Search Results (42)

Natural deep eutectic solvents (NaDESs) are emerging solvents for their yield when used for extraction of different molecules, including polyphenols. NaDESs are a cutting-edge technology that offers numerous advantages, including cheap cost, safety, effectiveness and environmental friendliness. However, due to NaDES’ high boiling point, the recovery and separation of compounds after the extraction is the bottleneck of the process. In this work, two affordable methods were tested for the recovery of phenolic compounds from three binary NaDESs (composed of choline chloride mixed separately with lactic acid, tartaric acid or glycerol as hydrogen bond donors): the antisolvent and the liquid–liquid extraction methods. The former was assessed by diluting the extracts with different aliquots of water, employed as antisolvent, which was ineffective. For the liquid–liquid extraction method, ethyl acetate (EtOAc), acetonitrile (ACN), 2-chlorobutane (2-CB) and 2-methyltetrahydrofuran (2-MeTHF) were compared. Except for ACN, all solvents were perfectly immiscible with the three NaDESs, forming biphasic systems that were analyzed by colorimetric assays and HPLC/MS. 2-MeTHF applied on a 10-fold water dilution of the NaDES extract reached recovery percentages higher than 90% for most of the non-anthocyanin phenols and good recovery (up to 80%) for some anthocyanins. 2-MeTHF appears to be the first known solvent capable of extracting anthocyanins from NaDESs. Finally, a two-step liquid–liquid extraction performed firstly with EtOAc and subsequently with 2-MeTHF is proposed for the separation of different phenolic fractions. Full article

Human milk contains a variety of biologically active molecules that are essential for infant growth and development, as well as indicators of maternal health. However, understanding the full potential of these molecules is challenging due to variations in their concentrations among mothers, potential degradation during sample handling and storage, and the limited accessibility of specific human milk analyses. This study aimed to evaluate the effectiveness of a freeze-dried preservative cocktail in maintaining the stability of key milk molecules during collection, transport, and storage. GenV participants (n = 96) were given a sample collection kit and followed the instructions to collect approximately 5 mL of breast milk, which was placed in a collection tube containing the preservative. The samples were mailed at ambient temperature to the GenV laboratory (Murdoch Children’s Research Institute, Melbourne, Victoria, Australia), where they were aliquoted into 1 mL tubes using a liquid handling system (Janus) and stored at −80 °C. These samples were randomly selected and sent to The University of Western Australia (Perth, Western Australia, Australia) on dry ice for biochemical analysis. The average collection day postpartum was 16 ± 14 (range 1–91 days), while the average postal receipt time was 5 ± 3 days (range 1–16 days), and samples were processed within 6 days of receipt (average 3 ± 2 days). The mean concentrations of key molecules—fat (48.6 ± 17.1 g/L), protein (15.5 ± 4.3 g/L), lactose (78.9 ± 13.9 g/L), glucose (0.17 ± 0.17 g/L), lysozyme (0.16 ± 0.16 g/L), and insulin (6.1 ± 4.9 μIU/mL)—were consistent with reported literature values. There were no statistically significant differences in molecular concentrations based on postal transit time, receipt, or processing delays (p > 0.05). These results demonstrate that the preservative cocktail effectively preserved the integrity of key molecules in human milk during handling, postal transport, and storage at ambient temperature. The findings support its use as a valuable tool for human milk research, enabling more flexible sample collection and handling without compromising the quality of the milk or the biochemical analysis. Future research should explore its application in broader contexts to further enhance the accuracy and reliability of milk composition studies across diverse research settings. Full article

