DICER ribonuclease removes harmful R-loops.

R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids. Now, we show that the RNase DICER also resolves R-loops. Biochemical analysis reveals that DICER acts by specifically cleaving the RNA within R-loops. Importantly, a DICER RNase mutant impaired in R-loop processing causes a strong accumulation of R-loops in cells. Our results thus not only reveal a function of DICER as an R-loop resolvase independent of DROSHA but also provide evidence for the role of multi-functional RNA processing factors in the maintenance of genome integrity in higher eukaryotes.

UAP56/DDX39B is a major cotranscriptional RNA-DNA helicase that unwinds harmful R loops genome-wide.

Nonscheduled R loops represent a major source of DNA damage and replication stress. Cells have different ways to prevent R-loop accumulation. One mechanism relies on the conserved THO complex in association with cotranscriptional RNA processing factors including the RNA-dependent ATPase UAP56/DDX39B and histone modifiers such as the SIN3 deacetylase in humans. We investigated the function of UAP56/DDX39B in R-loop removal. We show that UAP56 depletion causes R-loop accumulation, R-loop-mediated genome instability, and replication fork stalling. We demonstrate an RNA-DNA helicase activity in UAP56 and show that its overexpression suppresses R loops and genome instability induced by depleting five different unrelated factors. UAP56/DDX39B localizes to active chromatin and prevents the accumulation of RNA-DNA hybrids over the entire genome. We propose that, in addition to its RNA processing role, UAP56/DDX39B is a key helicase required to eliminate harmful cotranscriptional RNA structures that otherwise would block transcription and replication.

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.

Human THO-Sin3A interaction reveals new mechanisms to prevent R-loops that cause genome instability.

R-loops, formed by co-transcriptional DNA-RNA hybrids and a displaced DNA single strand (ssDNA), fulfill certain positive regulatory roles but are also a source of genomic instability. One key cellular mechanism to prevent R-loop accumulation centers on the conserved THO/TREX complex, an RNA-binding factor involved in transcription elongation and RNA export that contributes to messenger ribonucleoprotein (mRNP) assembly, but whose precise function is still unclear. To understand how THO restrains harmful R-loops, we searched for new THO-interacting factors. We found that human THO interacts with the Sin3A histone deacetylase complex to suppress co-transcriptional R-loops, DNA damage, and replication impairment. Functional analyses show that histone hypo-acetylation prevents accumulation of harmful R-loops and RNA-mediated genomic instability. Diminished histone deacetylase activity in THO- and Sin3A-depleted cell lines correlates with increased R-loop formation, genomic instability, and replication fork stalling. Our study thus uncovers physical and functional crosstalk between RNA-binding factors and chromatin modifiers with a major role in preventing R-loop formation and RNA-mediated genome instability.

The Fanconi Anemia Pathway Protects Genome Integrity from R-loops.

Co-transcriptional RNA-DNA hybrids (R loops) cause genome instability. To prevent harmful R loop accumulation, cells have evolved specific eukaryotic factors, one being the BRCA2 double-strand break repair protein. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we investigated the FA role in R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells from FANCD2 deficient mice, we show that the FA pathway removes R loops, and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci in untreated and MMC-treated cells are largely R loop dependent, suggesting that the FA functions at R loop-containing sites. We conclude that co-transcriptional R loops and R loop-mediated DNA damage greatly contribute to genome instability and that one major function of the FA pathway is to protect cells from R loops.

Insulin resistance and the metabolic syndrome are related to the severity of steatosis in the pediatric population with obesity.

BACKGROUND: To determine the factors associated with an increased risk for severe steatosis (SS) and establish the Homeostatic Model Assessment-Insulin Resistance (HOMA-IR) as a screening tool. METHODS: A cross-sectional study was performed in obese children to assess the relationship between the metabolic syndrome (MetS) and glucose metabolism alterations (GMA) and the risk for severe steatosis. RESULTS: A total of 94 children (51 males) aged from six to 14 years were included. Thirteen children (14.8%) had severe steatosis (SS). The anthropometric variables associated with SS included body mass index (BMI) (SS 34.1 vs non-SS 29.7, p = 0.005), waist circumference (cm) (100 vs 92.5, p = 0.015) and hip circumference (cm) (108 vs 100, p = 0.018). The blood parameters included alanine aminotransferase (ALT) (UI/dl) (27 vs 21, p = 0.002), gamma-glutamil transpeptidase (GGT) (UI/dl) (16 vs 15, p = 0.017), fasting glycemia (mg/dl) (96 vs 88, p = 0.006), fasting insulin (UI/dl) (25 vs 15.3, p < 0.001) and HOMA-IR score (7.1 vs 3.7, p < 0.001). Eighteen children with MetS were found to be at an increased risk for severe steatosis (odds ratio [OR] 11.36, p < 0.001). After receiver operating characteristic (ROC) curve analysis, the best area under the curve (AUC) was obtained for HOMA-R of 0.862. The HOMA-R 4.9 cut-off value had a 100% sensitivity (CI 95%: 96.2-100) and 67.9% specificity (CI 95%: 57.1-78.7) for severe steatosis. CONCLUSIONS: The presence of MetS and glucose metabolism alterations are risk factors for severe steatosis. The 4.9 cut-off value for HOMA-IR may be a risk factor for severe steatosis in obese children.

