately. This attraction was accompanied by increased digging behaviour compared to the odour of post-spawning females. CA and TCH exert their action through separate receptor mechanisms. These findings are consistent with a role for faeces – and bile acids therein – in chemical communication in this species, acting as an attractant for males to reproductive females.

Determining factors of durum wheat bread loaf volume and alveograph characteristics under optimal, drought and heat stress conditions

Durum wheat is an important crop, especially in Mediterrannean countries; around 25% of durum wheat world-wide, and 70–90% in the Middle East, is used for bread making. Climate change is causing increased periods of drought stress and higher temperatures which are affecting wheat quality. Alveograph extensibility is important for durum wheat loaf volume (LFV), as is the relationship between dough tenacity and extensibility. This study was done to identify the factors determining LFV and alveograph properties under optimal and stress conditions. Six durum wheat cultivars with the same glutenin composition were grown for two seasons under optimal, drought and heat stress conditions. Good LFV was consistently related with higher dough extensibility, irrespective of production conditions. A number of the LMW glutenin fractions, and the γ-gliadins were significantly reduced and β-gliadins were increased under both heat and drought stress. The large and total unextractable polymeric proteins and unextractable HMW glutenins were significantly reduced by heat stress. In a multiple regression of LFV (without alveograph values), solvent retention capacity and sodium dodecyl sulphate sedimentation were the best predictors of LFV under all conditions. Size-exclusion high-performance liquid chromatography fractions combined with flour protein content were good predictors of LFV only under optimal conditions.

Synergistic enhancement of glucagon-like peptide-1 release by γ-aminobutyric acid and L-phenylalanine in enteroendocrine cells-searching active ingredients in a water extract of corn zein protein.

This study investigated the glucagon-like peptide-1 (GLP-1)-releasing activity of an aqueous extract (ZeinS) from corn zein protein and aimed to identify the active compounds responsible for this activity. Glucagon-like peptide-1-releasing activity was evaluated using a murine enteroendocrine cell line (GLUTag). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on purified fractions of ZeinS to identify active molecules. ZeinS stimulated more GLP-1 secretion from GLUTag cells compared to zein hydrolysate. Fractions displaying biological activity were determined by solid-phase extraction and high-performance liquid chromatography (HPLC) fractionation. Subsequent LC-MS/MS analysis identified several amino acids in the active fractions of ZeinS. In particular, γ-aminobutyric acid (GABA) exhibited significant GLP-1-releasing activity both alone and synergistically with L-phenylalanine (Phe). Moreover, ZeinS-induced GLP-1 secretion was attenuated by antagonists for the GABA receptor and calcium sensing receptor. These results demonstrate that GABA and Phe identified in ZeinS synergistically stimulate GLP-1 secretion in enteroendocrine cells.

Ultra-high-performance liquid chromatography-tandem high-resolution elevated mass spectrometry profiling of anti-methicillin-resistant Staphylococcus aureus metabolites from the endophytic bacteria collected from the weeds of a previous dumpsite

The culturable endophytic bacteria from the weeds Cleome rutidosperma of the family Cleomaceae and Digitaria sanguinalis of the family Poaceae obtained from a previous dumpsite in Pampanga, Philippines have been assessed for their anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and the analytes with such activity should be identified. However, due to the limited amounts collected from the isolation process, 1.8 mg yield of compound 1 from the endophyte of C. rutidosperma and 1.2 mg of a mixture from the endophyte of D. sanguinalis were selected for LC-MSE analysis. The production of compounds from the culturable endophytic bacteria Pseudomonas aeruginosa— determined by gene-sequencing, an untargeted and data-independent analysis (DIA) by ultra-high performance liquid chromatography-high resolution-elevated energy mass spectrometry (UHPLC-HR-MSE) technique was employed to profile the metabolites present in the two high-performance liquid chromatography (HPLC) fractions. The analytes present from P. aeruginosa detected by UHPLC-HR-MSE isolated from C. rutidosperma was phenazine-1-carboxylic acid (1), and for D. sanguinalis were chamigrenal (2), dialkyl resorcinol (3), and a pyoverdine elicitor (4). This study proves that UHPLC-HR-MSE could identify the anti-MRSA constituents in P. aeruginosa from commensal weeds C. rutidosperma and D. sanguinalis. The UHPLC-HR-MSE could help strengthen metabolomics antibacterial research and its related applications from a future perspective. Application of metabolomics research using UHPLC-HR-MSE could enhance the rehabilitation of dumpsites by the microbial community present.

