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Papers by Leonard Hearne
Journal of Thermal Biology, 2008
Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-fiv... more Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-five mice were allocated to either chronic heat stress (34 1C) or control (24 1C) conditions for a period of 2 weeks from 47 to 60 d of age. Nineteen genes differentially expressed in liver were identified using DNA microarrays. Genes involved in the anti-oxidant pathway and metabolism were up-regulated. Genes involved in generation of reactive oxygen radicals and mitochondrial expressed genes were down-regulated. Enzyme activity measurements confirmed the array results. Mice exposed to chronic heat stress showed signs of increased oxidative stress in liver cells.
Journal of Animal Science
Fescue toxicosis is caused by consumption of toxins produced by an endophytic fungus, Neotyphodiu... more Fescue toxicosis is caused by consumption of toxins produced by an endophytic fungus, Neotyphodium coenophialum, in tall fescue [Lolium arundinaceum (Schreb.) Darbysh]. Microarray analysis was used to identify shifts in genetic expression associated with the affected physiological processes to identify potential targets for future pharmacological/toxicological intervention. Male rats (n = 24) were implanted with temperature transmitters, which measure core temperature every 5 min. After an 8-d recovery, the rats were fed an endophyte-free diet for 5 d. During the following 5-d treatment period, rats were fed either an endophyte-free or an endophyte-infected (91.5 microg of ergovaline.kg of BW(-1).d(-1)) diet. At the end of treatment, rats were euthanized and a sample of the liver was obtained. Feed conversion efficiency was calculated for both treatment groups. Serum prolactin concentrations were measured using ELISA. Liver tissue RNA was reverse transcribed and hybridized to an oli...
Fescue toxicosis affects wild and domestic animals consuming ergot alkaloids contained in tall fe... more Fescue toxicosis affects wild and domestic animals consuming ergot alkaloids contained in tall fescue forage infected with the endophytic fungus, Neotyphodium coenophialum. When animals are consuming infected fescue (E+) forage during periods of elevated ambient temperatures (summer), a range of phenotypic disorders collectively called summer slump is observed. It is characterized by hyperthermia, with an accompanying decrease in feed intake,
Theriogenology, 2009
Fertility losses in male mice occur approximately 18-28 d after heat stress. The objective of thi... more Fertility losses in male mice occur approximately 18-28 d after heat stress. The objective of this study was to identify gene expression differences in males highly versus lowly fertile after heat stress. Mature male mice were exposed to heat stress (35 AE 1 8C; n = 50) or thermoneutral (21 AE 1 8C; n = 10) conditions for 24 h (Day 0) and hemicastrated (Day 1) to collect tissue for gene expression analyses. Males were subjected to a mating test from Days 18 to 26 when variation in fertility was anticipated. A fertility index was used to rank heat-stressed males and identify those males resistant and susceptible to heat stress, respectively. Microarray analyses were conducted on testis tissues from control (n = 5), heat stress resistant (n = 5), and heat stress susceptible (n = 5) males, and 225 genes were observed to be differentially expressed (P < 0.05), including genes involved in chaperone (Canx, Hspcb1, and Tcp1) and catalytic (Fkpb6, Psma7, and Idh1) activity. Expression patterns of these genes were confirmed using realtime RT-PCR. Male progeny from selected sires were similarly divergent in fertility after heat stress. Testicular expression levels of Canx, Hspcb, and Tcp1 genes were determined in these progeny. Hspcb expression was moderately heritable (0.31 AE 0.25); however, expression patterns of Canx and Tcp1 were not heritable. #
Stem Cells and Development, 2008
To realize the full potential of human embryonic stem cells (hESCs), it is important to develop c... more To realize the full potential of human embryonic stem cells (hESCs), it is important to develop culture conditions that maintain hESCs in a pluripotent, undifferentiated state. A low O 2 atmosphere (~4% O 2 ), for example, prevents spontaneous differentiation and supports self-renewal of hESCs. To identify genes whose expression is sensitive to O 2 conditions, microarray analysis was performed on RNA from hESCs that had been maintained under either 4% or 20% O 2 . Of 149 genes differentially expressed, 42 were up-regulated and 107 down-regulated under 20% O 2 . Several of the down-regulated genes are most likely under the control of hypoxia-inducing factors and include genes encoding enzymes involved in carbohydrate catabolism and cellular redox state. Although genes associated with pluripotency, including OCT4, SOX2, and NANOG were generally unaffected, some genes controlled by these transcription factors, including LEFTY2, showed lowered expression under 20% O 2 , while a few genes implicated in lineage specifi cation were up-regulated. Although the differences between O 2 conditions were generally subtle, they were observed in two different hESC lines and at different passage numbers. The data are consistent with the hypothesis that 4% O 2 favors the molecular mechanisms required for the maintenance of pluripotency.
