The selection of proper reference genes and materials is critical in the design of PCR experiments, especially for differential expression studies. In this study, we propose a method to identify robust endogenous control miRNAs in the visceral adipose tissue of C57BL/6J mice with non-alcoholic fatty liver disease induced by alternating Western and control diets. This study outlines a comprehensive methodology for the analysis of microRNA endogenous controls using microfluidic cards in conjunction with miRNA profiling through small RNA sequencing and subsequent validation by quantitative PCR and the RefFinder algorithm. Criteria included were fold change, p-value, reads per million, and gene stability assessment. A set of six putative endogenous microRNAs was identified (miR-331-3p, let-7a-5p, miR-1839-5p, miR-151a-5p, let-7d-5p, and let-7c-5p). Subsequent validation and analysis using the RefFinder algorithm assessed the stability of the selected genes, and a combination of the three most stable endogenous miRNA controls (miR-331-3p, let-7a-5p, and miR-1839-5p) exhibiting consistent expression patterns with minimal variability was set. Given the absence of universal endogenous controls, individual evaluation of normalizers for each experiment is imperative for accurate miRNA expression measurements. This approach, which combines multiple techniques and assessments, provides a reliable strategy for identifying and validating endogenous controls in miRNA studies.

Clinical consequences of mineral and bone disorder (CKD-MBD) remain still a therapeutic and prognostic challenge. Moreover, osteoporosis was recently recognized as significant contributor to bone pathology in kidney patients. Thus, close multidisciplinary cooperation between nephrologists, osteologists and other medical specialities is mandatory. Centre for renal osteopathy and calciphylaxis (CROC) was established in our University Hospital in 2022 to provide the as best as possible clinical practice in this so difficult and important topic. Our aim is to review CROC´s three years of experience. CROC is a platform for cooperation of nephrology, clinical osteology, clinical biochemistry, endocrinology, otorhinolaryngology, and other medical specialities and focuses namely on these problems: In all situations, patients were diagnosed, treated, and monitored according to prospective protocols. Three years of working together allowed us to build up a dedicated and experienced multidisciplinary team, successfully covering all aspects of mineral and bone problems in CKD patients. However, further effort is mandatory to implement our experience in even broader clinical practice. Supported by MH CZ – DRO (UHHK, 00179906) and by COOPERATIO programs

OBJECTIVES:To describe the structure of vitamin D metabolome and investigate the possible cause of high serum levels of C3 epimers of 25-(OH)D in preterm infants, we compared the vitamin D metabolites in umbilical cord blood with serum samples taken at 28 days of age. METHODS:We analysed 40 preterm infants (29+0-32+6 weeks of gestation). Cholecalciferol, 25-(OH)D, and its C3-epimers were measured using liquid chromatography. A microsomal study with human liver and kidney microsomes was conducted to assess vitamin D metabolism. Identified metabolites were then examined in cord blood and serum samples. RESULTS:Cholecalciferol, 25-(OH)D, and its C3-epimers were significantly lower in cord blood compared to serum at 28 days of age (p<0.001 for all metabolites). Conversely, metabolites from the microsomal study (monohydroxylated-, dihydroxylated-, and mono-oxylated dihydroxylated-cholecalciferol and their C3-epimers) were significantly higher in cord blood (p<0.001 for all). CONCLUSIONS:Our findings indicate that cholecalciferol, 25-(OH)D, and its C3-epimers increase during the first month of life, suggesting functional biosynthesis and postnatal accumulation of these metabolites. Conversely, based on microsomal study results, it seems that biotransformation responsible for a degradation of vitamin D during the first month of life in preterm infants is functionally impaired.

