Combination Of High-performance Liquid Chromatography Research Articles

Plasma Oxylipins: A Potential Risk Assessment Tool in Atherosclerotic Coronary Artery Disease.

Background: While oxylipins have been linked to coronary artery disease (CAD), little is known about their diagnostic and prognostic potential.Objective: We tested whether plasma concentration of specific oxylipins may discriminate among number of diseased coronary arteries and predict median 5-year outcomes in symptomatic adults.Methods: Using a combination of high-performance liquid chromatography (HPLC) and quantitative tandem mass spectrometry, we conducted a targeted analysis of 39 oxylipins in plasma samples of 23 asymptomatic adults with low CAD risk and 74 symptomatic adults (≥70% stenosis), aged 38–87 from the Greater Portland, Oregon area. Concentrations of 22 oxylipins were above the lower limit of quantification in >98% of adults and were compared, individually and in groups based on precursors and biosynthetic pathways, in symptomatic adults to number of diseased coronary arteries [(1) n = 31; (2) n = 23; (3) n = 20], and outcomes during a median 5-year follow-up (no surgery: n = 7; coronary stent placement: n = 24; coronary artery bypass graft surgery: n = 26; death: n = 7).Results: Plasma levels of six quantified oxylipins decreased with the number of diseased arteries; a panel of five oxylipins diagnosed three diseased arteries with 100% sensitivity and 70% specificity. Concentrations of five oxylipins were lower and one oxylipin was higher with survival; a panel of two oxylipins predicted survival during follow-up with 86% sensitivity and 91% specificity.Conclusions: Quantification of plasma oxylipins may assist in CAD diagnosis and prognosis in combination with standard risk assessment tools.

Arsenic speciation in rice samples for trace level determination by high performance liquid chromatography-inductively coupled plasma-mass spectrometry

Six arsenic species, namely arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AsB) and arsenocholine (AsC) were speciated using a combination of high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC–ICP-MS). Under optimum chromatographic conditions, six arsenic species were well separated, and the performance of the combined system (HPLC–ICP-MS) for the species was determined. The limits of detection were calculated in the range of 0.14–0.29 ng/mL, and the corresponding quantification limits ranged between 0.45 and 0.97 ng mL−1 for the species. Spike recovery experiments performed on rice samples were used to validate the method’s applicability to complex matrices. The recovery results calculated ranged between 93 and 109%, validating the method’s applicability. Triplicate measurements for all spiked samples recorded percent relative standard deviation values below 10%.

Genital and systemic immune effects of the injectable, contraceptive norethisterone enanthate (NET-EN), in South African women.

Injectable hormonal contraceptives (IHC) have been associated with altered mucosal and systemic milieu which might increase HIV risk, but most studies have focused on DMPA and not NET-EN, despite the growing popularity and lower HIV risk associated with the latter in observational studies. We used high-performance liquid chromatography in combination with tandem triple quadrupole mass spectrometry (HPLC-LC-MS/MS) to measure steroid hormones in plasma samples of CAPRISA004 study participants. Concentrations of 48 cytokines were measured in the cervicovaginal lavage (CVL) and plasma, and their expression was compared between participants with detectable NET-EN (n=201) versus non-detectable IHC (n=90). Each log10 cytokine concentration was tested as an outcome in linear-mixed models, with NET-EN detection as the main explanatory variable. Multivariable models were adjusted for potential confounders. In bivariate analysis, detectable NET-EN was associated with reduced cervicovaginal M-CSF (P=0.008), GM-CSF (P=0.025) and G-CSF (P=0.039), and elevated levels MIF (P=0.008), IL-18 (P=0.011), RANTES (P=0.005) and IL-1Rα (P<0.001). Lower G-CSF (P=0.011) and elevated IL-1Rα (P=0.008) remained significant in adjusted models. Multivariable analyses of plasma samples obtained from NET-EN-detectable women showed a significant increase in IP-10 (P=0.026) and reductions in TNF-β (P=0.037), RANTES (P=0.009), and M-CSF (P<0.001). While similar growth factor reduction in CVL was noted for both DMPA and NET-EN, similar trends were not observed for endogenous progesterone. Detectable NET-EN was associated with reduced growth factors in the plasma and genital tract; particularly G-CSF and M-CSF. Our results suggest that while NET-EN is not inflammatory, it may have important immunological effects.