Congenital lupus with multiorgan involvement: a case report and review of literature.

Neonatal lupus erythematosus (NLE) is an autoimmune disease caused by transplacental antibodies that can damage fetal tissue and cause various findings. With the exception of congenital heart block, which can be easily recognized at birth because of neonatal cardiac monitoring during the delivery and immediately after birth, most cases of NLE are recognized within days to weeks of life, but fewer than 10 cases with findings present at birth have been reported. We report the case of a newborn with extensive cutaneous eruption at the time of birth and multisystemic involvement, including hematologic, respiratory, hepatic, and neurologic involvement.

Differential expression of C-reactive protein and serum amyloid A in different cell types in the lung tissue of chronic obstructive pulmonary disease patients.

BACKGROUND: Chronic systemic inflammatory syndrome has been implicated in the pathobiology of extrapulmonary manifestations of chronic obstructive pulmonary disease (COPD). We aimed to investigate which cell types within lung tissue are responsible for expressing major acute-phase reactants in COPD patients and disease-free ("resistant") smokers. METHODS: An observational case-control study was performed to investigate three different cell types in surgical lung samples of COPD patients and resistant smokers via expression of the C-reactive protein (CRP) and serum amyloid A (SAA1, SAA2 and SAA4) genes. Epithelial cells, macrophages and fibroblasts from the lung parenchyma were separated by magnetic microbeads (CD326, CD14 and anti-fibroblast), and gene expression was evaluated by RT-PCR. RESULTS: The sample consisted of 74 subjects, including 40 COPD patients and 34 smokers without disease. All three cell types were capable of synthesizing these biomarkers to some extent. In fibroblasts, gene expression analysis of the studied biomarkers demonstrated increased SAA2 and decreased SAA1 in patients with COPD. In epithelial cells, there was a marked increase in CRP, which was not observed in fibroblasts or macrophages. In macrophages, however, gene expression of these markers was decreased in COPD patients compared to controls. CONCLUSIONS: These results provide novel information regarding the gene expression of CRP and SAA in different cell types in the lung parenchyma. This study revealed differences in the expression of these markers according to cell type and disease status and contributes to the identification of cell types that are responsible for the secretion of these molecules.

Stroke Steadiness as a Determinant Factor of Performance in 100 m Freestyle in Young Swimmers.

The classical kinematic variables in swimming are based on the calculation of mean values. Stroke steadiness determines the relationship between the duration of all consecutive strokes throughout a test. The aims of the current investigation were to examine differences in stroke-to-stroke steadiness according to swimmers' performance level on both body sides (breathing and non-breathing) and to analyse the interrelationship with kinematics during a 100 m front-crawl test. Thirty-two young, experienced swimmers voluntarily participated in the present study and were divided into two groups, national level (n = 15) and local level (n = 17), according to their competitive status within the national age-rankings. All participants performed a 100 m maximal test in a 50 m pool where they were laterally recorded. Kinematic variables such as mean velocity, stroke rate, stroke length, and stroke index, as well as long-term steadiness and short-term steadiness, were calculated. The two 50 m sections were analysed independently. Significant differences were observed between the two groups in the classical kinematic variables and in stroke steadiness (p < 0.05). In addition, stroke steadiness showed moderately high correlations with velocity (r = [-0.61-(-0.749)]) and stroke index (r = [-0.356-(-0.582)]). Maintaining a more stable inter-stroke period appears to be a determinant of performance in young, high-level national swimmers.

C-reactive protein and serum amyloid a overexpression in lung tissues of chronic obstructive pulmonary disease patients: a case-control study.

