Marcia L. Hart

Comparative Evaluation of DNA Extraction Methods from Feces of Multiple Host Species for Downstream Next-Generation Sequencing

The gastrointestinal tract contains a vast community of microbes that to this day remain largely unculturable, making studies in this area challenging. With the newly affordable advanced sequencing technology, important breakthroughs in this exciting field are now possible. However, standardized methods of sample collection, handling, and DNA extraction have yet to be determined. To help address this, we investigated the use of 5 common DNA extraction methods on fecal samples from 5 different species. Our data show that the method of DNA extraction impacts DNA concentration and purity, successful NGS amplification, and influences microbial communities seen in NGS output dependent on the species of fecal sample and the DNA extraction method used. These data highlight the importance of careful consideration of DNA extraction method used when designing and interpreting data from cross species studies.

The utility of Apc-mutant rats in modeling human colon cancer.

Prior to the advent of genetic engineering in the mouse, the rat was the model of choice for investigating the etiology of cancer. Now, recent advances in the manipulation of the rat genome, combined with a growing recognition of the physiological differences between mice and rats, have reignited interest in the rat as a model of human cancer. Two recently developed rat models, the polyposis in the rat colon (Pirc) and Kyoto Apc Delta (KAD) strains, each carry mutations in the intestinal-cancer-associated adenomatous polyposis coli (Apc) gene. In contrast to mouse models carrying Apc mutations, in which cancers develop mainly in the small intestine rather than in the colon and there is no gender bias, these rat models exhibit colonic predisposition and gender-specific susceptibility, as seen in human colon cancer. The rat also provides other experimental resources as a model organism that are not provided by the mouse: the structure of its chromosomes facilitates the analysis of genomic events, the size of its colon permits longitudinal analysis of tumor growth, and the size of biological samples from the animal facilitates multiplexed molecular analyses of the tumor and its host. Thus, the underlying biology and experimental resources of these rat models provide important avenues for investigation. We anticipate that advances in disease modeling in the rat will synergize with resources that are being developed in the mouse to provide a deeper understanding of human colon cancer.

N-acetylcysteine amide, a promising antidote for acetaminophen toxicity.

Acetaminophen (N-acetyl-p-aminophenol, APAP) is one of the most widely used over the counter antipyretic and analgesic medications. It is safe at therapeutic doses, but its overdose can result in severe hepatotoxicity, a leading cause of drug-induced acute liver failure in the USA. Depletion of glutathione (GSH) is one of the initiating steps in APAP-induced hepatotoxicity; therefore, one strategy for restricting organ damage is to restore GSH levels by using GSH prodrugs. N-acetylcysteine (NAC), a GSH precursor, is the only currently approved antidote for an acetaminophen overdose. Unfortunately, fairly high doses and longer treatment times are required due to its poor bioavailability. In addition, oral and I.V. administration of NAC in a hospital setting are laborious and costly. Therefore, we studied the protective effects of N-acetylcysteine amide (NACA), a novel antioxidant with higher bioavailability, and compared it with NAC in APAP-induced hepatotoxicity in C57BL/6 mice. Our results showed that NACA is better than NAC at a low dose (106mg/kg) in preventing oxidative stress and protecting against APAP-induced damage. NACA significantly increased GSH levels and the GSH/GSSG ratio in the liver to 66.5% and 60.5% of the control, respectively; and it reduced the level of ALT by 30%. However, at the dose used, NAC was not effective in combating the oxidative stress induced by APAP. Thus, NACA appears to be better than NAC in reducing the oxidative stress induced by APAP. It would be of great value in the health care field to develop drugs like NACA as more effective and safer options for the prevention and therapeutic intervention in APAP-induced toxicity.

Differing Complex Microbiota Alter Disease Severity of the IL-10−/− Mouse Model of Inflammatory Bowel Disease

It is estimated that 1.4 million people in the United States suffer from Inflammatory Bowel Disease (IBD), with an overall annual health care cost of more than

The RNA-Binding Protein HuR Posttranscriptionally Regulates IL-2 Homeostasis and CD4+ Th2 Differentiation

1.7 billion. Although the exact etiology of this disease remains unknown, research suggests that it is a multifactorial disease associated with aberrant gastrointestinal microbial populations (dysbiosis). The C57BL/6 and C3H/HeJBir mouse strains with targeted mutations in the IL-10 gene are commonly used models to study IBD. However, anecdotally, disease phenotype can vary in severity from lab to lab. Moreover, studies using germfree and monocolonized mice have suggested that gut microbiota (GM) are critical to disease induction in these models. With recent studies suggesting variation in naturally occurring GM composition and complexity among mouse producers, we hypothesized that differences in these naturally occurring complex GM profiles may modulate disease severity in the IL-10−/− mouse model. To test this hypothesis, we use a technique referred to as complex microbiota targeted rederivation (CMTR) to transfer genetically identical C57BL/6 IL-10−/− and C3H/HeJBir IL-10−/− embryos into surrogate CD-1 or C57BL/6 dams from different commercial producers with varying microbiota complexity and composition. We found that disease severity significantly and reproducibly differed among mice in both IL-10−/− strains, dependent on differing maternally inherited GM. Furthermore, disease severity was associated with alterations in relative abundance of several physiologically relevant bacterial species. These findings suggest that the composition of the resident GM is a primary determinant of disease severity in IBD and provide proof-of-concept that CMTR can be used to investigate the contribution of contemporary complex GM on disease phenotype and reproducibility.