Therapeutic Potential of Peucedanum japonicum Thunb. and Its Active Components in a Delayed Corneal Wound Healing Model Following Blue Light Irradiation-Induced Oxidative Stress.

Blue light is reported to be harmful to eyes by inducing reactive oxygen species (ROS). Herein, the roles of Peucedanum japonicum Thunb. leaf extract (PJE) in corneal wound healing under blue light irradiation are investigated. Blue-light-irradiated human corneal epithelial cells (HCECs) show increased intracellular ROS levels and delayed wound healing without a change in survival, and these effects are reversed by PJE treatment. In acute toxicity tests, a single oral administration of PJE (5000 mg/kg) does not induce any signs of clinical toxicity or body weight changes for 15 days post-administration. Rats with OD (oculus dexter, right eye) corneal wounds are divided into seven treatment groups: NL (nonwounded OS (oculus sinister, left eye)), NR (wounded OD), BL (wounded OD + blue light (BL)), and PJE (BL + 25, 50, 100, 200 mg/kg). Blue-light-induced delayed wound healing is dose-dependently recovered by orally administering PJE once daily starting 5 days before wound generation. The reduced tear volume in both eyes in the BL group is also restored by PJE. Forty-eight hours after wound generation, the numbers of inflammatory and apoptotic cells and the expression levels of interleukin-6 (IL-6) largely increase in the BL group, but these values return to almost normal after PJE treatment. The key components of PJE, identified by high-performance liquid chromatography (HPLC) fractionation, are CA, neochlorogenic acid (NCA), and cryptochlorogenic acid (CCA). Each CA isomer effectively reverses the delayed wound healing and excessive ROS production, and their mixture synergistically enhances these effects. The expression of messenger RNAs (mRNAs) related to ROS, such as SOD1, CAT, GPX1, GSTM1, GSTP1, HO-1, and TRXR1, is significantly upregulated by PJE, its components, and the component mixture. Therefore, PJE protects against blue-light-induced delayed corneal wound healing via its antioxidative, anti-inflammatory, and antiapoptotic effects mechanistically related to ROS production.

Combining offline high performance liquid chromatography fractionation of peptides and intact proteins to enhance proteome coverage in bottom-up proteomics

Offline peptide separation (PS) using high-performance liquid chromatography (HPLC) is currently used to enhance liquid chromatography–tandem mass spectrometry (LC–MS/MS) detection of proteins. In search of more effective methods for enhancing MS proteome coverage, we developed a robust method for intact protein separation (IPS), an alternative first-dimension separation technique, and explored additional benefits that it offers. Comparing IPS to the traditional PS method, we found that both enhance detection of unique protein IDs to a similar magnitude, though in diverse ways. IPS was especially effective in serum, which has a small number of extremely high abundance proteins. PS was more effective in tissues with fewer dominating high-abundance proteins and was more effective in enhancing detection of post-translational modifications (PTMs). Combining the IPS and PS methods together (IPS+PS) was especially beneficial, enhancing proteome detection more than either method could independently. The comparison of IPS+PS versus six PS fractionation pools increased total number of proteins IDs by nearly double, while also significantly increasing number of unique peptides detected per protein, percent peptide sequence coverage of each protein, and detection of PTMs. This IPS+PS combined method requires fewer LC-MS/MS runs than current PS methods would need to obtain similar improvements in proteome detection, and it is robust, time- and cost-effective, and generally applicable to various tissue and sample types.