BACKGROUND: Although researchers have consistently demonstrated systemic inflammation in chronic obstructive pulmonary disease (COPD), its origin is yet unknown. We aimed to compare the lung bronchial and parenchymal tissues as potential sources of major acute-phase reactants in COPD patients and resistant smokers. METHODS: Consecutive patients undergoing elective surgery for suspected primary lung cancer were considered for the study. Patients were categorized as COPD or resistant smokers according to their spirometric results. Lung parenchyma and bronchus sections distant from the primary lesion were obtained. C-reactive protein (CRP) and serum amyloid A (SAA1, SAA2 and SAA4) gene expressions were evaluated by RT-PCR. Protein levels were evaluated in paraffin embedded lung tissues by immunohistochemistry and in serum samples by nephelometry. RESULTS: Our study included 85 patients with COPD and 87 resistant smokers. In bronchial and parenchymal tissues, both CRP and SAA were overexpressed in COPD patients. In the bronchus, CRP, SAA1, SAA2, and SA4 gene expressions in COPD patients were 1.89-fold, 4.36-fold, 3.65-fold, and 3.9-fold the control values, respectively. In the parenchyma, CRP, SAA1, and SAA2 gene expressions were 2.41-, 1.97-, and 1.76-fold the control values, respectively. Immunohistochemistry showed an over-stained pattern of these markers on endovascular cells of COPD patients. There was no correlation with serum protein concentration. CONCLUSIONS: These results indicate an overexpression of CRP and SAA in both bronchial and parenchymal tissue in COPD, which differs between both locations, indicating tissue/cell type specificity. The endothelial cells might play a role in the production of theses markers.

Synbiotic Supplementation Improves Quality of Life and Inmunoneuroendocrine Response in Patients with Fibromyalgia: Influence of Codiagnosis with Chronic Fatigue Syndrome.

Fibromyalgia (FM) and chronic fatigue syndrome (CFS) are two medical conditions in which pain, fatigue, immune/inflammatory dysregulation, as well as various mental health disorders predominate in the diagnosis, without evidence of a clear consensus on the treatment of FM and CFS. The main aim of this research was to analyse the possible effects of a synbiotic (Synbiotic, Gasteel Plus® (Heel España S.A.U.), through the study of pro-inflammatory/anti-inflammatory cytokines (IL-8/IL-10) and neuroendocrine biomarkers (cortisol and DHEA), in order to evaluate the interaction between inflammatory and stress responses mediated by the cytokine-HPA (hypothalamic-pituitary-adrenal) axis, as well as mental and physical health using body composition analysis, accelerometry and previously validated questionnaires. The participants were women diagnosed with FM with or without a diagnostic of CFS. Each participant was evaluated at baseline and after the intervention, which lasted one month. Synbiotic intervention decreased levels of perceived stress, anxiety and depression, as well as improved quality of life during daily activities. In addition, the synbiotic generated an activation of HPA axis (physiological cortisol release) that can compensate the increased inflammatory status (elevated IL-8) observed at baseline in FM patients. There were no detrimental changes in body composition or sleep parameters, as well as in the most of the activity/sedentarism-related parameters studied by accelerometry. It is concluded that synbiotic nutritional supplements can improve the dysregulated immunoneuroendocrine interaction involving inflammatory and stress responses in women diagnosed with FM, particularly in those without a previous CFS diagnostic; as well as their perceived of levels stress, anxiety, depression and quality of life.

Assessment of Intercostal Muscle Near-Infrared Spectroscopy for Estimating Respiratory Compensation Point in Trained Endurance Athletes.

This study aimed to assess the reliability and validity of estimating the respiratory compensation point (RCP) in trained endurance athletes by analyzing intercostal muscles' NIRS-derived tissue oxygenation dynamics. Seventeen experienced trail runners underwent an incremental treadmill protocol on two separate occasions, with a 7-day gap between assessments. Gas exchange and muscle oxygenation data were collected, and the oxygen saturation breakpoint (SmO2BP) measured in the intercostal muscles was compared to the RCP, which was identified by the increase in the VE/V.CO2 slope and the point at which the PetCO2 started to decrease. No statistically significant differences were observed between the two methods for any of the variables analyzed. Bland-Altman analysis showed significant agreement between the NIRS and gas analyzer methods for speed (r = 0.96, p < 0.05), HR (r = 0.98, p < 0.05), V.O2 relative to body mass (r = 0.99, p < 0.05), and %SmO2 (r = 0.98, p < 0.05). The interclass correlation coefficient values showed moderate to good reliability (0.60 to 0.86), and test-retest analysis revealed mean differences within the confidence intervals for all variables. These findings suggest that the SmO2BP measured using a portable NIRS device in the intercostal muscles is a reliable and valid tool for estimating the RCP for experienced trail runners and might be useful for coaches and athletes to monitor endurance training.

Incidence of hypertension in young transgender people after a 5-year follow-up: association with gender-affirming hormonal therapy.

In order to assess the risk of hypertension development, we performed a retrospective analysis of the clinical records of consecutive transgender patients who began gender-affirming hormonal therapy in our Outpatient Gender Identity Clinic with <30 years of age and had a follow-up >5 years. 149 transgender women treated with estradiol and 153 transgender men treated with testosterone were included; 129 of the transgender women received also androgen blockers (54 spironolactone, 49 cyproterone acetate and 26 LHRH agonists). The annual incidence of hypertension in young transgender men (1.18%) seemed comparable to that of the general population. In young transgender women, it seemed higher (2.14%); we found that the choice of androgen blocker had a remarkable effect, with a highly significant increase in patients treated with cyproterone acetate (4.90%) vs. the rest (0.80%); the adjusted hazard-ratio was 0.227 (p = 0.001). Correlation, logistic regression and mediation analyses were performed for the associations of the available clinical variables with the increase in systolic blood pressure and the onset of hypertension, but besides the use of cyproterone acetate, only the ponderal gain was found significant (Spearman's r: 0.361, p < 0.001); with a 36.7% mediation effect (31.2-42.3%). Cyproterone acetate has additional known risks, such as meningioma; although we cannot conclusively prove that it has a role in the development of hypertension, we conclude that the use of cyproterone acetate for this indication should be reconsidered.

Determination of inflammatory biomarkers in patients with COPD: a comparison of different assays.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an inflammatory pulmonary disorder with systemic inflammatory manifestations that are mediated by circulating acute-phase reactants. This study compared an enzyme-linked immunosorbent assay (ELISA) to a nephelometric technique for the measurement of serum C-reactive protein (CRP) and serum amyloid A (SAA) and investigated how the choice of assay influenced the estimation of inflammation in patients with stable COPD. METHODS: CRP and SAA concentrations measured by ELISA and nephelometry in 88 patients with COPD and 45 control subjects were used to evaluate the performance of these methods in a clinical setting. RESULTS: With both assays, the concentrations of CRP and SAA were higher in COPD patients than in controls after adjustment for age and sex. There was a moderate correlation between the values measured by ELISA and those measured by nephelometry (logCRP: r = 0.55, p < 0.001; logSAA: r = 0.40, p < 0.001). However, the concentrations of biomarkers determined by nephelometry were significantly higher than those obtained with ELISA for CRP (mean difference = 2.7 (9.4) mg/L) and SAA (mean difference = 0.31 (14.3) mg/L). CONCLUSION: Although the serum CRP and SAA concentrations measured by ELISA and nephelometry correlated well in COPD patients, the ELISA values tended to be lower for CRP and SAA when compared with nephelometric measurements. International standardization of commercial kits is required before the predictive validity of inflammatory markers for patients with COPD can be effectively assessed in clinical practice.

The Consumption of a Synbiotic Does Not Affect the Immune, Inflammatory, and Sympathovagal Parameters in Athletes and Sedentary Individuals: A Triple-Blinded, Randomized, Place-bo-Controlled Pilot Study.

This investigation aimed to identify the effect of a synbiotic in athletes and sedentary people, and their potential varying responses regarding the immune system, autonomic regulation and body composition. Twenty-seven participants were involved in the protocol: 14 sedentary and 13 semi-professional soccer players. Both groups were randomly divided into an experimental and control group. A synbiotic (Gasteel Plus®, Heel España S.A.U.) comprising a blend of probiotic strains, including Bifidobacterium lactis CBP-001010, Lactobacillus rhamnosus CNCM I-4036, and Bifidobacterium longum ES1, was administered to the experimental group, and a placebo was given to the control group for 30 days. Heart rate variability, body composition, and immune/inflammatory cytokines were determined. Statistically significant differences were observed between sedentary individuals and athletes in heart rate variability but not between the experimental and control groups. A difference between the athletic and sedentary group is observed with the influence of training on the effects of the synbiotic on the levels of fat mass and body-fold sum. No significant differences were shown in cytokines after the protocol study. No changes occur with the synbiotic treatment between the athlete and sedentary groups, while no negative effect was produced. Further research will be necessary to see chronic effects in the analyzed biomarkers.

