Non-target screening and prioritization of organic contaminants in seawater desalination and their ecological risk assessment.

The impact of desalination brine on the marine environment is a global concern. Regarding this, salinity is generally accepted as the major environmental factor in desalination concentrate. However, recent studies have shown that the influence of organic contaminants in brine cannot be ignored. Therefore, a non-targeted screening method based on comprehensive two-dimensional gas chromatography-quadrupole mass spectrometry (GC × GC-qMS) was developed for identifying organic contaminants in the desalination brine. A total of 404 compounds were tentatively identified from four seawater desalination plants (three reverse osmosis plants and one multiple effect distillation plant) in China. The identified compounds were prioritized based on their persistence, bioaccumulation, ecotoxicity, usage, and detection frequency. Twenty-one (21) compounds (seven phthalates, ten pesticides, four trihalomethanes) were then selected for further quantitative analysis and ecological risk assessment, including compounds from the priority list along with substances from the same chemical classes. Ecologically risky substances in brine include diisobutylphthalate and bis(2-Ethylhexyl) phthalate, atrazine and acetochlor, and bromoform. Most of the contaminants come from raw seawater, and no high risk contaminants introduced by the desalination process have been found except for disinfection by-products. In brine discharge management, people believed that all pollution in raw seawater was concentrated by desalination process. This study shows that not all pollutants are concentrated during the desalination process. In this study, the total concentration of pesticide in the brine increased by 58.42%. The concentration of ∑PAEs decreased by 13.65% in reverse osmosis desalination plants and increased by 10.96% in the multi-effect distillation plant. The concentration of trihalomethane increased significantly in the desalination concentrate. The change in the concentration of pollutants in the desalination concentrate was related to the pretreatment method and the chemical characteristics of the contaminants. The method and results given in this study hinted a new idea to identify and control the environmental impact factors of brine.

Thermogravimetric/Thermal-Mass Spectroscopy Insight into Oxidation Propensity of Various Mechanochemically Made Kesterite Cu2ZnSnS4 Nanopowders.

Thermogravimetry coupled with thermal analysis and quadrupole mass spectroscopy TGA/DTA-QMS were primarily used to assess the oxidation susceptibility of a pool of nanocrystalline powders of the semiconductor kesterite Cu2ZnSnS4 for prospective photovoltaic applications, which were prepared via the mechanochemically assisted synthesis route from two different precursor systems. Each system, as confirmed by XRD patterns, yielded first the cubic polytype of kesterite with defunct semiconductor properties, which, after thermal annealing at 500 °C under neutral gas atmosphere, was converted to the tetragonal semiconductor polytype. The TGA/DTA-QMS determinations up to 1000 °C were carried out under a neutral argon Ar atmosphere and under a dry, oxygen-containing gas mixture of O2:Ar = 1:4 (vol.). The mass spectroscopy data confirmed that under each of the gas atmospheres, a distinctly different, multistep evolution of such oxygen-bearing gaseous compounds as sulfur oxides SO2/SO3, carbon dioxide CO2, and water vapor H2O was taking place. The TGA/DTA changes in correlation with the nature of evolving gases helped in the elucidation of the plausible chemistry linked to kesterite oxidation, both in the stage of nanopowder synthesis/storage at ambient air conditions and during forced oxidation up to 1000 °C in the dry, oxygen-containing gas mixture.

Addressing challenges with evaluating hydrogen-selective membrane performance by quadrupole mass spectrometry.

Hydrogen separation using nanostructured membranes has gained research attention because of its potential to produce high-purity hydrogen by separating gases at the molecular level. Quadrupole mass spectrometry (QMS) is one method to evaluate these membranes' effectiveness in separating hydrogen from gas mixtures. However, quantifying gases in a mixture with QMS is challenging, especially when heavier gas ions interfere with a light gas ion, resulting in lower quantification accuracy. This study addresses this challenge by presenting a detailed calibration procedure that significantly improves hydrogen quantification accuracy up to a factor of 2.5. CO and CO2 were chosen as interfering gases because they are commonly released in conventional hydrogen production processes. By carefully evaluating the performance of these membranes, new opportunities for hydrogen separation may be realized.

Application of Simultaneous and Coupled Thermal Analysis Techniques in Studies on the Melting Process, Course of Pyrolysis and Oxidative Decomposition of Fused Triazinylacetohydrazides.

