Physical state of human papillomavirus type 16 in cervical intraepithelial lesions and cancers determined by two different quantitative real-time PCR methods

The aim of this study was to analyse the correlation between a new multiplex qPCR assay and a reference qPCR assay for assessment of the human papillomavirus (HPV16) load and the viral genome status. The study was performed on 100 HPV16 positive samples containing premalignant lesions and carcinomas. HPV16 E2 and E6 gene loads were assessed by two PCR methods. The load of E2 and E6 was normalized to the cell number by qPCR targeting the RNase P open reading frame. The physical state of the viral genome was determined as a ratio of E2/E6 copies number per cell. Among 100 samples analysed, there were no statistically significant differences in the E2 and E6 viral load evaluated by multiplex qPCR and qPCR, the correlation coefficients were 0.98 and 0.97, respectively. There were 19% of samples with the integrated, 73% with mixed and 8% with episomal state of viral genome detected by multiplex qPCR and 17%, 79%, 4%, respectively, found by qPCR. Prevalence of integrated and episomal forms estimated by multiplex qPCR was higher than the one obtained by qPCR (Chi2, p < 0.0001), but in samples with premalignant and malignant diagnoses no significant differences were demonstrated regardless of the methods used. Sensitivity and specificity of multiplex qPCR were 93.7% and 100% as compared with qPCR, the positive predictive value was 100%. In summary, the multiplex qPCR assay in respect of HPV16 load and the frequency of viral genome status was shown to be a sensitive and specific reference method. Simultaneous estimation of E2 and E6 genes in one reaction tube reduces the cost of testing

Decomposition of Flavonols in the Presence of Saliva

In this study, the LC-MS/MS was applied to explore the stability of four common dietary flavonols, kaempferol, quercetin, isorhamnetin, and myricetin, in the presence of hydrogen peroxide and saliva. In addition, the influence of saliva on the representative quercetin glycosides, rutin, quercitrin, hyperoside, and spiraeoside was examined. Our study showed that, regardless of the oxidative agent used, flavonols stability decreases with increasing B-ring substitution. The decomposition of analyzed compounds was based on their splitting by the opening the heterocyclic C-ring and realizing more simple aromatic compounds. The dead-end products corresponded to different benzoic acid derivatives derived from B-ring. Kaempferol, quercetin, isorhamnetin, and myricetin were transformed into 4-hydroxybeznoic acid, protocatechuic acid, vanillic acid, and gallic acid, respectively. Additionally, for quercetin and myricetin, two intermediate depsides and 2,4,6-trihydroxybenzoic acid derived from A-ring were detected. All analyzed glycosides were resistant to hydrolysis in the presence of saliva. Based on our data, saliva was proven to be a next oxidative agent which leads to the formation of corresponding phenolic acids. Hence, studies on flavonols&rsquo; metabolism should take into consideration that the flavonols decomposition starts in the oral cavity; hence, in subsequent parts of the human digestive tract, they could be present not in their parent form but as phenolic acids. Further analyses of the influence of saliva on flavonols glycosides need to be performed due to the possible interindividual fluctuations

HPLC with Post-Column Derivatization with Alizarin for Determination of OATD-02, an Anticancer Arginase Inhibitor in Clinical Development

The aim of this study was to develop an analytical method for selective determination of OATD-02 by high-performance liquid chromatography (HPLC) with post-column derivatization and fluorescence detection (FLD). OATD-02, a new boronic acid derivative, is a highly potent anticancer arginase inhibitor in clinical development. Chromatographic analysis of OATD-02 poses problems because this molecule has weak ultraviolet absorption. The derivatization reaction was based on the reaction between boronic acid from OATD-02 and alizarin solution. The optimized mobile phase consisted of a mixture of sodium bicarbonate in water and acetonitrile at a flow rate of 0.50 mL/min. Alizarin solution in methanol was delivered at a flow rate of 0.50 mL/min. The fluorescent complexes were detected by a fluorescence detector (excitation and emission wavelengths at 470 and 580 nm, respectively). The present method demonstrated proper values for selectivity, linearity, recovery (>99%), precision (RSD: 0.6%), sensitivity (LOD: 20 µg/mL and LOQ: 50 µg/mL), stability of solutions, and robustness

Application of Hydrophilic Interaction Liquid Chromatography for the Quantification of Flavonoids in Genista tinctoria Extract

