Liquid Chromatography-electrospray Mass Spectrometry Research Articles

Assay of Endocannabinoid Uptake.

Endocannabinoids at nanomolar physiological concentrations cross cellular membranes by facilitated diffusion, a process that can be studied by measuring transport kinetics and endocannabinoid trafficking employing radioligands and mass spectrometry. Here, we describe radiosubstrate-based assays using arachidonoyl[1-3H]ethanolamine and 2-arachidonoyl[1,2,3-3H]glycerol to measure cellular endocannabinoid uptake in a three-phase assay with human U937 cells. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS)-based lipidomics was used to interrogate the roles of serum and albumin for endocannabinoid trafficking in U937 cells.

Making high salt concentrations for optimal chromatography compatible with electrospray ionization mass spectrometry using an ion exchange membrane suppressor: Analysis of biomarkers for transporter protein inhibition as a case study

This study reports on the potential of using ion-exchange suppressor technology in liquid chromatography—electrospray ionization mass spectrometry workflows. The aim was to use high salt concentrations to improve separation performance, while overcoming the resulting significant ion suppression during electrospray ionization. As a case study, we apply suppressor technology to the hydrophilic interaction liquid chromatography separation and detection of taurine and glycochenodeoxycholate sulfate, endogenous biomarkers for organic anion transporter protein inhibition. The desired chromatographic selectivity was achieved applying 100 mM ion-pairing agent, while competing ions negatively affecting MS sensitivity were actively removed post-column from the solvent via a charged partially permeable membrane and replaced with protons, resulting in an up to 10-fold increase in detection sensitivity.

A Validated Stability-Indicating HPLC-PDA Method for Tolnaftate: Identification, Characterization and In Silico Toxicity Predictions of Major Degradation Products.

Simple and rapid stability indicating High Performance Liquid Chromatography-Photo Diode Array (HPLC-PDA) method was developed and validated for the estimation of tolnaftate in the presence of its forced degradation products. The method employed SunQSil C18 column (250mm × 4.6mm, 5μm) as stationary phase and acetonitrile:water (85:15, v/v) as mobile phase. Retention time of tolnaftate was 6.9min. Acid and alkali hydrolysis, oxidation, photo degradation and thermal degradation studies were carried out to evaluate the degradation behavior of tolnaftate. The developed and optimized method was validated as per International Conferences on Harmonization (ICH) guidelines. Limit of detection and limit of quantitation were found to be 0.092 and 0.276μg/mL, respectively. Linearity was observed in a concentration range of 0.276-6μg/mL with R2= 0.9936. %Recovery was found to be between 98.28% and 100.71%. The developed and validated Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) method was successfully applied for quantification of tolnaftate in in-house topical solution. Major base and oxidative degradation products were identified and characterized by liquid chromatography-electrospray ionization mass spectrometry. The probable mechanisms for the formation of degradation products were predicted based on the fragmentation pattern of degradation products. The in silico dermal penetration predictions and carcinogenicity of degradation products were evaluated by using QikProp and CarcinoPred-EL functionality.

Exploring the composition of Syringa reticulata subsp. amurensis seed and its underlying mechanism against chronic bronchitis

• The chemical compositions of Syringa reticulata subsp. amurensis seed were first determined with UPLC-ESI-MS. • Deciphering the underlying mechanism composition of Syringa reticulata subsp. amurensis seed against chronic bronchitis. • Six ingredients and seven targets were found against chronic bronchitis. The aim of this study was to analyze the bioactive components of Syringa reticulata subsp. amurensis (SRA) seeds, and the potential mechanism of SRA seed against chronic bronchitis is elucidated using network pharmacology. In total, 235 constituents were detected from SRA seed by ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS), and 28 active ingredients were identified through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database (TCMSP), which was classified into 10 different categories including flavonoids, diterpenoids, coumarins, alkaloids, isoflavones, phenylpropanoids, sesquiterpene, anthraquinones, phenols, and lignans. Among them, 6 components vestitol(V1), wogonin(W2), cheilanthifoline(C1), miltirone(M2), glabridin(G3), and myricanone (M3) were selected as potential key constituents of SRA seed against chronic bronchitis through network pharmacology analysis. Then the 7 key targets (PTGS2, TP53, TNF, VEGFA, IL1β, IL6, and JUN) were obtained based on the target prediction and protein-protein interaction (PPI). In conclusion, we use UPLC-ESI-MS analysis and network pharmacology to reveal the chronic bronchitis mechanisms of SRA seed, which provides new theoretical research ideas for the sustainable development of endangered multifunctional plants.

