-Despite its functional importance in various fundamental bioprocesses, the studies of N6-methyladenosine (m6A) in the heart are lacking. Here we show that, fat mass and obesity-associated (FTO), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling and regeneration. -We used clinical human samples, preclinical pig and mouse models and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO using AAV9 (in vivo), adenovirus (both in vivo and in vitro) and siRNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility and cardiac function post-ischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing (MeRIP-seq) to map transcriptome-wide m6A, and MeRIP qPCR assays to map and validate m6A in individual transcripts, in healthy and failing hearts and myocytes. -We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is carried out by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. Additionally, we demonstrate that FTO overexpression in mouse models of MI decreased fibrosis and enhanced angiogenesis. -Collectively, our study demonstrates the functional importance of FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

Objective:To compare the diagnostic performance of PET with the amyloid ligand Pittsburgh compound B (PiB-PET) to fluorodeoxyglucose (FDG-PET) in discriminating between Alzheimer disease (AD) and frontotemporal lobar degeneration (FTLD). Methods:Patients meeting clinical criteria for AD (n ϭ 62) and FTLD (n ϭ 45) underwent PiB and FDG-PET. PiB scans were classified as positive or negative by 2 visual raters blinded to clinical diagnosis, and using a quantitative threshold derived from controls (n ϭ 25). FDG scans were visually rated as consistent with AD or FTLD, and quantitatively classified based on the region of lowest metabolism relative to controls.Results: PiB visual reads had a higher sensitivity for AD (89.5% average between raters) than FDG visual reads (77.5%) with similar specificity (PiB 83%, FDG 84%). When scans were classified quantitatively, PiB had higher sensitivity (89% vs 73%) while FDG had higher specificity (83% vs 98%). On receiver operating characteristic analysis, areas under the curve for PiB (0.888) and FDG (0.910) were similar. Interrater agreement was higher for PiB ( ϭ 0.96) than FDG ( ϭ 0.72), as was agreement between visual and quantitative classification (PiB ϭ 0.88-0.92; FDG ϭ 0.64-0.68). In patients with known histopathology, overall classification accuracy (2 visual and 1 quantitative classification per patient) was 97% for PiB (n ϭ 12 patients) and 87% for FDG (n ϭ 10). Conclusions:PiB and FDG showed similar accuracy in discriminating AD and FTLD. PiB was more sensitive when interpreted qualitatively or quantitatively. FDG was more specific, but only when scans were classified quantitatively. PiB slightly outperformed FDG in patients with known histopathology. Neurology Differentiating Alzheimer disease (AD) and frontotemporal lobar degeneration (FTLD) has implications for prognosis and symptomatic treatment, 1,2 and is critical for the efforts to develop disease-specific therapies. Making an accurate diagnosis during life can be challenging given overlapping clinical features.3,4 MRI or fluorodeoxyglucose PET (FDG-PET) can improve diagnostic accuracy by demonstrating distinct topographic patterns of atrophy or hypometabolism (temporoparietal predominant in AD; frontal and anterior temporal involvement in FTLD), 5,6 but anatomic overlap between the diseases is increasingly apparent. 5,7 Consequently, many patients with pathologically confirmed FTLD are diagnosed with AD during

Iron homeostasis is highly regulated in organisms across evolutionary time scale as iron is essential for various cellular processes. In a computational screen, we identified the Yap/bZIP domain family in Candida clade genomes. Cap2/Hap43 is essential for C. albicans growth under iron-deprivation conditions and for virulence in mouse. Cap2 has an amino-terminal bipartite domain comprising a fungal-specific Hap4-like domain and a bZIP domain. Our mutational analyses showed that both the bZIP and Hap4-like domains perform critical and independent functions for growth under iron-deprivation conditions. Transcriptome analysis conducted under iron-deprivation conditions identified about 16% of the C. albicans ORFs that were differentially regulated in a Cap2-dependent manner. Microarray data also suggested that Cap2 is required to mobilize iron through multiple mechanisms; chiefly by activation of genes in three iron uptake pathways and repression of iron utilizing and iron storage genes. The expression of HAP2, HAP32, and HAP5, core components of the HAP regulatory complex was induced in a Cap2-dependent manner indicating a feed-forward loop. In a feed-back loop, Cap2 repressed the expression of Sfu1, a negative regulator of iron uptake genes. Cap2 was coimmunoprecipitated with Hap5 from cell extracts prepared from iron-deprivation conditions indicating an in vivo association. ChIP assays demonstrated Hap32-dependent recruitment of Hap5 to the promoters of FRP1 (Cap2-induced) and ACO1 (Cap2-repressed). Together our data indicates that the Cap2-HAP complex functions both as a positive and a negative regulator to maintain iron homeostasis in C. albicans.