Reproduction, 2007
Interferon-t (IFNT) is released by preimplantation conceptuses of ruminant species and prepares t... more Interferon-t (IFNT) is released by preimplantation conceptuses of ruminant species and prepares the mother for pregnancy. Although one important function is to protect the corpus luteum from the luteolytic activity of prostaglandin-F 2a, IFNT most likely regulates a range of other physiological processes in endometrium. Here, an immortalized cell line from ovine uterine luminal epithelial cells was treated with IFNT for either 8 or 24 h. RNA was subjected to cDNA microarray analysis, with RNA from untreated cells as the reference standard. Of 15 634 genes, 1274 (8%) were IFNT responsive at P!0.01 and 585 at P!0.001 to at least one treatment. Of the latter, 356 were up-regulated and 229 down-regulated. Increasing IFNT concentrations from 10 ng/ml to 10 mg/ml had minor effects, and most genes up-or down-regulated at 8 h were regulated similarly at 24 h. Although IFNT influences many genes implicated in antiviral activity and apoptosis, its action also likely regulates prostaglandin metabolism, growth factors and their receptors, apoptosis and the nuclear factor (NF)-kB cascade, extracellular matrix accretion, angiogenesis, blood coagulation, and inflammation. In particular, it increased mRNA concentrations of genes related to the vascular endothelial growth factor R2 pathway of angiogenesis and down-regulated ones associated with hypoxia. Two genes implicated in the antiluteolytic actions of IFNT (encoding cyclooxygenase-2 and the oxytocin receptor respectively) were downregulated in response to all treatments. IFNT targets a complex range of physiological processes during the establishment of pregnancy.
PLoS ONE, 2010
Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in ... more Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in North America and Europe. Little is known regarding how plants respond to insect attack of roots, thus complicating the selection for plant defense targets. Diabrotica virgifera virgifera is the most successful species in its genus and is the only Diabrotica beetle harboring an almost species-wide Wolbachia infection. Diabrotica virgifera virgifera are infected with Wolbachia and the typical gut flora found in soil-living, phytophagous insects. Diabrotica virgifera virgifera larvae cannot be reared aseptically and thus, it is not possible to observe the response of maize to effects of insect gut flora or other transient microbes. Because Wolbachia are heritable, it is possible to investigate whether Wolbachia infection affects the regulation of maize defenses. To answer if the success of Diabrotica virgifera virgifera is the result of microbial infection, Diabrotica virgifera virgifera were treated with antibiotics to eliminate Wolbachia and a microarray experiment was performed. Direct comparisons made between the response of maize root tissue to the feeding of antibiotic treated and untreated Diabrotica virgifera virgifera show down-regulation of plant defenses in the untreated insects compared to the antibiotic treated and control treatments. Results were confirmed via QRT-PCR. Biological and behavioral assays indicate that microbes have integrated into Diabrotica virgifera virgifera physiology without inducing negative effects and that antibiotic treatment did not affect the behavior or biology of the insect. The expression data and suggest that the pressure of microbes, which are most likely Wolbachia, mediate the downregulation of many maize defenses via their insect hosts. This is the first report of a potential link between a microbial symbiont of an insect and a silencing effect in the insect host plant. This is also the first expression profile for a plant attacked by a root-feeding insect.
PLANT PHYSIOLOGY, 2010
Previous systems analyses in plants have focused on a single developmental stage or time point, a... more Previous systems analyses in plants have focused on a single developmental stage or time point, although it is often important to additionally consider time-index changes. During seed development a cascade of events occurs within a relatively brief time-scale. We have collected protein and transcript expression data from five sequential stages of Arabidopsis thaliana seed development encompassing the period of reserve polymer accumulation. Protein expression profiling employed 2-dimensional gel electrophoresis coupled with tandem mass spectrometry, while transcript profiling used oligonucleotide microarrays. Analyses in biological triplicate yielded robust expression information for 523 proteins and 22,746 genes across the five developmental stages, and established 319 protein/transcript pairs for subsequent pattern analysis. General linear modeling was used to evaluate the protein/transcript expression patterns. Overall, application of this statistical assessment technique showed concurrence for a slight majority (56%) of expression pairs. Many specific examples of discordant protein/transcript expression patterns were detected, suggesting that this approach will be useful in revealing examples of post-transcriptional regulation. proteomics reveals Arabidopsis thaliana gene models and proteome dynamics. Science 320: 938-941 Baginsky S, Kleffmann T, von Zychlinski A, Gruissem W (2005) Analysis of shotgun proteomics and RNA profiling data from Arabidopsis thaliana chloroplasts. J Proteome Res 4: 637-640 Baud S, Wuilleme S, To A, Rochat C, Lepiniec L (2009) Role of WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis. Plant J 60: 933-947 Box GEP, Cox DR (1964) An analysis of transformations. J Royal Stat Soc Series B 26: 211-252 Chen G, Gharib TG, Huang CC, Taylor JM, Misek DE, Kardia SL, Giordano TJ, Iannettoni MD, Orringer MB, Hanash SM, Beer DG (2002) Discordant protein and mRNA expression in lung adenocarcinomas. Mol Cell Proteomics 4: 304-313 A (2007) Integrated proteomic and transcriptomic profiling of mouse lung development and Nmyc target genes. Mol Syst Biol 3: 1-15 Degerman R (1982) Ordered binary trees constructed through an application of Kendall's tau. Psychometrika 47: 523-527 Dempster AP, Laird NM, Rubin DB (1977) Maximum likelihood from incomplete data via the EM algorithm. J Royal Stat Soc Ser B 39: 1-38 