The effect of the structure of promising antioxidant agents with prospective medical use, i.e., unsubstituted and para-substituted annelated triazinylacetic acid hydrazides, on their melting points, thermal stabilities, pyrolysis and oxidative decomposition stages and the type of volatiles emitted under heating with the use of DSC and TG/DTG/FTIR/QMS methods was evaluated and discussed. The melting point of the investigated compounds increased with an enhanced number of electrons (directly correlated with their molecular weight). Melting enthalpy values were determined and presented for all the studied compounds. The pyrolysis and oxidative decomposition processes of the analysed molecules consisted of several poorly separated stages, which indicated a multi-step course of the decomposition reactions. It was found that the thermal stability of the tested compounds depended on the type of substituent at the para position of the phenyl moiety or its absence. In both atmospheres used (air and helium), the thermal stability increased in relation to R as follows: -CH3 ≤ -OCH3 < -H < -OC2H5. In an inert atmosphere, it was higher by approx. 8-18 °C than in an oxidative atmosphere. The pyrolysis was connected with the emission of NH3, HCN, HNCO, HCONH2, HCHO, CO2, CO and H2O in the case of all the tested compounds, regardless of the substituent attached. In the case of the derivative containing the para-CH3 group, para-toluidine was an additional emitted aromatic product. In turn, emissions of aniline and alcohol (methanol or ethanol) for compounds with the para-OCH3 and para-OC2H5 groups, respectively, were confirmed. In oxidative conditions, the release of NH3, NO, HCN, HNCO, HCONH2, CO2, H2O and cyanogen (for all the compounds) and para-toluidine (for the para-CH3 derivative), aniline (for para-OCH3, para-OC2H5 and unsubstituted derivatives) and acetaldehyde (for the para-OC2H5 derivative) were clearly observed. No alcohol emissions were recorded for either compound containing the para-OCH3- or para-OC2H5-substitututed phenyl ring. These results confirmed that the pyrolysis and oxidative decomposition of the investigated annelated triazinylacetohydrazides occurred according to the radical mechanism. Moreover, in the presence of oxygen, the reactions of volatiles and residues with oxygen (oxidation) and the combustion process additionally proceeded.

Novel bioanalytical strategy using isotope pattern deconvolution and ICP-QMS for the study of iron incorporation in erythrocytes: An insight to better assessment.

Iron is an essential element for human life and its nutritional status in the human body is directly linked to human health. More than 1015 atoms of iron per second are necessary for the maintenance of haemoglobin formation. To predict iron bioavailability three approaches are normally employed: (a) faecal recovery; (b) plasma appearance; and (c) erythrocyte incorporation (the most used). Isotope Pattern Deconvolution (IPD) is a mathematical tool that allows the isolation of distinct isotope signatures from mixtures of natural abundance and enriched tracers. In this work we propose a novel strategy to assess erythrocyte iron incorporation, based on the use of an iron stable isotope (57Fe) and the IPD concept. This strategy allows direct calculation of the exogenous concentration of 57Fe incorporated into RBCs after supplementation. In this way, to determine the mass of iron incorporated into erythrocytes, the unique prediction that must be made is the blood volume, estimate to reproduce the natural dilution of the tracer (57Fe) in the blood. This novel bioanalytical approach was applied for the measurements of iron incorporation and further iron absorption studies in humans, using a group of twelve healthy participants, that should be further evaluated for the assessment of other chemical elements that could be of health concerns and directly impact society.

Kinetic and Parametric Analysis of the Separation of Ultra-Small, Aqueous Superparamagnetic Iron Oxide Nanoparticle Suspensions under Quadrupole Magnetic Fields.

Superparamagnetic iron oxide nanoparticles (SPIONs) have gathered tremendous scientific interest, especially in the biomedical field, for multiple applications, including bioseparation, drug delivery, etc. Nevertheless, their manipulation and separation with magnetic fields are challenging due to their small size. We recently reported the coupling of cooperative magnetophoresis and sedimentation using quadrupole magnets as a promising strategy to successfully promote SPION recovery from media. However, previous studies involved SPIONs dispersed in organic solvents (non-biocompatible) at high concentrations, which is detrimental to the process economy. In this work, we investigate, for the first time, the magnetic separation of 20 nm and 30 nm SPIONs dispersed in an aqueous medium at relatively low concentrations (as low as 0.5 g·L-1) using our custom, permanent magnet-based quadrupole magnetic sorter (QMS). By monitoring the SPION concentrations along the vessel within the QMS, we estimated the influence of several variables in the separation and analyzed the kinetics of the process. The results obtained can be used to shed light on the dynamics and interplay of variables that govern the fast separation of SPIONs using inexpensive permanent magnets.