Lipidomic profile changes of yellow-feathered chicken meat during thermal processing based on UPLC-ESI-MS approach

In this work, ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC-ESI-MS) was applied to compare the lipidomics profile of chicken breast meat heated to various temperatures. A total of 445 lipids including phospholipids, diacylglycerols, triacylglycerols, glycoglycerolipids, and sphingolipids were detected in chicken muscle. Significant decreases in a list of characteristic unsaturated phospholipid species including phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were identified after heating, suggesting the occurrence of lipid oxidation, while significant increases in characteristic lysophospholipid species were found with increasing heating temperature, which were mainly derived from hydrolysis of phospholipids. Moreover, ether phospholipid and neutral lipid species exhibited remarkable changes during the heating process, and monoalk(en)yl diacylglycerol was first identified in chicken meat. The findings could contribute to the improved understanding of key lipids and biochemical reactions engaged in the heating of meat products.

Maceration and liquid-liquid extractions of phenolic compounds and antioxidants from Algerian olive oil mill wastewater.

Olive oil mill wastewater (OMW) is a major waste stream generated in olive oil industry. It is highly polluted due to phenolic compounds. The present study focused on the physicochemical properties of OMW as well as the quantitative and qualitative effects of two extraction methods of phenolic compounds which were liquid-liquid and maceration methods. Spectrophotometry and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) were adopted to quantify the phytochemical contents and the phenolic compounds. The extract obtained by the maceration method showed the highest yields of total polyphenol, flavonoid, and tannin contents. The LC-MS results revealed the presence of 16 phenolic compounds in the macerated, and only 12 phenolic compounds were found in the extract of the second method. Quinic acid was identified as the most abundant compound. Moreover, the macerated extracts possessed the highest antioxidant potential as evidenced by their strong ferric reducing antioxidant power (FRAP) and their 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical scavenging activities. The phytochemical contents, as well as the antioxidant potentials of OMW after extraction using maceration, were significantly greater than using liquid-liquid method. Therefore, maceration seemed to be the most effective method for extracting phenolic compounds from OMW. The OMW constitute a rich source of natural phenolic compounds that could be used as a potential source of natural antioxidants.

Accurate Determination of the Degree of Deacetylation of Chitosan Using UPLC–MS/MS

The mole fraction of deacetylated monomeric units in chitosan (CS) molecules is referred to as CS’s degree of deacetylation (DD). In this study, 35 characteristic ions of CS were detected using liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS/MS). The relative response intensity of 35 characteristic ion pairs using a single charge in nine CS samples with varying DDs was analyzed using 30 analytical methods. There was a good linear relationship between the relative response intensity of the characteristic ion pairs determined using ultrahigh performance (UP) LC–MS/MS and the DD of CS. The UPLC–MS/MS method for determining the DD of CS was unaffected by the sample concentration. The detection instrument has a wide range of application parameters with different voltages, high temperatures, and gas flow conditions. This study established a detection method for the DD of CS with high sensitivity, fast analysis, accuracy, stability, and durability.

What’s next after bevaciumab resistance? Targeting metabolic vulnerabilities in ovarian cancer (022)

What’s next after bevaciumab resistance? Targeting metabolic vulnerabilities in ovarian cancer (022)

Analysis of Oxidized 1-Palmitoyl-2-Arachidonoyl-Sn-Glycero-3 Phosphocholine Products in Uremic Patients by LC-ESI/MS

