Evaluation of greenness and analytical performances of separative methods for chiral separation of novel lactam-based P2RX7-antagonists.

In this work, a preparative supercritical fluid chromatography (SFC) method was first developed to separate a series of chiral compounds evaluated as lactam-based P2RX7 antagonists. Subsequently, high-performance liquid chromatography, SFC, and capillary electrophoresis (CE) were comparatively investigated as QC tools to determine the enantiomeric purity of the separated isomers, including analytical performance and greenness. The screening of the best conditions was carried out in liquid and SFC on the nine derivatives and the amylose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase was found to be highly efficient. The same screening was carried out in CE and very different conditions, either in acidic or basic background electrolyte and different cyclodextrins used as chiral selectors, allowed the separation of six of the nine derivatives. 1-((3,4-Dichlorophenyl)carbamoyl)-5-oxopyrrolidine-2-carboxylic acid (compound 1) was chosen as a probe, and its semi-preparative separation by SFC and enantiomeric verification using the three techniques are presented. Its limit of detection and limit of quantification are calculated for each method. Finally, the greenness of each quality control method was evaluated.

Quantification and stability assessment of urinary phenolic and acidic biomarkers of non-persistent chemicals using the SPE-GC/MS/MS method.

Nowadays, people are exposed to numerous man-made chemicals, many of which are ubiquitously present in our daily lives, and some of which can be hazardous to human health. Human biomonitoring plays an important role in exposure assessment, but complex exposure evaluation requires suitable tools. Therefore, routine analytical methods are needed to determine several biomarkers simultaneously. The aim of this study was to develop an analytical method for quantification and stability testing of 26 phenolic and acidic biomarkers of selected environmental pollutants (e.g., bisphenols, parabens, pesticide metabolites) in human urine. For this purpose, a solid-phase extraction coupled with gas chromatography and tandem mass spectrometry (SPE-GC/MS/MS) method was developed and validated. After enzymatic hydrolysis, urine samples were extracted using Bond Elut Plexa sorbent, and prior to GC, the analytes were derivatized with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA). Matrix-matched calibration curves were linear in the range of 0.1-1000 ng mL-1 with R > 0.985. Satisfactory accuracy (78-118%), precision (< 17%), and limits of quantification (0.1-0.5 ng mL-1) were obtained for 22 biomarkers. The stability of the biomarkers in urine was assayed under different temperature and time conditions that included freezing and thawing cycles. All tested biomarkers were stable at room temperature for 24 h, at 4 °C for 7 days, and at -20 °C for 18 months. The total concentration of 1-naphthol decreased by 25% after the first freeze-thaw cycle. The method was successfully used for the quantification of target biomarkers in 38 urine samples.

Protein overproduction alters exosome secretion in Chinese hamster ovary cells.

Despite the abundance of available cell lines, nearly 70% of all recombinant therapeutic proteins today are produced in Chinese hamster ovary (CHO) cells. The impact of protein overproduction on the secretion of exosomes by CHO cells has been investigated here. Increased secretion of extracellular vesicles (EVs) by protein overexpressing CHO cells was demonstrated with protein content assay, nanoparticle tracking analysis, and capillary electrophoresis. Our results revealed that a protein overproduction might induce EVs secretion, which might be accompanied by the sequestration and loading of overexpressed proteins into the exosomes. These findings are of vital importance for the manufacturing of therapeutics in CHO expression systems due to the risk of product loss during downstream processing of culture medium as well as the application of exosomes as nanocarriers of therapeutic proteins. The study indicates also the importance of culturing process control.

Reviewing the variability in urinary concentrations of non-persistent organic chemicals: evaluation across classes, sampling strategies and dilution corrections.