Experimental Studies on the Thermal Properties and Decomposition Course of a Novel Class of Heterocyclic Anticancer Drug Candidates.

The experimental studies on the thermal properties and decomposition course of a novel class of potential anticancer drugs (1-5) containing in their heterobicyclic structures the asymmetrical triazine template were performed with the use of differential scanning calorimetry (DSC) and simultaneous thermogravimetry/differential scanning calorimetry (TG/DTG/DSC) coupled online with Fourier transform infrared spectroscopy (FTIR) and quadrupole mass spectrometry (QMS) in inert and oxidizing conditions. All the compounds were thermally characterized in detail for the first time in this article. The DSC studies proved that the melting points of the tested compounds depended on the position and type of the substituent at the phenyl moiety, whereas they did not depend on the furnace atmosphere. All the tested polynitrogenated heterocycles proved to be molecules with high thermal stability in both atmospheres, and most of them (1, 3-5) were more stable in oxidizing conditions, which indicated the formation of a more thermally stable form of the compounds when interacting with oxygen. The simultaneous TG/FTIR/QMS analyses confirmed that their pyrolysis process occurred in one main stage resulting in the emission of volatiles such as NH3, HNCO, HCN, CO, CO2, H2O, NO2, aromatic amine derivatives, alkenes (for compounds 1-5), and HCl (for the compound 5). On the other hand, the oxidative decomposition process was more complicated and proceeded in two main stages leading to the emission of NH3, CO2, CO, HCN, HNCO, H2O, some aromatics (for compounds 1-5), HCl (for compounds 3-5) as well as the additional volatiles such as N2, NO2, NH2OH, and (CN)2. The type of the formed volatiles indicated that the decomposition process of the studied heterocycles under the influence of heating was initiated by the radical mechanism. Their decomposition was related to the symmetric cleavage of C-N and C-C bonds (inert conditions) and additional reaction of the volatiles and residues with oxygen (oxidizing conditions).

Quantitative detection of methylated SOCS-1 in schizophrenia and bipolar disorder considering SOCS-1 -1478CA/del polymorphism and clinical parameters.

BACKGROUND: We aimed to investigate the quantitative detection of methylated suppressor of cytokine signaling-1 (SOCS-1) in schizophrenia (SCZ) and bipolar disorder (BD), considering SOCS-1 -1478CA/del polymorphism and clinical parameters. METHODS: Our research is a case-control study in which 114 patients with SCZ, 86 patients with BD, and 80 volunteers as a healthy group participated. Bisulfite-converted DNA samples were analyzed using the real-time quantitative methylation-specific PCR (qMS-PCR) method to measure the methylation level of the SOCS-1 gene. In addition, SOCS-1 -1478CA/del gene polymorphism was analyzed with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: When the SOCS-1 promoter methylation levels of SCZ and BD patients were compared with the control group, the methylation levels of SCZ and BD were significantly lower than the control group. An earlier age of illness onset was significantly related to the SOCS-1 promoter hypermethylation in DNA samples of SCZ patients. Again, SOCS-1 promoter hypermethylation was significantly associated with the higher Young Mania Rating Scale (YMRS) score in BD patients. While the SOCS-1 CA/CA genotype frequency was significantly higher in the control group than in the BD group, the del/del genotype was significantly related to a higher frequency of rapid cycling and a lower frequency of family history in the BD patient group. CONCLUSION: In summary, the methylated SOCS-1 quantity in DNA samples of SCZ and BD patients were significantly lower than in control samples. Whereas the SOCS-1 -1478CA/del polymorphism was not related to SCZ, it may be associated with the BD.

Evaluation of the interchangeability between the new fully-automated affinity chrome-mediated immunoassay (ACMIA) and the Quantitative Microsphere System (QMS) with a CE-IVD-certified LC-MS/MS assay for therapeutic drug monitoring of everolimus after solid organ transplantation.

