TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells

TET1 is a 5-methylcytosine dioxygenase and its DNA demethylating activity has been implicated in pluripotency and reprogramming. However, the precise role of TET1 in DNA methylation regulation outside of developmental reprogramming is still unclear. Here, we show that overexpression of the TET1 catalytic domain but not full length TET1 (TET1-FL) induces massive global DNA demethylation in differentiated cells. Genome-wide mapping reveals that 5-hydroxymethylcytosine production by TET1-FL is inhibited as DNA methylation increases, which can be explained by the preferential binding of TET1-FL to unmethylated CpG islands (CGIs) through its CXXC domain. TET1-FL specifically accumulates 5-hydroxymethylcytosine at the edges of hypomethylated CGIs, while knockdown of endogenous TET1 induces methylation spreading from methylated edges into hypomethylated CGIs. We also found that gene expression changes after TET1-FL overexpression are relatively small and independent of its dioxygenase function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically prevents aberrant methylation spreading into CGIs in differentiated cells.     

MetaTM - a consensus method for transmembrane protein topology prediction

Background  

Transmembrane (TM) proteins are proteins that span a biological membrane one or more times. As their 3-D structures are hard to determine, experiments focus on identifying their topology (i. e. which parts of the amino acid sequence are buried in the membrane and which are located on either side of the membrane), but only a few topologies are known. Consequently, various computational TM topology predictors have been developed, but their accuracies are far from perfect. The prediction quality can be improved by applying a consensus approach, which combines results of several predictors to yield a more reliable result.     

Cholestasis induced nephrotoxicity: The role of endogenous opioids

AimsElevated levels of endogenous opioids play a pivotal role in several deleterious consequences of cholestasis. Renal dysfunction occurs in cholestasis but its exact mechanism is still unknown. In this study, we investigated the role of endogenous opioids in cholestasis induced nephrotoxicity.Main methodsThirty-five rats were divided into five groups. In groups 1 and 2 BDL rats received either daily subcutaneous 20 mg/kg of naltrexone or its vehicle, for 7 days after BDL. In groups 3 and 4, BDL or Sham rats received no injections. In group 5, normal rats received subcutaneous injections of 20 mg/kg/day of naltrexone for 7 days. At the 7th day, 24 h urine was collected to measure urinary N-acetyl-β-D-glucosaminidase (NAG) as an early marker of renal tubular injury. Kidney samples were then collected for light and electron microscopic studies.Key findingsBDL significantly increased NAG activity compared to sham groups. Naltrexone significantly reversed NAG activity to normal levels in BDL animals. Naltrexone treatment in BDL animals also significantly reversed ALT and AST to their normal levels. In light and electron microscopic studies, there were significant structural alterations in BDL samples, which were mostly prevented in naltrexone treated BDL animals.SignificanceSignificant changes in urinary NAG activity and renal morphology of cholestatic rats were reversed by naltrexone treatment. These results suggest a possible role for endogenous opioids in inducing cholestatic nephrotoxicity.     

CYP17, SRD5A2, CYP1B1, and CYP2D6 gene polymorphisms with prostate cancer risk in North Indian population

To investigate the involvement of the CYP17, SRD5A2, CYP1B1, and CYP2D6 variants with prostate cancer, a case-control study of 100 patients and an equal number of age-matched control men was conducted. There appears to be a nonsignificant increase with risk of prostate cancer for individuals carrying one copy of the CYP17 A2 allele (OR, 1.80; 95% CI, 0.99-3.29, P=0.05). The risk was increased in individuals having two A2 alleles (OR; 2.81, 95% CI, 1.06-7.40, P=0.03). Compared with men having the VV genotype of SRD5A2 gene, there was no significant association between the VL genotype and the risk of prostate cancer (OR; 0.54, 95% CI; 0.29-1.03, P=0.06). There was no difference in the occurrence of the genotype LL between controls and prostate cancer patients (OR; 0.90, 95% CI; 0.43-1.89, P=0.79). There was a nonsignificant increased risk of prostate cancer for individuals carrying the CYP1B1Leu/Val genotype (OR, 1.70, 95% CI, 0.91-3.17, P =0.09), which was increased in those having the Val/Val allele (OR, 3.38; 95% CI, 1.13-10.07, P=0.02). Relative to men homozygous for the wild-type allele in CYP2D6 gene, those heterozygous for the B allele had an odds ratio of 1.78 (95% CI, 0.76-4.17, P=0.18) for patients, and for homozygous individuals, it was 1.95 (0.55-6.93, P=0.30). These observations have suggested that the CYP17 A2/A2, CYP1B1 Val/Val, and CYP2D6 genotypes may be associated with an altered risk of prostate cancer, while the CYP2D6 and SRD5A2 V89L polymorphism have no association with its risk in the North Indian population.     

