Branchpoint translocation by fork remodelers as a general mechanism of R-loop removal.

Co-transcriptional R loops arise from stalling of RNA polymerase, leading to the formation of stable DNA:RNA hybrids. Unresolved R loops promote genome instability but are counteracted by helicases and nucleases. Here, we show that branchpoint translocases are a third class of R-loop-displacing enzyme in vitro. In cells, deficiency in the Fanconi-anemia-associated branchpoint translocase FANCM causes R-loop accumulation, particularly after treatment with DNA:RNA-hybrid-stabilizing agents. This correlates with FANCM localization at R-loop-prone regions of the genome. Moreover, other branchpoint translocases associated with human disease, such as SMARCAL1 and ZRANB3, and those from lower organisms can also remove R loops in vitro. Branchpoint translocases are more potent than helicases in resolving R loops, indicating their evolutionary important role in R-loop suppression. In human cells, FANCM, SMARCAL1, and ZRANB3 depletion causes additive effects on R-loop accumulation and DNA damage. Our work reveals a mechanistic basis for R-loop displacement that is linked to genome stability.

SMG8/SMG9 Heterodimer Loss Modulates SMG1 Kinase to Drive ATR Inhibitor Resistance.

Gastric cancer represents the third leading cause of global cancer mortality and an area of unmet clinical need. Drugs that target the DNA damage response, including ATR inhibitors (ATRi), have been proposed as novel targeted agents in gastric cancer. Here, we sought to evaluate the efficacy of ATRi in preclinical models of gastric cancer and to understand how ATRi resistance might emerge as a means to identify predictors of ATRi response. A positive selection genome-wide CRISPR-Cas9 screen identified candidate regulators of ATRi resistance in gastric cancer. Loss-of-function mutations in either SMG8 or SMG9 caused ATRi resistance by an SMG1-mediated mechanism. Although ATRi still impaired ATR/CHK1 signaling in SMG8/9-defective cells, other characteristic responses to ATRi exposure were not seen, such as changes in ATM/CHK2, γH2AX, phospho-RPA, or 53BP1 status or changes in the proportions of cells in S- or G2-M-phases of the cell cycle. Transcription/replication conflicts (TRC) elicited by ATRi exposure are a likely cause of ATRi sensitivity, and SMG8/9-defective cells exhibited a reduced level of ATRi-induced TRCs, which could contribute to ATRi resistance. These observations suggest ATRi elicits antitumor efficacy in gastric cancer but that drug resistance could emerge via alterations in the SMG8/9/1 pathway. SIGNIFICANCE: These findings reveal how cancer cells acquire resistance to ATRi and identify pathways that could be targeted to enhance the overall effectiveness of these inhibitors.

Intracellular and tissue specific expression of FTO protein in pig: changes with age, energy intake and metabolic status.

Genome-wide association studies in the FTO gene have identified SNPs correlating with obesity and type 2 diabetes. In mice, lack of Fto function leads to intrauterine growth retardation and lean phenotype, whereas in human it is lethal. The aim of this study in a pig model was to determine the localization of the FTO protein in different tissues and cell compartments, in order to investigate potential targets of FTO action. To better understand physiological role of FTO protein, its expression was studied in pigs of different age, metabolic status and nutrition, using both microscopic methods and Western blot analysis. For the first time, FTO protein was found in vivo in the cytoplasm, of not all, but specific tissues and cells e.g. in the pancreatic ß-cells. Abundant FTO protein expression was found in the cerebellum, salivary gland and kidney of adult pigs. No FTO protein expression was detected in blood, saliva, and bile, excluding its role in cell-to-cell communication. In the pancreas, FTO protein expression was positively associated with energy intake, whereas in the muscles it was strictly age-related. In IUGR piglets, FTO protein expression was much higher in the cerebellum and kidneys, as compared to normal birth body weight littermates. In conclusion, our data suggest that FTO protein may play a number of distinct, yet unknown intracellular functions due to its localization. Moreover, it may play a role in animal growth/development and metabolic state, although additional studies are necessary to clarify the detailed mechanism(s) of action.

ALKBH overexpression in head and neck cancer: potential target for novel anticancer therapy.