Evaluation of Ficus nitida Allelopathic Potential and the Most Efficient Application Method for Controlling Weeds Associated with Sunflower Plant

Allelopathy is a phenomenon by which plants positively or negatively affect neighboring plants by releasing allelopathic compounds. These allelochemicals are secondary metabolites found in different concentrations in shoots, roots, leaves, flowers, and even pollen grains. Allelochemicals have potential as natural bioherbicides for controlling weeds when applied in the form of extracts or through intercropping, cover cropping, and mulching. The present study was conducted to investigate the allelopathic potential of Ficus nitida leaves against Echinochloa crus-galli L. and Corchorus olitorius L weeds associated with sunflower plant via two application methods (mixing of F. nitida leaf powder with soil and foliar spray of F. nitida leaf powder alcoholic extract). Two pot experiments were carried out for the two summer seasons of 2020 and 2021 in the greenhouse of the National Research Centre (NRC), Dokki, Giza, Egypt. Nine treatments were applied in a completely randomized block design. Three treatments were applied before sowing, namely F. nitida leaf powder was mixed with the soil at rates of 15, 30, and 45 g/pot. The other three treatments of alcoholic leaf powder extract of F. nitida were sprayed twice on both plants and weeds at 10, 20, and 30% (w/v) concentrations. Additionally, three check treatments—healthy (sunflower only), unweeded, and both weeds only—were applied for comparison. The recorded results showed that F. nitida had an allelopathic bioherbicidal effect on both weeds. By increasing the concentration of F. nitida extract, the bioherbicidal potential increased. Moreover, the recorded results showed that foliar spray of alcoholic extract at 30% concentration was the superior application method for controlling weeds. Mixing of F. nitida leaf powder at 45 g/pot ranked second after this superior treatment. A noticeable result is that these two superior treatments improved sunflower growth parameters and yield traits. Quantitative estimation of phenolic compounds and flavonoids demonstrated that the concentration of these allelochemicals is higher in alcoholic extract than in water extract. Moreover, phenolic acid concentrations as detected by high-performance liquid chromatography fractionation are higher in alcoholic extract than in water extract.

Identification of bioactive components behind the antimicrobial activity of cow urine by peptide and metabolite profiling.

Cow urine possesses several bioactive properties but the responsible components behind these bioactivities are still far from identified. In our study, we tried to identify the possible components behind the antimicrobial activity of cow urine by exploring the peptidome and metabolome. We extracted peptides from the urine of Sahiwal cows belonging to three different physiological states viz heifer, lactation, and pregnant, each group consisting of 10 different animals. The peptides were extracted using the solid phase extraction technique followed by further extraction using ethyl acetate. The antimicrobial activity of the aqueous extract was evaluated against different pathogenic strains like Staphylococcus aureus, Escherichia coli, and Streptococcus agalactiae. The safety of urinary aqueous extract was evaluated by hemolysis and cytotoxicity assay on the BuMEC cell line. The urinary peptides were further fractionated using high-performance liquid chromatography (HPLC) to identify the fraction(s) containing the antimicrobial activity. The HPLC fractions and ethyl acetate extract were analyzed using nLC-MS/MS for the identification of the peptides and metabolites. A total of three fractions were identified with antimicrobial activity, and nLC-MS/MS analysis of fractions resulted in the identification of 511 sequences. While 46 compounds were identified in the metabolite profiling of organic extract. The urinary aqueous extract showed significant activity against E. coli as compared to S. aureus and S. agalactiae and was relatively safe against mammalian cells. The antimicrobial activity of cow urine is a consequence of the feeding habit. The metabolites of plant origin with several bioactivities are eliminated through urine and are responsible for their antimicrobial nature. Secondly, the plethora of peptides generated from the activity of endogenous proteases on protein shed from different parts of tissues also find their way to urine. Some of these sequences possess antimicrobial activity due to their amino acid composition.

Proteomic analysis of Taenia solium cysticercus and adult stages.

Taenia solium (T. solium) cysticercosis is a neglected parasitic zoonosis that occurs in developing countries. Since T. solium has a complex life cycle that includes eggs, oncospheres, cysticerci, and adults, presumably many proteins are produced that enable them to survive and establish an infection within the host. The objectives of this study were to perform a comparative proteomic analysis of two ontogenetic stages of T. solium (cysticerci and adult) and to analyze their differential expression of proteins. Methods proteins were separated by High Performance Liquid Chromatography (HPLC) fractionation, and protein samples were also digested in liquid and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS); the differentially expressed proteins were then processed by a bioinformatics analysis and verified by parallel reaction monitoring (PRM). Results we identified 2,481 proteins by label-free quantitative proteomics. Then differentially expressed proteins were screened under P values < 0.05 and 2 fold change, we found that 293 proteins up-regulated and 265 proteins down-regulated. Discussion through the bioinformatics analysis, we analyzed the differences types and functions of proteins in the Taenia solium and cysticercus, the data will provide reference value for studying the pathogenic mechanism of the two stages and the interaction with the host, and also support for further experimental verification.