Eng JK, McCormack AL, Yates III, JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5: 976-989 Friston K (2008) Hierarchical models in the brain. PLoS Comput Biol 4: e1000211 Goldberg RB, de Paiva G, Yadegari R (1994) Plant embryogenesis: Zygote to seed. Science 266: 605-614 Griffin TJ, Gygi SP, Ideker T, Rist B, Eng J, Hood L, Aebersold R (2002) Complementary Nicolas T (2009) Data mining, a tool for systems biology or a systems biology tool. J Comput Sci Syst Biol 2: 216-218 Nie L, Wu G, Culley DE, Scholten JC, Zhang W (2007) Integrative analysis of transcriptomic and proteomic data: challenges, solutions and applications. Crit Rev Biotechnol 27: 63-75 Ørntoft TF, Thykjaer T, Waldman FM, Wolf H, Celis JE (2002) Genome-wide study of gene copy numbers, transcripts, and protein levels in pairs of non-invasive and invasive human transitional cell carcinomas. Mol Cell Proteomics 1: 37-45 Pascal LE, True LD, Campbell DS, Deutsch EW, Risk M, Coleman IM, Eichner LJ, Nelson PS, Liu AY (2008) Correlation of mRNA and protein levels: cell type-specific gene expression of cluster designation antigens in the prostate. BMC Genomics 9: 246 Piques M, Schulze WX, Höhne M, Usadel B, Gibon Y, Rohwer J, Stitt M (2009) Ribosome and transcript copy numbers, polysome occupancy and enzyme dynamics in Arabidopsis. Molec Systems Biol 5: 314 Prioul JL, Méchin V, Lessard P, Thévenot C, Grimmer M, Chateau-Joubert S, Coates S, Hartings H, Kloiber-Maitz M, Murigneux A, Sarda X, Damerval C, Edwards KJ (2008) A joint transcriptomic, proteomic and metabolic analysis of maize endosperm development and starch filling. Plant Biotechnol J 6: 855-869 Rodgers JL, Nicewander WA (1988) Thirteen ways to look at the correlation coefficient. Amer Statis 42: 59-66
Molecular Plant-Microbe Interactions, 2007
Global analysis of gene expression changes in soybean (Glycine max) and Heterodera glycines (soyb... more Global analysis of gene expression changes in soybean (Glycine max) and Heterodera glycines (soybean cyst nematode [SCN]) during the course of infection in a compatible interaction was performed using the Affymetrix GeneChip soybean genome array. Among 35,611 soybean transcripts monitored, we identified 429 genes that showed statistically significant differential expression between uninfected and nematode-infected root tissues. These included genes encoding enzymes involved in primary metabolism; biosynthesis of phenolic compounds, lignin, and flavonoids; genes related to stress and defense responses; cell wall modification; cellular signaling; and transcriptional regulation. Among 7,431 SCN transcripts monitored, 1,850 genes showed statistically significant differential expression across different stages of nematode parasitism and development. Differentially expressed SCN genes were grouped into nine different clusters based on their expression profiles during parasitism of soybean roots. The patterns of gene expression we observed in SCN suggest coordinated regulation of genes involved in parasitism. Quantitative real-time reverse-transcription polymerase chain reaction confirmed the results of our microarray analysis. The simultaneous genome-wide analysis of gene expression changes in the host and pathogen during a compatible interaction provides new insights into soybean responses to nematode infection and the first profile of transcript abundance changes occurring in the nematode as it infects and establishes a permanent feeding site within a host plant root.
Journal of Thermal Biology, 2008
Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-fiv... more Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-five mice were allocated to either chronic heat stress (34 1C) or control (24 1C) conditions for a period of 2 weeks from 47 to 60 d of age. Nineteen genes differentially expressed in liver were identified using DNA microarrays. Genes involved in the anti-oxidant pathway and metabolism were up-regulated. Genes involved in generation of reactive oxygen radicals and mitochondrial expressed genes were down-regulated. Enzyme activity measurements confirmed the array results. Mice exposed to chronic heat stress showed signs of increased oxidative stress in liver cells.
Journal of Proteome Research, 2007
Near-isogenic sunflower lines containing 25% (inbred RHA280) and 48% (RHA801) oil by seed dry mas... more Near-isogenic sunflower lines containing 25% (inbred RHA280) and 48% (RHA801) oil by seed dry mass were comparatively analyzed in biological triplicate at 18 days after flowering using two-dimensional (both pI 3-10 and 4-7) Difference Gel Electrophoresis. Additionally, two inbred lines varying in oleic acid content, HA89 (18% oleic) and HA341 (89% oleic), were also analyzed in the same manner. Statistical analyses of these sunflower lines was performed beginning with fitting a mixed effects linear model to the log-transformed optical volume of each spot to account for gel variation, followed by testing the significance between varieties for mean transformed optical spot volumes. The p-values from the spot analysis procedures were then used to find the cutoff point for differential expression using a 10% false-discovery rate (FDR). Comparison of the oil content and oleic acid composition lines revealed 77 and 42 protein spots below the 10% FDR cutoff, respectively, and were therefore declared differentially expressed. Liquid chromatography-tandem mass spectrometry analysis of each of these protein spots resulted in assignments for 44 and 17 spots, respectively. Fructokinase, plastid phosphoglycerate kinase, and enolase proteins were determined to be up-regulated in the high oil line, while phosphofructokinase, cytosolic phosphoglucomutase, and cytsolic phosphoglycerate kinase were up-regulated in the low oil variety. Additionally, four activities involved in amino acid synthesis were up-regulated in the low oil variety in addition to 12S storage proteins and a protein similar to legumin storage protein. Interestingly, two 2-DE spots identified as 14-3-3 proteins were found to be up-regulated in high oleic acid variety. Alteration of glycolytic and amino acid biosynthetic enzymes, as well as storage protein levels, suggests seed oil content is tightly linked to carbohydrate metabolism and protein synthesis in a complex manner.