Patients with early age-of-onset Alzheimer's disease show more rapid progression, more generalized cognitive deficits and greater cortical atrophy and hypometabolism compared to late-onset patients at a similar disease stage. The biological mechanisms that underlie these differences are not well understood. The purpose of this study was to examine in vivo whether metabolic differences between early-onset and late-onset Alzheimer's disease are associated with differences in the distribution and burden of fibrillar amyloid-beta. Patients meeting criteria for probable Alzheimer's disease (National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's; Disease and Related Disorders Association criteria) were divided based on estimated age at first symptom (less than or greater than 65 years) into early-onset (n = 21, mean age-at-onset 55.2 +/- 5.9 years) and late-onset (n = 18, 72.0 +/- 4.7 years) groups matched for disease duration and severity. Patients underwent positron emission tomography with the amyloid-beta-ligand [(11)C]-labelled Pittsburgh compound-B and the glucose analogue [(18)F]-labelled fluorodeoxyglucose. A group of cognitively normal controls (n = 30, mean age 73.7 +/- 6.4) was studied for comparison. [(11)C]-labelled Pittsburgh compound-B images were analysed using Logan graphical analysis (cerebellar reference) and [(18)F]-labelled fluorodeoxyglucose images were normalized to mean activity in the pons. Group differences in tracer uptake were assessed on a voxel-wise basis using statistical parametric mapping, and by comparing mean values in regions of interest. To account for brain atrophy, analyses were repeated after applying partial volume correction to positron emission tomography data. Compared to normal controls, both early-onset and late-onset Alzheimer's disease patient groups showed increased [(11)C]-labelled Pittsburgh compound-B uptake throughout frontal, parietal and lateral temporal cortices and striatum on voxel-wise and region of interest comparisons (P < 0.05). However, there were no significant differences in regional or global [(11)C]-labelled Pittsburgh compound-B binding between early-onset and late-onset patients. In contrast, early-onset patients showed significantly lower glucose metabolism than late-onset patients in precuneus/posterior cingulate, lateral temporo-parietal and occipital corticies (voxel-wise and region of interest comparisons, P < 0.05). Similar results were found for [(11)C]-labelled Pittsburgh compound-B and [(18)F]-labelled fluorodeoxyglucose using atrophy-corrected data. Age-at-onset correlated positively with glucose metabolism in precuneus, lateral parietal and occipital regions of interest (controlling for age, education and Mini Mental State Exam, P < 0.05), while no correlations were found between age-at-onset and [(11)C]-labelled Pittsburgh compound-B binding. In summary, a comparable burden of fibrillar amyloid-beta was associated with greater posterior cortical hypometabolism in early-onset Alzheimer's disease. Our d...

SUMMARY Genetically hard-wired neural mechanisms must enforce behavioral reproductive isolation because interspecies courtship is rare even in sexually naïve animals of most species. We find that the chemoreceptor Gr32a inhibits male D. melanogaster from courting diverse fruit fly species. Gr32a recognizes non-volatile aversive cues present on these reproductively dead-end targets, and activity of Gr32a neurons is necessary and sufficient to inhibit interspecies courtship. Male-specific Fruitless (FruM), a master regulator of courtship, also inhibits interspecies courtship. Gr32a and FruM are not co-expressed, but FruM neurons contact Gr32a neurons, suggesting that these genes influence a shared neural circuit that inhibits inter-species courtship. Gr32a and FruM also suppress within-species intermale courtship, but we show that distinct mechanisms preclude sexual displays toward conspecific males and other species. Although this chemosensory pathway does not inhibit interspecies mating in D. melanogaster females, similar mechanisms appear to inhibit this behavior in many other male drosophilids.