Stable Isotope Analysis of Residual Pesticides via High Performance Liquid Chromatography and Elemental Analyzer-Isotope Ratio Mass Spectrometry.

To broaden the range of measurable pesticides for stable isotope analysis (SIA), we tested whether SIA of the anthranilic diamides cyantraniliprole (CYN) and chlorantraniliprole (CHL) can be achieved under elemental analyzer/isotope ratio mass spectrometry with compound purification in high-performance liquid chromatography (HPLC). Using this method, carbon isotope compositions were measured in pesticide residues extracted from plants (lettuce) grown indoors in potting soil that were treated with 500 mg/kg CHL and 250 mg/kg CYN and were followed up for 45 days. Our results show that the CYN and CHL standard materials did not have significant isotope differences before and after clean-up processing in HPLC. Further, when applied to the CYN product and CHL product in soil, stable isotope differences between the soil and plant were observed at <1.0‱ throughout the incubation period. There was a slight increase in the variability of pesticide isotope ratio detected with longer-term incubation (CHL, on average 1.5‱). Overall, we measured the carbon isotope ratio of target pesticides from HPLC fraction as the purification and pre-concentration step for environmental and biological samples. Such negligible isotopic differences in pesticide residues in soils and plants 45 days after application confirmed the potential of CSIA to quantify pesticide behavior in environments.

Enrichment and proteomic identification of Cryptosporidium parvum oocyst wall

BackgroundCryptosporidium parvum is a zoonotic parasitic protozoan that can infect a variety of animals and humans and is transmitted between hosts via oocysts. The oocyst wall provides strong protection against hostile environmental factors; however, research is limited concerning the oocyst wall at the proteomic level.MethodsA comprehensive analysis of the proteome of oocyst wall of C. parvum was performed using label-free qualitative high-performance liquid chromatography (HPLC) fractionation and mass spectrometry-based qualitative proteomics technologies. Among the identified proteins, a surface protein (CpSP1) encoded by the C. parvum cgd7_5140 (Cpcgd7_5140) gene was predicted to be located on the surface of the oocyst wall. We preliminarily characterized the sequence and subcellular localization of CpSP1.ResultsA total of 798 proteins were identified, accounting for about 20% of the CryptoDB proteome. By using bioinformatic analysis, functional annotation and subcellular localization of the identified proteins were examined for better understanding of the characteristics of the oocyst wall. To verify the localization of CpSP1, an indirect immunofluorescent antibody assay demonstrated that the protein was localized on the surface of the oocyst wall, illustrating the potential usage as a marker for C. parvum detection in vitro.ConclusionThe results provide a global framework about the proteomic composition of the Cryptosporidium oocyst wall, thereby providing a theoretical basis for further study of Cryptosporidium oocyst wall formation as well as the selection of targets for Cryptosporidium detection.Graphical

Addressing recent challenges in isotope ratio mass spectrometry: Development of a method applicable to 1-androstene-steroids, 6α-hydroxy-androstenedione, and androstatrienedione.

In 2020, the confirmation of the non-endogenous origin of several pseudo-endogenous steroids by means of isotope ratio mass spectrometry (IRMS) was recommended by the World Anti-Doping Agency (WADA), in addition to previously established target analytes for IRMS in sports drug testing. To date, however, IRMS-based methods validated in accordance with current WADA regulations have not been available. Therefore, the aim of this research project was the development and validation of a method to determine the carbon isotope ratios (CIR) of all newly considered pseudo-endogenous steroids, encompassing the anabolic androgenic steroids comprising a 1-ene-core structure (5α-androst-1-ene-3β,17β-diol, 5α-androst-1-ene-3,17-dione [1AD], 17β-hydroxy-5α-androst-1-en-3-one, 3α-hydroxy-5α-androst-1-ene-17-one [1AND], and 3β-hydroxy-5α-androst-1-ene-17-one [1EpiAND]), as well as steroids referred to as hormone and metabolic modulators (androsta-1,4,6-triene-3,17-dione [TRD] and its main metabolite 17β-hydroxy-androsta-1,4,6-triene-3-one) and 6α- and 6β-hydroxy-androst-4-ene-3,17-dione. With peak purity of target analytes being critical for IRMS analyses, a twofold high-performance liquid chromatography (HPLC)-based sample purification was employed, with all analytes being acetylated between the first and second HPLC fractionation. Using established gas chromatography/combustion/IRMS instrumentation, limits of quantification were estimated at 10 ng/ml for a 20 ml urine aliquot for all analytes, except for 1AND (20 ng/ml), and combined measurement uncertainties were estimated between 0.4‰ and 0.9‰. For proof-of-concept, samples collected after the single oral administration of a nutritional supplement containing 1AD and 1EpiAND were analyzed as well as existing excretion study urine samples obtained after the administration of 4-androstenedione and TRD. Based on the obtained results, the developed method was considered to be fit-for-purpose.