Endocrinology, 2006
In ruminants, conceptus interferon-(IFNT) alters maternal physiology to accommodate a pregnancy. ... more In ruminants, conceptus interferon-(IFNT) alters maternal physiology to accommodate a pregnancy. We hypothesized that the effectiveness of IFNT on extending corpus luteum (CL) life span in nonpregnant ewes would depend upon the dose and manner of administration and would be correlated with the response in gene expression in endometrium. We anticipated that IFNT, whether administered im or by uterine infusion, would mimic changes observed in pregnancy. Ewes were assigned to five treatments: 1) uterine infusion of saline; 2) uterine infusion of ovine IFNT4 (200 g/d); 3) saline im injection; 4) im injection of IFNT4 at low dose (200 g/d); and 5) high dose (2 mg/d). CL life span was increased in groups 2 and 5, but not in 1, 3, and 4. Endometrial RNA extracted from groups 1-5 on d 14 and from d 14 pregnant and nonbred (cy-clic) ewes was used to assess expression of 70 genes on microarrays. When pregnant and cyclic ewes were compared, 30 genes were up-regulated and nine down-regulated during pregnancy. Responses were slightly less in groups 2 and 5 but were much lower in group 4. The majority of the highly upregulated genes were associated with antiviral responses. Those down-regulated included ones for IGF-II, hypoxiainducible factor 1␣, oxytocin receptor, prostaglandin F synthase, and cyclooxygenase-2. Quantitative PCR for selected genes confirmed these data and revealed that similar gene expression changes occurred in the CL of pregnant and group 2 ewes. IFNT treatment mimics pregnancy, but relatively high doses of im-injected IFNT are required to elicit a full endometrial response.
Circulation, 1998
Background-Factor VII plays a pivotal role in coagulation. Factor VIIc levels were reported to be... more Background-Factor VII plays a pivotal role in coagulation. Factor VIIc levels were reported to be a risk factor for fatal coronary heart disease (CHD). Factor VIIc and VIIag levels were noted to be positively associated with plasma triglyceride (TG) levels and influenced by a VII gene polymorphism. The purpose of this study is to determine whether these associations are related to activated factor VII (factor VIIa). Methods and Results-Fasting and 3.5-hour postprandial samples from 216 cases with subclinical atherosclerosis and 341 matched controls selected from the ARIC cohort were assayed for levels of factors VIIa, VIIc, and VIIag and TG, and factor VII codon 353 gene polymorphism. The level of factor VIIa was higher in Arg/Arg than in Arg/GlnϩGln/Gln genotypes, and the difference was in accord with that of factors VIIag and VIIc. However, the factor VIIa difference was statistically insignificant. Factor VIIa values were not correlated with fasting or 3.5-hour postprandial TG levels, nor were they associated with subclinical atherosclerosis. Conclusions-Factor VIIa levels, like factor VIIag and VIIc levels, are influenced by factor VII gene codon 353 polymorphism. However, unlike factor VIIag or VIIc, factor VIIa is not influenced by TG levels; none of these is associated with subclinical atherosclerosis. (Circulation. 1998;98:2815-2821.)
Autonomic Neuroscience, 2009
We investigated the gender effects on transient pupillary light reflex (PLR) in healthy young adu... more We investigated the gender effects on transient pupillary light reflex (PLR) in healthy young adults between 18 and 22 years old. Both dark-adapted and light-adapted PLRs were measured using green and red stimuli of different intensities. The results indicate that females had significantly larger relative constriction amplitudes than males in a dark-adapted condition. This gender effect depends on the stimulus intensities. The relative constriction amplitude in female subjects increased faster than it did in the males with the stimulus intensity. We did not observe any significant gender differences in the other PLR parameters, including latency, constriction speed, and recovery speed.
Journal of Animal Science
Fescue toxicosis affects wild and domestic animals grazing fescue pasture infected with the endop... more Fescue toxicosis affects wild and domestic animals grazing fescue pasture infected with the endophytic fungus Neotyphodium coenophialum. Signs of fescue toxicosis include increased core body temperature and respiration rate and decreased milk yield and reproductive performance. Laboratory mice also exhibit symptoms of fescue toxicosis, as indicated by reduced growth rate and reproductive performance. Mice were used to study the effects of fescue toxicosis on hepatic gene expression. Twenty-seven mice were randomly allocated to a diet containing either 50% endophyte-infected (E+; 6 ppm ergovaline) or endophyte-free (E-) fescue seed for 2 wk under thermoneutral conditions. Liver genes differentially expressed due to fescue toxicosis were identified using DNA microarray. A 2-stage ANOVA of microarray data identified 36 differentially expressed genes between mice fed E+ and E- diets. Another analysis method, significance analysis of microarray, identified 9 genes as differentially expre...