Various biomonitoring studies have been carried out to investigate the exposure of populations by measuring non-persistent organic chemicals in urine. To accurately assess the exposure, study designs should be carefully developed to maximise reproducibility and achieve good characterization of the temporal variability. To test these parameters, the intraclass correlation coefficients (ICCs) are calculated from repeated measurements and range from poor (<0.4) to excellent (≥0.75). Several studies have reported ICCs based on diverse study designs, but an overview, including recommendations for future studies, was lacking. Therefore, this review aimed to collect studies describing ICCs of non-persistent organic chemicals, discuss variations due to study design and formulate recommendations for future studies. More than 60 studies were selected, considering various chemical classes: bisphenols, pyrethroids, parabens, phthalates, alternative plasticizers and phosphate flame retardants. The variation in ICCs for an individual chemical was high (e.g. ICC of propyl paraben = 0.28-0.91), showing the large impact of the study design and of the specific exposure sources. The highest ICCs were reported for parabens (median = 0.52), while lowest ICCs were for 3-phenoxybenzoic acid (median = 0.08) and bisphenol A (median = 0.20). Overall, chemicals that had an exposure source with high variation, such as the diet, showed lower ICCs than those with more stable exposure sources, such as indoor materials. Urine correction by specific gravity had an overall positive effect on reducing the variability of ICCs. However, this effect was mostly seen in the adult population, while specific compounds showed less variation with creatinine correction. Single samples might not accurately capture the exposure to most non-persistent organic chemicals, especially when small populations are sampled. Future studies that examine compounds with low ICCs should take adequate measures to improve accuracy, such as correcting dilution with specific gravity or collecting multiple samples for one participant.

Quality Control of Bacterial Extracellular Vesicles with Total Protein Content Assay, Nanoparticles Tracking Analysis, and Capillary Electrophoresis.

Extracellular vesicles (EVs) were isolated from Pectobacterium zantedeschiae culturing media using direct ultracentrifugation (UC), iodixanol cushion ultracentrifugation (ICUC), and iodixanol density gradient ultracentrifugation (IDGUC) techniques. The isolates were characterized with total protein content assay (bicinchoninic acid assay, BCA), nanoparticles tracking analysis (NTA), and capillary electrophoresis (CE). A satisfactory correlation (R2 > 0.94) between quantitative results obtained with BCA, NTA and CE was achieved only for isolates obtained with the IDGUC. The correlation between protein content and CE was proved to be related to the isolates' purity. The NTA was found unable to provide reliable information on EVs quantity in samples isolated with UC and ICUC, due to the co-isolated particulate impurities. Moreover, the work reports polysaccharides, used as culturing media components, as a potential source of bias of quantitation with total protein content assay and NTA. The study demonstrates the advantageous selectivity of CE in quality control of EVs and its ability to differentiate subpopulations of EVs of Pectobacterium.

Investigation of selected parameters of capillary zone electrophoresis method for analysis of isolates of outer membrane vesicles.

The capillary zone electrophoresis (CZE) has recently been proposed by our group as a novel technique for outer membrane vesicles (OMVs) characterization (J. Chromatography 1621 (2020) 461047). In present work the impact of selected parameters of CZE method on OMVs isolates analysis was assessed. It was shown that the extension of sample injection plug length significantly improves the detectability of macromolecular aggregates in CZE. Moreover, a negligible adsorption of OMVs to both uncoated and polymer-modified (poly(DMA-GMA-MAPS)) capillary walls was proven. Finally, the relaxation effect as well as deformation/polarization of vesicles were demonstrated to affect OMVs electrophoretic mobility. The significance of these findings was discussed.

Development and validation of a gas chromatography method coupled with flame ionization detector for quantitative analysis of fragrance allergens in aromas for e-cigarettes.