Proteome-wide analysis of lysine 2-hydroxyisobutyrylation in Frankliniella occidentalis

BackgroundLysine 2-hydroxyisobutyrylation (Khib) is a novel and conserved post-translational modification (PTM). Frankliniella occidentalis are economically important agricultural pests globally and also notorious for vectoring destructive plant viruses. To better study the disease transmission mechanism of F. occidentalis, it is necessary to conduct in-depth analysis of it. So far, no Khib modification of insects has been reported.ResultsIn this study, a proteome-wide analysis of Khib modifications in F. occidentalis was analyzed for the first time through the combination of high performance liquid chromatography fractionation technology and 2-hydroxyisobutyrylated peptide enrichment and other advanced technologies, 4093 Khib sites were identified on 1125 modified proteins. Bioinformatics and functional enrichment analyses showed that Khib-modified proteins were significantly enriched in many cell compartments and pathways, especially related to various cellular components and biological processes, and were more concentrated in ribosomes and proteasome subunits, involved in energy metabolism, protein synthesis and degradation, compared to the other nine species including Japonica rice, Homo sapiens, P. patens, Botrytis, Ustilaginoidea virens, Saccharomyces cerevisiae, T. gondii, C. albicans, and F. oxysporum. And Khib sites on virus-interacting insect proteins were discovered for the first time, such as cyclophilin and endoCP-GN.ConclusionsAfter three repeated experiments, we found a total of 4093 Khib sites on 1125 proteins. These modified proteins are mainly concentrated in ribosomes and proteasome subunits, and are widely involved in a variety of critical biological activities and metabolic processes of F. occidentalis. In addition, for the first time, Khib modification sites are found on the proteome of F. occidentalis, and these sites could be acted as for the virus interaction, including cyclophilin and endoCP-GN. The global map of 2-hydroxyisobutyrylation in thrips is an invaluable resource to better understand the biological processes of thrips and provide new means for disease control and mitigation of pest damage to crops.

Molecular Characterization of Estrogen Receptor Agonists during Sewage Treatment Processes Using Effect-Directed Analysis Combined with High-Resolution Full-Scan Screening.

Endocrine-disrupting potential was evaluated during the sewage treatment process using in vitro bioassays. Aryl hydrocarbon receptor (AhR)-, androgen receptor (AR)-, glucocorticoid receptor (GR)-, and estrogen receptor (ER)-mediated activities were assessed over five steps of the treatment process. Bioassays of organic extracts showed that AhR, AR, and GR potencies tended to decrease through the sewage treatment process, whereas ER potencies did not significantly decrease. Bioassays on reverse-phase high-performance liquid chromatography fractions showed that F5 (log KOW 2.5-3.0) had great ER potencies. Full-scan screening of these fractions detected two novel ER agonists, arenobufagin and loratadine, which are used pharmaceuticals. These compounds accounted for 3.3-25% of the total ER potencies and 4% of the ER potencies in the final effluent. The well-known ER agonists, estrone and 17β-estradiol, accounted for 60 and 17% of the ER potencies in F5 of the influent and primary treatment, respectively. Fourier transform ion cyclotron resonance mass spectrometry analysis showed that various molecules were generated during the treatment process, especially CHO and CHOS (C: carbon, H: hydrogen, O: oxygen, and S: sulfur). This study documented that widely used pharmaceuticals are introduced into the aquatic environments without being removed during the sewage treatment process.