A simple liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI/MS) method has been developed to analyze oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products on the lipoproteins of uremic patients. The native PAPC standard was in vitro oxidized by the Fenton reaction, and the ox-PAPC products were analyzed by LC- ESI/MS. For LC, a C8 column and a mobile phase (acetonitrile-isopropanol containing 0.1% formic acid (70:30, v/v)) were selected. For ESI/MS, the optimal conditions included sheath gas pressure (10 psi), capillary temperature (270 °C), and injection time (1000 ms). The identification of ox-PAPC products on human lipoproteins was based on the extracted ion chromatograms (EICs) and the ESI-MS spectra of the in vitro oxidation products of PAPC standard. The EICs and ESI-MS spectra showed good repeatability and sensitivity. A total of 21 ox-PAPC products was determined. Linear analysis has been performed for the phospholipid standard, 1, 2-Di-O-hexadecyl-sn-glycero-3-phosphocholine (PC(O-16:0/O-16:0)). The linear range was 5.0–100.0 µg/mL, and the coefficient of determination (R2) was 0.989. The concentration limit of detection (LOD) was 1.50 µg/mL, and the concentration limit of quantitation (LOQ) was 4.54 µg/mL. The selected 21 ox-PAPC products have been identified and quantified in very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) of uremic and healthy subjects. Interestingly, the results showed that the levels of 18 products in VLDL, one product in LDL, and 19 products in HDL were significantly higher for uremic patients than healthy controls. This simple LC-ESI/MS method might accelerate the searching for biomarkers of uremia in the future.

Investigation of Protein Corona Formed around Biologically Produced Gold Nanoparticles.

Although there are several research articles on the detection and characterization of protein corona on the surface of various nanoparticles, there are no detailed studies on the formation, detection, and characterization of protein corona on the surface of biologically produced gold nanoparticles (AuNPs). AuNPs were prepared from Fusarium oxysporum at two different temperatures and characterized by spectrophotometry, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDS). The zeta potential of AuNPs was determined using a Zetasizer. AuNPs were incubated with 3 different concentrations of mouse plasma, and the hard protein corona was detected first by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then by electrospray liquid chromatography–mass spectrometry (LC-MS). The profiles were compared to AuNPs alone that served as control. The results showed that round and oval AuNPs with sizes below 50 nm were produced at both temperatures. The AuNPs were stable after the formation of the protein corona and had sizes larger than 86 nm, and their zeta potential remained negative. We found that capping agents in the control samples contained small peptides/amino acids but almost no protein(s). After hard protein corona formation, we identified plasma proteins present on the surface of AuNPs. The identified plasma proteins may contribute to the AuNPs being shielded from phagocytizing immune cells, which makes the AuNPs a promising candidate for in vivo drug delivery. The protein corona on the surface of biologically produced AuNPs differed depending on the capping agents of the individual AuNP samples and the plasma concentration.

A Pyridinium Derivatization Reagent for Highly Sensitive Detection of Poly(carboxylic acid)s Using Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry.

Short-chain fatty acids are difficult to analyze with high sensitivity using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) owing to the high polarity of their carboxyl groups. Various derivatization methods have been developed; however, most are effective only for monocarboxylic acids and not for those having multiple carboxyl groups. Therefore, we successfully attempted to synthesize a derivatization reagent that could analyze both mono- and poly(carboxylic acid)s with high sensitivity. We optimized our derivatization reagent by modifying the structure of the reaction site, hydrophobicity of the derivatized compound, and linker structure connecting the reaction site to the permanently charged substructure. The reactivity toward carboxyl groups was improved by employing a piperidine moiety as the reaction site, and the ESI efficiency was improved by the highly hydrophobic and permanently charged triphenylpyridinium group. Furthermore, the incorporation of an alkyl linker enabled polylabeling. When the optimized reagent was applied to mono-, di-, tri-, and tetracarboxylic acids, the ESI efficiency increased with polylabeling; thus, our derivatization reagent outperforms existing derivatization methods and enables the analysis of poly(carboxylic acid)s with high sensitivity. Since this derivatization reagent can be applied to most carboxyl-containing compounds, it can be widely used for lipidomics, proteomics, and metabolomics.

The key role of hydrophobicity in the determination of pharmaceuticals by liquid chromatography–electrospray ionization-mass spectrometry under the interference of natural organic matter

The determination of trace-level pharmaceuticals in water is generally performed using liquid chromatography combined with mass spectrometry, which is susceptible to interference from non-target substances, such as natural organic matter (NOM). In this study, the interference of NOM on the determination of 20 typical pharmaceuticals using solid-phase extraction followed by ultra-performance liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (UPLC-ESI-tqMS) was investigated with a combined consideration of recoveries, matrix effects, and process efficiencies. The results showed that the recoveries of most pharmaceuticals were not significantly affected by NOM concentrations of 1-50mg/L. The matrix effects and process efficiencies decreased linearly with increasing logarithmic NOM concentrations, and the changes in matrix effects and process efficiencies both exhibited negative linear correlations with the pharmaceuticals' hydrophobicity (logKow). This result indicated that the determination of hydrophilic pharmaceuticals suffered from more severe NOM interference, as NOM entered the ESI source together with hydrophilic pharmaceuticals after UPLC separation and subsequently weakened the ionization efficiency of these pharmaceuticals. According to the correlations between logKow and the changes in matrix effects and process efficiencies, the pharmaceutical determination in positive/negative ESI modes with logKow ≤ 3.80/4.27 is considered to be significantly affected by NOM, accompanied by > 20% changes in matrix effects and process efficiencies.