This study aimed to examine the effects of crude garden cress seed oil (CGCSO) on frozen–thawed boar sperm qualities. Semen ejaculates (n = 12) were collected and further divided into six equal aliquots based on CGCSO concentrations (0, 0.5, 1, 1.5, 2, and 2.5% v/v) in the freezing extender. Semen samples were processed and cryopreserved utilizing the traditional liquid nitrogen vapor technique. Subsequently, semen samples were thawed in a thermos with warm water at 50 °C for 12 s and evaluated for sperm morphology using scanning electron microscopy, sperm motility using a CASA, sperm viability, acrosome integrity, mitochondrial function, MDA level, total antioxidant capacity (TAC), glutathione peroxidase (GSH-Px), and catalase (CAT) activity. The results indicated that 1% CGCSO resulted in superior post-thaw sperm characteristics, including enhanced sperm morphology, motility, viability, acrosome integrity, and mitochondrial function. Particularly, the total motile sperm increased by 16.5%, progressive motile sperm increased by 13.0%, viability improved by 15.1%, acrosome integrity increased by 14%, and mitochondrial function improved by 14.1% compared to the control group. CGCSO treatment at 1% and 1.5% exhibited the lowest level of MDA (45.73 ± 11.2 and 45.73 ± 11.3 µmol/L, respectively) compared to the other groups. The CGCSO-supplemented groups showed higher values of TAC, GSH-Px, and CAT than the control group but not significantly. Full article

In the first part of this study, the electrochemical polymerization of two compounds, 3,5-dihydroxybenzoic acid and 2′,6′-dihydroxyacetophenone, was compared in dimethyl sulfoxide solvent on platinum and glassy carbon electrodes. The voltammograms obtained showed remarkable differences between the two monomers and between the two electrode materials. The acetophenone derivative formed electropolymer remnants at the electrodes, while in the case of the benzoic acid derivative, practically no passivation occurred, and the scanning electron microscopic results reinforced this. A few stackings adsorbed only after electropolymerization from a highly concentrated solution of dihydroxybenzoic acid. As a modifying layer on the platinum and glassy carbon electrodes, the prepared films from 2′,6′-dihydroxyacetophenone were tested for tributylamine in acetonitrile and in an aqueous solution of a redox-active compound, hydroquinone, during the stirring of the solution. More stable amperometric current signals could be reached with modified platinum than with glassy carbon, and the significant influence of the organic washing liquid after deposition was established via the study of noise level. In this respect, acetone was the best choice. The amperometric signals with the modified platinum obtained upon the addition of aliquots of the stock solution resulted in a 3.29 μM detection limit. Full article

Oxytocin and cortisol (OXY and CORT) are hormones related to stress, cognitive, and social behaviors. Their detection is relevant to epidemiological studies aimed at investigating the effects of stressor factors on human life. The aim of this study was to develop and validate an assay for the measurement of OXY and CORT in saliva samples using liquid chromatography/tandem mass spectrometry (LC-MS/MS) in the presence of deuterated analogs. A 500 mL aliquot of oral fluid, obtained by the centrifugation of a chewed swab, was purified by solid-phase extraction. Analytes were then separated using C18 reversed-phase chromatography, subjected to positive electrospray ionization, and then quantified using a triple-quadrupole mass detector in multiple-reaction monitoring mode. The limits of quantification and the linear dynamic ranges were 2.0 × 10⁻³ and 0.5 nmol/L, and up to 1.0 × 10⁻¹ and 20 nmol/L for OXY and CORT, respectively. Inter- and intra-run precision, expressed as relative standard deviation, was <7%, and accuracy was within 93–104% of the theoretical concentrations. The evaluation of matrix effects showed that the use of internal standards controlled sources of bias. The high sensitivity of the method allowed the quantification of OXY and CORT in the salivary samples of both adults and children: levels of CORT ranged from 0.6 to 18.5 nmol/L, while OXY levels were two orders of magnitude lower (from 1.7 × 10⁻³ to 1.1 × 10⁻² nmol/L). To our knowledge, this is the first method that can analyze, in the same chromatographic run, both hormones in saliva samples. Full article

