Susan Venable

Gene expression profile of long-lived Ames dwarf mice and Little mice.

Ames dwarf mice (Prop1df/df) and Little mice (Ghrhrlit/lit) are used as models of delayed aging and show significant increases in lifespan (50% and 25%, respectively) when compared with their wild‐type siblings. To gain further insight into the molecular basis for the extended longevity of these mice, we used oligonucleotide microarrays to measure levels of expression of over 14 000 RNA transcripts in liver during normal aging at 3, 6, 12 and 24 months. We found that the Prop1df/df and Ghrhrlit/lit genotypes produce dramatic alterations in gene expression, which are predominantly maintained at all ages. We found 1125 genes to be significantly affected by the Prop1df/df genotype and 1152 genes were significantly affected by the Ghrhrlit/lit genotype; 547 genes were present in both gene lists and showed parallel changes in gene expression, suggesting common mechanisms for the extended longevity in these mutants. Some of the functional gene classes most affected in these mutants included: amino acid metabolism, TCA cycle, mitochondrial electron transport, fatty acid, cholesterol and steroid metabolism, xenobiotic metabolism and oxidant metabolism. We found that the Prop1df/df genotype, and to a minor extent the Ghrhrlit/lit genotype, also produced complex alterations in age‐dependent changes in gene expression as compared with wild‐type mice. In some cases these alterations reflected a partial delay or deceleration of age‐related changes in gene expression as seen in wild‐type mice but they also introduced age‐related changes that are unique for each of these mutants and not present in wild‐type mice.

Rapid prenatal diagnosis using uncultured amniocytes and oligonucleotide array CGH

Oligonucleotide‐based array comparative genomic hybridization (array CGH) is an established method for detecting chromosomal abnormalities. Here, we explored the feasibility of using DNA extracted from uncultured amniocytes in amniotic fluid for array CGH on an oligonucleotide array platform.

MRG15 activates the B-myb promoter through formation of a nuclear complex with the retinoblastoma protein and the novel protein PAM14

The MORF4-RelatedGene on chromosome 15 (MRG15) is a member of a novel family of genes originally identified in studies to reveal cell senescence-inducing factors. MRG15 contains several predicted protein motifs, including a nuclear localization signal, a helix-loop-helix region, a leucine zipper, and a chromodomain. These motifs are commonly associated with transcription factors, suggesting that MRG15 may likewise function as a transcriptional regulator. To examine the potential function(s) of MRG15, we sought to identify cellular factors associated with thisMRG family member. In this regard, we have found that both the retinoblastoma tumor suppressor (Rb) and a novel nuclear protein PAM14 (Protein Associated with MRG,14 kDa) specifically associate with MRG15. We have further demonstrated that these interactions require the helix-loop-helix and leucine zipper domains of MRG15. Interestingly we have found all three proteins present in a multiprotein complex, suggesting that at least some of their functions may be interdependent. Although the functions of PAM14 have yet to be elucidated, Rb has several well characterized activities, including repression of E2F-activated promoters such as that of B-myb. Significantly we have demonstrated that MRG15 blocks the Rb-induced repression of this promoter, leading toB-myb promoter activation. Collectively these results suggest that MRG15 regulates transcription through interactions with a cellular protein complex containing Rb and PAM14.

Host Response to Probiotics Determined by Nutritional Status of Rotavirus-infected Neonatal Mice

Objectives: Beneficial microbes and probiotics are promising agents for the prevention and treatment of enteric and diarrheal diseases in children; however, little is known about their in vivo mechanisms of action. We used a neonatal mouse model of rotavirus diarrhea to gain insight into how probiotics ameliorate acute gastroenteritis. Methods: Rotavirus-infected mice were treated with 1 of 2 strains of human-derived Lactobacillus reuteri. We assessed intestinal microbiome composition with 16S metagenomic sequencing, enterocyte migration and proliferation with 5-bromo-2′-deoxyuridine, and antibody and cytokine concentrations with multiplex analyses of intestinal explant cultures. Results: Probiotics reduced diarrhea duration, improved intestinal histopathology, and enhanced intestinal microbiome richness and phylogenetic diversity. The magnitude of reduction of diarrhea by probiotics was strain specific and influenced by nutritional status. L reuteri DSM 17938 reduced diarrhea duration by 0, 1, and 2 days in underweight, normal weight, and overweight pups, respectively. The magnitude of reduction of diarrhea duration correlated with increased enterocyte proliferation and migration. Strain ATCC PTA 6475 reduced diarrhea duration by 1 day in all of the mice without increasing enterocyte proliferation. Both probiotic strains decreased concentrations of proinflammatory cytokines, including macrophage inflammatory protein-1&agr; and interleukin-1&bgr;, in all of the animals, and increased rotavirus-specific antibodies in all but the underweight animals. Body weight also influenced the host response to rotavirus, in terms of diarrhea duration, enterocyte turnover, and antibody production. Conclusions: These data suggest that probiotic enhancement of enterocyte proliferation, villus repopulation, and virus-specific antibodies may contribute to diarrhea resolution, and that nutritional status influences the host response to both beneficial microbes and pathogens.