Retinal Pigment Epithelium in Human Donor Eyes Contains Higher Levels of Bisretinoids Including A2E in Periphery than Macula

PurposeWith age, human retinal pigment epithelium (RPE) accumulates bisretinoid fluorophores that may impact cellular function and contribute to age-related macular degeneration (AMD). Bisretinoids are comprised of a central pyridinium, dihydropyridinium, or cyclohexadiene ring. The pyridinium bisretinoid A2E has been extensively studied, and its quantity in the macula has been questioned. Age-changes and distributions of other bisretinoids are not well characterized. We measured levels of three bisretinoids and oxidized A2E in macula and periphery in human donor eyes of different ages.MethodsEyes (N = 139 donors, 61 women and 78 men, aged 40–80 years) were dissected into 8 mm diameter macular and temporal periphery punches. Using liquid chromatography – electrospray ionization – mass spectrometry (LC-ESI-MS) and an authentic synthesized standard, we quantified A2E (ng). Using LC-ESI-MS and a 50-eye-extract of A2E, we semiquantified A2E and 3 other compounds (eye extract equivalent units [EEEUs): A2-glycerophosphoethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE), and monofuranA2E (MFA2E).ResultsA2E quantities in ng and EEEUs were highly correlated (r = 0.97, P < 0.001). From 262 eyes, 5 to 9-fold higher levels were observed in the peripheral retina than in the macula for all assayed compounds. A2E, A2DHPE, and MFA2E increased with age, whereas A2GPE remained unaffected. No significant right-left or male-female differences were detected.ConclusionsSignificantly higher levels were observed in the periphery than in the macula for all assayed compounds signifying biologic differences between these regions. Levels of oxidized A2E parallel native A2E and not the distribution of retinal illuminance. Data will assist with the interpretion of clinical trial outcomes of agents targeting bisretinoid-related pathways.

Hepatic Metabolomics Analysis of Hybrid Grouper (Epinephelus fuscoguttatus♀×Epinephelus polyphekadion♂) Fed With Quercetin and Sodium Quercetin-5′-Sulfonates

Combining feeding trials and metabolomics analyses of tissues and biofluids could shed light on nutrient effects and changes in feed intake. In the present study, hybrid grouper (F1 hybrid Epinephelus fuscoguttatus♀×Epinephelus polyphekadion♂) was used as the marine fish model to quantify the impacts of quercetin and sodium quercetin-5′-sulfonates on serum biochemistry and the status of hepatic lipid accumulation and the changes of metabolites in the liver using ultra-performance liquid chromatography-electrospray triple quadrupole mass spectrometry (UPLC-QTRAP/MS/MS). The study shows that total protein, albumin, alkaline phosphatase and glucose were not significantly different among the three groups (P &amp;gt; 0.05). Total cholesterol, triglyceride, and alanine aminotransferase of fish fed quercetin, and sodium quercetin-5′-sulfonates were significantly lower than fish fed without additives (P &amp;lt; 0.05). Low-density lipoprotein cholesterol and aspartate aminotransferase of fish fed quercetin were significantly lower than that of fish fed without additives and sodium quercetin-5′-sulfonates (P &amp;lt; 0.05). The high-density lipoprotein cholesterol of fish fed quercetin, and sodium quercetin-5′-sulfonates were significantly higher than that of fish fed without additives (P &amp;lt; 0.05). Lipid accumulation in the quercetin and sodium quercetin-5′-sulfonates groups decreased significantly. Quercetin and sodium quercetin-5′-sulfonates were effective by increasing hypolipidemic and hepatoprotective compounds that are known for reducing blood lipid levels and liver fat accumulation. As a result of this study, we provide international data for metabolic adaptations during the additives feeding using the fish liver as the study model. By understanding the metabolic effects of these feed additives, this study provides a first step toward understanding the molecular mechanisms of these additives and how they function.