Journal of Thermal Biology, 2008
Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-fiv... more Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-five mice were allocated to either chronic heat stress (34 1C) or control (24 1C) conditions for a period of 2 weeks from 47 to 60 d of age. Nineteen genes differentially expressed in liver were identified using DNA microarrays. Genes involved in the anti-oxidant pathway and metabolism were up-regulated. Genes involved in generation of reactive oxygen radicals and mitochondrial expressed genes were down-regulated. Enzyme activity measurements confirmed the array results. Mice exposed to chronic heat stress showed signs of increased oxidative stress in liver cells.
Journal of Animal Science
Fescue toxicosis is caused by consumption of toxins produced by an endophytic fungus, Neotyphodiu... more Fescue toxicosis is caused by consumption of toxins produced by an endophytic fungus, Neotyphodium coenophialum, in tall fescue [Lolium arundinaceum (Schreb.) Darbysh]. Microarray analysis was used to identify shifts in genetic expression associated with the affected physiological processes to identify potential targets for future pharmacological/toxicological intervention. Male rats (n = 24) were implanted with temperature transmitters, which measure core temperature every 5 min. After an 8-d recovery, the rats were fed an endophyte-free diet for 5 d. During the following 5-d treatment period, rats were fed either an endophyte-free or an endophyte-infected (91.5 microg of ergovaline.kg of BW(-1).d(-1)) diet. At the end of treatment, rats were euthanized and a sample of the liver was obtained. Feed conversion efficiency was calculated for both treatment groups. Serum prolactin concentrations were measured using ELISA. Liver tissue RNA was reverse transcribed and hybridized to an oli...
Fescue toxicosis affects wild and domestic animals consuming ergot alkaloids contained in tall fe... more Fescue toxicosis affects wild and domestic animals consuming ergot alkaloids contained in tall fescue forage infected with the endophytic fungus, Neotyphodium coenophialum. When animals are consuming infected fescue (E+) forage during periods of elevated ambient temperatures (summer), a range of phenotypic disorders collectively called summer slump is observed. It is characterized by hyperthermia, with an accompanying decrease in feed intake,
Theriogenology, 2009
Fertility losses in male mice occur approximately 18-28 d after heat stress. The objective of thi... more Fertility losses in male mice occur approximately 18-28 d after heat stress. The objective of this study was to identify gene expression differences in males highly versus lowly fertile after heat stress. Mature male mice were exposed to heat stress (35 AE 1 8C; n = 50) or thermoneutral (21 AE 1 8C; n = 10) conditions for 24 h (Day 0) and hemicastrated (Day 1) to collect tissue for gene expression analyses. Males were subjected to a mating test from Days 18 to 26 when variation in fertility was anticipated. A fertility index was used to rank heat-stressed males and identify those males resistant and susceptible to heat stress, respectively. Microarray analyses were conducted on testis tissues from control (n = 5), heat stress resistant (n = 5), and heat stress susceptible (n = 5) males, and 225 genes were observed to be differentially expressed (P < 0.05), including genes involved in chaperone (Canx, Hspcb1, and Tcp1) and catalytic (Fkpb6, Psma7, and Idh1) activity. Expression patterns of these genes were confirmed using realtime RT-PCR. Male progeny from selected sires were similarly divergent in fertility after heat stress. Testicular expression levels of Canx, Hspcb, and Tcp1 genes were determined in these progeny. Hspcb expression was moderately heritable (0.31 AE 0.25); however, expression patterns of Canx and Tcp1 were not heritable. #
Stem Cells and Development, 2008
To realize the full potential of human embryonic stem cells (hESCs), it is important to develop c... more To realize the full potential of human embryonic stem cells (hESCs), it is important to develop culture conditions that maintain hESCs in a pluripotent, undifferentiated state. A low O 2 atmosphere (~4% O 2 ), for example, prevents spontaneous differentiation and supports self-renewal of hESCs. To identify genes whose expression is sensitive to O 2 conditions, microarray analysis was performed on RNA from hESCs that had been maintained under either 4% or 20% O 2 . Of 149 genes differentially expressed, 42 were up-regulated and 107 down-regulated under 20% O 2 . Several of the down-regulated genes are most likely under the control of hypoxia-inducing factors and include genes encoding enzymes involved in carbohydrate catabolism and cellular redox state. Although genes associated with pluripotency, including OCT4, SOX2, and NANOG were generally unaffected, some genes controlled by these transcription factors, including LEFTY2, showed lowered expression under 20% O 2 , while a few genes implicated in lineage specifi cation were up-regulated. Although the differences between O 2 conditions were generally subtle, they were observed in two different hESC lines and at different passage numbers. The data are consistent with the hypothesis that 4% O 2 favors the molecular mechanisms required for the maintenance of pluripotency.