Cloning and characterization of 29 tetranucleotide and two dinucleotide polymorphic microsatellite loci from the endangered marbled murrelet (Brachyramphus marmoratus)

We developed 31 novel, polymorphic microsatellite loci in the marbled murrelet (Brachyramphus marmoratus), a critically endangered seabird. Variability was tested on 15 individuals from the Santa Cruz, California population, with each locus characterized by two to 12 alleles. Observed levels of heterozygosity ranged from 0.13 to 0.93. These loci provide a valuable means of assessing the population structure and demographic parameters of this species, which may be critical to its conservation across a fragmented habitat.     

Modulation of fatty acid transport and metabolism by maternal obesity in the human full-term placenta

Knowledge of the consequences of maternal obesity in human placental fatty acids (FA) transport and metabolism is limited. Animal studies suggest that placental uptake of maternal FA is altered by maternal overnutrition. We hypothesized that high maternal body mass index (BMI) affects human placental FA transport by modifying expression of key transporters. Full-term placentas were obtained by vaginal delivery from normal weight (BMI, 18.5-24.9 kg/m(2)) and obese (BMI > 30 kg/m(2)) women. Blood samples were collected from the mother at each trimester and from cord blood at delivery. mRNA and protein expression levels were evaluated with real-time RT-PCR and Western blotting. Lipoprotein lipase (LPL) activity was evaluated using enzyme fluorescence. In vitro linoleic acid transport was studied with isolated trophoblasts. Our results demonstrated that maternal obesity is associated with increased placental weight, decreased gestational age, decreased maternal high-density lipoprotein (HDL) levels during the first and third trimesters, increased maternal triglyceride levels during the second and third trimesters, and increased maternal T3 levels during all trimesters, and decreased maternal cholesterol (CHOL) and low-density lipoprotein (LDL) levels during the third trimester; and increased newborn CHOL, LDL, apolipoprotein B100, and T3 levels. Increases in placental CD36 mRNA and protein expression levels, decreased SLC27A4 and FABP1 mRNA and protein and FABP3 protein expression, and increased LPL activity and decreased villus cytotrophoblast linoleic acid transport were also observed. No changes were seen in expression of PPARA, PPARD, or PPARG mRNA and protein. Overall this study demonstrated that maternal obesity impacts placental FA uptake without affecting fetal growth. These changes, however, could modify the fetus metabolism and its predisposition to develop diseases later in life.     

Boronated phosphonium salts containing arylboronic acid, closo-carborane, or nido-carborane: synthesis, X-ray diffraction, in vitro cytotoxicity, and cellular uptake

The preparation of boronated triaryl and tetraaryl phosphonium salts of the type [PPh₃CH₂R]Br [R is 4-boronophenyl (1), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-yl)phenyl (2), 3-boronophenyl (3), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-yl)phenyl (4), 2-boronophenyl (5), 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-yl)phenyl (6), and closo-1,2-carboran-1-yl (7)] is described. These compounds were prepared by the reaction of triphenylphosphine with benzylic bromides or 1-bromomethyl-closo-1,2-carborane in acetonitrile solution at 85 °C. The zwitterionic nido-7,8-carborane derivative PPh₃CH₂C₂B₉H₁₁ (8) was prepared by treatment of 7 with cesium fluoride in refluxing ethanol. All compounds were fully characterized by multinuclear (¹H, ¹¹B, ¹³C, and ³¹P) 1D- and 2D-NMR spectroscopy, electrospray ionization mass spectrometry, and elemental analysis, and single-crystal X-ray structures were determined for compounds 1, 3, 7, and 8. The cytotoxicities and boron uptake of selected derivatives were investigated in vitro using human glioblastoma (T98G) and canine kidney tubule (MDCK II) cells. The zwitterionic species 8 was found to be the least cytotoxic agent while also delivering the greatest amount of boron to the T98G cells, peaking at 9.15 ± 2.65 μg B/mg protein.     