Aromas can give smell and/or flavor to a variety of products in the cosmetic and food industry. They are also used as e-cigarette additives. Some of those substances are recognized as fragrance allergens and can cause allergic reactions so there is a need to control their use. Gas chromatography is the method of choice for analyzing fragrance allergens because of their low boiling points. This study aimed to develop and validate a robust and simple method for the analysis of fragrance allergens in aromas for e-cigarettes. A method using gas chromatography coupled with a flame ionization detector was developed for 25 fragrance allergens. Optimized parameters were sample diluent, internal standard, and carrier gas. The output method was the one previously developed and optimized. The linearity of the method was >0.994 over the range of 0.5-40 µg/mL. Accuracy and precision were within the acceptance range. Limits of detection and quantification were determined, and calibration curves were plotted. The method was applied to three real samples of aromas. Thirteen fragrance allergens were detected. Concentrations varied in the range of dozens to thousands of µg/mL showed that concentrations of fragrances in aromas for e-cigarettes can be high and varies among products.

Parameters of ovarian reserve in relation to urinary concentrations of parabens.

BACKGROUND: Parabens are synthetic chemicals commonly used in cosmetics, pharmaceuticals, food and beverage processing as antimicrobial preservatives. In experimental animals, parabens exposure was associated with adverse effects on female reproduction. Despite the widespread use of parabens little is known about their effect on female fecundity. The objective of the current analysis was to evaluate the associations of urinary parabens concentrations with parameters of ovarian reserve among women undergoing treatment in a fertility clinic. METHODS: Five hundred eleven female aged 25-39 years who attended the infertility clinic in central region of Poland for diagnostic purposes were recruited between September 2014 and February 2019. Urinary concentrations of parabens were measured by a validated gas chromatograohy ion-tap mass spectrometry method. Parameters of ovarian reserve were: antral follicle count (AFC), anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH) and estradiol (E2) levels. RESULTS: The geometric mean of specific gravity adjusted urinary concentrations of methyl (MP), ethyl (EP), propyl (PP), butyl (BP) and izobutyl paraben (iBuP) were 107.93 µg/L, 12.9 µg/L, 18.67 µg/L, 5.02 µg/L and 2.80 µg/L. Urinary concentrations of PP in the third quartile of exposure ((50-75] percentyl) were inversely associated with antral follicle count (p = 0.048), estradiol level (p = 0.03) and positively with FSH concentration (p = 0.026). MP, EP, BP and iBuP parabens were not associated any with parameters of ovarian reserve. CONCLUSIONS: Chronic exposure to PP may potentially contributing to reduced fecundity and impair fertility. As this is one of the first study to investigate the potential effect of parabens on ovarian reserve further epidemiological studies with longer duration of observation are needed.

Migration time shift of analytes in micellar electrokinetic chromatography induced by stacking.

A significant shift of migration time of nonretained compounds (ascorbic acid and cysteine) in micellar electrokinetic chromatography was observed under variation of sample matrix composition. The shift was affected by borate buffer concentration in sample matrix, sample injection time, and pH of BGE (80 mM SDS, Tris/HCl). Surprisingly, longer migration time of analyte was recorded at higher pH of separation buffer. These observations were linked to transient isotachophoresis process. Computer simulation with Simul5 software was conducted to support this hypothesis. The manuscript documents rarely reported in the literature phenomenon of isotachophoresis in micellar electrokinetic chromatography. The analytical potential of described observations was also discussed.

Evaluation of sample injection precision in respect to sensitivity in capillary electrophoresis using various injection modes.

A comparative study was conducted to assess the injection precision in capillary electrophoresis for cationic analytes (arecoline, codeine, papaverine). The precision was measured in respect to methods sensitivity in various injection modes in capillary electrophoresis: standard hydrodynamic injection (3.45 kPa for 6 s), large volume sample stacking (3.45 kPa for 40 s), and field-amplified sample injection (10 kV for 65 s). All measurements were conducted for aqueous solutions of standards to minimize the errors linked to the sample preparation step. The methods were submitted to precision assessment at three concentration levels: at the limit of quantification, three-fold and ten-fold of limit of quantification. The results were compared to those from high-performance liquid chromatography as a reference technique. The field-amplified sample injection method was shown to provide greatest sensitivity (quantification limits down to 4 ng/mL for all three tested compounds) but the lowest precision. High-performance liquid chromatography was established as the most reliable technique (coefficient of variation in all intraday experiments was below 1%). It was also shown that with a use of large volume sample injection technique, similar sensitivity as in high-performance liquid chromatography can be easily reached.