AIM:This study evaluated urinary calcium (Ca) and phosphorus (P) excretion in very low birth weight infants ( < 1500 g, VLBW) without severe morbidity, treated with methylxanthines. METHODS:Eighty-one VLBW infants were analysed. Urinary Ca and P and their creatinine ratios were measured biweekly until day 56. Serum parathyroid hormone (PTH), 25-hydroxyvitamin D [25-(OH)D], total Ca, P and alkaline phosphatase (ALP) were assessed. Bone densitometry was performed before discharge. RESULTS:In 254 urine samples, urinary Ca and P excretion differed significantly from their creatinine ratios. Vitamin D status influenced urinary mineral excretion, likely via PTH regulation, but did not affect serum Ca, P, or ALP. Normative data were established only for infants with serum 25-(OH)D > 50 nmol/L. Due to age-related variability, a single reference range was not possible. CONCLUSIONS:We provide age-specific normative data for urinary Ca and P excretion in healthy VLBW infants with sufficient vitamin D. Urinary mineral excretion varies significantly with postnatal age, emphasising the need for individualised monitoring to prevent metabolic bone disease of prematurity. Vitamin D status is crucial for calcium-phosphorus homeostasis, with sufficient levels linked to increased Ca excretion and relative P depletion from the 2 month of life.

Background: COVID-19 is a viral disease notorious for frequent worldwide outbreaks. It is difficult to control, thereby resulting in overload of the healthcare system. A possible solution to prevent overcrowding is rapid triage of patients, which makes it possible to focus care on the high-risk patients and minimize the impact of crowding on patient prognosis. Methods: The triage algorithm assessed self-sufficiency, oximetry, systolic blood pressure, and the Glasgow coma scale. Compliance with the triage protocol was defined as fulfillment of all protocol steps, including assignment of the correct level of care. Triage was considered successful if there was no change in the scope of care (e.g., unscheduled hospital admission, transfer to different level of care) or if there was unexpected death within 48 hours. Results: A total of 929 patients were enrolled in the study. Triage criteria were fulfilled in 825 (88.8%) patients. Within 48 hours, unscheduled hospital admission, transfer to different level of care, or unexpected death occurred in 56 (6.0%), 6 (0.6%), and 5 (0.5%) patients, respectively. The risk of unscheduled hospital admission or transfer to different level of care was significantly increased if triage criteria were not fulfilled [13.1% vs. 76.1%, RR 5.8 (3.8-8.3), p < 0.001; 0.5% vs. 5.2%, RR 11.4 (2.3-57.7), p = 0.036, respectively]. Conclusion: The proposed algorithm for triage of patients with proven COVID-19 is a simple, fast, and reliable tool for rapid sorting for outpatient treatment, hospitalization on a standard ward, or assignment to an intensive care unit.

PURPOSE:The primary goal was to estimate reference values of parathyroid hormone (PTH) in very low birth weight infants without severe neonatal morbidity. A secondary objective was to assess the relationship between PTH serum levels and selected laboratory markers of bone metabolism. METHODS:Ninety two infants with birth weight less than 1500 g met the inclusion criteria of the study. Serum levels of PTH, 25-hydroxyvitamin-D [25(OH)D], C3-epi-25(OH)D, total calcium, phosphorus, and alkaline phosphatase, and urinary levels of calcium, phosphorus, and creatinine were examined on day 14 and subsequently every 2 weeks until discharge. RESULTS:Of the total 167 serum samples examined for PTH levels in infants without 25(OH)D deficiency the estimated range was 0.9-11.9 pmol/l (8.5-112.3 pg/mL). During the first month, no statistically significant correlation was observed between PTH level and that of 25(OH)D, C3-epimers of 25(OH)D, S-Ca, S-P, or ALP, nor with urinary excretion of calcium and phosphorus. From the second month of life, there was a moderately significant correlation between PTH and 25(OH)D (Rho = -0.40, P =< .001), between PTH and calcium/creatinine ratio (Rho = -0.56, P = < .001), and between PTH and phosphorus/creatinine ratio (Rho = 0.51, P = < .001). CONCLUSIONS:The physiological range for PTH levels for preterm neonates without 25(OH)D deficiency was estimated as 0.9-11.9 pmol/l (8.5-112.3 pg/mL). It seems that elevation of serum PTH above this range can be considered as hyperparathyroidism in very low birth weight infants.

