Surrogate Dams Research Articles

Fetal programming by the parental microbiome of offspring behavior, and DNA methylation and gene expression within the hippocampus.

The microorganisms colonizing the gastrointestinal tract of animals, collectively referred to as the gut microbiome, affect numerous host behaviors dependent on the central nervous system (CNS). Studies comparing germ-free mice to normally colonized mice have demonstrated influences of the microbiome on anxiety-related behaviors, voluntary activity, and gene expression in the CNS. Additionally, there is epidemiologic evidence supporting an intergenerational influence of the maternal microbiome on neurodevelopment of offspring and behavior later in life. There is limited experimental evidence however directly linking the maternal microbiome to long-term neurodevelopmental outcomes, or knowledge regarding mechanisms responsible for such effects. Here we show that that the maternal microbiome has a dominant influence on several offspring phenotypes including anxiety-related behavior, voluntary activity, and body weight. Adverse outcomes in offspring were associated with features of the maternal microbiome including bile salt hydrolase activity gene expression ( bsh ), abundance of certain bile acids, and hepatic expression of Slc10a1 . In cross-foster experiments, offspring resembled their birth dam phenotypically, despite faithful colonization in the postnatal period with the surrogate dam microbiome. Genome-wide methylation analysis of hippocampal DNA identified microbiome- associated differences in methylation of 196 loci in total, 176 of which show conserved profiles between mother and offspring. Further, single-cell transcriptional analysis revealed accompanying differences in expression of several differentially methylated genes within certain hippocampal cell clusters, and vascular expression of genes associated with bile acid transport. Inferred cell-to-cell communication in the hippocampus based on coordinated ligand-receptor expression revealed differences in expression of neuropeptides associated with satiety. Collectively, these data provide proof-of-principle that the maternal gut microbiome has a dominant influence on the neurodevelopment underlying certain offspring behaviors and activities, and selectively affects genome methylation and gene expression in the offspring CNS in conjunction with that neurodevelopment.

Report on a Milking Mule: Milk Qualitative Characteristics during Lactation.

Despite their inability to reproduce naturally, mules can host embryos and be surrogate dams. The aim of this investigation was to increase our knowledge of the qualitative composition of mule's milk and its variations throughout the whole lactation period-namely, from 6 h after foaling to 180 days in milk (DIM). Milk was obtained from a mule dam that had foaled after receiving a mule embryo transfer. For each sample, the gross, mineral, and fatty acid composition was evaluated. The average quality of the mule milk was as follows: protein 1.97 g 100 mL-1, fat 0.90 g 100 mL-1, and ash 0.39 g 100 mL-1. Saturated fatty acids made up, on average, 50.00 g 100 g-1 of fat. Monounsaturated and polyunsaturated fatty acids made up half of the total fatty acid content (31.80 g 100 g-1 and 18.2 g 100 g-1 of fat, respectively). Linoleic acid and linolenic acid were the main polyunsaturated fatty acids in the milk. The milk composition changed throughout lactation. Dry matter, protein, fat, and ash decreased significantly from early lactation (6 h to 14 DIM). The n3 polyunsaturated fatty acids decreased at the end of lactation. The changes in milk composition throughout lactation are probably due to adaptations to the growth requirements of the foal.

Impact of cesarean section on metabolic syndrome components in offspring rats.

Epidemiological evidence suggests an association between CS and offspring metabolic syndrome (MetS), but whether a causal relationship exists is unknown. In this study, timed-mated Wistar rat dams were randomly assigned to cesarean section(CS), vaginal delivery (VD), and surrogate groups. The offspring from both CS and VD groups were reared by surrogate dams until weaning, and weaned male offspring from both groups were randomly assigned to receive normal diet (ND) or high-fat/high-fructose diet (HFF) ad libitum for 39 weeks. By the end of study, CS-ND offspring gained 17.8% more weight than VD-ND offspring, while CS-HFF offspring gained 36.4% more weight than VD-HFF offspring. Compared with VD-ND offspring, CS-ND offspring tended to have increased triglycerides (0.27 mmol/l, 95% CI, 0.05 to 0.50), total cholesterol (0.30 mmol/l, -0.08 to 0.68), and fasting plasma glucose (FPG) (0.30 mmol/l, -0.01 to 0.60); more pronounced differences were observed between CS-HFF and VD-HFF offspring in these indicators (triglyceride, 0.66 mmol/l, 0.35 to 0.97; total cholesterol, 0.46 mmol/l, 0.13 to 0.79; and FPG, 0.55 mmol/l, 0.13 to 0.98). CS offspring were more prone to adverse metabolic profile and HFF might exacerbate this condition, indicating the association between CS and MetS is likely to be causal. Whether the observed associations between CS and MetS in non-randomized human studies are causally relevant remains undetermined. Compared with vaginally born offspring rats, CS born offspring gained more body weight and tended to have compromised lipid profiles and abnormal insulin sensitivity, suggesting a causal relationship between CS and MetS that may be further amplified by a high-fat/high-fructose diet. Due to the high prevalence of CS births globally, greater clinical consideration must be given to the potential adverse effects of CS, and whether these risks should be made known to patients in clinical practice merits evaluation.