Forty to fifty per cent of skeletal mass, accumulated during childhood and adolescence, is influenced by sunlight exposure, physical activity, lifestyle, endocrine status, nutrition and gender. In view of scarce data on association of nutrition and lifestyle with hypovitaminosis D in Indian children and adolescents, an in-depth study on 3127 apparently healthy Delhi schoolgirls (6 -18 years) from the lower (LSES, n 1477) and upper socioeconomic strata (USES, n 1650) was carried out. These girls were subjected to anthropometry and clinical examination for hypovitaminosis D. Girls randomly selected from the two strata (LSES, n 193; USES, n 211) underwent detailed lifestyle, dietary, biochemical and hormonal assessment. Clinical vitamin D deficiency was noted in 11·5 % girls (12·4 % LSES, 10·7 % USES). USES girls had significantly higher BMI than LSES counterparts. Prevalence of biochemical hypovitaminosis D (serum 25-hydroxyvitamin D , 50 nmol/l) was seen in 90·8 % of girls (89·6 % LSES, 91·9 % USES, NS). Mean intake of energy, protein, fat, Ca, vitamin D and milk/milk products was significantly higher in USES than LSES girls. Conversely, carbohydrate, fibre, phytate and cereal intakes were higher in LSES than USES girls. Physical activity and time spent outdoors was significantly higher in LSES girls (92·8 v. 64 %, P¼ 0·000). Significant correlation between serum 25-hydroxyvitamin D and estimated sun exposure (r 0·185, P¼ 0·001) and percentage body surface area exposed (r 0·146, P¼ 0·004) suggests that these lifestyle-related factors may contribute significantly to the vitamin D status of the apparently healthy schoolgirls. Hence, in the absence of vitamin D fortification of foods, diet alone appears to have an insignificant role.

No abstract

Intron-containing genes are often transcribed more efficiently than nonintronic genes. The effect of introns on transcription of genes is an evolutionarily conserved feature, being exhibited by such diverse organisms as yeast, plants, flies, and mammals. The mechanism of intron-mediated transcriptional activation, however, is not entirely clear. To address this issue, we inserted an intron in INO1, which is a nonintronic gene, and deleted the intron from ASC1, which contains a natural intron. We then compared transcription of INO1 and ASC1 genes in the presence and absence of an intron. Transcription of both genes was significantly stimulated by the intron. The introns have a direct role in enhancing transcription of INO1 and ASC1 because there was a marked increase in nascent transcripts from these genes in the presence of an intron. Intron-mediated enhancement of transcription required a splicing competent intron. Interestingly, both INO1 and ASC1 were in a looped configuration when their genes contained an intron. Intron-dependent gene looping involved a physical interaction of the promoter and the terminator regions. In addition, the promoter region interacted with the 5′ splice site and the terminator with the 3′ splice site. Intron-mediated enhancement of transcription was completely abolished in the looping defective sua7-1 strain. No effect on splicing, however, was observed in sua7-1 strain. On the basis of these results, we propose a role for gene looping in intron-mediated transcriptional activation of genes in yeast.chromosome conformation capture | RNA polymerase II T he protein encoding genes in eukaryotes differ from their prokaryotic counterparts in having noncoding intervening regions called introns, which are removed by splicing to generate mature mRNA. Since their discovery in 1977, there has been considerable debate regarding the functional role of introns in eukaryotes (1). It is widely believed that introns increase proteomic complexity by facilitating expression of multiple proteins from a single gene by alternative splicing (2). In budding yeast, where more than 95% of genes are without introns and there are very few instances of alternative splicing, introns do not contribute significantly to the proteomic diversity (3). The presence of introns in all eukaryotes, despite the high cost of maintaining them and the existence of the elaborate splicing machinery needed to remove them, suggest that introns are playing a more fundamental and evolutionarily conserved role in eukaryotic cells.One role of introns that has been remarkably conserved among diverse organisms, and which confers an additional advantage to eukaryotic genes, is their effect on efficiency of gene expression (4-6). Introns significantly enhance the transcriptional output of genes that harbor them. The expression level of intronless transgenes in mammalian cells is often 10-100 times lower than their intron-containing counterparts (5). The inclusion of just one intron near the 5′ end of the gene increases transcription of th...