Introduction Cervical cancer causes approximately 350,000 deaths each year. The availability of sensitive and specific diagnostic tests to detect cervical cancer in its early stages is essential to improve survival rates. Methods In this study, we compared two strategies for selecting endogenous controls: miRNA profiling by small-RNA sequencing and a commercially available microfluidic card with 30 recommended endogenous controls preloaded by the manufacturer. We used the RefFinder algorithm and coefficient of variation to select endogenous controls. We selected the combination of miR-181a-5p and miR-423-3p as the most optimal normalizer. In the second part of this study, we determined the differential expression (between tumor/non-tumor groups) of microRNA in cervical cancer FFPE tissue samples. We determined the comprehensive miRNA expression profile using small-RNA sequencing technology and verified the results by real-time PCR. We determined the relative expression of selected miRNAs using the 2 -ΔΔCt method. Results We detected statistically significant upregulation of miR-320a-3p, miR-7704, and downregulation of miR-26a-5p in the tumor group compared to the control group. The combination of these miRNAs may have the potential to be utilized as a diagnostic panel for cervical cancer. Using ROC curve analysis, the proposed panel showed 93.33% specificity and 96.97% sensitivity with AUC = 0.985. Conclusions We proposed a combination of miR-181a-5p and miR-423-3p as optimal endogenous control and detected potentially significant miRNAs (miR-320a-3p, miR-7704, miR-26a-5p). After further validation of our results, these miRNAs could be used in a diagnostic panel for cervical cancer.

Introduction: Our study aimed to investigate the effect of zonisamide (ZNS) on bone metabolism in the rat model. Methods: Eight-week-old rats were divided into four groups. The sham-operated control group (SHAM) and the control group after orchidectomy (ORX) received the standard laboratory diet (SLD). The experimental group after orchidectomy (ORX+ZNS) and the sham-operated control group (SHAM+ZNS) received SLD enriched with ZNS for 12 weeks. Bone marker concentrations in serum of receptor activator of nuclear factor kappa B ligand, PINP, and osteoprotegerin, and the levels of sclerostin and bone alkaline phosphatase in bone homogenate, were measured using an enzyme-linked immunosorbent assay. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. The femurs were used for biomechanical testing. Results: We found a statistically significant reduction in BMD and biomechanical strength 12 weeks after orchidectomy of the rats (ORX). After ZNS administration to orchidectomized rats (ORX+ZNS) and the sham-operated control rats (SHAM+ZNS), there were no statistically significant changes in BMD, bone turnover markers, or biomechanical properties as compared with the ORX group and SHAM group. Conclusions: The results suggest that administration of ZNS to rats exerts no negative effect on BMD, bone metabolism markers, or biomechanical properties.

Osteoporosis is a chronic disease with high unmet medical need. It is characterized by low bone mass and deteriorated bone architecture, leading to increased risk of fragility fractures, with vertebral and hip fractures representing the highest risk of morbidity and mortality. The baseline therapeutic approach to osteoporosis treatment has been based on adequate intake of calcium and supplementation of vitamin D. In this review, we focus on two approved monoclonal antibodies, romosozumab and denosumab, which have been shown to be efficient and safe options to prevent patient fractures. Romosozumab is a humanized monoclonal antibody IgG2 isotype that extracellularly binds sclerostin with high affinity and specificity. Denosumab is a fully human monoclonal antibody IgG2 isotype that binds RANK ligand (RANKL) and prevents the interaction of RANKL with its receptor RANK. Denosumab is an antiresorptive that has been used for more than a decade, and romosozumab has recently been approved for clinical practice worldwide.