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control.

The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the myostatin gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.

Supplier-origin mouse microbiomes significantly influence locomotor and anxiety-related behavior, body morphology, and metabolism

The mouse is the most commonly used model species in biomedical research. Just as human physical and mental health are influenced by the commensal gut bacteria, mouse models of disease are influenced by the fecal microbiome (FM). The source of mice represents one of the strongest influences on the FM and can influence the phenotype of disease models. The FM influences behavior in mice leading to the hypothesis that mice of the same genetic background from different vendors, will have different behavioral phenotypes. To test this hypothesis, colonies of CD-1 mice, rederived via embryo transfer into surrogate dams from four different suppliers, were subjected to phenotyping assays assessing behavior and physiological parameters. Significant differences in behavior, growth rate, metabolism, and hematological parameters were observed. Collectively, these findings show the profound influence of supplier-origin FMs on host behavior and physiology in healthy, genetically similar, wild-type mice maintained in identical environments.

Modification of Fecal Bacteria Counts and Blood Immune Cells in the Offspring of BALB/c and C57BL/6 Mice Obtained through Interstrain Mouse Embryo Transfer.

The reproducibility of results obtained with rodent models depends on the genetic purity of the strain and the stability of the environment. However, another potential factor is changes in the gut microbiota due to the transmission of mother's bacteria during embryo transfer. In this study, we demonstrate the transmission of the microbiota and immune cell blood phenotype to the offspring of 2 strains, C57BL/6JNskrc and BALB/cJNskrc, from surrogate dams of different genotypes. Interstrain embryo transfer resulted in a change in the number of Enterococcus spp. organisms, as shown by quantitative PCR analysis. The number of blood leukocytes was also affected, as estimated by flow cytometry. The number of blood leukocytes, including B cells and helper T cells, and the number of Enterococcus spp. organisms in male C57BL/6JNskrc offspring born to BALB/cJNskrc surrogate dams became similar to those of male BALB/cJNskrc mice born to BALB/cJNskrc dams. Likewise, the same parameters of male BALB/cJNskrc mice born to C57BL/6JNskrc dams became similar to those of male C57BL/6JNskrc offspring. Researchers should be aware of the possible transmission of the dam's microbiota and immune cell phenotypes to the experimental strains when planning embryo transfer experiments, because these factors could affect the experimental outcomes or the reproducibility of experimental results.

The gut microbiota modulates differential adenoma suppression by B6/J and B6/N genetic backgrounds in ApcMin mice

Tumor multiplicity in the ApcMin (Min) mouse model of CRC is a classic quantitative trait that is subject to complex genetic and environmental factors, and therefore serves as an ideal platform to study modifiers of disease. While disparate inbred genetic backgrounds have well-characterized modifying effects on tumor multiplicity, it is unclear whether more closely related backgrounds such as C57BL/6J and C57BL6/N differentially modify the phenotype. Furthermore, it is unknown whether the complex gut microbiota (GM) influences the effects of these background strains. We assessed tumor multiplicity in F1 mice generated from the original Min colony from the McArdle Laboratory at the University of Wisconsin (C57BL/6JMlcr-ApcMin) crossed with either C57BL/6J or C57BL/6N wild-type mice. We also used complex microbiota targeted rederivation to rederive B6NB6JMF1-ApcMin embryos using surrogate dams harboring complex GMs from two different sources to determine the effects of complex GM. Both B6/J and B6/N backgrounds significantly repressed tumor multiplicity. However, the B6/N background conferred a stronger dominant suppressive effect than B6/J. Moreover, we observed that complex GM likely modulated B6/N-mediated adenoma repression such that two distinct communities conferred differential tumor multiplicity in isogenic B6NB6JMF1-ApcMin mice. Although we cannot rule out possible maternal effects of embryo transfer, we show that B6/J and B6/N have modifier effects on Min, and these effects are further altered by the complex GM. Foremost, strict attention to genetic background and environmental variables influencing the GM is critical to enhance reproducibility in models of complex disease traits.