ATR-FTIR Spectroscopy, HPLC Chromatography, and Multivariate Analysis for Controlling Bee Pollen Quality in Some Algerian Regions.

Bee pollen collected by honeybees (Apis mellifera) is one of the bee products, and it is as valuable as honey, propolis, royal jelly, or beebread. Its quality varies according to its geographic location or plant sources. This study aimed to apply rapid, simple, and accurate analytical methods such as attenuated total reflectance Fourier transform infrared spectroscopy (ATR–FTIR) and high-performance liquid chromatography (HPLC) along with chemometrics analysis to construct a model aimed at discriminating between different pollen samples. In total, 33 samples were collected and analyzed using principal component analysis (PCA), hierarchical clustering analysis (HCA), and partial least squares regression (PLS) to assess the differences and similarities between them. The PCA score plot based on both HPLC and ATR–FTIR revealed the same discriminatory pattern, and the samples were divided into four major classes depending on their total content of polyphenols. The results revealed that spectral data obtained from ATR–FTIR acquired in the region (4000–500 cm–1) were further subjected to a standard normal variable (SNV) method that removes scattering effects from spectra. However, PCA, HCA, and PLS showed that the best PLS model was obtained with a regression coefficient (R2) of 0.9001, root-mean-square estimation error (RMSEE) of 0.0304, and root-mean-squared error cross-validation (RMSEcv) of 0.036. Discrimination between the three species has also been possible by combining the pre-processed ATR–FTIR spectra with PCA and PLS. Additionally, the HPLC chromatograms after pre-treatment (SNV) were subjected to unsupervised analysis (PCA–HCA) and supervised analysis (PLS). The PLS model confers good results by factors (R2 = 0.98, RMSEE = 8.22, and RMSEcv = 27.86). Prospects for devising bee pollen quality assessment methods include utilizing ATR–FTIR and HPLC in combination with multivariate methods for rapid authentication of the geographic location or plant sources of bee pollen.

Influence of drying process on furostanoside and spirostanoside profiles of Paridis Rhizoma by combination of HPLC, UPLC and UPLC-QTOF-MS/MS analyses

Drying method is one of the important factors affecting quality of traditional Chinese medicine. To study the effect of shaded drying and hot air drying on steroidal saponins of Paridis Rhizoma (PR), high performance liquid chromatography (HPLC) analysis was used to investigate the difference of Paris polyphylla var. chinensis (PPC) samples treated by different methods, and then, a rapid and reliable ultra-high performance liquid chromatography (UPLC) method was established to quantitatively analyze the content change of ten steroidal saponins. Hot air drying at 50 ℃ could obviously improve the content of polyphyllin Ⅶ, 17-hydroxygracillin and polyphyllin H, which were major steroidal saponins in PPC. Based on that, the main component changes induced by different drying methods were further analyzed using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS), and the structural identification of varied components revealed that hot air drying could promote the transformation of proto-pennogenyl glycosides to pennogenyl glycosides. This phenomenon was also found in other plants of genus Paris rich in diosgenyl glycosides. The present study provided a useful method for improving quality of PR and valuable information for TCM containing steroidal saponins.