Environmental exposure to parabens and sperm chromosome disomy.

Parabens are widely used as antimicrobial preservatives in cosmetics, pharmaceuticals, food and beverage processing due to their board spectrum of activity, inertness, and low cost. The study population consisted of 156 men under 45 years of age who attended the infertility clinic for diagnostic purposes with normal semen concentration of 15-300 mln/ml. Participants were interviewed and provided a semen sample. The parabens concentrations: ethyl paraben (EP), butyl paraben (BP), methyl paraben (MP), and iso-butyl paraben (iBuP) were analyzed in the urine using a validated gas chromatography ion-tap mass spectrometry method. The positive association was found between urinary level of BP and XY18 disomy (p = 0.045) and PP and disomy of chromosome 13 (p = 0.007). This is the first study to examine these relationships, and replication of our findings is needed before the association between parabens concentration in urine and aneuploidy can be fully defined. These findings may be of concern due to increased parabens use.

Time-dependent changes in antioxidative enzyme expression and photosynthetic activity of Chlamydomonas reinhardtii cells under acute exposure to cadmium and anthracene.

Heavy metals (HM) and polycyclic aromatic hydrocarbons (PAHs) are present in the freshwater environment at concentrations that can be hazardous to the biota. Among HMs and PAHs, cadmium (Cd) and anthracene (ANT) are the most prevalent and toxic ones. The response of Chlamydomonas cells to Cd and ANT at concentrations that markedly reduced the growth of algal population was investigated in this study. At such concentrations, both cadmium and anthracene were recognized as oxidative stress inducers, since high concentration of H2O2 in treated cultures was observed. Therefore, as a part of the "molecular phase" of the cell response to this stress, we examined the time-dependent expression of genes encoding the main antioxidative enzymes: superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as the activity of these enzymes in cells, with special attention paid to chloroplastic and mitochondrial isoforms of SOD. To characterize the cell response at the "physiological level", we examined the photosynthetic activity of stressed cells via analysis of chlorophyll a fluorescence in vivo. In contrast to standard ecotoxicity studies in which the growth end-points are usually determined, herein we present time-dependent changes in algal cell response to Cd- and ANT-induced stress. The most significant effect(s) of the toxicants on photosynthetic activity was observed in the 6th hour, when strong depression of PI parameter value, an over 50 percent reduction of the active reaction center fraction (RC0) and a 3-fold increase in non-photochemical energy dissipation (DI0/RC) were noted. At the same time, the increase (up to 2.5-fold) in mRNA transcript of SOD and CAT genes, followed by the enhancement in the enzyme activity was observed. The high expression of the Msd 3 gene in treated Chlamydomonas cells probably complements the partial loss of chloroplast Fe-SOD and APX activity, while catalase and Mn-SOD 5 seem to be the major enzymes responsible for mitochondrion protection. The progressive increase in SOD and CAT activities seems to be involved in the recovery of photosynthesis within 12-24h after the application of the toxicants.

Zinc in drug-naïve patients with short-illness-duration first episode major depressive disorder: impact on psychopathological features.

OBJECTIVE: In major depressive disorder (MDD) hypozincaemia associated with symptoms severity, melancholia, anxiety and treatment-resistance is reported. Data linking zinc with specific psychopathological dimensions is limited. METHODS: Plasma zinc was analyzed in this cross-sectional case-control study on 20 non-late-life adult, treatment-naïve MDD patients with short-illness-duration first affective episode and 20 matched healthy controls together with psychometric evaluations including Hamilton Rating Scale for Depression (HAMD-17) and Spielberger State-Trait Anxiety Inventory (STAI). RESULTS: No significant difference in zinc levels was found between MDD subjects and controls. No significant correlations were observed between zinc concentration and the total HAMD-17 score as well as with the specific core depression, insomnia, anxiety and somatic psychopathological dimensions or STAIX-1 and STAIX-2 scores. CONCLUSION: The study provides evidence for unchanged plasma zinc concentration at early stage of MDD and failed to demonstrate any correlation between plasma zinc and psychopathological features including severity of symptoms and specific psychopathological dimensions in MDD.