The History of Transgenesis.

A transgenic mouse carries within its genome an artificial DNA construct (transgene) that is deliberately introduced by an experimentalist. These animals are widely used to understand gene function and protein function. When addressing the history of transgenic mouse technology, it is apparent that a number of basic science research areas laid the groundwork for success. These include reproductive science, genetics and molecular biology, and micromanipulation and microscopy equipment. From reproductive physiology came applications on how to optimize mouse breeding, how to superovulate mice to produce zygotes for DNA microinjection or preimplantation embryos for combination with embryonic stem (ES) cells, and how to return zygotes and embryos to a pseudopregnant surrogate dam for gestation and birth. From developmental biology, it was learned how to micromanipulate embryos for morula aggregation and blastocyst microinjection and how to establish germline competent ES cells. From genetics came the foundational principles governing the inheritance of genes, the interactions of gene products, and an understanding of the phenotypic consequences of genetic mutations. From molecular biology came a panoply of tools and reagents that are used to clone DNA transgenes, to detect the presence of transgenes, to assess gene expression by measuring transcription, and to detect proteins in cells and tissues. Technical advances in light microscopes, micromanipulators, micropipette pullers, and ancillary equipment made it possible for experimentalists to insert thin glass needles into zygotes or embryos under controlled conditions to inject DNA solutions or ES cells. To fully discuss the breadth of contributions of these numerous scientific disciplines to a comprehensive history of transgenic science is beyond the scope of this work. Examples will be used to illustrate scientific developments central to the foundation of transgenic technology and that are in use today.

Assessment of microchimerism following somatic cell nuclear transfer and natural pregnancies in goats.

Microchimerism is defined as the presence of a small population of cells or DNA in 1 organism originated from a genetically different organism. It is well established that this phenomenon occurs in humans and mice as cells are exchanged between mother and fetus during gestation. Currently, no information is available about the presence of maternal microchimerism in goats, and the only published study is limited to an evaluation of fetal and fetal-fetal microchimerism in blood samples following natural breeding. In order to determine whether bidirectional fetal-maternal cell or DNA trafficking occurs in goats, we assessed: 1) fetal microchimerism in surrogates that gave birth to somatic cell nuclear transfer (SCNT)-derived transgenic offspring (n = 4), 2) maternal microchimerism following natural breeding of SCNT-derived transgenic does with a nontransgenic buck (n = 4), and 3) fetal-fetal microchimerism in nontransgenic twins of transgenic offspring (n = 3). Neomycin-resistance gene (NEO) gene was selected as the marker to detect the presence of the αMHC-TGF-β1-Neo transgene in kidney, liver, lung, lymph node, and spleen. We found no detectable maternal or fetal-fetal microchimerism in the investigated tissues of nontransgenic offspring. However, fetal microchimerism was detected in lymph node tissue of one of the surrogate dams carrying a SCNT pregnancy. These results indicate occurrence of cell trafficking from fetus to mother during SCNT pregnancies. The findings of this study have direct implications on the use and disposal of nontransgenic surrogates and nontransgenic offspring.

The Long-Term Impairment in Redox Homeostasis Observed in the Hippocampus of Rats Subjected to Global Perinatal Asphyxia (PA) Implies Changes in Glutathione-Dependent Antioxidant Enzymes and TIGAR-Dependent Shift Towards the Pentose Phosphate Pathways: Effect of Nicotinamide.

We have recently reported that global perinatal asphyxia (PA) induces a regionally sustained increase in oxidized glutathione (GSSG) levels and GSSG/GSH ratio, a decrease in tissue-reducing capacity, a decrease in catalase activity, and an increase in apoptotic caspase-3-dependent cell death in rat neonatal brain up to 14 postnatal days, indicating a long-term impairment in redox homeostasis. In the present study, we evaluated whether the increase in GSSG/GSH ratio observed in hippocampus involves changes in glutathione reductase (GR) and glutathione peroxidase (GPx) activity, the enzymes reducing glutathione disulfide (GSSG) and hydroperoxides, respectively, as well as catalase, the enzyme protecting against peroxidation. The study also evaluated whether there is a shift in the metabolism towards the penthose phosphate pathway (PPP), by measuring TIGAR, the TP53-inducible glycolysis and apoptosis regulator, associated with delayed cell death, further monitoring calpain activity, involved in bax-dependent cell death, and XRCC1, a scaffolding protein interacting with genome sentinel proteins. Global PA was induced by immersing fetus-containing uterine horns removed by a cesarean section from on term rat dams into a water bath at 37°C for 21min. Asphyxia-exposed and sibling cesarean-delivered fetuses were manually resuscitated and nurtured by surrogate dams. Animals were euthanized at postnatal (P) days 1 or 14, dissecting samples from hippocampus to be assayed for glutathione, GR, GPx (all by spectrophotometry), catalase (Western blots and ELISA), TIGAR (Western blots), calpain (fluorescence), and XRCC1 (Western blots). One hour after delivery, asphyxia-exposed and control neonates were injected with either 100μl saline or 0.8mmol/kg nicotinamide, i.p., shown to protect from the short- and long-term consequences of PA. It was found that global PA produced (i) a sustained increase of GSSG levels and GSSG/GSH ratio at P1 and P14; (ii) a decrease of GR, GPx, and catalase activity at P1 and P14; (iii) a decrease at P1, followed by an increase at P14 of TIGAR levels; (iv) an increase of calpain activity at P14; and (v) an increase of XRCC1 levels, but only at P1. (vi) Nicotinamide prevented the effect of PA on GSSG levels and GSSG/GSH ratio, and on GR, GPx, and catalase activity, also on increased TIGAR levels and calpain activity observed at P14. The present study demonstrates that the long-term impaired redox homeostasis observed in the hippocampus of rats subjected to global PA implies changes in GR, GPx, and catalase, and a shift towards PPP, as indicated by an increase of TIGAR levels at P14.

Success of embryo transfer in mice with freshly collected and cryopreserved two-cell embryos with different genetic backgrounds correlated with the number of transferred embryos: A 5-year retrospective analysis.

Embryo transfer of pre-implantation embryos to surrogate dams is a key technique for the hygienic sanitation of strains, cryopreservation, in vitro fertilization, genetic modification and engineering. However, the effects of several parameters, such as the number of transferred embryos, on the success of embryo transfer are not well studied. In this retrospective study, we reanalysed 1320 embryo transfers of two-cell embryos originating from genetically altered donors, which were performed under routine conditions in our facility over a period of 5 years. Of them, 453 embryo transfers were done with freshly collected embryos and 867 transfers were performed with cryopreserved embryos. Despite the fact that the genetic background of the embryo donors was quite heterogeneous, we found that the transfer of ≥ 21 embryos reduced the success of embryo transfers for freshly collected embryos in correlation with the number of pregnancies and born pups, whereas this was not the case for transfer in the cryopreservation group. Most pregnancies were achieved after embryo transfer of 10-20 freshly collected embryos (90.4%), which dropped to 37.5% if more embryos were transferred. The highest pregnancy rates in the cryopreservation group were achieved if 15-17 embryos were transferred (62.9%). Despite the fact that the precise substrains were only rarely defined, we confirmed that beside the number of transferred embryos, the genetic background of the donors had an influence on the success of embryo transfer. Significantly more embryos in a C57BL/6 background developed to term than embryos on a BALB/c background.

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

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.

Abstract 4076: Gut microbiota and metabolite-driven phenotype modulation in a mouse model of colorectal cancer