The emptying rate of specific nutrients in enteral formulas is poorly understood, despite the importance of controlling the emptying rate in tube-fed patients. Because of their viscosity, thickened formulas are widely used to avoid gastric reflux and reduce the burden on caregivers. This study examined how thickeners in enteral formulas affected the gastric emptying rates of proteins and carbohydrates. A semi-dynamic gastric model was used to prepare and digest test enteral formulas that contained either no thickeners or agar (0.2%). The amounts of protein and carbohydrates in each emptied aliquot were determined, and the emptying rate was calculated. We found that agar accelerated protein emptying, and an exploratory experiment with agar (0.5%) suggested the possibility of concentration dependence. Additionally, experiments using gellan gum (0.08%), guar gum (0.2%), or carrageenan (0.08%, 0.2%) suggested that protein emptying could vary depending on the thickener type and that carrageenan might slow it. These results could help with the appropriate selection of thickeners added to liquid foods based on the patient’s metabolic profile to manage nutrition, not only for tube-fed patients but also for those with oropharyngeal dysphagia or diabetes. Full article

We have been encouraging practicing gynecologists to adopt molecular diagnostics tests, PCR, and cancer biomarkers, as alternatives enabled by these platforms, to traditional Papanicolaou and colposcopy tests, respectively. An aliquot of liquid-based cytology was used for the molecular test [high-risk HPV types, (HR HPV)], another for the PAP test, and one more for p16/Ki67 dual-stain cytology. A total of 4499 laboratory samples were evaluated, and we found that 25.1% of low-grade samples and 47.9% of high-grade samples after PAP testing had a negative HR HPV-PCR result. In those cases, reported as Pap-negative, 22.1% had a positive HR HPV-PCR result. Dual staining with p16/Ki67 biomarkers in samples was positive for HR HPV, and 31.7% were also positive for these markers. Out of the PCR results that were positive for any of these HR HPV subtypes, n 68.3%, we did not find evidence for the presence of cancerous cells, highlighting the importance of performing dual staining with p16/Ki67 after PCR to avoid unnecessary colposcopies. The encountered challenges are a deep-rooted social reluctance in Mexico to abandon traditional Pap smears and the opinion of many specialists. Therefore, we still believe that colposcopy continues to be a preferred procedure over the dual-staining protocol. Full article

Sperm cryopreservation is a procedure widely used to store gametes for later use, to preserve fertility in patients prior to gonadotoxic treatments or surgery, and for sperm donation programs. The purpose of the study was to assess the impact of cryopreservation on human sperm transcriptome. Semen samples were collected from 13 normospermic men. Each sample was divided into two aliquots. The total RNA was immediately extracted from one aliquot. The second aliquot was frozen and total RNA was extracted after a week of storage in liquid nitrogen. The RNA samples were randomized in four pools, each of six donors, and analyzed by microarrays. The paired Significance Analysis of Microarray was performed. We found 219 lower abundant transcripts and 28 higher abundant transcripts in cryopreserved sperm than fresh sperm. The gene ontology analysis disclosed that cryopreservation alters transcripts of pathways important for fertility (i.e., spermatogenesis, sperm motility, mitochondria function, fertilization, calcium homeostasis, cell differentiation, and early embryo development), although the increase of some transcripts involved in immune response can compensate for the harmful effects of freezing. Full article

Oxidative stress is involved in the development, progression, and complications of diabetes mellitus (DM). Oxidative modification of human serum albumin’s cysteine-34 is a marker for oxidative stress-related pathological conditions. We aimed to evaluate the redox state of albumin in patients with DM to investigate possible correlations with age, diabetes duration, and disease control status. Plasma aliquots were collected from 52 participants (26 type 1 and 26 type 2 DM). Patients were divided into two groups according to their glycated hemoglobin levels less than or equal to and greater than 58 mmol/L. Albumin redox state was assessed with high-performance liquid chromatography by fractionating it into human mercaptalbumin (HMA) and human nonmercaptalbumin 1 and 2 (HNA1 and HNA2). Albumin redox fractions were differently related to the age of study participants. In age-matched T1DM and T2DM groups, the albumin redox state was essentially the same. Irreversibly oxidized HNA2 was positively correlated with diabetes duration, especially in the T1DM group. HNA was increased in people with an increased HbA1c (>58 mmol/mol). Our results support the hypothesis that oxidative stress plays a crucial role in DM pathogenesis and emphasize the importance of diabetes control on systemic oxidative burden. Full article