Hydrophilic interaction chromatography (HILIC) was employed to investigate chromatographic behavior of selected flavonoids from their different subgroups differing in polarity. Chromatographic measurements were performed on two different HILIC columns: unmodified silica (Atlantis-HILIC) and zwitterionic sulfoalkylbetaine (SeQuant ZIC-HILIC). Separation parameters such as content and type of organic modifier were studied. On ZIC column retention factors were observed to be inversely proportional to the buffer content in the mobile phase, which is the typical partitioning mechanism. In the case of bare silica column more or less apparent dual retention mechanism was observed, depending on the water component content in the mobile phase. ZIC-HILIC showed better selectivity (in comparison to silica column) with the detection limit of 0.01 mg/L (only for rutin was 0.05 mg/L). Finally, this chromatographic procedure was validated and applied for the determination of some flavonoids in Genista tinctoria L. extract

Determining Potassium Bromate in the Inhalable Aerosol Fraction in Workplace Air with Ion Chromatography

Background: The article presents the results of studies performed in order to develop a new method of airborne potassium bromate(V) determination at workplaces. Methods: The method is based on a collection of the inhalable fraction of potassium bromate(V) using the IOM Sampler, then extraction of bromates with deionized water and chromatographic analysis of the obtained solution. The analysis was performed using ion chromatography with conductometric detection. The tests were performed on a Dionex IonPac®AS22 analytic column (250 × 4 mm, 6 μm) with AG22 precolumn (50 × 4 mm 11 μm). Results: The method provides for potassium bromate(V) determination within the concentration range of 0.043 ÷ 0.88 mg/m3 for an air sample of 0.72 m3 in volume, i.e., 0.1–2 times the exposure limit value as proposed in Poland. The method was validated in accordance with PN-EN 482. The obtained validation data are as follows: measuring range: 3.1–63.4 μg/mL, limit of detection (LOD) = 0.018 μg/mL and limit of quantification (LOQ) = 0.053 μg/mL. The developed method has been tested in the work environment, on laboratory employees having contact with potassium bromate(V). Conclusion: The analytical method allowed the determination of the inhalable fraction of airborne potassium bromate(V) at workplaces and can be used to assess occupational exposure

The Civil City Framework for the Implementation of Nature-Based Smart Innovations: Right to a Healthy City Perspective

This paper aims to expand the current debate concerning the implementation of health-related innovations by employing the rights-based approach. Specifically, we address the challenges related to the implementation of technological and Nature-Based Solutions (NBS) from the right to the city perspective. As a result, we present a comprehensive Civil City Framework that involves the synergic use of ICT tools and participation models to mobilize grassroots potential for building healthier cities, while ensuring equity and respect for diverse needs. We explain the participatory and technological aspects of implementing and monitoring innovative NBS, involving data gathering through environmental sensors, personal wristbands, and smartphone apps. Finally, we highlight opportunities and challenges stemming from the use of health- and environment-monitoring technologies. To build such a vision one needs to reconceive the city as commons, enabling collective action of city residents and cooperation between different local stakeholders. This can be achieved by embedding a pentahelix approach of multi-governance in urban planning, based on a close interaction between five key stakeholders, including public authorities, industry and business sector, academia, civil society organizations, and individual citizens

Antioxidant Activity of Sulfate Metabolites of Chlorogenic Acid

This study aimed to determine the antioxidant properties of the sulfate monoesters of ferulic, caffeic, dihydroferulic and dihydrocaffeic acids, the main metabolites of chlorogenic acids. These compounds are not commercially available, so they were synthesized in the laboratory. The LC-MS/MS analysis allowed for the full characterization of these derivatives, which has made them reliable standards for further research. Purified metabolites including ferulic acid-4-O-sulfate, caffeic acid-4-O-sulfate and caffeic acid-3-O-sulfate, dihydrocaffeic acid-4-O-sulfate and caffeic acid-3-O-sulfate were examined for their antioxidant capacities and compared to their precursor compounds using Folin&ndash;Ciocalteu, CUPRAC (cupric ion&mdash;reducing) and DPPH&bull; (2,2-diphenyl-1-picrylhydrazyl) methods. This study shows that hydrogenation of caffeic and ferulic acids into dihydrocaffeic and dihydroferulic acids has a positive influence on their reducing properties. Moreover, all synthesized sulfate monoesters exhibited very weak antioxidant properties compared to precursor compounds. The presented results show that the transformation of phenolic acids via sulfation leads to the inhibition of antioxidant properties due to the blockage of hydroxyl groups