Aptamers Against the β-Conglutin Allergen: Insights into the Behavior of the Shortest Multimeric(Intra)Molecular DNA G-Quadruplex

In previous work, a 93-mer aptamer was selected against the anaphylactic allergen, β-conglutin and truncated to an 11-mer, improving the affinity by two orders of magnitude, whilst maintaining the specificity. This 11-mer was observed to fold in a G-quadruplex, and preliminary results indicated the existence of a combination of monomeric and higher-order structures. Building on this previous work, in the current study, we aimed to elucidate a deeper understanding of the structural forms of this 11-mer and the effect of the structure on its binding ability. A battery of techniques including polyacrylamide gel electrophoresis, high-performance liquid chromatography in combination with electrospray ionization time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight, thermal binding analysis, circular dichroism and nuclear magnetic resonance were used to probe the structure of both the 11-mer and the 11-mer flanked with TT- at either the 5′ or 3′ end or at both ends. The TT-tail at the 5′ end hinders stacking effects and effectively enforces the 11-mer to maintain a monomeric form. The 11-mer and the TT- derivatives of the 11-mer were also evaluated for their ability to bind its cognate target using microscale thermophoresis and surface plasmon resonance, and biolayer interferometry confirmed the nanomolar affinity of the 11-mer. All the techniques utilized confirmed that the 11-mer was found to exist in a combination of monomeric and higher-order structures, and that independent of the structural form present, nanomolar affinity was observed.

Investigations of polymer samples of polyamide 11 concerning the content of monomer, oligomers, and the oxidation stabilizer Irganox 1098 by utilizing inverse gradient HPLC in combination with a triple detection system (diode array detection/mass spectrometry/charged aerosol detection)

The aim of this work was to develop a method to comprehensively characterize extracts from polyamide 11 powder samples used in the laser sintering process. Firstly, high resolution mass spectrometry was applied to identify low molecular compounds in the material. Secondly, by means of an inverse gradient high-performance liquid chromatography (HPLC) in combination with a triple detection system consisting of a diode array detector (DAD), a mass spectrometer (MS), and a charged aerosol detector (CAD), a method was established to determine the amount of monomer and oligomers up to the linear tetramer and cyclic pentamer. Furthermore, the oxidation stabilizer identified as Irganox 1098 was determined. The DAD was utilized for the quantification of the UV-active oxidation stabilizer. Thus, to be able to extract the low molecular weight compounds, various protocols were studied and finally accelerated solvent extraction was used. As there is a lack of commercial standards of polyamide 11 related oligomers, these compounds were detected with the CAD, taking advantage of its uniform response behavior in conjunction with an inverse gradient HPLC protocol. In addition, the MS was utilized as a qualitative detector for peak identification. The information regarding low molecular weight compounds are required as the powder is subject to the thermal loading of the sintering process, which influences the composition of the low molecular weight compounds. Thus, the method was applied to new and oven-aged polyamide 11 samples. Finally, it was possible to monitor the change of low molecular weight compounds in stressed polyamide 11 powder samples.

Methods for the Preparation of Blood Serum Samples in the Assessment of the Pharmacokinetics of Protein Drugs by the Example of Viscumin

A comparative evaluation of methods for preparing blood serum samples for determination of the lectin content of the mistletoe Viscum Album, viscumin, by high-performance liquid chromatography in combination with high-resolution mass-spectrometry detection has been carried out. Based on the results of the analysis of the lectin hydrolyzate, a specific peptide fragment with m/z 791.388 suitable for subsequent quantitative determination was selected. Isolation of viscumin from serum components was carried out without the use of specific antibodies. The study used methods employing various spin columns and the method of protein precipitation with 1% TCA (trichloroacetic acid) in IPA (isopropyl alcohol). Testing the methods of depletion of blood serum proteins was based on the determination of the degree of extraction of lectin from model serum samples. As a result of our research, we have developed a technique for the quantitative analysis of viscumin in blood serum by the most efficient method of sample preparation using the ProteoMiner spin columns. The technique allows to determine the protein concentration up to 5·10-4 mg/ml with the extraction degree (2.7 ± 0.6) %.

Quality control and long-term stability study of ginger from different geographical origins using chemometrics.