Analyse comparative de 3 logiciels de reconstruction itérative de tomoscintigraphie monophotonique myocardique

ObjectiveThis study makes a comparative analysis of the quality reconstruction of three software: 2D ordered subset expectation maximisation (2D OSEM), 3D ordered subsets expectation maximisation (Flash 3D) and Wallis.Patients and methodsThe data from myocardial scintigraphy acquisitions of 50 patients (38 men and 12 women; average age 61 ± 9 years) were successively reconstructed using three myocardial perfusion SPECT algorithms (Flash 3D, OSEM 2D and Wallis). Different combinations of iterations and subsets were considered. For Wallis, only the cut-off frequency was considered. The image's quality was assessed by determining the maximum contrast and the signal to noise ratio.ResultsThe Wallis software provided a higher signal to noise ratio compared to Flash 3D and OSEM 2D at stress and rest. The Wallis signal to noise ratio increased by a factor 1.93 (P = 0.0010) and 2.28 at stress (P = 0.0009); 1.50 (P = 0.0011) and 2.84 at rest (P = 0.0024) compared to respectively Flash 3D and OSEM 2D. Flash 3D provided better signal to noise ratio than OSEM 2D at stress and at rest. The difference in medians and interquartile ranges of the signal-to-noise ratio in post-stress were 22 % and 54 %, respectively between Flash 3D and OSEM 2D. At rest, the difference between the two methods in signal to noise ratio was 32 % ± 0.,29.ConclusionWallis algorithm was improve image quality with better signal to noise ratio compared to the reference method of Siemens Flash 3D. For both Flash 3D and OSEM 2D methods, the combination with 8 subsets and 12 iterations provided the best contrast and signal to noise ratio.     

Combination of obesity and co-morbidities leads to unfavorable outcomes in COVID-19 patients

ObjectiveObesity has been described as a significant independent risk factors of COVID-19. We aimed to study the association between obesity, co-morbidities and clinical outcomes of COVID-19.MethodsClinical data from 417 patients were collected retrospectively from the Al Kuwait Hospital, Ministry of Health and Prevention (MOHAP), Dubai, United Arab Emirates, who were admitted between March and June 2020. Patients were divided according to their body mass index (BMI). Various clinical outcomes were examined: presenting symptoms, severity, major co-morbidities, ICU admission, death, ventilation, ARDS, septic shock and laboratory parameters.ResultsThe average BMI was 29 ± 6.2 kg/m². BMI alone was not associated with the outcomes examined. However, class II obese patients had more co-morbidities compared to other groups. Hypertension was the most significant co-morbidity associated with obesity. Patients with BMI above the average BMI (29 kg/m²) and presence of underlying co-morbidities showed significant increase in admission to ICU compared to patients below 29 kg/m² and underlying co-morbidities (21.7% Vs. 9.2%), ARDS development (21.7% Vs. 10.53%), need for ventilation (8.3% Vs. 1.3%), and mortality (10% Vs. 1.3%).ConclusionsOur data suggests that presence of underlying co-morbidities and high BMI work synergistically to affect the clinical outcomes of COVID-19.     

Assessment of Tropism and Effectiveness of New Primate-Derived Hybrid Recombinant AAV Serotypes in the Mouse and Primate Retina

Adeno-associated viral vectors (AAV) have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4^−/− mouse which is a model for Stargardt disease and in the Pde6b^rd1/rd1 mouse) in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4^−/− mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments.