DNMT3B in vitro knocking-down is able to reverse embryonal rhabdomyosarcoma cell phenotype through inhibition of proliferation and induction of myogenic differentiation

Aberrant DNA methylation has been frequently observed in many human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. To date, the expression and function of the de novo DNA methyltransferase (DNMT) 3B in RMS have not yet been investigated. Our study show for the first time a significant up-regulation of DNMT3B levels in 14 RMS tumour samples and 4 RMS cell lines in comparison to normal skeletal muscle. Transfection of RD and TE671 cells, two in vitro models of embryonal RMS (ERMS), with a synthetic DNMT3B siRNA decreased cell proliferation by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the checkpoint regulators p21 and p27. DNMT3B depletion also impaired RB phosphorylation status and decreased migratory capacity and clonogenic potential. Interestingly, DNMT3B knock-down was able to commit ERMS cells towards myogenic terminal differentiation, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Finally, inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, giving evidence that DNMT3B is a down-stream molecule of this oncogenic pathway.Taken together, our data indicate that altered expression of DNMT3B plays a key role in ERMS development since its silencing is able to reverse cell cancer phenotype by rescuing myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel therapeutic strategy against RMS

Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition

BACKGROUND: Rhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumour in children and adolescents. Aberrant expression of Anaplastic Lymphoma Kinase (ALK) and MET gene has been implicated in the malignant progression of RMS, especially in the alveolar subtype. This observation suggests that crizotinib (PF-02341066), a kinase inhibitor against ALK and MET, may have a therapeutic role in RMS, although its antitumour activity in this malignancy has not yet been studied. METHODS: RH4 and RH30 alveolar RMS (ARMS) cell lines were treated with crizotinib and then assessed by using proliferation, viability, migration and colony formation assays. Multiple approaches, including flow cytometry, immunofluorescence, western blotting and siRNA-based knock-down, were used in order to investigate possible molecular mechanisms linked to crizotinib activity. RESULTS: In vitro treatment with crizotinib inhibited ALK and MET proteins, as well as Insulin-like Growth Factor 1 Receptor (IGF1R), with a concomitant robust dephosphorylation of AKT and ERK, two downstream kinases involved in RMS cell proliferation and survival. Exposure to crizotinib impaired cell growth, and accumulation at G2/M phase was attributed to an altered expression and activation of checkpoint regulators, such as Cyclin B1 and Cdc2. Crizotinib was able to induce apoptosis and autophagy in a dose-dependent manner, as shown by caspase-3 activation/PARP proteolytic cleavage down-regulation and by LC3 activation/p62 down-regulation, respectively. The accumulation of reactive oxygen species (ROS) seemed to contribute to crizotinib effects in RH4 and RH30 cells. Moreover, crizotinib-treated RH4 and RH30 cells exhibited a decreased migratory/invasive capacity and clonogenic potential. CONCLUSIONS: These results provide a further insight into the molecular mechanisms affected by crizotinib in ARMS cells inferring that it could be a useful therapeutic tool in ARMS cancer treatment

Sex-Specific Effects of Adiponectin on Carotid Intima-Media Thickness and Incident Cardiovascular Disease

Background-Plasma adiponectin levels have previously been inversely associated with carotid intima-media thickness (IMT), a marker of subclinical atherosclerosis. In this study, we used a sex-stratified Mendelian randomization approach to investigate whether adiponectin has a causal protective influence on IMT. Methods and Results-Baseline plasma adiponectin concentrationwas tested for association with baseline IMT, IMT progression over 30 months, and occurrence of cardiovascular events within 3 years in 3430 participants (women, n=1777; men, n=1653) with high cardiovascular risk but no prevalent disease. Plasma adiponectin levels were inversely associated with baseline mean bifurcation IMT after adjustment for established risk factors (beta=-0.018, Pless than0.001) in men but not in women (beta=-0.006, P=0.185; P for interaction=0.061). Adiponectin levels were inversely associated with progression of mean common carotid IMT in men (beta=-0.0022, P=0.047), whereas no association was seen in women (0.0007, P=0.475; P for interaction=0.018). Moreover, we observed that adiponectin levels were inversely associated with coronary events in women (hazard ratio 0.57, 95% CI 0.37 to 0.87) but not in men (hazard ratio 0.82,95% CI0.54 to 1.25). Agenescore of adiponectin-raisingalleles in6loci, reported recently inalarge multi-ethnic metaanalysis, was inversely associated with baseline mean bifurcation IMT in men (beta=-0.0008, P=0.004) but not in women (beta=-0.0003, P=0.522; P for interaction=0.007). Conclusions-This report provides some evidence for adiponectin protecting against atherosclerosis, with effects being confined to men; however, compared with established cardiovascular risk factors, the effect of plasma adiponectin was modest. Further investigation involving mechanistic studies is warranted.Funding Agencies|European Commission [QLG1-CT-2002-00896]; Swedish Heart-Lung Foundation; Swedish Research Council [8691, 0593]; Knut and Alice Wallenberg Foundation; Foundation for Strategic Research; Stockholm County Council [592229]; Karolinska Institutet; Stockholm County Council; European Union Framework Programme 7 for the Innovative Medicine Initiative [IMI/115006]; Academy of Finland [110413]; British Heart Foundation [RG2008/08, RG2008/014]; Italian Ministry of Health (Ricerca Corrente); Uppsala University; Uppsala University Hospital; Swedish Research Council for Infrastructures; Swedish Heart-Lung Foundation [20120600, 20130399]; Tore Nilsson foundation; Gamla Tjanarinnor foundation; Thurings foundation; Stiftelsen for Gamla Tjanarinnor; Ake Wiberg foundation; Tore Nilssons foundation; Magnus Bergvall Foundation; Foundation for Old Servants; Ministry of Education and Culture in Finland; Vasterbotten County Council; Swedish Heart and Lung Foundation; National Excellence Program [TAMOP 4.2.4.A/1-11-1-2012-0001]; European Union; European Social Fund; UK Medical Research Council [K013351]; Economic and Social Research Council; Academy of Finland; University College London Genetics Institute</p

Soluble CD93 Is Involved in Metabolic Dysregulation but Does Not Influence Carotid Intima-Media Thickness

Type 2 diabetes and cardiovascular disease are complex disorders involving metabolic and inflammatory mechanisms. Here we investigated whether sCD93, a group XIV c-type lectin of the endosialin family, plays a role in metabolic dysregulation or carotid intima-media thickness (IMT). Although no association was observed between sCD93 and IMT, sCD93 levels were significantly lower in subjects with type 2 diabetes (n = 901, mean ± SD 156.6 ± 40.0 ng/mL) compared with subjects without diabetes (n = 2,470, 164.1 ± 44.8 ng/mL, P < 0.0001). Genetic variants associated with diabetes risk (DIAGRAM Consortium) did not influence sCD93 levels (individually or combined in a single nucleotide polymorphism score). In a prospective cohort, lower sCD93 levels preceded the development of diabetes. Consistent with this, a cd93-deficient mouse model (in addition to apoe deficiency) demonstrated no difference in atherosclerotic lesion development compared with apoe(-/-) cd93-sufficient littermates. However, cd93-deficient mice showed impaired glucose clearance and insulin sensitivity (compared with littermate controls) after eating a high-fat diet. The expression of cd93 was observed in pancreatic islets, and leaky vessels were apparent in cd93-deficient pancreases. We further demonstrated that stress-induced release of sCD93 is impaired by hyperglycemia. Therefore, we propose CD93 as an important component in glucometabolic regulation