Reproduction, 2007
Interferon-t (IFNT) is released by preimplantation conceptuses of ruminant species and prepares t... more Interferon-t (IFNT) is released by preimplantation conceptuses of ruminant species and prepares the mother for pregnancy. Although one important function is to protect the corpus luteum from the luteolytic activity of prostaglandin-F 2a, IFNT most likely regulates a range of other physiological processes in endometrium. Here, an immortalized cell line from ovine uterine luminal epithelial cells was treated with IFNT for either 8 or 24 h. RNA was subjected to cDNA microarray analysis, with RNA from untreated cells as the reference standard. Of 15 634 genes, 1274 (8%) were IFNT responsive at P!0.01 and 585 at P!0.001 to at least one treatment. Of the latter, 356 were up-regulated and 229 down-regulated. Increasing IFNT concentrations from 10 ng/ml to 10 mg/ml had minor effects, and most genes up-or down-regulated at 8 h were regulated similarly at 24 h. Although IFNT influences many genes implicated in antiviral activity and apoptosis, its action also likely regulates prostaglandin metabolism, growth factors and their receptors, apoptosis and the nuclear factor (NF)-kB cascade, extracellular matrix accretion, angiogenesis, blood coagulation, and inflammation. In particular, it increased mRNA concentrations of genes related to the vascular endothelial growth factor R2 pathway of angiogenesis and down-regulated ones associated with hypoxia. Two genes implicated in the antiluteolytic actions of IFNT (encoding cyclooxygenase-2 and the oxytocin receptor respectively) were downregulated in response to all treatments. IFNT targets a complex range of physiological processes during the establishment of pregnancy.
PLoS ONE, 2010
Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in ... more Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in North America and Europe. Little is known regarding how plants respond to insect attack of roots, thus complicating the selection for plant defense targets. Diabrotica virgifera virgifera is the most successful species in its genus and is the only Diabrotica beetle harboring an almost species-wide Wolbachia infection. Diabrotica virgifera virgifera are infected with Wolbachia and the typical gut flora found in soil-living, phytophagous insects. Diabrotica virgifera virgifera larvae cannot be reared aseptically and thus, it is not possible to observe the response of maize to effects of insect gut flora or other transient microbes. Because Wolbachia are heritable, it is possible to investigate whether Wolbachia infection affects the regulation of maize defenses. To answer if the success of Diabrotica virgifera virgifera is the result of microbial infection, Diabrotica virgifera virgifera were treated with antibiotics to eliminate Wolbachia and a microarray experiment was performed. Direct comparisons made between the response of maize root tissue to the feeding of antibiotic treated and untreated Diabrotica virgifera virgifera show down-regulation of plant defenses in the untreated insects compared to the antibiotic treated and control treatments. Results were confirmed via QRT-PCR. Biological and behavioral assays indicate that microbes have integrated into Diabrotica virgifera virgifera physiology without inducing negative effects and that antibiotic treatment did not affect the behavior or biology of the insect. The expression data and suggest that the pressure of microbes, which are most likely Wolbachia, mediate the downregulation of many maize defenses via their insect hosts. This is the first report of a potential link between a microbial symbiont of an insect and a silencing effect in the insect host plant. This is also the first expression profile for a plant attacked by a root-feeding insect.
PLANT PHYSIOLOGY, 2010
Previous systems analyses in plants have focused on a single developmental stage or time point, a... more Previous systems analyses in plants have focused on a single developmental stage or time point, although it is often important to additionally consider time-index changes. During seed development a cascade of events occurs within a relatively brief time-scale. We have collected protein and transcript expression data from five sequential stages of Arabidopsis thaliana seed development encompassing the period of reserve polymer accumulation. Protein expression profiling employed 2-dimensional gel electrophoresis coupled with tandem mass spectrometry, while transcript profiling used oligonucleotide microarrays. Analyses in biological triplicate yielded robust expression information for 523 proteins and 22,746 genes across the five developmental stages, and established 319 protein/transcript pairs for subsequent pattern analysis. General linear modeling was used to evaluate the protein/transcript expression patterns. Overall, application of this statistical assessment technique showed concurrence for a slight majority (56%) of expression pairs. Many specific examples of discordant protein/transcript expression patterns were detected, suggesting that this approach will be useful in revealing examples of post-transcriptional regulation. proteomics reveals Arabidopsis thaliana gene models and proteome dynamics. Science 320: 938-941 Baginsky S, Kleffmann T, von Zychlinski A, Gruissem W (2005) Analysis of shotgun proteomics and RNA profiling data from Arabidopsis thaliana chloroplasts. J Proteome Res 4: 637-640 Baud S, Wuilleme S, To A, Rochat C, Lepiniec L (2009) Role of WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis. Plant J 60: 933-947 Box GEP, Cox DR (1964) An analysis of transformations. J Royal Stat Soc Series B 26: 211-252 Chen G, Gharib TG, Huang CC, Taylor JM, Misek DE, Kardia SL, Giordano TJ, Iannettoni MD, Orringer MB, Hanash SM, Beer DG (2002) Discordant protein and mRNA expression in lung adenocarcinomas. Mol Cell Proteomics 4: 304-313 A (2007) Integrated proteomic and transcriptomic profiling of mouse lung development and Nmyc target genes. Mol Syst Biol 3: 1-15 Degerman R (1982) Ordered binary trees constructed through an application of Kendall's tau. Psychometrika 47: 523-527 Dempster AP, Laird NM, Rubin DB (1977) Maximum likelihood from incomplete data via the EM algorithm. J Royal Stat Soc Ser B 39: 1-38 