Boar sperm is sensitive to particular conditions during cryopreservation, resulting in an extreme reduction in fertilizing ability due to damage to the sperm membranes. PKMPH contains bioactive peptides that have antioxidant and antimicrobial activities. There is no information on the use of palm-kernel-meal-derived bioactive peptides for boar semen cryopreservation. This study aimed to examine the effects of bioactive peptides from PKMPH on post-thawed boar sperm quality. Boar semen ejaculates (n = 17) were collected and divided into six equal aliquots based on PKMPH concentrations (0, 1.25, 2.5, 5, 10, and 15 µg/mL) in a freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, the frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm motility using a computer-assisted sperm analyzer and for sperm viability, acrosome integrity, mitochondrial function, and lipid peroxidation by measuring the level of malondialdehyde (MDA). The results demonstrate that the supplementation of PKMPH with 2.5 µg/mL afforded superior post-thawed sperm qualities, such as increased total motility, viability, acrosome integrity, and mitochondrial function by 10.7%, 12.3%, 18.3%, and 12.7%, respectively, when compared to the control group. PKMPH at a concentration of 2.5 µg/mL showed the lowest level of MDA (40.6 ± 2.0 µMol/L) compared to the other groups. In conclusion, adding PKMPH peptides at 2.5 µg/mL to the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in higher post-thawed sperm quality. Full article

Thymoquinone nanoparticles (TQNPs) are broadly utilized in numerous pharmaceutical applications. In the present study, we tested the effects of TQNP supplementation on sperm quality and kinematics, acrosome exocytosis, oxidative biomarkers, apoptosis-like and morphological changes of frozen–thawed buffalo sperm, as well as the fertilizing capacity. Semen was collected from buffalo bulls, diluted (1:10; semen/extender), and divided into five aliquots comprising various concentrations of TQNP 0 (CON), 12.5 (TQNP12.5), 25 (TQNP25), 37.5 (TQNP37.5), and 50 (TQNP50) µg/mL, and then cryopreserved and stored in liquid nitrogen (−196 °C). The results revealed that TQNPs (25 to 50 µg/mL) provided the most optimal results in terms of membrane integrity (p < 0.001) and progressive motility (p < 0.01). In contrast, TQNP50 resulted in a greater post-thawed sperm viability (p = 0.02) compared with other groups. The addition of TQNPs to the extender had no discernible effects on sperm morphology measures. Sperm kinematic motion was significantly improved in the TQNP50 group compared to the control group (p < 0.01). TQNPs effectively reduced the content of H₂O₂ and MDA levels and improved the total antioxidant capacity of post-thawed extended semen (p < 0.01). The addition of TQNP significantly increased the number of intact acrosomes (p < 0.0001) and decreased the number of exocytosed acrosomes (p < 0.0001). A significant reduction in apoptosis-like changes was observed in TQNP groups. The non-return rates of buffalo cows inseminated with TQNP50-treated spermatozoa were higher than those in the control group (p < 0.05; 88% vs. 72%). These findings suggested that the freezing extender supplemented with TQNPs could effectively enhance the cryotolerance and fertility of buffalo sperm. Full article

The detection of nucleic acids as specific markers of infectious diseases is commonly implemented in molecular biology laboratories. The translation of these benchtop assays to a lab-on-a-chip format demands huge efforts of integration and automation. The present work is motivated by a strong requirement often posed by molecular assays that combine isothermal amplification and CRISPR/Cas-based detection: after amplification, a 2–8 microliter aliquot of the reaction products must be taken for the subsequent reaction. In order to fulfill this technical problem, we have designed and prototyped a microfluidic device that is able to meter and aliquot in the required range during the stepped assay. The operation is achieved by integrating a porous material that retains the desired amount of liquid after removing the excess reaction products, an innovative solution that avoids valving and external actuation. The prototypes were calibrated and experimentally tested to demonstrate the overall performance (general fluidics, metering, aliquoting, mixing and reaction). The proposed aliquoting method is fully compatible with additional functions, such as sample concentration or reagent storage, and could be further employed in alternative applications beyond molecular diagnosis. Full article

