Stearoyl-CoA desaturase (SCD) is a central lipogenic enzyme catalyzing the synthesis of monounsaturated fatty acids, mainly oleate (C18:1) and palmitoleate (C16:1), which are components of membrane phospholipids, triglycerides, wax esters, and cholesterol esters. Several SCD isoforms (SCD1-3) exist in the mouse. Here we show that mice with a targeted disruption of the SCD1 isoform have reduced body adiposity, increased insulin sensitivity, and are resistant to diet-induced weight gain. The protection from obesity involves increased energy expenditure and increased oxygen consumption. Compared with the wild-type mice the SCD1؊͞؊ mice have increased levels of plasma ketone bodies but reduced levels of plasma insulin and leptin. In the SCD1؊͞؊ mice, the expression of several genes of lipid oxidation are up-regulated, whereas lipid synthesis genes are down-regulated. These observations suggest that a consequence of SCD1 deficiency is an activation of lipid oxidation in addition to reduced triglyceride synthesis and storage.S tearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. It catalyzes the introduction of the cis double bond in the ⌬9 position of fatty acyl-CoA substrates. The preferred desaturation substrates are palmitoyl-CoA and stearoyl-CoA, which are converted to palmitoleoyl-CoA (16:1) and oleoyl-CoA (18:1), respectively (1-4). These fatty acids are requisite components of membrane phospholipids, triglycerides, cholesterol esters, and wax esters (5-7). Effects on composition of phospholipids ultimately determine membrane fluidity, and the effects on the composition of cholesterol esters and triglycerides can affect lipoprotein metabolism and adiposity. SCD expression is sensitive to dietary factors including polyunsaturated fatty acids, cholesterol and vitamin A, hormonal changes (i.e., insulin and glucagon), developmental processes, temperature changes, thiazolinediones, metals, alcohol, peroxisomal proliferators, and phenolic compounds (3). High SCD activity has been implicated in a wide range of disorders including diabetes, atherosclerosis, cancer, obesity, and viral infection (3,(8)(9)(10)(11)(12)(13).The existence of multiple SCD isoforms in mice (6, 14-18) and rats makes it difficult to determine the role of each isoform in lipid metabolism. New insights into the physiological role of the SCD1 gene and its endogenous products came from recent studies of the asebia mouse strains (ab j and ab 2j ) that have naturally occurring mutations in SCD1 (17-19) as well as a laboratory mouse model with a targeted disruption (SCD1Ϫ͞Ϫ) (6). We used these animal models to show that SCD1Ϫ͞Ϫ mice are deficient in hepatic triglycerides and cholesterol esters (7,20). The levels of palmitoleate (16:1) and oleate (18:1) are reduced, whereas palmitate and stearate are increased in the lipid fractions of SCD1Ϫ͞Ϫ mice. On a high carbohydrate diet supplemented with triolein, the cholesterol ester levels are corrected but the triglyceride levels are not reversed to the ...

Supplementary data are available at Bioinformatics online.

We consider the problem of inferring fold changes in gene expression from cDNA microarray data. Standard procedures focus on the ratio of measured uorescent intensities at each spot on the microarray, but to do so is to ignore the fact that the variation of such ratios is not constant. Estimates of gene expression changes are derived within a simple hierarchical model that accounts for measurement error and uctuations in absolute gene expression levels. Signi cant gene expression changes are identi ed by deriving the posterior odds of change within a similar model. The methods are tested via simulation and are applied to a panel of Escherichia coli microarrays.