Eng JK, McCormack AL, Yates III, JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5: 976-989 Friston K (2008) Hierarchical models in the brain. PLoS Comput Biol 4: e1000211 Goldberg RB, de Paiva G, Yadegari R (1994) Plant embryogenesis: Zygote to seed. Science 266: 605-614 Griffin TJ, Gygi SP, Ideker T, Rist B, Eng J, Hood L, Aebersold R (2002) Complementary Nicolas T (2009) Data mining, a tool for systems biology or a systems biology tool. J Comput Sci Syst Biol 2: 216-218 Nie L, Wu G, Culley DE, Scholten JC, Zhang W (2007) Integrative analysis of transcriptomic and proteomic data: challenges, solutions and applications. Crit Rev Biotechnol 27: 63-75 Ørntoft TF, Thykjaer T, Waldman FM, Wolf H, Celis JE (2002) Genome-wide study of gene copy numbers, transcripts, and protein levels in pairs of non-invasive and invasive human transitional cell carcinomas. Mol Cell Proteomics 1: 37-45 Pascal LE, True LD, Campbell DS, Deutsch EW, Risk M, Coleman IM, Eichner LJ, Nelson PS, Liu AY (2008) Correlation of mRNA and protein levels: cell type-specific gene expression of cluster designation antigens in the prostate. BMC Genomics 9: 246 Piques M, Schulze WX, Höhne M, Usadel B, Gibon Y, Rohwer J, Stitt M (2009) Ribosome and transcript copy numbers, polysome occupancy and enzyme dynamics in Arabidopsis. Molec Systems Biol 5: 314 Prioul JL, Méchin V, Lessard P, Thévenot C, Grimmer M, Chateau-Joubert S, Coates S, Hartings H, Kloiber-Maitz M, Murigneux A, Sarda X, Damerval C, Edwards KJ (2008) A joint transcriptomic, proteomic and metabolic analysis of maize endosperm development and starch filling. Plant Biotechnol J 6: 855-869 Rodgers JL, Nicewander WA (1988) Thirteen ways to look at the correlation coefficient. Amer Statis 42: 59-66
Molecular Plant-Microbe Interactions, 2007
Global analysis of gene expression changes in soybean (Glycine max) and Heterodera glycines (soyb... more Global analysis of gene expression changes in soybean (Glycine max) and Heterodera glycines (soybean cyst nematode [SCN]) during the course of infection in a compatible interaction was performed using the Affymetrix GeneChip soybean genome array. Among 35,611 soybean transcripts monitored, we identified 429 genes that showed statistically significant differential expression between uninfected and nematode-infected root tissues. These included genes encoding enzymes involved in primary metabolism; biosynthesis of phenolic compounds, lignin, and flavonoids; genes related to stress and defense responses; cell wall modification; cellular signaling; and transcriptional regulation. Among 7,431 SCN transcripts monitored, 1,850 genes showed statistically significant differential expression across different stages of nematode parasitism and development. Differentially expressed SCN genes were grouped into nine different clusters based on their expression profiles during parasitism of soybean roots. The patterns of gene expression we observed in SCN suggest coordinated regulation of genes involved in parasitism. Quantitative real-time reverse-transcription polymerase chain reaction confirmed the results of our microarray analysis. The simultaneous genome-wide analysis of gene expression changes in the host and pathogen during a compatible interaction provides new insights into soybean responses to nematode infection and the first profile of transcript abundance changes occurring in the nematode as it infects and establishes a permanent feeding site within a host plant root.
Journal of Thermal Biology, 2008
Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-fiv... more Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-five mice were allocated to either chronic heat stress (34 1C) or control (24 1C) conditions for a period of 2 weeks from 47 to 60 d of age. Nineteen genes differentially expressed in liver were identified using DNA microarrays. Genes involved in the anti-oxidant pathway and metabolism were up-regulated. Genes involved in generation of reactive oxygen radicals and mitochondrial expressed genes were down-regulated. Enzyme activity measurements confirmed the array results. Mice exposed to chronic heat stress showed signs of increased oxidative stress in liver cells.
Journal of Proteome Research, 2007
Near-isogenic sunflower lines containing 25% (inbred RHA280) and 48% (RHA801) oil by seed dry mas... more Near-isogenic sunflower lines containing 25% (inbred RHA280) and 48% (RHA801) oil by seed dry mass were comparatively analyzed in biological triplicate at 18 days after flowering using two-dimensional (both pI 3-10 and 4-7) Difference Gel Electrophoresis. Additionally, two inbred lines varying in oleic acid content, HA89 (18% oleic) and HA341 (89% oleic), were also analyzed in the same manner. Statistical analyses of these sunflower lines was performed beginning with fitting a mixed effects linear model to the log-transformed optical volume of each spot to account for gel variation, followed by testing the significance between varieties for mean transformed optical spot volumes. The p-values from the spot analysis procedures were then used to find the cutoff point for differential expression using a 10% false-discovery rate (FDR). Comparison of the oil content and oleic acid composition lines revealed 77 and 42 protein spots below the 10% FDR cutoff, respectively, and were therefore declared differentially expressed. Liquid chromatography-tandem mass spectrometry analysis of each of these protein spots resulted in assignments for 44 and 17 spots, respectively. Fructokinase, plastid phosphoglycerate kinase, and enolase proteins were determined to be up-regulated in the high oil line, while phosphofructokinase, cytosolic phosphoglucomutase, and cytsolic phosphoglycerate kinase were up-regulated in the low oil variety. Additionally, four activities involved in amino acid synthesis were up-regulated in the low oil variety in addition to 12S storage proteins and a protein similar to legumin storage protein. Interestingly, two 2-DE spots identified as 14-3-3 proteins were found to be up-regulated in high oleic acid variety. Alteration of glycolytic and amino acid biosynthetic enzymes, as well as storage protein levels, suggests seed oil content is tightly linked to carbohydrate metabolism and protein synthesis in a complex manner.