Ginger rhizome (Zingiber officinale) is a well-known spice and medicinal plant that is widely used in the Egyptian market as a spice, flavor and medicinal herb for different diseases. Since it is not cultivated as rhizomes in Egypt, ginger is imported from other countries, which may impact its quality. In this study, UV spectroscopy and high-performance liquid chromatography (HPLC) were applied as efficient available techniques for the discrimination and quality control of ginger collected from different geographical origins in combination with chemometrics. In addition, HPLC was applied to investigate the stability of ginger samples upon storage for 3 years to trace the changes in their main active constituents. Data obtained from both UV and HPLC in combination with Principal Component Analysis (PCA) displayed proper discrimination of the samples according to their geographical origins. Regarding the stability study, ginger samples showed a significant decrease in quality after storage for 3 years, in which significant variation in the main pungent principles (6-, 8-, 10-gingerols and 6-shogaol) were observed. PCA failed to discriminate between ginger samples after long-time storage, so the applied model could discriminate between ginger samples before and after storage. UV and HPLC in combination with chemometrics can be applied as a successful tool for the study of quality, stability and geographical discrimination of ginger. © 2020 Society of Chemical Industry.

Shotgun Proteomics and Mass Spectrometry as a Tool for Protein Identification and Profiling of Bio-Carrier-Based Therapeutics on Human Cancer Cells.

Shotgun proteomics has been widely applied to study proteins in complex biological samples. Combination of high-performance liquid chromatography with mass spectrometry has allowed for comprehensive protein analysis with high resolution, sensitivity, and mass accuracy. Prior to mass spectrometry analysis, proteins are extracted from biological samples and subjected to in-solution trypsin digestion. The digested proteins are subjected for clean-up and injected into the liquid chromatography-mass spectrometry system for peptide mass identification. Protein identification is performed by analyzing the mass spectrometry data on a protein search engine software such as PEAKS studio loaded with protein database for the species of interest. Results such as protein score, protein coverage, number of peptides, and unique peptides identified will be obtained and can be used to determine proteins identified with high confidence. This method can be applied to understand the proteomic changes or profile brought by bio-carrier-based therapeutics in vitro. In this chapter, we describe methods in which proteins can be extracted for proteomic analysis using a shotgun approach. The chapter outlines important in vitro techniques and data analysis that can be applied to investigate the proteome dynamics.

Determination of Total Antioxidant Capacities of Algal Pigments in Seaweed by the Combination of High-Performance Liquid Chromatography (HPLC) with A Cupric Reducing Antioxidant Capacity (CUPRAC) Assay

This study mainly aimed to analyze the essential antioxidant algal pigments present in marine algae to quantify the total antioxidant capacity (TAC) of these pigment constituents with CUPRAC (cupric ion reducing antioxidant capacity) and ABTS spectrophotometric methods, and to correlate the TAC values combined with high-performance liquid chromatography (HPLC) results. S. vulgare, C. barbata brown and F. petiolata green algae species demonstrated the highest antioxidant activity with the values of 12.11 ± 0.01; 18.56 ± 0.01; and 13.98 ± 0.02 (mmol trolox (TR) g−1 dw) obtained by the CUPRAC method. The individual antioxidant constituents of acetonic seaweed extracts were detected and quantified by HPLC on a C30 column using a modified mobile phase of gradient elution comprised of methanol-acetonitrile (50:50, v/v) with 0.1% (v/v) triethylamine (TEA) and acetone and ultraviolet detection for caroteneoids at 450 nm and for chlorophylls at 650 nm. The theoretical TAC values of seaweed samples were calculated by multiplying individual concentrations of pigments determined by HPLC with the spectrophotometric trolox equivalent or β-carotene equivalent antioxidant capacity (TEAC or CEAC) coefficient of each identified pigment and summing up this values. Among the studied seaweeds, those with the highest carotenoid content determined by HPLC were S. vulgare (51.77 µg/g), H. incurva (32.89 µg/g) and F. petiolata (200.12 µg/g), respectively. On the other hand, those with the highest total chlorophyll content were F. petiolata (1694.67 µg/g), S. vulgare (127.07 µg/g), and C. barbata (100.78 µg/g), respectively. As expected, fucoxanthin was present as the main carotenoid accumulated in the brown algae species presented in the study.