Abstract Colorectal cancer (CRC) is a multifactorial disease that develops as a result of multiple genetic mutations, often including loss of the tumor suppressor Adenomatous Polyposis Coli (APC) gene. More recently, additional factors such as the host gut microbiota (GM) have also demonstrated an important role in tumor initiation and progression, yet our understanding of most commensal bacteria remains limited. We observed significant differences in intestinal tumor multiplicity between two ApcMin sub-strain mouse colonies; C57BL/6J-ApcMin (B6-Min/J) mice from The Jackson Laboratory had relatively lower adenoma burden while C57BL/6JD-ApcMin (B6-Min/D) mice from the University of Wisconsin develop twice the number of adenomas. To determine the underlying causes of this phenotypic variability, we used complex microbiota targeted rederivation (CMTR) to rederive embryos of the two Apc mutant sub-strains onto surrogate dams harboring distinct GM from two different vendors (GMJAX and GMHSD), generating four ApcMin groups: B6-Min/JGMJAX, B6-Min/JGMHSD, B6-Min/DGMJAX, and B6-Min/DGMHSD. We observed significant increases in intestinal tumor multiplicity in both B6-Min groups harboring the GMHSD compared to their GMJAX counterparts, suggesting that the GM accounts for a significant portion of variability in tumor phenotype between the ApcMin sub-strains. We characterized the GMJAX and GMHSD microbial populations using 16S rRNA gene sequencing to identify potentially causative and protective bacterial taxa. Relative abundances of several taxa including Bilophila sp., Desulfovibrio sp., and Akkermansia muciniphila, were significantly elevated in GMHSD suggesting pro-tumorigenic roles, while the family Peptococcaceae was more abundant in GMJAX, indicating a potential protective role. To establish the functional output of these distinct GM profiles contributing to tumor development, we used liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to evaluate differences in the intestinal metabolome of the four experimental groups. Notably, we found that the microbiome differences due to colonization with either GMJAX or GMHSD primarily accounted for distinct metabolomic profiles, whereas host genetic differences between the sub-strains had a minimal role in shaping the metabolomes. We detected 31 putative metabolites that were significantly modulated by the GM in both the B6-Min/J and B6-Min/D sub-strains, and were therefore predictive of the relative tumor burden. Moving forward, these metabolites have implications for both biomarker discovery and potential therapeutic and preventative targets. Overall, these results provide insight regarding environmental factors influencing tumorigenesis, and further demonstrate how host-environment interactions can modulate a given disease phenotype. Citation Format: Jacob E. Moskowitz, Zhentian Lei, Susheel Busi, Marcia Hart, Craig Franklin, Lloyd Sumner, James Amos-Landgraf. Gut microbiota and metabolite-driven phenotype modulation in a mouse model of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4076.

Abstract 2400: Complex gut microbiota modulate rat colon adenoma susceptibility, metabolites, and host gene expression

Abstract We used a multi-omics approach to identify signatures of differential susceptibility in the F344/NTac-Apc+/Pirc (Pirc) rat model of familial adenomatous polyposis. We assessed how different gut microbiomes (GMs) effect the metabolome and host transcriptome to reveal mechanisms of susceptibility. We previously found that differences in the GM are causative determinants of cancer susceptibility using complex microbiota targeted rederivation (CMTR), in which isogenic Pirc rat embryos are transferred into surrogate dams each harboring distinct specific pathogen free gut microbiota: GM-1 and GM-2. Fecal samples collected from CMTR Pirc rats at 1 month of age were analyzed using ultra-high performance liquid chromatography mass spectrometry (UHPLC/MS) to assess the impact of bacterial metabolites on disease susceptibility. At 6 months of age animals were sacrificed, tumor burden assessed, and normal colonic epithelium (NE) and tumor (T) tissues were collected for RNASeq analysis. Metabolomic analysis identified several features that were unique to either GM-1 or GM-2, and principal component analysis revealed a significant separation of the groups. Based on mass-charge ratios, 117 features were significantly different between the groups, of which 28 were putatively identifiable metabolites through the HMDB (Human Metabolite Database) and METLIN databases. Hierarchical cluster analysis of the 1 month metabolome data based on Euclidean distance measurements (Ward's algorithm) showed separation of samples based on tumor burden at sacrifice. Metabolites present prior to observable disease onset at 1 month were correlated with adenoma burden 5 months later. RNAseq between GM-1 and GM-2 tissues identified 2173 differentially expressed genes (DEGs) in the normal epithelium, and 3406 DEGs between tumor groups (FDR < 0.05). Ordinate analysis confirmed both NE and T samples were significantly distinct between rats harboring the two GM profiles. Integrated pathway (IP) analysis of metabolome and RNASeq data based on detected features showed that bile acid biosynthesis was enriched in GM-1, identifying 14 target genes affecting this pathway. Metabolome data from serum samples from an independent group of 1 month old Pirc and wildtype rats with GM-1 also identified metabolites from bile acid biosynthesis elevated in Pirc serum. Six out of the 14 target genes from the IP analysis showed significant differences (FDR < 0.05) between GM-1 and GM-2 in the NE and T tissues indicating a potential role of metabolites modulating both NE and T gene expression. DEG pathway analysis indicated increased fatty acid metabolism and mucin biosynthesis in the high susceptibility group, and apoptosis and calcium signaling pathways increased in the resistant group. These data show the utility of using metabolomics for early detection of adenomas and provide insights into the mechanism of how the GM modulates colon cancer. Citation Format: Susheel Bhanu Busi, Zhentian Lei, Lloyd Sumner, James Amos-Landgraf. Complex gut microbiota modulate rat colon adenoma susceptibility, metabolites, and host gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2400.