From Prediction to Function Using Evolutionary Genomics: Human-Specific Ecotypes of Lactobacillus reuteri Have Diverse Probiotic Functions

The vertebrate gut symbiont Lactobacillus reuteri has diversified into separate clades reflecting host origin. Strains show evidence of host adaptation, but how host–microbe coevolution influences microbial-derived effects on hosts is poorly understood. Emphasizing human-derived strains of L. reuteri, we combined comparative genomic analyses with functional assays to examine variations in host interaction among genetically distinct ecotypes. Within clade II or VI, the genomes of human-derived L. reuteri strains are highly conserved in gene content and at the nucleotide level. Nevertheless, they share only 70–90% of total gene content, indicating differences in functional capacity. Human-associated lineages are distinguished by genes related to bacteriophages, vitamin biosynthesis, antimicrobial production, and immunomodulation. Differential production of reuterin, histamine, and folate by 23 clade II and VI strains was demonstrated. These strains also differed with respect to their ability to modulate human cytokine production (tumor necrosis factor, monocyte chemoattractant protein-1, interleukin [IL]-1β, IL-5, IL-7, IL-12, and IL-13) by myeloid cells. Microarray analysis of representative clade II and clade VI strains revealed global regulation of genes within the reuterin, vitamin B12, folate, and arginine catabolism gene clusters by the AraC family transcriptional regulator, PocR. Thus, human-derived L. reuteri clade II and VI strains are genetically distinct and their differences affect their functional repertoires and probiotic features. These findings highlight the biological impact of microbe:host coevolution and illustrate the functional significance of subspecies differences in the human microbiome. Consideration of host origin and functional differences at the subspecies level may have major impacts on probiotic strain selection and considerations of microbial ecology in mammalian species.

Transcriptional Profiling of Bipotential Embryonic Liver Cells to Identify Liver Progenitor Cell Surface Markers

The ability to purify to homogeneity a population of hepatic progenitor cells from adult liver is critical for their characterization prior to any therapeutic application. As a step in this direction, we have used a bipotential liver cell line from 14 days postcoitum mouse embryonic liver to compile a list of cell surface markers expressed specifically by liver progenitor cells. These cells, known as bipotential mouse embryonic liver (BMEL) cells, proliferate in an undifferentiated state and are capable of differentiating into hepatocyte‐like and cholangiocyte‐like cells in vitro. Upon transplantation, BMEL cells are capable of differentiating into hepatocytes and cholangiocytes in vivo. Microarray and Gene Ontology (GO) analysis of gene expression in the 9A1 and 14B3 BMEL cell lines grown under proliferating and differentiating conditions was used to identify cell surface markers preferentially expressed in the bipotential undifferentiated state. This analysis revealed that proliferating BMEL cells express many genes involved in cell cycle regulation, whereas differentiation of BMEL cells by cell aggregation causes a switch in gene expression to functions characteristic of mature hepatocytes. In addition, microarray data and protein analysis indicated that the Notch signaling pathway could be involved in maintaining BMEL cells in an undifferentiated stem cell state. Using GO annotation, a list of cell surface markers preferentially expressed on undifferentiated BMEL cells was generated. One marker, Cd24a, is specifically expressed on progenitor oval cells in livers of diethyl 1,4‐dihydro‐2,4,6‐trimethyl‐3,5‐pyridinedicarboxylate‐treated animals. We therefore consider Cd24a expression a candidate molecule for purification of hepatic progenitor cells.

Anti-inflammatory Properties of Gastric-derived Lactobacillus plantarum XB7 in the Context of Helicobacter pylori Infection

Helicobacter pylori colonization of the gastric epithelium induces interleukin‐8 (IL‐8) production and inflammation leading to host cell damage. We searched for gastric‐derived Lactobacillus with the ability to suppress H. pylori‐induced inflammation.

Studies demonstrating the complexity of regulation and action of the growth inhibitory gene SDI1.

The identification of the DNA synthesis inhibitory gene SDI1 by investigators studying cell senescence, tumor suppression, cell cycle control and differentiation suggest a key regulatory role for this gene. To better understand the growth regulatory activity of this gene we proceeded with the experiments described here. The data demonstrate that SDI1 is an important downstream effector of p53, but here we report that it can also cause inhibition of DNA synthesis in several immortal human cell lines, independent of p53 or Rb status. Levels of SDI1 mRNA expression in immortal cells are consistently much lower than that of normal cells, indicating that immortalization is not compatible with high expression of SDI1. These results highlight the complex nature of regulation of this gene and its mode of action.

Fusobacterium Nucleatum Subspecies Animalis Influences Proinflammatory Cytokine Expression and Monocyte Activation in Human Colorectal Tumors

Chronic infection and associated inflammation have long been suspected to promote human carcinogenesis. Recently, certain gut bacteria, including some in the Fusobacterium genus, have been implicated in playing a role in human colorectal cancer development. However, the Fusobacterium species and subspecies involved and their oncogenic mechanisms remain to be determined. We sought to identify the specific Fusobacterium spp. and ssp. in clinical colorectal cancer specimens by targeted sequencing of Fusobacterium 16S ribosomal RNA gene. Five Fusobacterium spp. were identified in clinical colorectal cancer specimens. Additional analyses confirmed that Fusobacterium nucleatum ssp. animalis was the most prevalent F. nucleatum subspecies in human colorectal cancers. We also assessed inflammatory cytokines in colorectal cancer specimens using immunoassays and found that expression of the cytokines IL17A and TNFα was markedly increased but IL21 decreased in the colorectal tumors. Furthermore, the chemokine (C-C motif) ligand 20 was differentially expressed in colorectal tumors at all stages. In in vitro co-culture assays, F. nucleatum ssp. animalis induced CCL20 protein expression in colorectal cancer cells and monocytes. It also stimulated the monocyte/macrophage activation and migration. Our observations suggested that infection with F. nucleatum ssp. animalis in colorectal tissue could induce inflammatory response and promote colorectal cancer development. Further studies are warranted to determine if F. nucleatum ssp. animalis could be a novel target for colorectal cancer prevention and treatment. Cancer Prev Res; 10(7); 398–409. ©2017 AACR.