LATEST INFORMATION ON THE DISTRIBUTION OF FUSARIUM LANGSETHIAE, THE PRODUCER OF T-2 AND HT-2 TOXINS, IN RUSSIA

The annual monitoring of grain contamination with Fusarium fungi and the identification of their species composition showed the widespread distribution of F. langsethiae producing dangerous T-2 and HT-2 toxins in the Northwestern and Central regions of Russia. Mycological analysis of grain samples harvested in 2018–2019 allowed revealing the new places of F. langsethiae distribution, including Urals. The top infection rate of the oats grain by F. langsethiae in 2019 reached 14 %. The identification of F. langsethiae strains was supported by PCR with species-specific primers. The analysis of toxic metabolites in F. langsethiae by the combination of high-performance liquid chromatography and tandem mass spectrometry revealed the high level of T-2 and HT-2 toxins. The considerable total amounts of T-2 and HT-2 toxins (165–1230 μg/kg) were found in the grain samples infected with this species. Further clarification of the geographical area of F. langsethiae and the study of its intraspecific diversity are needed to understand the distribution of this toxin-producing fungus.

Intravenous iron preparations transiently generate non-transferrin-bound iron from two proposed pathways

Intravenous iron-carbohydrate complex preparations (IVIP) are noninterchangeable pro-drugs: their pharmacokinetics (PK) varies determined by semi-crystalline iron core and carbohydrate shell structures, influences pharmacodynamics (PD) and thus efficacy and safety. Examining PK/PD relationships of three IVIP we identify a two-pathway model of transient non-transferrin-bound iron (NTBI) generation following single dose administration. Twenty-eight hypoferremic non-anemic patients randomized to 200 mg iron as ferric carboxymaltose (Fe-carboxymaltose), iron sucrose (Fe-sucrose), iron isomaltoside 1000 (Fe-isomaltoside- 1000), n=8/arm, or placebo, n=4, on a 2-week PK/PD study, had samples analysed for total serum iron, IVIP-iron, transferrin-bound iron (TBI) by high-performance liquid chromatography in combination with inductively coupled plasma mass spectrometry (HPLC-ICP-MS), transferrin saturation (TSAT), serum ferritin (s-Ferritin) by standard methods, NTBI and hepcidin as published before. IVIP-dependent increases in these parameters returned to baseline in 48-150 hours (h), except for s-Ferritin and TSAT. NTBI was low with Fe-isomaltoside-1000 (0.13 μM at 8 h), rapidly increased with Fe-sucrose (0.8 μM at 2 h, 1.25 μM at 4 h), and delayed for Fe-carboxymaltose (0.57 μM at 24 h). NTBI area-under-curve (AUC) were 7-fold greater for Fe-carboxymaltose and Fe-sucrose than for Fe-isomaltoside-1000. Hepcidin peak time varied, but not AUC or mean levels. s-Ferritin levels and AUC were highest for Fe-carboxymaltose and greater than placebo for all IVIP. We propose two mechanisms for the observed NTBI kinetics: rapid and delayed NTBI appearance consistent with direct (circulating IVIP-to-plasma) and indirect (IVIP-to-macrophage-to-plasma) iron release based on IVIP plasma half-life and s-Ferritin dynamics. IVIP generate different, broadly stability- and PK-dependent, NTBI and s-Ferritin signatures, which may influence iron bioavailability, efficacy and safety. Longer-term studies should link NTBI exposure to subsequent safety and efficacy parameters and potential clinical consequences.