TMT-Based Quantitative Proteomic Analysis Reveals the Response of Tomato (Solanum lycopersicum L.) Seedlings to Ebb-and-Flow Subirrigation

Ebb-and-flow subirrigation (EFI) is a water-saving and environmentally friendly irrigation method that can effectively improve water use efficiency and promote plant growth. In this study, we elucidated the effects of ebb-and-flow subirrigation on the protein levels in tomato roots in comparison with top sprinkle irrigation (TSI) and used an integrated approach involving tandem mass tag (TMT) labeling, high-performance liquid chromatography (HPLC) fractionation, and mass-spectrometry (MS)-based analysis. A total of 8510 quantifiable proteins and 513 differentially accumulated proteins (DAPs) were identified, of which the expressions of 283 DAPs were up-regulated, and 230 DAPs were down-regulated in the EFI vs. TSI treatment comparison. According to proteomic data, we performed a systematic bioinformatics analysis of all the identified proteins and DAPs. The DAPs were most significantly associated with the terms ‘metabolic process’, ‘anchored component of membrane’, ‘oxidoreductase activity’, ‘phenylpropanoid biosynthesis’, and ‘biosynthesis of secondary metabolites’ according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment (KEGG) analysis. The 272 DAPs were classified into 12 subcellular components according to their subcellular localization. Furthermore, the activities of SOD, POD, CAT, GR, and APX in tomato roots were remarkably increased under EFI, while the MDA content was decreased compared with TSI. Correlation analysis among activities of enzymes and their related DAPs showed that 30 DAPs might be responsible for the regulation of these enzymes. The results showed that ebb-and-flow subirrigation could induce a series of DAPs responses in tomato roots to be adapted to the new mode of water supply.

Fagonia cretica: Identification of compounds in bioactive gradient high performance liquid chromatography fractions against multidrug resistant human gut pathogens.

Plants are alternative source of natural medicines due to secondary active metabolites. Fagonia cretica extracts and Gradient High-Pressure Liquid Chromatography fractionations were checked against multidrug-resistant gastrointestinal pathogens including, Salmonella typhi, Escherichia coli and Shigella flexneri. ESI-MS/MS analysis of bioactive HPLC fractions was performed to elucidate antibacterial compounds. F. cretica extracts exhibited potential antibacterial activity. Twenty-four (24) HPLC fractions were obtained from methanol, ethanol and aqueous extracts of F. cretica. Eighteen (18) fractions showed antibacterial activity, while no activity was observed by the remaining six (6) fractions. HPLC fractions, F1 (25g ± 0.20 mm) and F2 (15f ± 0.12 mm) of aqueous extract exhibited activity against multidrug resistant GI pathogens. Gallic acid, quinic acid, cyclo-l-leu-l-pro, vidalenolone, liquirtigenin, rosmarinic acid and cerebronic acid were identified in F1 fraction of aqueous extract, while succinic acid, cyclo (l-Leul-Pro) and liquirtigenin were identified in F2 fraction of aqueous extract through ESI-MS/MS analysis. F. cretica extracts and HPLC fractions showed potential activity against MDR GI pathogens. Vidalenolone, Cyclo-1-leu-1-pro and Cerebronic acid are first time reported in F. cretica. Further characterization of bioactive compounds from F. cretica may be helpful to elucidate antibacterial therapeutic molecules.

Characterization of Humulus lupulus glycosides with porous graphitic carbon and sequential high performance liquid chromatography quadrupole time-of-flight mass spectrometry and high performance liquid chromatography fractionation

Monoterpenes contribute to the characteristic aroma of several hop varieties and may occur as nonvolatile glycosides. Upon hydrolysis, the volatile terpenes are released from the glycoside precursors. Little is known, however, about the glycoside composition of hops. Seven pentose-hexose monoterpene alcohol glycosides from dried Humulus lupulus L. cv. Citra cones were isolated using high performance liquid chromatography separation and fractionation on a reverse phase phenyl-hexyl column. Further evaluation of each isolated fraction through HPLC qTOF MS with porous graphitic carbon (PGC) showed that the seven isolated monoterpenyl glycoside fractions could be further resolved into 20 isomers. Isolation on phenyl-hexyl followed by separation on PGC was needed to distinguish each isomer present. Additionally, the hop cones were screened for potential aroma glycosides. Using the PGC column combined with a database of over 900 potential glycosides, the identification of 21 additional monoterpene-polyol, norisoprenoid, volatile phenol, and aliphatic alcohol glycosides is reported.