OBJECTIVES: This study aims to evaluate the interchangeability between the Siemens Healthineers' "EVRO" new affinity chrome-mediated immunoassay (ACMIA/EVRO) and Thermo Fisher Scientific's "EVER" Quantitative Microsphere System (QMS/EVER) with Chromsystems' CE-IVD-certified "MassTox" liquid-chromatography/tandem-mass spectrometry (LC-MS/MS) assay for the therapeutic drug monitoring of everolimus. METHODS: A single lot of reagent, calibrators and controls were used for each assay. A total of 67 whole blood samples (n=67) from patients receiving solid organ transplant were analyzed (n=31 with kidney transplant and n=36 with liver transplant); Passing-Bablok regression and Bland-Altman difference plot were used to evaluate bias and individual agreement; LC-MS/MS analysis was used to measure the actual concentrations of calibrators and controls compared to the assigned value. RESULTS: ACMIA/EVRO did not show any systematic bias compared to LC-MS/MS (intercept=0.244 ng/mL, 95% CI: -0.254 to 0.651 ng/mL). Nevertheless, significant proportional bias (slope=1.511, 95% CI: 1.420 to 1.619) associated to a combined bias of 44.8% (95% CI: 41.2-48.3%) was observed. Conversely, QMS/EVER did not show any bias at both systematic (intercept=-0.151 ng/mL, 95% CI: -0.671 to 0.256 ng/mL) and proportional level (slope=0.971, 95% CI: 0.895 to 1.074) with a non-statistically significant combined bias of -3.6% (95% CI: -8.4-1.1%). Based on a concentration of calibrators and controls above the assigned value for both the analytical methods, in the ACMIA/EVRO a correction which was approximately one-third of the correction for the QMS/EVER was observed. CONCLUSIONS: ACMIA/EVRO but not QMS/EVER shows a lack of interchangeability with the CE-IVD-certified LC-MS/MS assay. We hypothesize that, as the ACMIA/EVRO uses an anti-sirolimus antibody, the under-corrected assigned value in the assay calibrators was not sufficient to reproduce the everolimus metabolites cross-reactivity occurring in real samples.

Practical considerations for laboratories: Implementing a holistic quality management system.

A laboratory quality management system (LQMS) is an essential element for the effective operation of research, clinical, testing, or production/manufacturing laboratories. As technology continues to rapidly advance and new challenges arise, laboratories worldwide have responded with innovation and process changes to meet the continued demand. It is critical for laboratories to maintain a robust LQMS that accommodates laboratory activities (e.g., basic and applied research; regulatory, clinical, or proficiency testing), records management, and a path for continuous improvement to ensure that results and data are reliable, accurate, timely, and reproducible. A robust, suitable LQMS provides a framework to address gaps and risks throughout the laboratory path of workflow that could potentially lead to a critical error, thus compromising the integrity and credibility of the institution. While there are many LQMS frameworks (e.g., a model such as a consensus standard, guideline, or regulation) that may apply, ensuring that the appropriate framework is adopted based on the type of work performed and that key implementation steps are taken is important for the long-term success of the LQMS and for the advancement of science. Ultimately, it ensures accurate results, efficient operations, and increased credibility, enabling protection of public health and safety. Herein, we explore LQMS framework options for each identified laboratory category and discuss prerequisite considerations for implementation. An analysis of frameworks' principles and conformity requirements demonstrates the extent to which they address basic components of effective laboratory operations and guides optimal implementation to yield a holistic, sustainable framework that addresses the laboratory's needs and the type of work being performed.

Factor Analysis of Quality Management Systems Implementation in Healthcare: An Online Survey.

This paper investigates the views of healthcare researchers and professionals on the implementation of the Quality Management System (QMS) approach using a 5-point Likert scale survey. Researchers and healthcare professionals who observed or participated in QMS implementation were surveyed. Multiple channels, including occupational societies, social networking, i.e., LinkedIn, hospital's directories, award recipients, academic researchers, and professional connections, made it possible to reach this particular sample. Participants were surveyed using a series of questions with a total of 56 questions. The survey was administrated through the web portal of Qualtrics and then analyzed both on Qualtrics and SPSS software packages. Descriptive Statistics, Exploratory Factor Analysis (EFA), and Linear Regression were employed to draw conclusions. The final sample group consisted of 71 participants representing a range of healthcare settings. EFA was conducted, producing a model of 10 emergent factors and an outcome for total improvement. Regression modeling revealed the Critical Success Factors (CSFs) and the interaction between emergent factors. The results indicated that QMS Implementation Culture, Structure, and Managerial Training are critical to the QMS implementation success. This research helps quality professionals enhance their ability to prioritize elements affecting the successful implementation of the QMS.