Comparison of molecular properties of saturate and aromatic fractions of Turkish and Iraqi crude oil residues

The chemical characterization of crude oil fractions is important for the petroleum research and complex structure of crude oil residue keeps studies ongoing. An analytical method which gives the composition of residue is not available yet and a combination of facile methods that elucidates the structure of residue is the most important in the petroleum research.This work reveals the detailed analyses of saturate and aromatic fractions of the selected crude oil atmospheric residues (ARs). Molecular structure of the saturate and the aromatic fractions was investigated by a combination of High-Performance Liquid Chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy techniques. In this study, saturate and aromatic fractions were physically divided into sub-fractions by HPLC and sub-fractions were examined in depth according to the hydrogen type using 1H NMR. Moreover, AR maltenes and their saturates were analyzed by High Temperature-Simulated Distillation (HT-SIMDIS), in order to deduce carbon number (CN) ranges of the AR distillation cut points in weight percentages. The study revealed the distribution of aliphatic and aromatic hydrogen types in sub-fractions of the saturates and the aromatics of the selected ARs. Mono, di- and tri-aromatic hydrogen types of each sub-fraction were also disclosed. For all sub-fractions, total aromaticity increases in the order of increasing elution sequence. Celikli AR maltene fraction has molecules that have CN in the range of 14–30 or less for half of its total weight and contains mostly low boiling structures.

The combination of HPLC and biological analysis to determine the quality markers and its structural composition of Eclipta prostrata L.

The diversity and complexity of components are important reasons for the unstable efficacy and safety of Chinese materia medica (CMM) in quality control. The good and stable quality control may be related to the quality markers with structural composition of multi-components. In the present study, we take Eclipta prostrata L. as a representative example. The 11 samples were collected from the different areas in China, and the discrepancy in bioactivity and chemical composition of these samples were compared. DOX (doxorubicin hydrochloride)-induced ICR mice were established for in vivo nephrotic syndrome experiments. The biochemical indicators including 24-h urine protein, triglyceride (TG), etc. were measured and the pathological change of kidney tissue was observed. MPC-5 cells damage model was induced to compare the difference of these samples in bio-activity. High-performance liquid chromatography (HPLC) method for 11 EEP (extract of Eclipta prostrata L.) samples were performed to analyse the content of the quality markers. In vivo experiments, EEP could mitigate the content or activity of urine protein, TG, etc. compared with the positive group (TG content was 2.53 ± 0.39 mmol/L, urinary protein quantification on 35th day was 16.79 ± 2.32 mg). In vitro experiments, CCE (coumestans component of Ecliptae) and EEP had equivalent effects on biochemical indicators such as cell viability, etc. According to the HPLC analysis, the content of wedelolactone was 45.88% and demethylwedelolactone was 23.74% in CCE. The CCE with a ratio of 2:1 can be considered as a quality marker of Eclipta prostrata L.. This research may provide a perspective for quality control of CMM.

Lipid composition and abundance in the reproductive and alimentary tracts of female Haemonchus contortus

BackgroundLipids play essential structural and functional roles in the biology of animals. Studying the composition and abundance of lipids in parasites should assist in gaining a better understanding of their molecular biology, biochemistry and host-parasite interactions.MethodsHere, we used a combination of high-performance liquid chromatography and mass spectrometric analyses, combined with bioinformatics, to explore the lipid composition and abundance in the reproductive (Rt) and alimentary (At) tracts of Haemonchus contortus.ResultsWe identified and quantified 320 unique lipid species representing four categories: glycerolipids, glycerophospholipids, sphingolipids and steroid lipids. Glycerolipids (i.e. triradylglycerols) and glycerophospholipids (i.e. glycerophosphocholines) were the most commonly and abundant lipid classes identified and were significantly enriched in Rt and At, respectively. We propose that select parasite-derived lipids in Rt and At of adult female H. contortus are required as an energy source (i.e. triradylglycerol) or are involved in phospholipid biosynthesis (i.e. incorporated fatty acids) and host-parasite interactions (i.e. phospholipids and lysophospholipids).ConclusionsThis work provides a first foundation to explore lipids at the organ-specific and tissue-specific levels in nematodes, and to start to unravel aspects of lipid transport, synthesis and metabolism, with a perspective on discovering new intervention targets.

LC-QTOF-MS and 1H NMR Metabolomics Verifies Potential Use of Greater Omentum for Klebsiella pneumoniae Biofilm Eradication in Rats.