Nabumetone and flufenamic acid pose a serious risk to aquatic plants: A study with Chlamydomonas reinhardtii as a model organism.

The aquatic environment is constantly under threat due to the release of numerous pollutants. Among them, pharmaceuticals constitute a huge and diverse group. Non-steroidal anti-inflammatory drugs (NSAIDs) are increasingly found in water bodies, but knowledge about their potential toxicity is still low. In particular, there is a lack of information about their influences on aquatic plants and algae. We estimated the susceptibility of the microalgae Chlamydomonas reinhardtii to nabumetone (NBT) and flufenamic acid (FFA), focusing on photosynthesis. Due to the differences in the structures of these compounds, it was assumed that these drugs would have different toxicities to the tested green algae. The hypothesis was confirmed by determining the effective concentration values, the intensity of photosynthesis, the intensity of dark respiration, the contents of photosynthetic pigments, the fluorescence of chlorophyll a in vivo (OJIP test), and cell ultrastructure analysis. Assessment of the toxicity of the NSAIDs was extended by the calculation of an integrated biomarker response index (IBR), which is a valuable tool in ecotoxicological studies. The obtained results indicate an over six times higher toxicity of NBT compared to FFA. After analysis of the chlorophyll a fluorescence in vivo, it was found that NBT inhibited electron transport beyond the PS II. FFA, unlike NBT, lowered the intensity of photosynthesis, probably transforming some reaction centers into "silent centers", which dissipate energy as heat. The IBR estimated based on photosynthetic parameters suggests that the toxic effect of FFA results mainly from photosynthesis disruption, whereas NBT significantly affects other cellular processes. No significant alteration in the ultrastructure of treated cells could be seen, except for changes in starch grain number and autophagic vacuoles that appeared in FFA-treated cells. To the best of our knowledge, this is the first work reporting the toxic effects of NBT and FFA on unicellular green algae.

Photoprotection and Antiaging Activity of Extracts from Honeybush (Cyclopia sp.)-In Vitro Wound Healing and Inhibition of the Skin Extracellular Matrix Enzymes: Tyrosinase, Collagenase, Elastase and Hyaluronidase.

Cyclopia sp. (honeybush) is an African shrub known as a rich source of polyphenols. The biological effects of fermented honeybush extracts were investigated. The influence of honeybush extracts on extracellular matrix (ECM) enzymes responsible for the skin malfunction and aging process-collagenase, elastase, tyrosinase and hyaluronidase-was analysed. The research also included assessment of the in vitro photoprotection efficiency of honeybush extracts and their contribution to the wound healing process. Antioxidant properties of the prepared extracts were evaluated, and quantification of the main compounds in the extracts was achieved. The research showed that the analysed extracts had a significant ability to inhibit collagenase, tyrosinase and hyaluronidase and a weak influence on elastase activity. Tyrosinase was inhibited effectively by honeybush acetone (IC50 26.18 ± 1.45 µg/mL), ethanol (IC50 45.99 ± 0.76 µg/mL) and water (IC50 67.42 ± 1.75 µg/mL) extracts. Significant hyaluronidase inhibition was observed for ethanol, acetone and water extracts (IC50 were 10.99 ± 1.56, 13.21 ± 0.39 and 14.62 ± 0.21µg/mL, respectively). Collagenase activity was inhibited effectively by honeybush acetone extract (IC50 42.5 ± 1.05 µg/mL). The wound healing properties of the honeybush extracts, estimated in vitro in human keratinocytes (HaCaTs), were indicated for water and ethanol extracts. In vitro sun protection factor (SPF in vitro) showed medium photoprotection potential for all the honeybush extracts. The quantity of polyphenolic compounds was estimated with the use of high-performance liquid chromatography equipped with diode-array detection (HPLC-DAD), indicating the highest mangiferin contents in ethanol, acetone and n-butanol extracts, while in the water extract hesperidin was the dominant compound. The antioxidant properties of the honeybush extracts were estimated with FRAP (2,4,6-Tris(2-pyridyl)-s-triazine) and DPPH (2,2-diphenyl-1-picrylhydrazyl) tests, indicating their strong antioxidant activity, similar to ascorbic acid for the acetone extract in both tests. The wound healing abilities, estimation of SPF in vitro and the direct influence on selected enzymes (elastase, tyrosinase, collagenase and hyaluronidase) of the tested honeybush extracts were analysed for the first time, indicating a high potential of these well-known herbal tea for antiaging, anti-inflammation, regeneration and protection of the skin.