Abstract Objectives The primary objective was to determine levels of C3-epi-25(OH)D in very low birth weight infants. The secondary objective was to evaluate the possible influence of preterm birth, intrauterine growth restriction (IUGR), and season of birth on the production of C3-epimers. Methods A total of 127 infants with birth weight less than 1,500 g met the inclusion criteria of the study. We examined 25-hydroxyvitamin-D [25(OH)D] levels and C3-epi-25(OH)D in maternal serum before labor, and in cord blood and infants’ serum on days 14 and 28, and at discharge. Results The mean levels (±SD) of C3-epi-25(OH)D of the cord, on day 14, on day 28, and at discharge were 2.2 (2.9), 7.7 (5.5), 11.7 (7.6) and 14.9 (11.7) nmol/L respectively. The proportion of total 25(OH)D as the C3-epimer was 6.9% (cord), 16.3% (day 14), 22.4% (day 28) and 23.3% (discharge). A statistically significant correlation between 25(OH)D and C3-epi-25(OH)D can be demonstrated from birth. The severity of immaturity and IUGR did not affect the production of C3-epimers. In summer/autumn vs. winter/spring, the mean (SD) percentage of total 25(OH)D as the C3-epimer significantly differs only in maternal serum samples and umbilical cord samples (p value <0.001). Conclusions The production of C3-epi-25(OH)D is functional even in the most immature newborns, has fetal origins, and is largely dependent on circulating 25(OH)D. At the end of the first month of life, C3-epimers make up more than 20% of 25(OH)D.

Background: The pathogenesis of psoriasis vulgaris involves changes in DNA molecules, genomic instability, telomere attrition, and epigenetic alterations among them. These changes are also considered important mechanisms of aging in cells and tissues. Objective: This study dealt with oxidation damage, telomere length and methylation status in DNA originating from peripheral blood of 41 psoriatic patients and 30 healthy controls. Methods: Oxidative damage of serum DNA/RNA was determined immunochemically. Real-time PCR was used for the analysis of the telomere length. ELISA technique determined levels of 5-methylcytosine in blood cells' DNA. Results: Oxidative damage of serum DNA/RNA was higher in patients than in controls (median, 3758 vs. 2286 pg/mL, p < 0.001). A higher length of telomeres per chromosome was found in patients whole-cell DNA than in controls (3.57 vs. 3.04 kilobases, p = 0.011). A negative corre-lation of the length of telomeres with an age of the control subjects was revealed (Spearman's rho =-0.420, p = 0.028). Insignificantly different levels of 5-methylcytosine in patients and con-trols were observed (33.20 vs. 23.35%, p = 0.234). No influences of sex, smoking, BMI, PASI score, and metabolic syndrome on the methylation status were found. Study limitations: i) A relatively small number of the participants, particularly for reliable subgroup analyses, ii) the Caucasian origin of the participants possibly influencing the results of the parameters determined, and iii) Telomerase activity was not directly measured in serum or blood cells. Conclusion: The study demonstrated increased levels of oxidized DNA/RNA molecules in the serum of patients with exacerbated psoriasis vulgaris. The results were minimally influenced by sex, the presence of metabolic syndrome, or cigarette smoking. In the psoriatic blood cells' DNA, the authors observed longer telomeres compared to healthy controls, particularly in females. Insignificantly higher global DNA methylation in psoriasis cases compared to the controls indicated marginal clinical importance of this epigenetic test performed in the blood cells' DNA. (c) 2022 Sociedade Brasileira de Dermatologia. Published by Elsevier Espana, S.L.U. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