Metal free, one pot 1,6-Conjugate Addition of Diol on para-Quinone Methide followed by ipso cyclization: An Approach to Spiro 1,4-Dioxane cyclohexadienone Derivatives.

A novel one-pot transition metal-free and Brönsted acid mediated 1,6-conjugate addition of bisnuleophilic diol on biselectrophilc para-quinone methide followed by ipso cyclization assisted by NBS has been developed under mild reaction conditions, offers a new approach to synthesize spiro 1,4-dioxane cyclohexadienone derivatives. This strategy features broad substrate scope of p-QMs with high functional group tolerance and good yields of spirocyclic scaffolds (60-92 %). N-Bromo succinimide an important role in an ipso spirocyclization with high efficiency.

Implementing an intensified mentorship approach towards accelerated medical laboratory accreditation in 10 counties in Kenya.

Background: Despite Kenya's roll-out of the Strengthening Laboratory Management Towards Accreditation programme in 2010, most laboratories had not made significant or tangible improvements towards accreditation by 2016. In April 2016, the University of Maryland, Baltimore enrolled 27 facilities in the standard Strengthening Laboratory Management Towards Accreditation programme. Objective: This study aimed to describe and evaluate the implementation of an intensified mentorship strategy on laboratory accreditation. Methods: In October 2017, the University of Maryland, Baltimore implemented intensive mentorship in 27 hospital laboratories in Nairobi, Kiambu, Meru, Embu, Muranga, Nyeri, Laikipia, Nyandarua, Tharaka-Nithi, and Kirinyaga counties in Kenya. Laboratories were paired with competent mentors whose skills were matched to facility gaps. Baseline and follow-up assessments were done between April 2016 and March 2019 using the World Health Organization's Stepwise Laboratory Quality Improvement Process Towards Accreditation Checklist and overall scores of the 12 Quality System Essentials and star ratings (from zero to five, based on scores) used to evaluate the effectiveness of the intensified mentorship. Results: In September 2017, 14 laboratories scored zero stars, three scored one star, eight scored two stars, one scored three stars, and one laboratory was accredited. By March 2019, eight laboratories were accredited, five scored four stars, 10 scored three stars, three scored two stars, and only one scored one star. The average score change with the intensified approach was 81.5 versus 53.9 for the standard approach. Conclusion: The intensified mentorship strategy resulted in fast-tracked progress towards laboratory accreditation and can be adopted in similar resource-limited settings.

Determination of Rare Earth Elements by Inductively Coupled Plasma-Tandem Quadrupole Mass Spectrometry With Nitrous Oxide as the Reaction Gas.

Nitrous oxide (N2O) was investigated as the reaction gas for the determination of rare earth elements (REEs) by inductively coupled plasma-tandem quadrupole mass spectrometry (ICP-QMS/QMS). The use of N2O as the reaction gas apparently improved the yields of m M16O+ for Eu and Yb in the reaction cell. As a result, the sensitivities for measurement of Eu and Yb were apparently improved in comparison to those obtained with O2 as the reaction gas. A high sensitivity measurement of the whole set of REEs was achieved, providing a typical sensitivity of 300,000 CPS mL/ng for REEs measured with an isotope having isotopic abundance close to 100%. The use of N2O as the reaction gas helped suppress Ba-related spectral interferences with the measurement of Eu, permitting the measurement of Eu in a natural sample without mathematic correction of spectral interferences. The detection limits (unit, pg/mL) for 14 REEs (except for Pm) from La to Lu were 0.028, 0.018, 0.006, 0.026, 0.006, 0.010, 0.017, 0.006, 0.016, 0.010, 0.016, 0.004, 0.023, and 0.012, respectively. The validity of the present method was confirmed by determining REEs in river water-certified reference materials, namely, SLRS-3 and SLRS-4.

Acute methyl jasmonate exposure results in major bursts of stress volatiles, but in surprisingly low impact on specialized volatile emissions in the fragrant grass Cymbopogon flexuosus.