Green alga Chlamydomonas reinhardtii can effectively remove diclofenac from the water environment - A new perspective on biotransformation.

The use of unicellular algae to remove xenobiotics (including drugs) from wastewaters is one of the rapidly developing areas of environmental protection. Numerous data indicate that for efficient phycoremediation three processes are important, i.e. biosorption, bioaccumulation, and biotransformation. Although biosorption and bioaccumulation do not raise any serious doubts, biotransformation is more problematic since its products can be potentially more toxic than the parent compounds posing a threat to organisms living in a given environment, including organisms that made this transformation. Thus, two questions need to be answered before the proper algae strain is chosen for phycoremediation, namely what metabolites are produced during biotransformation, and how resistant is the analyzed strain to a mixture of parent compound and metabolites that appear over the course of culture? In this work, we evaluated the remediation potential of the model green alga Chlamydomonas reinhardtii in relation to non-steroidal anti-inflammatory drugs (NSAIDs), as exemplified by diclofenac. To achieve this, we analysed the susceptibility of C. reinhardtii to diclofenac as well as its capability to biosorption, bioaccumulation, and biotransformation of the drug. We have found that even at a relatively high concentration of diclofenac the algae maintained their vitality and were able to remove (37.7%) DCF from the environment. A wide range of phase I and II metabolites of diclofenac (38 transformation products) was discovered, with many of them characteristic rather for animal and bacterial biochemical pathways than for plant metabolism. Due to such a large number of detected products, 18 of which were not previously reported, the proposed scheme of diclofenac transformation by C. reinhardtii not only significantly contributes to broadening the knowledge in this field, but also allows to suggest possible pathways of degradation of xenobiotics with a similar structure. It is worth pointing out that a decrease in the level of diclofenac in the media observed in this study cannot be fully explained by biotransformation (8.4%). The mass balance analysis indicates that other processes (total 22%), such as biosorption, a non-extractable residue formation, or complete decomposition in metabolic cycles can be involved in the diclofenac disappearance, and those findings open the prospects of further research.

Development of the thin film solid phase microextraction (TF-SPME) method for metabolomics profiling of steroidal hormones from urine samples using LC-QTOF/MS.