Particulate organic nitrates at Mount Tai in winter and spring: Variation characteristics and effects of mountain-valley breezes and elevated emission sources

Particulate organic nitrates, among the major components of secondary organic aerosols and fine particles, play important roles in regional nitrogen cycle, ozone budget, and cloud condensation nuclei formation. However, the pollution characteristics of particulate organic nitrates at mountain areas and the effects of anthropogenic pollutant transport remain poorly understood. In this study, field sampling and measurements were conducted at a high-elevation mountain site over North China Plain in winter and spring. Total five kinds of particulate organic nitrates in fine particles were determined by ultra-high performance liquid chromatography-electrospray mass spectrometry. The average total concentrations of particulate organic nitrates were 330 ± 121 ng m-3 and 247 ± 63 ng m-3 in winter and spring. The monoterpene-derived organic nitrates were the dominant components in both seasons with their contribution higher than 70%, accounting for 1.2 ± 0.8% and 2.0 ± 1.0% in organic aerosols in winter and spring, respectively. The significantly higher levels of particulate organic nitrates in winter than spring was ascribed to the strong effects of mountain-valley breezes and coal combustion plumes. The increasing concentrations of NOx and particulate matters brought by the valley breeze at daytime facilitated the formation of MHN215, OAKN359, and OAHN361, while the rising SO2 abundance and the sulfate aerosols transported by elevated emission sources affected the formation of MDCN247 at nighttime.

Identification of Antifungal Compounds from Piper Plants Against Fusarium oxysporum: An Untargeted Metabolite Profiling-Based Approach

The phytopathogen Fusarium oxysporum produces considerable losses in economically important crops, making alternative control measures urgently required. Piper plants are widely distributed in tropical regions, and they are also known to produce metabolites with biological activity against infectious agents. As part of our continuous search for antifungals, 18 Piper-derived ethanolic extracts were evaluated by their in vitro effect on F oxysporum mycelial growth inhibition. The total content of phenol and flavonoid measurements and liquid chromatography-electrospray ionization-mass spectrometry analysis served as the chemical characterization of the investigated extracts. Piper pulchrum, Piper barcoense, and Piper tuberculatum exhibited the highest mycelial growth inhibition (&gt;74%). The integration of chemical fingerprints and bioactivity datasets led to recognizing 4 bioactive candidates among extracts through single- Y orthogonal partial least squares regression and univariate statistics. These candidates were 2 amides (1,3), an alkyl lactone (2), and a prenylated benzoquinone (4), subsequently isolated and identified by nuclear magnetic resonance spectroscopy. These isolated compounds exhibited reasonable antifungal activity (IC50 &lt; 50 µM). The findings indicated that the correlative association is advantageous for identifying bioactive metabolites within active extracts.

Dereplication of Phenolics from Cardiospermum corindum by Countercurrent Chromatography Combined with Liquid Chromatography-Electrospray Mass Spectrometry

Dereplication of Phenolics from Cardiospermum corindum by Countercurrent Chromatography Combined with Liquid Chromatography-Electrospray Mass Spectrometry

Assessment of variable agroclimatic impact on dissipation kinetics of ready-mix fungicide formulation in green chili for harmonization of food safety