INTRODUCTION:High indoxyl sulfate (IS) concentration is a serious problem for patients with CKD increasing the risk of cardiovascular diseases and CKD progression. Thus, the methods of decreasing the toxin concentrations are highly desired. The study aimed to discover the role of selected intestine-related factors on IS concentration.METHODS:We evaluated the impact of ABCG2 and ABCC2 polymorphisms influencing activity and protein intake by normalized protein catabolic rate. Additionally, we examined the relation of IS and uric acid (UA) that can share common elimination transporters. A monocentric, prospective, open cohort pilot study was performed on 108 patients undergoing dialysis treatment.RESULTS:The positive effect of residual diuresis on the reduction of IS levels was confirmed (p = 0.005). Also, an increase in IS depending on the dietary protein intake was confirmed (p = 0.040). No significant correlation between ABC gene polymorphisms was observed either, suggesting the negligible role of ABCG2 and ABCC2 in the elimination of IS in small bowel. The significant difference was observed for UA where ABCG2 421C>A (rs72552713) gene polymorphism was higher (505.3 μmol/L) in comparison with a wild-type genotype (360.5 μmol/L).CONCLUSION:No evidence of bowel elimination pathway via ABCC2 and ABCG2 transporters was found in renal replacement therapy patients.

Angiotensin-converting enzyme inhibitors (ACEis) have been used to treat anthracycline (ANT)-induced cardiac dysfunction, and they appear beneficial for secondary prevention in high-risk patients. However, it remains unclear whether they truly prevent ANT-induced cardiac damage and provide long-lasting cardioprotection. The present study aimed to examine the cardioprotective effects of perindopril on chronic ANT cardiotoxicity in a rabbit model previously validated with the cardioprotective agent dexrazoxane (DEX) with focus on post-treatment follow-up (FU). Chronic cardiotoxicity was induced by daunorubicin (DAU; 3 mg/kg/week for 10 weeks). Perindopril (0.05 mg/kg/day) was administered before and throughout chronic DAU treatment. After the completion of treatment, significant benefits were observed in perindopril co-treated animals, particularly full prevention of DAU-induced mortality and prevention or significant reductions in cardiac dysfunction, plasma cardiac troponin T (cTnT) levels, morphological damage, and most of the myocardial molecular alterations. However, these benefits significantly waned during 3 weeks of drug-free FU, which was not salvageable by administering a higher perindopril dose. In the longer (10-week) FU period, further worsening of left ventricular function and morphological damage occurred together with heart failure (HF)-related mortality. Continued perindopril treatment in the FU period did not reverse this trend but prevented HF-related mortality and reduced the severity of the progression of cardiac damage. These findings contrasted with the robust long-lasting protection observed previously for DEX in the same model. Hence, in the present study, perindopril provided only temporary control of ANT cardiotoxicity development, which may be associated with the lack of effects on ANT-induced and topoisomerase II β (TOP2B)-dependent DNA damage responses in the heart.

We compared the efficiency of real-time PCR analysis of FII (c.*97G>A, G20210A) and FV Leiden (c.1601G>A) thrombophilic mutations in the samples obtained from venous blood treated with various anti coagulant agents (EDTA, heparin, and sodium fluoride with potassium oxalate), or from clotted venous blood; one hundred samples of wild-type subjects were tested. Genomic DNA extracts and whole blood specimens modified by 90 °C heating were analysed by real-time PCR analysis; cycle threshold values were subsequently evaluated. Real-time PCR analysis for the FII gene assay performed in DNA extracts from EDTA blood samples revealed a median Ct value of 19.3. Similar Ct values were apparent in the DNA extracts obtained from the heparinized blood and sodium fluoride with potassium oxalatetreated samples: 18.5 and 18.9, respectively. Significantly higher Ct values were found in extracts from clotted blood with medians of 20.6 (tubes with inert separation gel) and 20.5 (tubes without the gel, both P < 0.001). The data on the FV real-time PCR analysis were very comparable to the FII assay. In the modified whole blood, the samples treated with heparin salts showed significantly lower Ct values (P < 0.001) in both assays when compared with the samples with EDTA, sodium fluoride with potassium oxalate, and with the samples with clotted blood. Our results indicate that real-time PCR analyses of thrombophilic mutations were not negatively influenced by the presence of heparin salts in collection tubes. Blood samples with various anticoagulants might be exchangeable for each other when DNA analysis of thrombophilic mutations is required.