High expression of SGK1 in thrombosis of acute ST-segment elevation myocardial infarction: Based on proteomics analysis of intracoronary thrombosis

Introduction and objectivesThrombosis is involved in the onset and progression of ST-segment elevation myocardial infarction (STEMI). The aim of this study was to explore the expression level of serum- and glucocorticoid-inducible kinase 1 (SGK1) in intracoronary thrombus and the relationship between them. MethodsWe identified 30 patients who received treatment in the Affiliated Hospital of Hangzhou Normal University between May 2018 to May 2020 and who underwent thrombus aspiration and percutaneous coronary intervention for ST-segment-elevation myocardial infarction. Additionally, we selected 30 patients with normal coronary angiography for the control group. We analyzed thrombus protein expression profiling by combining tandem mass tag labeling, high-performance liquid chromatography fractionation and mass spectrometry quantification approach. ResultsThe differentially regulated protein profiles revealed the alteration of serine-threonine/tyrosine-protein kinase, which was upregulated significantly in the experimental group. We selected SGK1 downstream factor for validation and found that the expression of SGK1 in the thrombus of STEMI was significantly increased. Immunohistochemistry (IHC) showed significantly increased expression of SGK1 in the thrombus of STEMI patients compared with the control group (17.21±2.36 vs. 4.14±1.17%, p<0.05). Similar findings were observed in the Western blot analysis (p<0.05). IHC showed that SGK1 expressed in a region similar to that of the platelets. ConclusionThis is the first quantitative proteomics study to assess thrombus in patients with STEMI. The expression of SGK1 in thrombus was significantly higher in patients with acute STEMI than in the control group. SGK1 might be involved in the platelet physiological process.

Hyperthermia enhances the efficacy of chemotherapeutic drugs in heat-sensitive cells through interfering with DNA damage repair.

BackgroundHyperthermic intraperitoneal chemotherapy (HIPEC) has been shown to be clinically effective, but the mechanisms by which hyperthermia enhances the sensitivity of cells to chemotherapeutic drugs has not yet been elucidated.MethodsTo identify the key molecules involved in thermochemotherapy, this study used mass spectrometry (MS)-based quantitative proteomics technology to analyze the effects of thermochemotherapy on the heat-sensitive ovarian cancer cell line A2780. We divided the A2780 cell line into four groups, one group served as blank control, and the other three groups were stimulated by oxaliplatin, stimulated by hyperthermia at 42 ℃, and stimulated by hyperthermia combined with oxaliplatin. Samples were then collected for tandem mass tag (TMT) labeling, high-performance liquid chromatography fractionation, and MS-based quantitative proteomics for analysis The differentially expressed proteins were quantitatively compared and identified, and Gene Ontology (GO) assessment and cluster analyses were performed. Finally, the above MS results were verified again by Western blotting experiments.ResultsA total of 349 differentially expressed proteins were identified between cells treated with chemotherapy alone (group B) and cells treated with a combination of chemotherapy and hyperthermia (group D). There were 145 upregulated proteins and 204 downregulated proteins. Among the top 20 proteins with significantly different expression levels, nearly two-thirds were involved in DNA damage repair. These proteins were subsequently verified by Western blot analysis. Indeed, consistent with MS data, the expression of the RBL1 protein was significantly upregulated in cells treated with thermochemotherapy (group D) compared to cells treated with chemotherapy alone (group B).ConclusionsIn heat-sensitive ovarian cancer cells, the damage repair of tumor cell DNA is disturbed by hyperthermia, making it unable to fully repair when damaged by chemotherapeutic drugs. As a result, hyperthermia enhances the efficacy of chemotherapeutic drugs. RBL1, as a tumor suppressor gene, may be associated with the repair of DNA damage, and thus it may be a key target for hyperthermia to enhance the sensitivity of thermosensitive cells to chemotherapeutic drugs.