Multi-locational supervised field trials were conducted with ready-mix formulation of two fungicides, tricyclazole (18%) + mancozeb (62%) in green chili (Capsicum annuum) to assess the impacts of variable weather conditions on the residual profile for more realistic food safety evaluation. Liquid chromatography-Electrospray Ionization-Mass Spectrometry [LC-ESI(+ve)-MS/MS] and Ultraviolet Visible (UV-Vis) spectrophotometer have been employed for tricyclazole and mancozeb determination respectively. Tricyclazole was extracted in acetone: water (9:1) with dispersive solid phase extraction (d-SPE) cleaned up whereas mancozeb was digested in concentrated HCl and SnCl2. The developed method was successfully validated as per SANTE/12682/2019 guidelines. On green chilli, 225 + 775 g a.iha−1(active ingredient per hectare) (1250 g formulation-ha−1) and 450 + 1550 g a.iha−1 (active ingredient per hectare) (2500 g formulation-ha−1) of a combined formulation of tricyclazole (18%) and mancozeb (62%), WP, were employed as the recommended dose (T1) and double the recommended dose (T2), respectively. The initial depositions of tricyclazole were varied from 0.47 to 0.74 and 1.30–2.33 mg kg−1 and for mancozeb ranged from 0.93 to 1.31 and 2.68–4.13 mg kg−1 at the respective doses. The respective half-life (days) values of tricyclazole and mancozeb were 1.65–2.41 and 1.20–1.66 irrespective of doses and locations. The maximum residue limit (MRL) of 0.05 mg kg−1 for tricyclazole in green chili has been proposed for Food Safety and Standard Authority of India (FASSAI), India with recommended pre-harvest intervals (PHI) of 12 days. The third location (Anand Agricultural University, Anand, Gujarat) with the least rainfall and humidity has recorded the highest initial deposition, persistence, half-life, and PHI. The ready-mix fungicide has also increased fresh yield up to 22–34%. All these data may provide valuable information on the safe use and promotion of fungicides mixture.

Optimization of a method to detect levothyroxine and related compounds in serum and urine by liquid chromatography coupled to triple quadrupole mass spectrometry

Thyroid hormones and their derivatives are structurally related to the non-essential amino acid tyrosine (4-hydroxyphenylalanine). However, there are physicochemical differences that make it difficult to apply an analytical method for their simultaneous detection. This work focused on the optimization of a method using liquid chromatography-electrospray ionization mass spectrometry to measure eight compounds related to levothyroxine (T4). In addition, the influence of the additives to the mobile phase, the solvents for liquid-liquid extraction and the influence of the hydrolysis of the conjugated analytes were studied.Optimization of MRM transitions and collision energy for analytes and capillary voltage, nebulizer gas pressure, nozzle voltage, sheath gas flow, sheath gas temperature, drying gas flow and drying gas temperature for ionization source was done. The recovery of analytes was studied using five solvents and six solvent systems to introduce them into the liquid-liquid extraction and matrix cleanup steps. Different additives to the mobile phase were evaluated as well as the effectiveness of enzymatic and chemical hydrolysis.The best MRM transitions and source parameters were settled in order to generate an optimized instrumental method. The addition of ammonium formate, ammonium fluoride, and acetic acid to the mobile phase showed no improvement in responses compared to classic 0.1% formic acid. The use of tert-butyl methyl ether: isopropanol (75: 25, V: V) showed a suitable recovery of analytes to perform a liquid-liquid extraction, and n-hexane might be an appropriate solvent if a cleanup step is needed. The stability of T3, T4 and thyronine, checked after the hydrolysis with extracts of E. coli and H. pomatia showed good results, contrary to chemical hydrolysis that showed a total degradation of T3 and T4.

CyPA interacts with SERPINH1 to promote extracellular matrix production and inhibit epithelial-mesenchymal transition of trophoblast via enhancing TGF-β/Smad3 pathway in preeclampsia

We previously reported that cyclophilin A (CyPA) production is upregulated in preeclampsia (PE). Moreover, CyPA is known to induce PE-like features in pregnant mice and impair trophoblast invasiveness. In this study, we further illustrated the role of CyPA in PE. RNA-seq analysis, RT-qPCR, immunohistochemical (IHC) staining, and western blotting of mouse placentae revealed that CyPA increased the levels of extracellular matrix (ECM) proteins, such as collagen I and fibronectin, and activated the TGF-β/Smad3 signaling pathway. Additionally, CyPA inhibited the expression of genes involved in epithelial-mesenchymal transition (EMT) (e.g., E-cadherin, N-cadherin, and vimentin) in mouse placentae. We then constructed stable overexpressing and knock-down CyPA cell models (using HTR8/SVneo cells) to clarify the molecular mechanism. We found that CyPA regulated the levels of ECM-related proteins and the EMT process through the TGF-β/Smad3 pathway. We also identified SERPINH1 as a putative CyPA-binding protein, using liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. SERPINH1 was found to be upregulated in the placentae of PE. Silencing SERPINH1 expression reversed the upregulation of ECM proteins and inhibition of the EMT process induced by the overexpression of CyPA. These findings revealed the functions of CyPA in the impaired invasiveness of trophoblasts in PE and indicated that CyPA and SERPINH1 may represent promising targets for the treatment of PE.