The Effect of a Casein and Gluten-Free Diet on the Epigenetic Characteristics of FoxP3 in Patients With Hashimoto's Thyroiditis.

Background Hashimoto's thyroiditis (HT) is an autoimmune thyroid disease characterized by inflammation and dysfunction of the thyroid gland, resulting in hypothyroidism, it results in impaired thyroid hormone generation and mimics hypothyroidism. The disease involves complex interactions among genetic, environmental, and epigenetic factors, particularly affecting the regulation of T regulatory (Treg) cells, including CD4 + foxp3 + T cells. Treg cells, defined as CD4 + T cells, rely on the expression of the foxp3 transcription factor, which is crucial for their development and differentiation. Disruptions in this regulation can lead to immune dysregulation and potential proinflammatory responses. The study focuses on investigating the impact of dietary patterns on the epigenetic changes in the foxp3 gene, a key player in the development of HT. The primary aim was to evaluate how eliminating gluten and casein proteins from dietary regimens may influence the methylation levels of the foxp3 gene, considering the potential link between these dietary components and the triggering of autoimmune diseases. Methods An epigenetic analysis of the foxp3 gene in HT patients who were strictly following a dietary plan compared with the control group. For the epigenetic study, a methylation analysis experiment was conducted.  Results Our findings revealed a notable reduction in foxp3 gene methylation levels among HT patients who adhered to a diet excluding casein and gluten. The control maintained normal dietary guidelines and showed no significant alterations in methylation levels. Discussion The laboratory values showed a decrease in methylation levels of the foxp3 gene, with statistical significance indicated as *p<0.005, **p<0.001, ***p<0.0001, suggesting a potential enhancement in its expression which could have profound implications for immune system regulation. Disruptions in the foxp3 pathway are crucial in the development of autoimmune disorders, where altered activity hinders the regulation of T cell (Treg) development, ultimately contributing to conditions like HT disease. These findings imply that nutritional interventions, especially for individuals with HT, could potentially be a strategy for mitigating autoimmunity through epigenetic mechanisms.

Investigating the role of let-7a microRNA in cisplatin sensitivity of A549 lung cancer cells.

Lung cancer (LC) is a major cause of death worldwide, and cisplatin is commonly used as a chemotherapeutic drug for the treatment of LC. However, high doses of cisplatin can reduce its efficacy, leading to the need for new methods to increase LC cell sensitivity to this drug molecule. To overcome this problem, it is important to discover new methods to increase the sensitivity of LC cells to cisplatin. In this study, we investigated the use of anti-let-7a, a microRNA, to enhance the cisplatin sensitivity in A549 LC cells by comparing its effects with the commonly used oncogenes akt1 and pik3ca. The A549 cell line was transfected with anti-let-7a, and its effects were analyzed using functional assays. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay was used for the measurement of cell viability, and gene expression levels of cell death-associated genes, were analyzed by using quantitative real-time PCR (qRT-PCR). Results showed that anti-let-7a downregulation decreased the viability of A549 cells significantly compared to the control group in the presence of cisplatin. Moreover, the single treatment of cells with anti-let-7a and cisplatin resulted in significant changes in gene expression levels, with the increased expression of pro-apoptotic genes and decreased expression of anti-apoptotic genes. Moreover, anti-let-7a treatment was found to increase the response of A549 cells to cisplatin by reducing the expression of oncogenes akt1 and pik3ca. This study suggests that anti-let-7a treatment may enhance the A549 LC cell sensitivity to cisplatin by modulating the expression of akt1 and pik3ca genes, making it a promising therapeutic target for LC treatment.

Sex-dependent multimodal response profiles to psychosocial stress.

INTRODUCTION: Sex differences in stress reactions are often reported in the literature. However, the sex-dependent interplay of different facets of stress is still not fully understood. Particularly in neuroimaging research, studies on large samples combining different indicators of stress remain scarce. MATERIALS AND METHODS: In a functional magnetic resonance imaging study, a sample of 140 healthy participants (67 females using oral contraceptives) underwent a standardized stress induction protocol, the ScanSTRESS. During the experiment, salivary cortisol and subjective ratings were obtained at multiple time points and heart rate was recorded. RESULTS: Sex differences emerged in different facets of the stress response:Women reacted with enhanced subjective feelings of stress and increases in heart rate, while men showed more pronounced neural activation in stress-related brain regions such as the inferior frontal gyrus and insula. Subjective feelings of stress and (para) hippocampal activity were negatively related in women,whereas a slightly positive association was observed in men. DISCUSSION: These results provide further insight in the sex-specific stress response patterns. Moreover, they emphasize the role of the hippocampus in the regulation of the stress response. This paves the way for the identification of sex-dependent vulnerability factors that can, in the future, be implemented in the prevention and treatment of stress-related disorders.

UHPLC-ESI-MS/MS assay for quantification of endocannabinoids in cerebrospinal fluid using surrogate calibrant and surrogate matrix approaches.