Phosphoinositides are a family of lipid signalling molecules that regulate many cellular functions in eukaryotes. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2), the central component in the phosphoinositide signalling circuitry, is generated primarily by type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIalpha, PIPKIbeta and PIPKIgamma). In addition to functions in the cytosol, phosphoinositides are present in the nucleus, where they modulate several functions; however, the mechanism by which they directly regulate nuclear functions remains unknown. PIPKIs regulate cellular functions through interactions with protein partners, often PtdIns4,5P2 effectors, that target PIPKIs to discrete subcellular compartments, resulting in the spatial and temporal generation of PtdIns4,5P2 required for the regulation of specific signalling pathways. Therefore, to determine roles for nuclear PtdIns4,5P2 we set out to identify proteins that interacted with the nuclear PIPK, PIPKIalpha. Here we show that PIPKIalpha co-localizes at nuclear speckles and interacts with a newly identified non-canonical poly(A) polymerase, which we have termed Star-PAP (nuclear speckle targeted PIPKIalpha regulated-poly(A) polymerase) and that the activity of Star-PAP can be specifically regulated by PtdIns4,5P2. Star-PAP and PIPKIalpha function together in a complex to control the expression of select mRNAs, including the transcript encoding the key cytoprotective enzyme haem oxygenase-1 (refs 8, 9) and other oxidative stress response genes by regulating the 3'-end formation of their mRNAs. Taken together, the data demonstrate a model by which phosphoinositide signalling works in tandem with complement pathways to regulate the activity of Star-PAP and the subsequent biosynthesis of its target mRNA. The results reveal a mechanism for the integration of nuclear phosphoinositide signals and a method for regulating gene expression.

BackgroundHuman pluripotent stem cells offer the best available model to study the underlying cellular and molecular mechanisms of human embryonic lineage specification. However, it is not fully understood how individual stem cells exit the pluripotent state and transition towards their respective progenitor states.ResultsHere, we analyze the transcriptomes of human embryonic stem cell-derived lineage-specific progenitors by single-cell RNA-sequencing (scRNA-seq). We identify a definitive endoderm (DE) transcriptomic signature that leads us to pinpoint a critical time window when DE differentiation is enhanced by hypoxia. The molecular mechanisms governing the emergence of DE are further examined by time course scRNA-seq experiments, employing two new statistical tools to identify stage-specific genes over time (SCPattern) and to reconstruct the differentiation trajectory from the pluripotent state through mesendoderm to DE (Wave-Crest). Importantly, presumptive DE cells can be detected during the transitory phase from Brachyury (T)+ mesendoderm toward a CXCR4+ DE state. Novel regulators are identified within this time window and are functionally validated on a screening platform with a T-2A-EGFP knock-in reporter engineered by CRISPR/Cas9. Through loss-of-function and gain-of-function experiments, we demonstrate that KLF8 plays a pivotal role modulating mesendoderm to DE differentiation.ConclusionsWe report the analysis of 1776 cells by scRNA-seq covering distinct human embryonic stem cell-derived progenitor states. By reconstructing a differentiation trajectory at single-cell resolution, novel regulators of the mesendoderm transition to DE are elucidated and validated. Our strategy of combining single-cell analysis and genetic approaches can be applied to uncover novel regulators governing cell fate decisions in a variety of systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1033-x) contains supplementary material, which is available to authorized users.

No abstract

Over 15% of the data sets catalogued in the Gene Expression Omnibus Database involve RNA samples that have been pooled before hybridization. Pooling affects data quality and inference, but the exact effects are not yet known because pooling has not been systematically studied in the context of microarray experiments. Here we report on the results of an experiment designed to evaluate the utility of pooling and the impact on identifying differentially expressed genes. We find that inference for most genes is not adversely affected by pooling, and we recommend that pooling be done when fewer than three arrays are used in each condition. For larger designs, pooling does not significantly improve inferences if few subjects are pooled. The realized benefits in this case do not outweigh the price paid for loss of individual specific information. Pooling is beneficial when many subjects are pooled, provided that independent samples contribute to multiple pools.

DNA microarrays provide for unprecedented large-scale views of gene expression and, as a result, have emerged as a fundamental measurement tool in the study of diverse biological systems. Statistical questions abound, but many traditional data analytic approaches do not apply, in large part because thousands of individual genes are measured with relatively little replication. Empirical Bayes methods provide a natural approach to microarray data analysis because they can significantly reduce the dimensionality of an inference problem while compensating for relatively few replicates by using information across the array. We propose a general empirical Bayes modelling approach which allows for replicate expression profiles in multiple conditions. The hierarchical mixture model accounts for differences among genes in their average expression levels, differential expression for a given gene among cell types, and measurement fluctuations. Two distinct parameterizations are considered: a model based on Gamma distributed measurements and one based on log-normally distributed measurements. False discovery rate and related operating characteristics of the methodology are assessed in a simulation study. We also show how the posterior odds of differential expression in one version of the model is related to the ratio of the arithmetic mean to the geometric mean of the two sample means. The methodology is used in a study of mammary cancer in the rat, where four distinct patterns of expression are possible.