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42\ub74% vs 44\ub72%; absolute difference \u20131\ub769 [\u20139\ub758 to 6\ub711] p=0\ub767; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5\u20138] vs 6 [5\u20138] cm H2O; p=0\ub70011). ICU mortality was higher in MICs than in HICs (30\ub75% vs 19\ub79%; p=0\ub70004; adjusted effect 16\ub741% [95% CI 9\ub752\u201323\ub752]; p&lt;0\ub70001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0\ub780 [95% CI 0\ub775\u20130\ub786]; p&lt;0\ub70001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status. Funding: No funding

Usefulness of cyanide-nitroprusside test in detecting incomplete recessive heterozygotes for cystinuria: a standardized dilution procedure

We present the results of a cyanide-nitroprusside test (CNT) after a standardized dilution procedure of urine samples and report the efficiency of this method in detecting heterozygotes for cystinuria when applied on an open pediatric population. In the preliminary study we assayed by quantitative determination of amino acids 162 urine samples from a hospital population identifying 24 type III heterozygotes and 2 type II heterozygotes for cystinuria. The classic CNT gave 38 false positive results and 5 false negative results showing a sensitivity and specificity of 0.808 and 0.721, respectively. When progressively diluted, all samples of heterozygotes remained CNT positive up to a creatinine concentration of 90 mg/dl. At this level of dilution 31 out of 38 false positive turned to negative, thus obtaining a specificity of 0.922 without a lowering of the sensitivity in detecting heterozygotes. The standardized dilution at 90 mg/dl of creatinine concentration was applied to 74.7% of a population of 1024 schoolchildren. In this way 163 out of 210 positive results were eliminated and thus the specificity of CNT rose from 0.789 to 0.953. On the basis of these results, the method proposed can be regarded as reliable and useful for a screening program in detecting heterozygotes for cystinuria

RENAL TUBULAR DAMAGE IN FETUSES WITH INTRAUTERINE GROWTH-RETARDATION

Fetal hypoxemia is one of the most frequent causes of intrauterine growth retardation (IUGR). In chronic fetal hypoxia, peripheral blood flow and blood flow to the kidneys is reduced to maintain heart, brain and adrenal perfusion, the 'brainsparing effect'. In kidneys the cells of the proximal tubules seem to be most sensitive to hypoxia caused by reduced blood flow. Damage to the cells of the proximal tubules can be easily diagnosed by urinary levels of N-acetyl-β-D-glucosaminidase (NAG), an enzyme present in high concentrations in these cells. The aim of the present study was to define the levels of NAG in the amniotic fluid, to diagnose damage to the cells of the proximal renal tubules in fetuses, and to correlate them with a detectable brain-sparing effect. The study was conducted on a total of 55 pregnancies: 9 pregnancies were complicated by IUGR, and the remaining 46 normal pregnancies formed the control group. Higher levels of NAG were detected in the amniotic fluid from the IUGR-complicated pregnancies (p < 0.025). In particular, fetuses with IUGR had high levels of NAG in the amniotic fluid in 8 of 9 cases (+ 2 SD compared with controls), while 1 had normal concentrations. In the 8 cases with high concentrations of NAG in the amniotic fluid, velocimetric Doppler study documented a brain-sparing effect, which was not present in the 1 fetus with normal NAG levels. In conclusion, high levels of NAG in the amniotic fluid may identify in uterus fetuses with renal damage. These individuals are in need of stricter peri- and postnatal care as, in the case of perinatal anoxia, this particular condition may be one of the causes concurrent in the onset of postnatal acute renal failure

Additional file 1: of Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition

ALK and MET knock-down by RNA interference. A: RH4 and RH30 cells were transfected with either scramble control siRNA (NC siRNA) or ALK siRNA. Cells were harvested 48 h after transfection and ALK, AKT (phosphorylated and total protein) and ERK (phosphorylated and total protein) levels were analysed by Western blotting. B: RH4 and RH30 cells were transfected with either scramble negative control siRNA (NC siRNA) or MET siRNA. Cells were harvested 48 h after transfection and MET, AKT (phosphorylated and total protein) and ERK (phosphorylated and total protein) levels were analysed by Western blotting. Tubulin was used as loading control in all experiments. (TIFF 258 kb