Abstract 4987: Biofilm-producing sulfate-reducing bacteria suppress tumor burden in a rat model of colon cancer

Abstract Human epidemiological and animal model studies have shown that the presence of colon cancer is associated with certain microbiota. Previously, we re-derived genetically identical embryos of the Pirc (F344/NTac-Apc+/Pirc) rat model of familial adenomatous polyposis using three strains of surrogate dams (F344/NHsd, LEW/SsNHsd, and Crl:SD), each harboring distinct gut microbiota (GM) that modulated adenoma susceptibility. Bacterial relative abundances as determined by 16S rDNA sequencing showed several taxa correlating with suppression of both tumor growth and phenotype penetrance as early as 1 month of age. One taxon associated with reduced adenoma burden was a sulfate-reducing bacterium, viz. Desulfovibrio spp. To determine if Desulfovibrio was responsible for differences in tumor burden, Pirc rats harboring complex GM were gavaged at 14 and 15 days of age with two genetic isolates of Desulfovibrio (~10^8 colony forming units, CFU): biofilm-forming DvH-MT and biofilm-deficient DvH-MO. We found that the rats stably colonized with the biofilm-forming DvH-MT strain, with the bacteria present at 4 months of age. Adenoma burden at sacrifice showed that the biofilm-competent strain significantly reduced colonic adenoma size (t-test, p<0.05), compared to the biofilm-deficient, DvH-MO-treated rats. To understand how the biofilm-forming capacity of the DvH-MT strain modulated adenoma burden, the genomes of the DvH-MT and DvH-MO strains were sequenced and 12 variants were identified between the two strains. In the DvH-MO strain one variant in the DVU1017 type-1 secretion system ABC transporter binding protein gene was shown to be required for biofilm-formation by Desulfovibrio in vitro. This gene was inactivated in the parent DvH-MT strain and the new construct was designated JWT716. To determine the presence and proximity of Desulfovibrio to adenomas in the colon, we modified the parental DvH-MT to express the fluorescent protein, dTomato and designated the fluorescent strain as JWT733. Pirc rats were treated with the biofilm-forming, JWT733 and the biofilm-deficient, JWT716. 3-months post-treatment we confirmed via qRT-PCR the presence of the biofilm-forming, JWT733 in fecal samples. JWT716 was undetectable in the fecal samples from most rats. Using a narrow-band filter to detect dTomato, we confirmed JWT733 colonization in the treated rats by colonoscopy. At 4 months of age, the rats were sacrificed and adenoma burden was estimated. We found that the biofilm-forming Desulfovibrio suppressed tumor burden in the colon compared to rats treated with the biofilm-deficient strain (t-test, p<0.05). This implicates the role of biofilm formation and potentially sulfate-reduction by this bacterium in the suppression of tumor multiplicity. In this model of colon cancer, Desulfovibrio alone or in concert may be residing in a niche in the intestinal mucus layer creating a beneficial or protective biofilm. Citation Format: Susheel Bhanu Busi, Kara B. De Leon, Judy D. Wall, James M. Amos-Landgraf. Biofilm-producing sulfate-reducing bacteria suppress tumor burden in a rat model of colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4987.

Regionally Impaired Redox Homeostasis in the Brain of Rats Subjected to Global Perinatal Asphyxia: Sustained Effect up to 14 Postnatal Days.

The present report evaluates the effect of global perinatal asphyxia on several parameters of oxidative stress and cell viability in rat brain tissue sampled at an extended neonatal period up to 14days, a period characterised by intensive neuritogenesis, synaptogenesis, synaptic consolidation, pruning and delayed cell death. Perinatal asphyxia was induced by immersing foetus-containing uterine horns removed by a caesarean section from on term rat dams into a water bath at 37°C for 21min. Asphyxia-exposed and sibling caesarean-delivered foetuses were manually resucitated and nurtured by surrogate dams for 1 to 14 postnatal (P) days. Brain samples (mesencephalon, telencephalon and hippocampus) were assayed for glutathione (reduced and oxidated levels; spectrophotometry), tissue reducing capacity (potassium ferricyanide reducing assay, FRAP), catalase (the key enzyme protecting against oxidative stress and reactive oxygen species, Western blots and ELISA) and cleaved caspase-3 (the key executioner of apoptosis, Western blots) levels. It was found that global PA produced a regionally specific and sustained increase in GSSG/GSH ratio, a regionally specific decrease in tissue reducing capacity and a regionally and time specific decrease of catalase activity and increase of cleaved caspase-3 levels. The present study provides evidence for regionally impaired redox homeostasis in the brain of rats subjected to global PA, an effect observed up to P14, mainly affecting mesencephalon and hippocampus, suggesting a sustained oxidative stress after the posthypoxia period. The oxidative stress observed postnatally can in part be associated to a respiratory apneic-like deficit, since there was a statistically significant decrease in respiration frequency in AS compared to CS neonates, also up to P14, together with the signs of a decreased peripheral blood perfusion (pink-blue skin colour in AS, compared to the pink colour observed in all CS neonates). It is proposed that PA implies a long-term metabolic insult, triggered by the length of hypoxia, the resuscitation/reoxigenation manoevres, but also by the developmental stage of the affected brain regions, and the integrity of cardiovascular and respiratory physiological functions, which are fundamental for warrantying a proper development.