Today, the measurement of ¹⁴C in environmental samples is of particular interest, as it enables the assessment of the impact caused by nuclear activities and the fossil fuel industry on the environment. In order to assure the quality of ¹⁴C measurement results, the strategy to enlarge the validation of three radioanalytical methods in environmental samples using liquid scintillation spectrometry—the direct counting of water, bubbling of water and combustion of solids—is presented. Due certain difficulties, such as the lack of quality control materials and the scarcity of proficiency test and intercomparison exercises, especially in solid samples, a set of water and soil samples were prepared for the purpose by tracing them with known quantities of a ¹⁴C standard solution at two activity levels. Aliquots were subjected to the corresponding method and their activity concentration was calculated. Finally, uncertainty, detection limit, accuracy, precision, repeatability and linearity were analysed. The acceptance criteria for the quality parameters were previously established according to ISO 13528:2015 standard and Eurachem Laboratory Guide to Method Validation. In all the methods, the studied parameters fall within the acceptance range, so they are validated. The quality of the results in real samples is controlled through field validation. Full article

Insects on corpses could be a useful tool for the detection of exogenous substances such as drugs of abuse. The identification of exogenous substances in carrion insects is critical for proper estimation of the postmortem interval. It also provides information about the deceased person that may prove useful for forensic purposes. High-performance liquid chromatography coupled with Fourier transform mass spectrometry is a highly sensitive analytical technique that can identify substances even at very low concentrations, such as in the case of searching for exogenous substances in larvae. In this paper, a method is proposed for the identification of morphine, codeine, methadone, 6-monoacetylmorphine (6-MAM) and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in the larvae of Lucilia sericata, a common carrion fly widely distributed in temperate areas of the world. The larvae, which were reared on a pig meat substrate, were killed once they reached their third stage by immersion in hot water at 80 °C and aliquoted into 400 mg samples. The samples were fortified with 5 ng of morphine, methadone and codeine. After solid-phase extraction, the samples were processed with a liquid chromatograph coupled to a Fourier transform mass spectrometer. This qualitative method has been validated and tested on larvae from a real case. The results lead to the correct identification of morphine, codeine, methadone and their metabolites. This method could prove useful in cases where toxicological analysis must be conducted on highly decomposed human remains, where biological matrices are very limited. Furthermore, it could help the forensic pathologist to better estimate the time of death, as the growth cycle of carrion insects can undergo changes if exogenous substances are taken. Full article

Various types of advanced glycation end-products (AGEs) have been identified and studied. I have reported a novel slot blot analysis to quantify two types of AGEs, glyceraldehyde-derived AGEs, also called toxic AGEs (TAGE), and 1,5-anhydro-D-fructose AGEs. The traditional slot blot method has been used for the detection and quantification of RNA, DNA, and proteins since around 1980 and is one of the more commonly used analog technologies to date. However, the novel slot blot analysis has been used to quantify AGEs from 2017 to 2022. Its characteristics include (i) use of a lysis buffer containing tris-(hydroxymethyl)-aminomethane, urea, thiourea, and 3-[3-(cholamidopropyl)-dimetyl-ammonio]-1-propane sulfonate (a lysis buffer with a composition similar to that used in two-dimensional gel electrophoresis-based proteomics analysis); (ii) probing of AGE-modified bovine serum albumin (e.g., standard AGE aliquots); and (iii) use of polyvinylidene difluoride membranes. In this review, the previously used quantification methods of slot blot, western blot, immunostaining, enzyme-linked immunosorbent assay, gas chromatography–mass spectrometry (MS), matrix-associated laser desorption/ionization–MS, and liquid chromatography–electrospray ionization–MS are described. Lastly, the advantages and disadvantages of the novel slot blot compared to the above methods are discussed. Full article