Methyl jasmonate (MeJA) is an airborne hormonal elicitor that induces a fast rise of emissions of characteristic stress marker compounds methanol and green leaf volatiles (GLV), and a longer-term release of volatile terpenoids, but there is limited information of how terpene emissions respond to MeJA in terpene-storing species. East-Indian lemongrass (Cymbopogon flexuosus), an aromatic herb with a large terpenoid storage pool in idioblasts, was used to investigate the short- (0-1 h) and long-term (1-16 h) responses of leaf net assimilation rate (A), stomatal conductance (Gs) and volatile emissions to MeJA concentrations ranging from moderate to lethal. Both A and Gs were increasingly inhibited with increasing MeJA concentration in both short and long term. MeJA exposure resulted in a rapid elicitation, within 1 h after exposure, of methanol and GLV emissions. Subsequently, a secondary rise of GLV emissions was observed, peaking at 2 h after MeJA exposure for the highest and at 8 h for the lowest application concentration. The total amount and maximum emission rate of methanol and the first and second GLV emission bursts were positively correlated with MeJA concentration. Unexpectedly, no de novo elicitation of terpene emissions was observed through the experiment. Although high MeJA application concentrations led to visible lesions and desiccation in extensive leaf regions, this did not result in breakage of terpene-storing idioblasts. The study highlights an overall insensitivity of lemongrass to MeJA and indicates that differently from mechanical wounding, MeJA-driven cellular death does not break terpene-storing cells. Further studies are needed to characterize the sensitivity of induced defense responses in species with strongly developed constitutive defenses.

Golden and Silver-Golden Chitosan Hydrogels and Fabrics Modified with Golden Chitosan Hydrogels.

Golden and silver-golden chitosan hydrogels and hydrogel-modified textiles of potential biomedical applications are investigated in this work. The hydrogels are formed by reactions of chitosan with HAuCl4·xH2O. For above the critical concentration of chitosan (c*), chitosan-Au hydrogels were prepared. For chitosan concentrations lower than c*, chitosan-Au nano- and microgels were formed. To characterise chitosan-Au structures, sol-gel analysis, UV-Vis spectrophotometry and dynamic light scattering were performed. Au concentration in the hydrogels was determined by the flame atomic absorption spectrophotometry. Colloidal chitosan-Au solutions were used for the modification of fabrics. The Au content in the modified fabrics was quantified by inductively coupled plasma mass spectrometry technique. Scanning electron microscopy with energy dispersion X-ray spectrometer was used to analyse the samples. Reflectance spectrophotometry was applied to examine the colour of the fabrics. The formation of chitosan-Au-Ag hydrogels by the competitive reaction of Au and Ag ions with the chitosan macromolecules is reported.

Advances in spray products for skin regeneration.

To date, skin wounds are still an issue for healthcare professionals. Although numerous approaches have been developed over the years for skin regeneration, recent advances in regenerative medicine offer very promising strategies for the fabrication of artificial skin substitutes, including 3D bioprinting, electrospinning or spraying, among others. In particular, skin sprays are an innovative technique still under clinical evaluation that show great potential for the delivery of cells and hydrogels to treat acute and chronic wounds. Skin sprays present significant advantages compared to conventional treatments for wound healing, such as the facility of application, the possibility to treat large wound areas, or the homogeneous distribution of the sprayed material. In this article, we review the latest advances in this technology, giving a detailed description of investigational and currently commercially available acellular and cellular skin spray products, used for a variety of diseases and applying different experimental materials. Moreover, as skin sprays products are subjected to different classifications, we also explain the regulatory pathways for their commercialization and include the main clinical trials for different skin diseases and their treatment conditions. Finally, we argue and suggest possible future trends for the biotechnology of skin sprays for a better use in clinical dermatology.

Kidney-Derived Methylated PAX2 Sequences in the Urine of Healthy Subjects as a Convenient Model for Optimizing Methylation-Based Liquid biopsy.