Chips off the Old Block: How a Father's Preconception Exposures Might Affect the Health of His Children.

Chips off the Old Block: How a Father's Preconception Exposures Might Affect the Health of His Children.

Abstract 2880: Modulating disease susceptibility in a model of human colon cancer by microbiome rederivation

Abstract To understand the impact of differences in complex microbiota on colon cancer susceptibility, we utilized the Pirc (Polyposis In Rat Colon) model of familial human colon cancer to correlate the longitudinal changes of the gut microbiota with early adenoma development. Recent human epidemiologic studies have shown associations of gut microbiota differences in patients with colorectal cancer vs. controls. To test if differences in the gut microbiota modulate susceptibility, we used the F344/NTac-ApcPirc/+ rat model and altered the microbiome through embryo rederivation using three distinct strains of surrogate dams: F344/NHsd, LEW/SsNHsd, and Crl:SD, all harboring different commensal microbiota. Fecal samples from the pups and dams were collected at various time points. Extracted DNA was sequenced using the Illumina MiSeq platform for the 16S rRNA gene V4 hypervariable region. Adenoma development was monitored periodically via colonoscopy and terminal tumor counts and histology were performed at 180 days. We found the complex microbiota of the surrogates to be distinct from one another and that the pups resembled their respective dams. At the terminal time-point, rats harboring the LEW microbiota had significantly reduced colonic tumor burden (number and size) compared to both F344 (p<0.001) and SD (p<0.05). Colonic tumors were also absent in 2 of the 15 rederived pups with the LEW microbiota reducing the Pirc phenotype penetrance from 100% to 87%. At weaning, we found the LEW microbiota to be unique with a high percentage of Prevotella copri. Pups with the LEW microbiota also differed significantly from the other pups by an additional 8 operational taxonomic units (OTUs). At 6 months of age, the relative abundance of P. copri, and 5 additional OTUs correlated with decreased tumor burden in these pups (Spearman’s, p≤0.05). Conversely, higher prevalence of family Peptococcaceae and Akkermansia muciniphila were positively correlated (p<0.05) with increased tumor number. Histological analysis of colons from the LEW and SD offspring showed significant goblet cell hyperplasia (p<0.002) in the distal colon of the LEW pups. No aberrant crypt foci (ACF) or microadenomas were found in the two LEW pups that did not harbor colonic adenomas, indicating a possible inhibition of initiation. Immunohistochemical staining for Ki67 showed that the proliferation index of both the normal epithelium and tumors in the SD group was trending significantly higher compared to the LEW rats. Our results indicate that microbial alteration through rederivation affects colon cancer susceptibility. The prevalence or lack of specific OTUs at weaning can potentially serve as predictors of tumor burden at later stages. More importantly, the study points towards differences in the microbiota of the offspring acting as modulators of disease phenotype affecting initiation or progression of colon cancer. Citation Format: James Amos-Landgraf, Susheel Busi, Aaron Ericsson, Marina McCoy, Taybor Parker, Rebecca Schehr, Miriam Hankins, Craig Franklin, Elizabeth Bryda. Modulating disease susceptibility in a model of human colon cancer by microbiome rederivation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2880. doi:10.1158/1538-7445.AM2015-2880

Ribosomes 'muscle up' postnatal muscle growth.