In the present study, the development and optimization of a thin film solid phase microextraction method (TF-SPME) was conducted for metabolomics profiling of eight steroid compounds (androsterone, dihydrotestosterone, dihydroepiandrosterone, estradiol, hydroxyprogesterone, pregnenolone, progesterone and testosterone) from urine samples. For optimization of extraction method, two extraction sorbents (PAN-C18 and PS-DVB) were used as they are known to be effective for isolation of low-polarity analytes. The stages of sample extraction and analyte desorption were considered as the most crucial steps in the process. Regarding the selection of the most suitable desorption solution, six different mixtures were analyzed. As a result, the mixture of ACN: MeOH (1:1, v/v) was chosen in terms of the highest analytes' abundances that were achieved using the chosen solvent. Besides other factors were examined such as the volume of desorption solvent and the time of both extraction and desorption processes. The analytical determination was carried out using the ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry detection in electrospray ionization and positive polarity in a scan mode (UHPLC-ESI-QTOF/MS). The developed and optimized TF-SPME method was validated in terms of such parameters as extraction efficiency, recovery as well as matrix effect. As a result, the extraction efficiency and recovery were in a range from 79.3% to 99.2% and from 88.8% to 111.8%, respectively. Matrix effect, calculated as coefficient of variation was less than 15% and was in a range from 1.4% to 11.1%. The values of both validation parameters (recovery and matrix effect) were acceptable in terms of EMA criteria. The proposed TF-SPME method was used successfully for isolation of steroids hormones from pooled urine samples before and after enzymatic hydrolysis of analytes.

Urinary concentration of selected nonpersistent endocrine disrupting chemicals-reproductive outcomes among women from a fertility clinic.

Parabens and benzophenones are compounds widely used in cosmetics and personal care products. Although human exposure is widespread there is a limited number of epidemiological studies assessing the relationship between exposure to these chemicals and female reproductive health. The aim of the study is to explore the relationship between paraben and benzophenone concentrations and reproductive outcomes among women attending a fertility center. This prospective cohort included 450 women undergoing in vitro treatment (IVF) at fertility clinic in Poland. The validated gas chromatography ion-tap mass spectrometry to assess concentrations of parabens in urine (methyl (MP), ethyl (EP), propyl (PP), butyl paraben (BP)) and benzophenone-3 (BP-3) was used. To explore the relationship between concentrations of examined chemicals and reproductive outcomes (methaphase II (MII) oocyte yield, total oocyte yield, implantation rate, fertilization rate, clinical pregnancy, live births), multivariable generalized linear mixed model was used for the analysis. Increased exposure to butyl paraben was associated with a significant decrease in MII oocyte count (p = 0.007) when exposure to BP was treated as the continuous variable. Additionally, the exposure to BP in the highest quartile of exposure also decreases MII oocyte count (p = 0.02) compared to the lowest quartile. Urinary concentrations of BP were not related to total oocyte count, fertilization and implantation rate, clinical pregnancy, and live birth when the exposure variable was continuous variable or in the quartiles of exposure. Exposure to MP, EP, PP, the sum of examined parabens, and benzophenone-3 were not related to any of the examined reproductive outcomes. Exposure to butyl paraben was associated with a decrease in MII oocyte count among women attending fertility clinic rinsing concerns that exposure may have a potential adverse impact on embryological outcomes. The results emphasize the importance to reduce chemicals in the environment in order to minimize exposure. As this is the first study showing such an association, further research is needed to confirm these novel results in other populations.

Isolation of Citrus lemon extracellular vesicles: Development and process control using capillary electrophoresis.

A new and scalable method for the isolation of extracellular vesicles (EV) from Citrus lemon juice samples was developed. The methodology included preliminary preconcentration of the sample using ultrafiltration (UF) followed by size-exclusion chromatography (SEC) purification and final preconcentration of the eluates. Transmission electron microscopy and proteomic analysis showed that isolates contained exosome-like vesicles, exocyst-positive organelle (EXPO), and microvesicles. The efficiency of certain isolation steps was evaluated with total protein content assay (bicinchoninic acid assay, BCA), nanoparticles tracking analysis (NTA), and capillary electrophoresis (CE). A good correlation between CE, BCA, and NTA results was shown. The application of CE enabled the detection of soluble contaminants, macromolecular aggregates, and vesicles' heterogeneity. The fluorescent staining of encapsulated nucleic acids was proposed for the identity confirmation of EV detected in CE. The study demonstrates the CE as a comprehensive tool for monitoring of the EV isolation process.