Identification of robust cardiac reference genes in a mouse model of cardiometabolic disease

Cardiovascular disease is linked to obesity, the metabolic syndrome, and altered 24hour (circadian) rhythms. Although the underlying mechanisms remain undefined, transcriptome analysis in the heart is beginning to provide important insights into the cardiometabolic pathogenesis. The reliability and accuracy of real-time quantitative PCR generated gene expression data is largely dependent on the selection of suitable reference genes (RG), which must be constitutively expressed regardless of cardio-metabolic disease state and time of day. However, many studies do not employ the appropriate selections strategies. In this study we determined the expression stability of seven candidate RGs (GAPDH, YWHAZ, B2M, EIF4A2, ATP5?, ACTB and CYC1) in a mouse model of diet-induced metabolic syndrome in both the day and night, using geNorm qBasePLUS software. RG expression varied in hearts of normal fed versus high fat fed mice, and was also dependant on the time of day. When all experimental variables were considered YWHAZ and ACTB were ranked the most stable and therefore the most suitable genes for generating comparative gene expression data in heart tissue from murine models of cardiometabolic disease. This study provides important information for reference gene selection, and will aid further transcriptome investigations into heart organ functio

Activation of AMPA Receptors in the Suprachiasmatic Nucleus Phase-Shifts the Mouse Circadian Clock In Vivo and In Vitro

The glutamatergic neurotransmission in the suprachiasmatic nucleus (SCN) plays a central role in the entrainment of the circadian rhythms to environmental light-dark cycles. Although the glutamatergic effect operating via NMDAR (N-methyl D-aspartate receptor) is well elucidated, much less is known about a role of AMPAR (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor) in circadian entrainment. Here we show that, in the mouse SCN, GluR2 and GluR4 AMPAR subtypes are abundantly expressed in the retinorecipient area. In vivo microinjection of AMPA in the SCN during the early subjective night phase-delays the behavioral rhythm. In the organotypic SCN slice culture, AMPA application induces phase-dependent phase-shifts of core-clock gene transcription rhythms. These data demonstrate that activation of AMPAR is capable of phase-shifting the circadian clock both in vivo and in vitro, and are consistent with the hypothesis that activation of AMPA receptors is a critical step in the transmission of photic information to the SCN

Deep sequencing reveals the complex and coordinated transcriptional regulation of genes related to grain quality in rice cultivars

<p>Abstract</p> <p>Background</p> <p>Milling yield and eating quality are two important grain quality traits in rice. To identify the genes involved in these two traits, we performed a deep transcriptional analysis of developing seeds using both massively parallel signature sequencing (MPSS) and sequencing-by-synthesis (SBS). Five MPSS and five SBS libraries were constructed from 6-day-old developing seeds of Cypress (high milling yield), LaGrue (low milling yield), Ilpumbyeo (high eating quality), YR15965 (low eating quality), and Nipponbare (control).</p> <p>Results</p> <p>The transcriptomes revealed by MPSS and SBS had a high correlation co-efficient (0.81 to 0.90), and about 70% of the transcripts were commonly identified in both types of the libraries. SBS, however, identified 30% more transcripts than MPSS. Among the highly expressed genes in Cypress and Ilpumbyeo, over 100 conserved <it>cis </it>regulatory elements were identified. Numerous specifically expressed transcription factor (TF) genes were identified in Cypress (282), LaGrue (312), Ilpumbyeo (363), YR15965 (260), and Nipponbare (357). Many key grain quality-related genes (i.e., genes involved in starch metabolism, aspartate amino acid metabolism, storage and allergenic protein synthesis, and seed maturation) that were expressed at high levels underwent alternative splicing and produced antisense transcripts either in Cypress or Ilpumbyeo. Further, a time course RT-PCR analysis confirmed a higher expression level of genes involved in starch metabolism such as those encoding ADP glucose pyrophosphorylase (AGPase) and granule bound starch synthase I (GBSS I) in Cypress than that in LaGrue during early seed development.</p> <p>Conclusion</p> <p>This study represents the most comprehensive analysis of the developing seed transcriptome of rice available to date. Using two high throughput sequencing methods, we identified many differentially expressed genes that may affect milling yield or eating quality in rice. Many of the identified genes are involved in the biosynthesis of starch, aspartate family amino acids, and storage proteins. Some of the differentially expressed genes could be useful for the development of molecular markers if they are located in a known QTL region for milling yield or eating quality in the rice genome. Therefore, our comprehensive and deep survey of the developing seed transcriptome in five rice cultivars has provided a rich genomic resource for further elucidating the molecular basis of grain quality in rice.</p

Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons

The mammalian suprachiasmatic nucleus (SCN) is a master circadian pacemaker. It is not known which SCN neurons are autonomous pacemakers or how they synchronize their daily firing rhythms to coordinate circadian behavior. Vasoactive intestinal polypeptide (VIP) and the VIP receptor VPAC(2) (encoded by the gene Vipr2) may mediate rhythms in individual SCN neurons, synchrony between neurons, or both. We found that Vip(−/−) and Vipr2(−/−) mice showed two daily bouts of activity in a skeleton photoperiod and multiple circadian periods in constant darkness. Loss of VIP or VPAC(2) also abolished circadian firing rhythms in approximately half of all SCN neurons and disrupted synchrony between rhythmic neurons. Critically, daily application of a VPAC(2) agonist restored rhythmicity and synchrony to VIP(−/−) SCN neurons, but not to Vipr2(−/−) neurons. We conclude that VIP coordinates daily rhythms in the SCN and behavior by synchronizing a small population of pacemaking neurons and maintaining rhythmicity in a larger subset of neurons

Variation in stability of housekeeping genes in healthy and adhesion-related mesothelium

ObjectiveTo investigate the stability of various housekeeping genes (HKG) within healthy versus scarred peritoneal mesothelium. The use of HKG as internal controls for quantitative real-time polymerase chain reaction (qRT-PCR) studies is based on the assumption of their inherent stability. However, recent evidence suggests that this is not true for all HKG and that stability may be tissue specific and affected by certain pathologies.DesignPaired mesothelial (n = 10) and adhesion tissue samples (n = 10) were taken during laparoscopic surgery. The stability of 12 candidate reference genes in the mesothelial tissues were evaluated; these include ATP5b, SDHA, CYC1, 18S rRNA, RPL13A, ACTB, YWHAZ, TOP1, UBC, EIF4A2, GAPDH, and B2M.SettingHospital.Patient(s)Female patients undergoing laparoscopic gynecological surgery were recruited from the Princess Anne Hospital, United Kingdom.Intervention(s)Assessment of HKG expression stability using geNorm algorithm software.Main Outcome Measure(s)Stability measure (M) generated by geometric averaging of multiple target genes and mean pairwise variation of genes.Result(s)The most stable HKGs observed across both healthy and adhesion-related mesothelium were found to be ACTB, YWHAZ, and CYC1. ACTB had a higher expression in healthy mesothelium compared with in peritoneal adhesion tissue.Conclusion(s)This study indicates that ACTB, YWHAZ, and CYC1 are the appropriate internal controls for qRT-PCR analysis in mesothelial gene expression studies. Published discrepancies in gene expression studies using the mesothelium may therefore be due in part to inappropriate HKG selection<br/

Diet-induced maternal obesity alters ovarian morphology and gene expression in the adult mouse offspring

ObjectiveTo examine the effects of high-fat (HF) diet-induced maternal obesity on follicular population and gene expression in adult offspring ovaries.DesignExperimental mouse study. Setting Laboratory. Animal(s) Mice on HF diet. Intervention(s) Female C57BL/6J mice were fed an HF or standard chow (C) diet 6 weeks before conception, through pregnancy and lactation. Offspring were fed the C or HF diet from weaning, creating the HF/HF, HF/C, C/HF, C/C offspring groups.Main Outcome Measure(s)Follicular counts and gene expression in adult offspring ovaries. Result(s) Prenatal exposure to maternal HF nutrition resulted in the reduction of primordial, antral, and Graafian follicle numbers in offspring ovaries (both HF/C and HF/HF). Expression levels of genes involved in apoptosis (FoXO3a), follicular growth and development (Gdf9), and circadian rhythms generation (Clock and Bmal1) were elevated in the ovaries of HF/C and HF/HF offspring, while expression of the circadian clock genes Cry1 and Per1 were lower in HF/HF ovaries.Conclusion(s)Maternal obesity during pregnancy has long-term deleterious consequences on follicular growth and development in the adult offspring ovaries, which may impact their reproductive potential. © 2014 by the American Congress of Rehabilitation Medicine