Proper nutrition ensures that all necessary nutrients are available to meet the body's demands to sustain growth, physiological specialization and maintenance. Survival depends on it. The consequences of malnutrition can be devastating, especially during the early postnatal period. In the developed world, one in three children suffers from short stature due to malnutrition, and if current trends continue, within the next decade more than 450 million children will be affected globally (Onis et al. 1993). Moreover, perinatal malnutrition is associated with persistent infections due to weakened innate and adaptive immunity, and is linked to the development of diseases like diabetes later in life. The long-term effects of delayed early growth resulting from malnutrition at critical periods of development can lead to derailments in both organ structure and function. This scenario can have far reaching consequences beyond the affected individual; at the community level the burden of malnutrition hinders productivity, food production, and socioeconomic and political development (Schaible & Kaufmann, 2007). Skeletal muscle size is highly sensitive to nutritional status. An increase in skeletal muscle protein synthesis following a nutritional ingestion is necessary for protein accretion, muscle growth and maintenance. Muscle protein synthesis rates are mainly determined by the cellular content of ribosomes (Millward et al. 1973), and because ribosomal RNA synthesis is rate limiting for ribosome formation, any insults that interfere with it will in turn affect muscle growth. How muscle ribosome quantity and function responds to food, or lack of it, remains a poorly defined area in physiology and nutrition, one that has tremendous relevance for clinical practice. In this issue of The Journal of Physiology, Fiorotto and coworkers revisited the role of a nutritional insult in postnatal muscle development, and identified a critical period when low dietary protein can have long-lasting consequences on muscle mass. They also provided a possible mechanistic explanation for this observation. In their study, lactating newborn mice were exposed to a low protein diet via surrogate dams. What Fiorotto et al. found was that the timing of a nutritional insult is a critical determinant of postnatal muscle development. When pups were exposed to a protein-deficient diet between postnatal days 1 and 11, but returned to a well-fed dam, no obvious developmental problems ensued. However, when the nutritional insult occurred between postnatal days 11 and 22, muscle growth deficits lasted for at least 18 months. This was despite animals being provided an ad libitum complete (i.e. protein containing) diet following the period of protein-deprived lactation. The critical factor identified using this elegant experimental approach was the ‘timing’ in which pups were exposed to the nutritional insult. A diet low in protein affected the translational capacity of the muscle upon refeeding with a complete diet. Muscle protein synthesis in the compromised pups was lower due to a defective production of muscle ribosomes in response to feeding. One observation that partially explains the inability of the muscle to increase ribosome content was the blunted upregulation of the ribosomal gene transcriptional co-activator UBF, which appears to be necessary for enhanced ribosome synthesis following feeding. A number of important questions arise from this interesting study. What is happening in the mouse between postnatal days 11 and 22 that is so critical for skeletal muscle development? Why was the timing so specific? How much translational capacity is needed to ensure proper growth, and why a small deficit in ribosome production at a ‘key’ developmental stage has such long-term effects? Even though differences in protein synthesis rates and ribosomal response to feeding were altered, these parameters could catch up at later stages and yet the muscular deficit persisted for a long time. Could translation of specific mRNAs or expression of non-coding regulatory RNAs be altered during postnatal days 11–22, so that even if protein synthesis rates and ribosome content are restored later in life muscle development is still compromised? This new study by Fiorotto et al. reveals a novel aspect of the interaction between nutrition, organismal development, the trophic state of the muscle, and the long-term consequences of altered nutrition. The finding that timing of nutritional delivery is of critical importance can have tremendous implications for the treatment of infant malnourishment, development and functioning in society. The study revealed a ‘window of opportunity for nutritional intervention’, which if missed, could result in a diminished ability of muscle ribosomes to increase in response to feeding, and consequently long-term deficits in muscle mass. The results reported by Fiorotto et al. are of potential significance for the human population. If failure to provide adequate protein in the diet at key developmental stages also results in faltered muscular development in humans, efforts should focus on delivering the right aid at the right time to infants in underprivileged nations so that long-term physical and cognitive impairments associated with malnutrition can be eradicated.

Effects of using the analgesic tramadol in mice undergoing embryo transfer surgery.

Embryo transfer is a surgical technique that is widely used in reproductive biotechnology. Despite the ethical obligation to relieve animals' post-operative pain, analgesia is not routinely provided after embryo transfer surgery because it has been suggested that analgesics may be detrimental to embryo survival. Studies suggest, however, that the potential for adverse effects varies depending on the type of analgesic used and the timing of its administration. The authors carried out a study to determine whether pre-operatively administered tramadol, a synthetic analogue of codeine, influenced birth rate, litter survival or the post-operative body weights of surrogate dams. Compared with controls that were not given any analgesic, surrogate dams given tramadol had similar birth rates and similar body weights at all time points. The tramadol-treated surrogate dams showed a statistically significant increase in the number of offspring that survived to weaning. The authors conclude that pre-operatively administered tramadol does not harm the success rate of embryo transfer surgery and even may improve litter survival.