Although urine-based liquid biopsy has received considerable attention, there is a lack of a simple model to optimize assay parameters, including cell-free DNA (cfDNA) extraction, bisulfite modification, and bis-DNA recovery after conversion for methylation analysis in urine. The primary aim of this work was to establish a practical model by developing a quantitative methylation-sensitive PCR (qMS-PCR) assay for PAX2 based on hypermethylated PAX2 cfDNA that could be detected in healthy human urine. We first studied the methylation status of PAX2 in kidney tissues and whole blood, followed by an assessment of commercial kits for bisulfite conversion and bis-DNA recovery. Furthermore, we investigated the influence of urine storage and collection conditions on the preservation of methylated PAX2 in urine samples by qMS-PCR. As expected, PAX2 methylation was identified in urine but not in blood. Two commercial kits (CellCook and Zymo Research) had similar conversion efficiency and bis-DNA recovery. Urine storage for up to 5 days did not change PAX2 methylation estimates. Overall, cold storage of urine samples and the CellCook urine container maintained higher levels of methylated PAX2 compared to urine kept at room temperature and the conventional tubes, respectively. These findings highlight the importance of using the correct approaches/kits and optimizing experimental conditions as a diagnostic tool in the clinical setting. Our study provides insights on the development of urine-based liquid biopsy with DNA methylation as a universal biomarker.

Urinary Volatomic Expression Pattern: Paving the Way for Identification of Potential Candidate Biosignatures for Lung Cancer.

The urinary volatomic profiling of Indian cohorts composed of 28 lung cancer (LC) patients and 27 healthy subjects (control group, CTRL) was established using headspace solid phase microextraction technique combined with gas chromatography mass spectrometry methodology as a powerful approach to identify urinary volatile organic metabolites (uVOMs) to discriminate among LC patients from CTRL. Overall, 147 VOMs of several chemistries were identified in the intervention groups-including naphthalene derivatives, phenols, and organosulphurs-augmented in the LC group. In contrast, benzene and terpenic derivatives were found to be more prevalent in the CTRL group. The volatomic data obtained were processed using advanced statistical analysis, namely partial least square discriminative analysis (PLS-DA), support vector machine (SVM), random forest (RF), and multilayer perceptron (MLP) methods. This resulted in the identification of nine uVOMs with a higher potential to discriminate LC patients from CTRL subjects. These were furan, o-cymene, furfural, linalool oxide, viridiflorene, 2-bromo-phenol, tricyclazole, 4-methyl-phenol, and 1-(4-hydroxy-3,5-di-tert-butylphenyl)-2-methyl-3-morpholinopropan-1-one. The metabolic pathway analysis of the data obtained identified several altered biochemical pathways in LC mainly affecting glycolysis/gluconeogenesis, pyruvate metabolism, and fatty acid biosynthesis. Moreover, acetate and octanoic, decanoic, and dodecanoic fatty acids were identified as the key metabolites responsible for such deregulation. Furthermore, studies involving larger cohorts of LC patients would allow us to consolidate the data obtained and challenge the potential of the uVOMs as candidate biomarkers for LC.

Phytochemical Composition, Antioxidant and Antiproliferative Activities of Citrus hystrix, Citrus limon, Citrus pyriformis, and Citrus microcarpa Leaf Essential Oils against Human Cervical Cancer Cell Line.

The essential oil derived from Citrus plants has long been used for medicinal purposes, due to its broad spectrum of therapeutic characteristics. To date, approximately 162 Citrus species have been identified, and many investigational studies have been conducted to explore the pharmacological potential of Citrus spp. oils. This study investigated the volatile constituents of essential oil distilled from the leaves of C. hystrix, C. limon, C. pyriformis, and C. microcarpa, using gas chromatography-quadrupole mass spectrometry. A total of 80 secondary compounds were tentatively identified, representing 84.88-97.99% of the total ion count and mainly comprising monoterpene (5.20-76.15%) and sesquiterpene (1.36-27.14%) hydrocarbons, oxygenated monoterpenes (3.91-89.52%) and sesquiterpenes (0.21-38.87%), and other minor chemical classes (0.10-0.52%). In particular, 27 compounds (1.19-39.06%) were detected across all Citrus species. Principal component analysis of the identified phytoconstituents and their relative quantities enabled differentiation of the Citrus leaf oils according to their species, with the loading variables contributing to these metabolic differences being identified. The Citrus leaf oils were tested for their antioxidant and antiproliferative activities using 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The results indicated that C. limon displayed the highest DPPH radical scavenging ability (IC50 value of 29.14 ± 1.97 mg/mL), while C. hystrix exhibited the lowest activity (IC50 value of 279.03 ± 10.37 mg/mL). On the other hand, all the Citrus oils exhibit potent antiproliferative activities against the HeLa cervical cancer cell line, with IC50 values of 11.66 µg/mL (C. limon), 20.41 µg/mL (C. microcarpa), 25.91 µg/mL (C. hystrix), and 87.17 µg/mL (C. pyriformis).