Bacterial wound infections are a common problem associated with surgical interventions. In particular, biofilm-forming bacteria are hard to eradicate, and alternative methods of treatment based on covering wounds with vascularized flaps of tissue are being developed. The greater omentum is a complex organ covering the intestines in the abdomen, which support wound recovery following surgical procedures and exhibit natural antimicrobial activity that could improve biofilm eradication. We investigated changes in rats’ metabolome following Klebsiella pneumoniae infections, as well as the greater omentum’s ability for Klebsiella pneumoniae biofilm eradication. Rats received either sterile implants or implants covered with Klebsiella pneumoniae biofilm (placed in the peritoneum or greater omentum). Metabolic profiles were monitored at days 0, 2, and 5 after surgery using combined proton nuclear magnetic resonance (1H NMR) and high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC–QTOF-MS) measurements of urine samples followed by chemometric analysis. Obtained results indicated that grafting of the sterile implant to the greater omentum did not cause major disturbances in rats’ metabolism, whereas the sterile implant located in the peritoneum triggered metabolic perturbations related to tricarboxylic acid (TCA) cycle, as well as choline, tryptophan, and hippurate metabolism. Presence of implants colonized with Klebsiella pneumoniae biofilm resulted in similar levels of metabolic perturbations in both locations. Our findings confirmed that surgical procedures utilizing the greater omentum may have a practical use in wound healing and tissue regeneration in the future.

A Rapid Method for Analysis of cDNA Synthesis Using Ion-Pair Reversed-Phase High Performance Liquid Chromatography

We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library construction including all enzymatic steps usually required, but replaces radioactive labelling of nucleic acids with fluorescence detection. The separation and quantification steps all involve ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). This quantitative method replaces the use of autoradiography and size exclusion chromatography with combined ion-pair reversed-phase high performance liquid chromatography and in line fluorescence detection. The result of this approach is combination of speed with the generation of reproducible, high quality cDNA libraries.

Soluble Inorganic Arsenic Species in Atmospheric Submicron Particles in Two Polish Urban Background Sites

This paper presents results of the research on soluble inorganic As(III) and As(V) bound to submicron atmospheric particles (PM1) in two Polish urban background sites (Zabrze and Warsaw). The purpose of the research was to give some insight on the susceptibility to leaching of PM1-bound arsenic species from easily water-soluble compounds, i.e., considered potentially bioavailable based on its daily and seasonal changes. Quantitative analysis for 120 PM1 samples (collected from 24 June 2014 to 8 March 2015) was performed by using a high-performance liquid chromatography in combination with inductively coupled plasma mass spectrometry. The mean seasonal concentrations of dominant soluble As specie—As(V)—ranged from 0.27 ng/m3 in the summer season in Warsaw to 2.41 ng/m3 in the winter season in Zabrze. Its mean mass shares in total As were 44% in Warsaw and 75% in Zabrze in the winter and 18% and 48%, respectively, in the summer. Obtained results indicated fossil fuel combustion as the main source of PM1-bound As(V) and road traffic emission as its minor sources. In opposite to As(V), soluble As(III) was not clearly seasonally variable. In both seasons, its mean concentrations were higher in Zabrze than in Warsaw. As(III) concentrations were not preferentially shaped by an exact emission from road traffic in both cities.

Proteomics of arthropod soluble olfactory proteins.

Shotgun proteomics is a very sensitive bottom-up approach used to study complex mixtures of proteins through a combination of high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS). This technique has been successfully applied to the study of olfactory appendixes of insects and other arthropods. Since extracting soluble proteins from tissues and processing them for shotgun proteomics is quite straightforward, the technique has proved to be very suitable for studying soluble olfactory proteins, such as odorant binding proteins (OBPs), chemosensory proteins (CSPs) and Niemann-Pick type 2 proteins. Here, we describe the main principles and methods at the basis of shotgun proteomics, including its use to quantify proteins through label-free quantification. We also provide operational protocols for the experimental workflow steps, i.e., insect dissection, protein extraction, protein enzymatic digestion, sample purification, HPLC-MS analysis, search of mass spectral data against polypeptide databases and first steps in the analysis of the search results.