The nine identified human homologues of E. coli AlkB 2-oxoglutarate (2OG) and Fe(II)-dependent dioxygenase, ALKBH1-8 and FTO, display different substrate specificities and diverse biological functions. Here we discovered the combined overexpression of members of the ALKBH family in head and neck squamous cell carcinomas (HNSCC). We found direct correlation of ALKBH3 and FTO expression with primary HNSCC tumor size. We observed unidentified thus far cytoplasmic localization of ALKBH2 and 5 in HNSCC, suggesting abnormal role(s) of ALKBH proteins in cancer. Further, high expression of ALKBHs was observed not only in HNSCC, but also in several cancerous cell lines and silencing ALKBH expression in HeLa cancer cells resulted in dramatically decreased survival. Considering the discovered impact of high expression of ALKBH proteins on HNSCC development, we screened for ALKBH blockers among newly synthetized anthraquinone derivatives and demonstrated their potential to support standard anticancer therapy.

Evaluation of left ventricular function in overweight children and teenagers with arterial hypertension and white coat hypertension.

BACKGROUND: Obesity in childhood is strongly associated with elevated arterial blood pressure and risk of hypertension. The aim of the study was the evaluation of left ventricular (LV) function in hypertensive and white coat hypertensive overweight children and teenagers. METHODS: The study group consisted of 74 overweight patients aged 10.3 ± 3.1 years (range: 6-16 years) diagnosed as hypertensive in standard blood pressure measurement. The control group consisted of 31 normotensive and normoweight children. Ambulatory blood pressure monitoring (ABPM) and echocardiographic assessment of the LV mass and function were performed in all participants. RESULTS: Using ABPM hypertension was confirmed in 20 (27%) children. In the 54 (73%) remaining children white coat hypertension was diagnosed. The analysis of echocardiographic parameters revealed higher LV mass index (LVMI) in hypertensive overweight than in normotensive normoweight children (47.5 ± 9.2 g/m2.7 vs. 39.8 ± 12.1 g/m2.7; p < 0.05) and no difference between overweight hypertensive and white coat hypertension-hypertensive groups. The deceleration time of mitral early filling (DCT) was longer in hypertensive normoweight children than in normotensive overweight patients (219.5 ± 110.3 ms vs. 197.8 ± 65.8 ms; p < 0.05). A significant correlation between systolic blood pressure load (SBPL) and DCT (r = 0.57) and moderate correlation between SBPL and LVMI (r: 0.48) as well as between LVMI and isovolumetric relaxation time (r = 0.37) were found. CONCLUSIONS: In overweight children the diagnosis of hypertension should be confirmed in ABPM because of the high prevalence of white coat hypertension. Periodic echocardiographic examinations should be recommended in overweight children with increased SBPL and decreased systolic nocturnal deep because of the possibility of LV function impairment.

Structure and Function of Enterocyte in Intrauterine Growth Retarded Pig Neonates.

The intestine of intrauterine growth retarded (IUGR) neonates showed different morphology compared to neonates born with normal body weight (NBW). The aim of the present study was to investigate the ultrastructure and proteomic profile of the gut epithelium in IUGR pig neonates with special attention to the digestive and absorptive function. Intestine tissue samples were investigated in 7-day-old IUGR and NBW littermate piglets using histometry, immunofluorescence, scanning electron microscopy (SEM), and mass spectrometry analysis. IUGR piglets have shown reduced mucosa and muscularis thickness and an enhanced number of foetal type enterocytes (FTE). SEM studies have shown the lack of the characteristic large-size vacuole in IUGR's enterocytes. Delayed removal of FTE in IUGR neonates was probably due to the inhibited apoptosis in the apical part of villi and increased apoptosis and reduced mitosis in the crypt region. In the expression of proteins in the intestinal mucosa such as hexokinase I, histones, and prelamin A/C, carbamoyl phosphate was reduced in IUGR neonates. Finally, IUGR intestines showed higher expression of HSPA9 and HSPA5 as apoptosis markers. The data indicate modifications of gut mucosa in IUGRs that may result in slower gut mucosa maturation and reduced utilisation of nutrient as compared to NBW pig neonates.

1,N6-α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase.