Development of outbred CD1 mouse colonies with distinct standardized gut microbiota profiles for use in complex microbiota targeted studies

Posttranscriptional gene regulation by RNA-binding proteins, such as HuR (elavl1), fine-tune gene expression in T cells, leading to powerful effects on immune responses. HuR can stabilize target mRNAs and/or promote translation by interacting with their 3′ untranslated region adenylate and uridylate–rich elements. It was previously demonstrated that HuR facilitates Th2 cytokine expression by mRNA stabilization. However, its effects upon IL-2 homeostasis and CD4+ Th2 differentiation are not as well understood. We found that optimal translation of Il2ra (CD25) required interaction of its mRNA with HuR. Conditional HuR knockout in CD4+ T cells resulted in loss of IL-2 homeostasis and defects in JAK–STAT signaling, Th2 differentiation, and cytokine production. HuR-knockout CD4+ T cells from OVA-immunized mice also failed to proliferate in response to Ag. These results demonstrate that HuR plays a pivotal role in maintaining normal IL-2 homeostasis and initiating CD4+ Th2 differentiation.

Abstract LB-032: A rat knockout of p53 that exhibits high rates of osteosarcoma and menigial sarcoma with frequent metastasis

Studies indicate that the gut microbiota (GM) can significantly influence both local and systemic host physiologic processes. With rising concern for optimization of experimental reproducibility and translatability, it is essential to consider the GM in study design. However, GM profiles can vary between rodent producers making consistency between models challenging. To circumvent this, we developed outbred CD1 mouse colonies with stable, complex GM profiles that can be used as donors for a variety of GM transfer techniques including rederivation, co-housing, cross-foster, and fecal microbiota transfer (FMT). CD1 embryos were surgically transferred into CD1 or C57BL/6 surrogate dams that varied by GM composition and complexity to establish four separate mouse colonies harboring GM profiles representative of contemporary mouse producers. Using targeted 16S rRNA amplicon sequencing, subsequent female offspring were found to have similar GM profiles to surrogate dams. Furthermore, breeding colonies of CD1 mice with distinct GM profiles were maintained for nine generations, demonstrating GM stability within these colonies. To confirm GM stability, we shipped cohorts of these four colonies to collaborating institutions and found no significant variation in GM composition. These mice are an invaluable experimental resource that can be used to investigate GM effects on mouse model phenotype.

Gut Dysbiosis and Neurobehavioral Alterations in Rats Exposed to Silver Nanoparticles

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Somatic mutations in the Tp53 tumor suppressor gene are the most commonly seen genetic alterations in cancer, and germline mutations in Tp53 predispose individuals to a variety of early-onset cancers. Development of appropriate translational animal models that carry mutations in Tp53 and recapitulate human disease are important for drug discovery, biomarker development, and disease modeling. Current Tp53 mouse and rat models have significant phenotypic and genetic limitations and often do not recapitulate certain aspects of human disease or have genetic limitations. We used a marker- assisted speed congenic approach to transfer a well-characterized Tp53 mutant allele from an outbred Sprague Dawley (SD) rat stock to the genetically inbred Fischer 344 rat to create the F344-Tp53tm1(EGFP-Pac)QlyRrrc rat strain. On the F344 genetic background, the tumor spectrum shifted, with the primary tumor types being osteosarcomas (32%) and meningeal sarcomas (32%), compared to the hepatic hemangiosarcoma (80% incidence in homozygotes) and lymphoma (55% incidence in heterozygotes) identified in the original outbred stock model. The Fischer model is more consistent with the early onset of bone and central nervous system sarcomas found in humans with germline Tp53 mutations. Osteosarcomas represented 36% and 31% of tumors that developed, in homozygous and heterozygous animals, respectively, and were highly representative of the human disease radiographically and histologically, with tumors harbored primarily on long bones with frequent pulmonary metastases. The inbred F344-Tp53tm1(EGFP-Pac)QlyRrrc knockout rat is a powerful model to investigate compelling questions surrounding the development of osteosarcomas and meningeal sarcomas. The inbred genetic background allows for easy maintenance of the model and introgression of additional mutations that already exist on the F344 background. We crossed the F344-Tp53tm1(EGFP-Pac)QlyRrrc rats to the F344-ApcPirc rat strain to examine the effects on tumor development. Tp53; Pirc double heterozygotes showed no difference in colonic or overall intestinal tumor number. Homozygous Tp53 heterozygous for the Pirc allele exhibited early onset pancreatic adenocarcinoma and rhabdomyosarcoma. There was also a significant absence of female homozygous Tp53 knockouts regardless of Apc status. Importantly, the rapid onset of osteosarcomas in the F344-Tp53tm1(EGFP-Pac)QlyRrrc fills a current void in animal models that recapitulate human pediatric osteosarcomas and may facilitate studies to identify therapeutic targets. Citation Format: James Amos-Landgraf, Sarah Hansen, Marcia Hart, Susheel Busi, Kevin Jones, Elizabeth Bryda. A rat knockout of p53 that exhibits high rates of osteosarcoma and menigial sarcoma with frequent metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-032.