Endocannabinoids are endogenous lipids with the main function recognized to act as neuromodulators through their cannabinoid receptors. Dysregulation of the endocannabinoid system is implicated in various pathologies, such as inflammatory and neurodegenerative diseases. In this study we describe a sensitive UHPLC-MS/MS method for the analysis of trace levels of 7 endocannabinoids in cerebrospinal fluid samples. The analytes covered comprised 1- and 2-arachidonoylglycerol 1- and 2-AG (which were analysed as sum due to their interconversion), 2-arachidonylglycerol ether 2-AGE, anandamide AEA, N-linoleoyl ethanolamide LEA, N-palmitoyl ethanolamide PEA and N-oleoyl ethanolamide OEA. Analytes were extracted from the biofluid by a simple monophasic procedure involving protein precipitation with acetonitrile (MeCN). The analytical method is based on chromatographic separation of the analytes with solid-core (core-shell, superficially porous) particle column Cortecs C18+ . Gradient elution with changing proportion of water and acetonitrile and constant concentration of formic acid provided reasonable separation of analytes, close elution of analytes and their internal standards and minimized matrix effects in biological samples. For specific detection of the endocannabinoids a triple-quadrupole tandem mass spectrometer with electrospray ionisation (ESI) and selected reaction monitoring (SRM) mode was used, and it provided good assay selectivity. The developed method required a minute volume of the biological samples (50 µL) and achieved excellent sensitivity (the lower limit of detection was between 4.15 and 30.18 pM of the biological sample). Linear calibration was achieved in the range from 25 to 10,545 pM for AEA, 90-3802 pM for 1-AG, 90-724 pM for 2-AG, 12-5226 pM for LEA, 33-13,942 for OEA, 34-23,850 pM for 2-AGE, 72-30,190 for PEA and 10-4218 for AEA-d4 in CSF. The method was validated and revealed relative errors in the range of - 14.7 to + 12.3% at LLOQ and - 14.1 to + 14.2% for the remaining validation range. Precisions were in the acceptable range (< 20% RSD at LLOQ, and <15% for the remaining levels) as well. It was finally used to quantify endocannabinoids in human cerebrospinal fluid obtained from 118 donors. Accurate quantification of endogenous compounds in biological samples was achieved by using two different principal approaches (surrogate matrix for AEA, 2-AG, OEA, 2-AGE, LEA and PEA, and surrogate calibrant for AEA only) and they were evaluated by use of the Passing-Bablok regression. Concentrations (median) of CSF samples of patients suffering from CNS infection and controls were found to be around 160 pM for 1- and 2-AG, 86 pM for AEA, 62 for 2-AGE, 58 for LEA, 93 pM for PEA, and 83 pM for OEA.

Fast accurate quantification of salivary cortisol and cortisone in a large-scale clinical stress study by micro-UHPLC-ESI-MS/MS using a surrogate calibrant approach.

Cortisol and cortisone are common markers for stress and thus preferentially analyzed in matrices that allow non-invasive sampling such as saliva. Though the major drawback of immunoassays is lack of specificity due to cross reactivities, they are still most commonly used for quantification of steroid hormones. To overcome such problems, sensitive methods based on liquid chromatography-mass spectrometry are becoming more and more accepted as the golden standard for steroid bioanalysis as they achieve accurate quantification at trace levels for multiple analytes in the same run. Along this line, the aim of this study was the development of a new microflow UHPLC-ESI-MS/MS method for the measurement of salivary cortisol and cortisone, which due to its microflow regime provides enhanced sensitivity and is more ecofriendly. The developed method implemented sample preparation by Solid-Phase Extraction (SPE) in a 96-well plate format. Data acquisitions were carried out in MRM (multiple reaction monitoring) mode. The quantitative determination of endogenous compounds in saliva remains a challenge since analyte-free matrix is lacking. Hence, a surrogate calibrant approach with cortisol-d4 andcortisone-13C3 was applied for the target compounds in the presented method. A number of factors were optimized and the method validated. The lower limit of quantitation (LLOQ) was 72 and 62 pg mL-1for cortisol and cortisone, respectively. Linear calibration was achieved in the range from 0.062 to 75.5 ng mL-1for cortisol-d4 and 0.072 to 44 ng mL-1forcortisone-13C3. The performance of the method was also evaluated via proficiency test for salivary cortisol. Finally, it was applied successfully to evaluate cortisol and cortisone concentrations in multiple batches in routine clinical stress study samples (4056 total injections with 1983 study samples). Moreover, the instrument performance (in particular retention time variability) within each batch, between different batches and lot-to-lot of 5 investigated capillary columns over time is described. The work documents that micro-UHPLC-ESI-MS/MS is suitable and robust enough to carry out a full clinical study with greater than 1000s of samples over an extended period if adequate internal standards can be used.

Cystic fibrosis of pancreas and nephrotic syndrome: a rare association / 소아과

Cystic fibrosis (CF) is a genetic disease with autosomal recessive inheritance and is common in Caucasian people. The prevalence of this disease is between 1/2,000 and 1/3,500 live births, and the incidence varies between populations. Although the CF transmembrane conductance regulator gene is expressed in the kidneys, renal involvement is rare. With advances in the treatment of CF, life expectancy has increased, and some previously unobserved disease associations are now seen in patients with CF. It is important to follow patients with CF for possible abnormalities that may accompany CF. In this paper, we present two rare cases of CF accompanied by nephrotic syndrome.