1,N6-α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein - an environmental pollutant and endocellular oxidative stress product. Escherichia coli AlkB dioxygenase belongs to the superfamily of α-ketoglutarate (αKG)- and iron-dependent dioxygenases which remove alkyl lesions from bases via an oxidative mechanism, thereby restoring native DNA structure. Here, we provide in vivo and in vitro evidence that HPA is mutagenic and is effectively repaired by AlkB dioxygenase. HPA generated in plasmid DNA caused A → C and A → T transversions and, less frequently, A → G transitions. The lesion was efficiently repaired by purified AlkB protein; the optimal pH, Fe(II), and αKG concentrations for this reaction were determined. In vitro kinetic data show that the protonated form of HPA is preferentially repaired by AlkB, albeit the reaction is stereoselective. Moreover, the number of reaction cycles carried out by an AlkB molecule remains limited. Molecular modeling of the T(HPA)T/AlkB complex demonstrated that the R stereoisomer in the equatorial conformation of the HPA hydroxyl group is strongly preferred, while the S stereoisomer seems to be susceptible to AlkB-directed oxidative hydroxylation only when HPA adopts the syn conformation around the glycosidic bond. In addition to the biochemical activity assays, substrate binding to the protein was monitored by differential scanning fluorimetry allowing identification of the active protein form, with cofactor and cosubstrate bound, and monitoring of substrate binding. In contrast FTO, a human AlkB homolog, failed to bind an ssDNA trimer carrying HPA.

Effects of changes in intracellular iron pool on AlkB-dependent and AlkB-independent mechanisms protecting E.coli cells against mutagenic action of alkylating agent.

An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH(+) strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg(+) reversion induction after λ>320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg(+) revertants in AB1157 alkB(+)hemH(-)/pMW1 strain was 40 and 26% reduced comparing to the alkB(+)hemH(-) and alkB(+)hemH(+)/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ>320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg(+) reversion was observed in irradiated and MMS-treated hemH(-)alkB(-) cells comparing to the hemH(+)alkB(-) strain. Also, the level of Arg(+) revertants in the irradiated and MMS treated hemH(-) alkB(-) mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH(-) intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs.

Bland-White-Garland syndrome - a rare and serious cause of failure to thrive.

PATIENT: Male, 0 FINAL DIAGNOSIS: Bland-White-Garland syndrome Symptoms: Cardiomegaly, feeding problems Medication: - Clinical Procedure: Reimplantation of the left coronary artery to the aorta Specialty: Pediatrics and Neonatology. OBJECTIVE: Rare disease. BACKGROUND: Bland-White-Garland syndrome (BWGS) is a very rare disease characterized by anomalous origin of the left coronary artery from the pulmonary trunk (ALCAPA). WBGS affects 1 in every 300 000 live births. Children typically present with dyspnea, pallor, and failure to thrive. Without surgical repair, most of these children die during the first months of life. CASE REPORT: This case report describes 3-month-old boy admitted to the hospital because of feeding problems. The boy was born at term, with birth weight 3200 g, and was 10 points in Apgar score. He was breast-fed from birth. From the seventh week of age, his mother observed his increasing difficulties with feeding. Physical examination revealed pale skin, diminished heart sounds, tachycardia, cardiomegaly, and hepatomegaly. Results of urine and blood tests and ultrasonography of the central nervous system and abdomen were normal. The chest radiography showed cardiomegaly and electrocardiogram revealed anterolateral myocardial infarction. On echocardiography, an anomalous left coronary artery arising from the pulmonary artery was found. The life-saving treatment of choice was immediate surgical reimplantation of the left coronary artery to the aorta. CONCLUSIONS: Children with congenital heart disease are often prone to malnutrition, but in rare cases failure to thrive and breast-feeding problems can be the first symptoms of life-threatening diseases like myocardial infarction secondary to Bland-White-Garland syndrome (BWGS).

[Therapeutic difficulties in a 3 year-old boy with Kawasaki disease]. / Problemy terapeutyczne u 3-letniego chlopca z choroba Kawasaki.

A case of a three year-old by with delayed diagnosis of disease is presented. Treatment with immunoglobulins and methylpreduisolone was affective, however, no regression in coronary artery lesions was observed. The diagnosis and treatment of Kawasaki disease are discussed.