Endocrinology, 2006
In ruminants, conceptus interferon-(IFNT) alters maternal physiology to accommodate a pregnancy. ... more In ruminants, conceptus interferon-(IFNT) alters maternal physiology to accommodate a pregnancy. We hypothesized that the effectiveness of IFNT on extending corpus luteum (CL) life span in nonpregnant ewes would depend upon the dose and manner of administration and would be correlated with the response in gene expression in endometrium. We anticipated that IFNT, whether administered im or by uterine infusion, would mimic changes observed in pregnancy. Ewes were assigned to five treatments: 1) uterine infusion of saline; 2) uterine infusion of ovine IFNT4 (200 g/d); 3) saline im injection; 4) im injection of IFNT4 at low dose (200 g/d); and 5) high dose (2 mg/d). CL life span was increased in groups 2 and 5, but not in 1, 3, and 4. Endometrial RNA extracted from groups 1-5 on d 14 and from d 14 pregnant and nonbred (cy-clic) ewes was used to assess expression of 70 genes on microarrays. When pregnant and cyclic ewes were compared, 30 genes were up-regulated and nine down-regulated during pregnancy. Responses were slightly less in groups 2 and 5 but were much lower in group 4. The majority of the highly upregulated genes were associated with antiviral responses. Those down-regulated included ones for IGF-II, hypoxiainducible factor 1␣, oxytocin receptor, prostaglandin F synthase, and cyclooxygenase-2. Quantitative PCR for selected genes confirmed these data and revealed that similar gene expression changes occurred in the CL of pregnant and group 2 ewes. IFNT treatment mimics pregnancy, but relatively high doses of im-injected IFNT are required to elicit a full endometrial response.
Circulation, 1998
Background-Factor VII plays a pivotal role in coagulation. Factor VIIc levels were reported to be... more Background-Factor VII plays a pivotal role in coagulation. Factor VIIc levels were reported to be a risk factor for fatal coronary heart disease (CHD). Factor VIIc and VIIag levels were noted to be positively associated with plasma triglyceride (TG) levels and influenced by a VII gene polymorphism. The purpose of this study is to determine whether these associations are related to activated factor VII (factor VIIa). Methods and Results-Fasting and 3.5-hour postprandial samples from 216 cases with subclinical atherosclerosis and 341 matched controls selected from the ARIC cohort were assayed for levels of factors VIIa, VIIc, and VIIag and TG, and factor VII codon 353 gene polymorphism. The level of factor VIIa was higher in Arg/Arg than in Arg/GlnϩGln/Gln genotypes, and the difference was in accord with that of factors VIIag and VIIc. However, the factor VIIa difference was statistically insignificant. Factor VIIa values were not correlated with fasting or 3.5-hour postprandial TG levels, nor were they associated with subclinical atherosclerosis. Conclusions-Factor VIIa levels, like factor VIIag and VIIc levels, are influenced by factor VII gene codon 353 polymorphism. However, unlike factor VIIag or VIIc, factor VIIa is not influenced by TG levels; none of these is associated with subclinical atherosclerosis. (Circulation. 1998;98:2815-2821.)
Autonomic Neuroscience, 2009
We investigated the gender effects on transient pupillary light reflex (PLR) in healthy young adu... more We investigated the gender effects on transient pupillary light reflex (PLR) in healthy young adults between 18 and 22 years old. Both dark-adapted and light-adapted PLRs were measured using green and red stimuli of different intensities. The results indicate that females had significantly larger relative constriction amplitudes than males in a dark-adapted condition. This gender effect depends on the stimulus intensities. The relative constriction amplitude in female subjects increased faster than it did in the males with the stimulus intensity. We did not observe any significant gender differences in the other PLR parameters, including latency, constriction speed, and recovery speed.
Journal of Animal Science
Fescue toxicosis affects wild and domestic animals grazing fescue pasture infected with the endop... more Fescue toxicosis affects wild and domestic animals grazing fescue pasture infected with the endophytic fungus Neotyphodium coenophialum. Signs of fescue toxicosis include increased core body temperature and respiration rate and decreased milk yield and reproductive performance. Laboratory mice also exhibit symptoms of fescue toxicosis, as indicated by reduced growth rate and reproductive performance. Mice were used to study the effects of fescue toxicosis on hepatic gene expression. Twenty-seven mice were randomly allocated to a diet containing either 50% endophyte-infected (E+; 6 ppm ergovaline) or endophyte-free (E-) fescue seed for 2 wk under thermoneutral conditions. Liver genes differentially expressed due to fescue toxicosis were identified using DNA microarray. A 2-stage ANOVA of microarray data identified 36 differentially expressed genes between mice fed E+ and E- diets. Another analysis method, significance analysis of microarray, identified 9 genes as differentially expre...