Dysfunction in the Cystic Fibrosis Transmembrane Regulator in Chronic Obstructive Pulmonary Disease as a Potential Target for Personalised Medicine.

In recent years, numerous pathways were explored in the pathogenesis of COPD in the quest for new potential therapeutic targets for more personalised medical care. In this context, the study of the cystic fibrosis transmembrane conductance regulator (CFTR) began to gain importance, especially since the advent of the new CFTR modulators which had the potential to correct this protein's dysfunction in COPD. The CFTR is an ion transporter that regulates the hydration and viscosity of mucous secretions in the airway. Therefore, its abnormal function favours the accumulation of thicker and more viscous secretions, reduces the periciliary layer and mucociliary clearance, and produces inflammation in the airway, as a consequence of a bronchial infection by both bacteria and viruses. Identifying CFTR dysfunction in the context of COPD pathogenesis is key to fully understanding its role in the complex pathophysiology of COPD and the potential of the different therapeutic approaches proposed to overcome this dysfunction. In particular, the potential of the rehydration of mucus and the role of antioxidants and phosphodiesterase inhibitors should be discussed. Additionally, the modulatory drugs which enhance or restore decreased levels of the protein CFTR were recently described. In particular, two CFTR potentiators, ivacaftor and icenticaftor, were explored in COPD. The present review updated the pathophysiology of the complex role of CFTR in COPD and the therapeutic options which could be explored.

The THO Complex as a Paradigm for the Prevention of Cotranscriptional R-Loops.

Different proteins associate with the nascent RNA and the RNA polymerase (RNAP) to catalyze the transcription cycle and RNA export. If these processes are not properly controlled, the nascent RNA can thread back and hybridize to the DNA template forming R-loops capable of stalling replication, leading to DNA breaks. Given the transcriptional promiscuity of the genome, which leads to large amounts of RNAs from mRNAs to different types of ncRNAs, these can become a major threat to genome integrity if they form R-loops. Consequently, cells have evolved nuclear factors to prevent this phenomenon that includes THO, a conserved eukaryotic complex acting in transcription elongation and RNA processing and export that upon inactivation causes genome instability linked to R-loop accumulation. We revise and discuss here the biological relevance of THO and a number of RNA helicases, including the THO partner UAP56/DDX39B, as a paradigm of the cellular mechanisms of cotranscriptional R-loop prevention.

Evaluation of lung parenchyma, blood vessels, and peripheral blood lymphocytes as a potential source of acute phase reactants in patients with COPD.

Background: Previous studies have shown that the arterial wall is a potential source of inflammatory markers in COPD. Here, we sought to compare the expression of acute phase reactants (APRs) in COPD patients and controls both at the local (pulmonary arteries and lung parenchyma) and systemic (peripheral blood leukocytes and plasma) compartments. Methods: Consecutive patients undergoing elective surgery for suspected primary lung cancer were eligible for the study. Patients were categorized either as COPD or control group based on the spirometry results. Pulmonary arteries and lung parenchyma sections, peripheral blood leukocytes, and plasma samples were obtained from all participants. Gene expression levels of C-reactive protein (CRP) and serum amyloid A (SAA1, SAA2, and SAA4) were evaluated in tissue samples and peripheral blood leukocytes by reverse transciption-PCR. Plasma CRP and SAA protein levels were measured by enzyme-linked immunosorbent assays. Proteins were evaluated in paraffin-embedded lung tissues by immunohistochemistry. Results: A total of 40 patients with COPD and 62 controls were enrolled. We did not find significant differences in the gene expression between COPD and control group. Both CRP and SAA were overexpressed in the lung parenchyma compared with pulmonary arteries and peripheral blood leukocytes. The expression of SAA was significantly higher in the lung parenchyma than in the pulmonary artery (2-fold higher for SAA1 and SAA4, P=0.015 and P<0.001, respectively; 8-fold higher for SAA2, P<0.001) and peripheral blood leukocytes (16-fold higher for SAA1, 439-fold higher for SAA2, and 5-fold higher for SAA4; P<0.001). No correlation between plasma levels of inflammatory markers and their expression in the lung and peripheral blood leukocytes was observed. Conclusions: The expression of SAA in lung parenchyma is higher than in pulmonary artery and peripheral blood leukocytes. Notably, no associations were noted between lung expression of APRs and their circulating plasma levels, making the leakage of inflammatory proteins from the lung to the bloodstream unlikely. Based on these results, other potential sources of systemic inflammation in COPD (eg, the liver) need further scrutiny.