Interstrain Differences Research Articles

A transposon present in specific strains of Bacillus subtilis negatively affects nutrient‐ and dodecylamine‐induced spore germination

Spore germination shows a large inter-strain variability. Spores of certain Bacillus subtilis strains, including isolates from spoiled food products, exhibit different germination behavior from spores of the well-studied model organism Bacillus subtilis 168, often for unknown reasons. In this study, we analyzed spore germination efficiencies and kinetics of seventeen B. subtilis strains with previously sequenced genomes. A subsequent gene-trait matching analysis revealed a correlation between a slow germination phenotype and the presence of a mobile genetic element, i.e., a Tn1546-like transposon. A detailed investigation of the transposon elements showed an essential role of a specific operon (spoVA2mob ) in inhibiting spore germination with nutrients and with the cationic surfactant dodecylamine. Our results indicate that this operon negatively influences release of Ca-DPA by the SpoVA channel and may additionally alter earlier germination events, potentially by affecting proteins in the spore inner membrane. The spoVA2mob operon is an important factor that contributes to inter-strain differences in spore germination. Screening for its genomic presence can be applied for identification of spores that exhibit specific properties that impede spore eradication by industrial processes.

Elucidation of the Metabolic Network of Helicobacter pylori J99 and Malaysian Clinical Strains by Phenotype Microarray.

Background Helicobacter pylori colonizes almost half of the human population worldwide. H. pylori strains are genetically diverse, and the specific genotypes are associated with various clinical manifestations including gastric adenocarcinoma, peptic ulcer disease (PUD), and nonulcer dyspepsia (NUD). However, our current knowledge of the H. pylori metabolism is limited. To understand the metabolic differences among H. pylori strains, we investigated four Malaysian H. pylori clinical strains, which had been previously sequenced, and a standard strain, H. pylori J99, at the phenotypic level.Materials and MethodsThe phenotypes of the H. pylori strains were profiled using the Biolog Phenotype Microarray system to corroborate genomic data. We initiated the analyses by predicting carbon and nitrogen metabolic pathways from the H. pylori genomic data from the KEGG database. Biolog PM aided the validation of the prediction and provided a more intensive analysis of the H. pylori phenomes.ResultsWe have identified a core set of metabolic nutrient sources that was utilized by all strains tested and another set that was differentially utilized by only the local strains. Pentose sugars are the preferred carbon nutrients utilized by H. pylori. The amino acids l‐aspartic acid, d‐alanine, and l‐asparagine serve as both carbon and nitrogen sources in the metabolism of the bacterium.ConclusionThe phenotypic profile based on this study provides a better understanding on the survival of H. pylori in its natural host. Our data serve as a foundation for future challenges in correlating interstrain metabolic differences in H. pylori.

Semi-automated registration-based anatomical labelling, voxel based morphometry and cortical thickness mapping of the mouse brain

BackgroundMorphoanatomical MRI methods have recently begun to be applied in the mouse. However, substantial differences in the anatomical organisation of human and rodent brain prevent a straightforward extension of clinical neuroimaging tools to mouse brain imaging. As a result, the vast majority of the published approaches rely on tailored routines that address single morphoanatomical readouts and typically lack a sufficiently-detailed description of the complex workflow required to process images and quantify structural alterations. New methodHere we provide a detailed description of semi-automated registration-based procedures for voxel based morphometry, cortical thickness estimation and automated anatomical labelling of the mouse brain. The approach relies on the sequential use of advanced image processing tools offered by ANTs, a flexible open source toolkit freely available to the scientific community. ResultsTo illustrate our procedures, we described their application to quantify morphological alterations in socially-impaired BTBR mice with respect to normosocial C57BL/6J controls, a comparison recently described by us and other research groups. We show that the approach can reliably detect both focal and large-scale grey matter alterations using complementary readouts. Comparison with existing methodsNo detailed operational workflows for mouse imaging are available for direct comparison with our methods. However, empirical assessment of the mapped inter-strain differences is in good agreement with the findings of other groups using analogous approaches. ConclusionThe detailed operational workflows described here are expected to help the implementation of rodent morphoanatomical methods by non-expert users, and ultimately promote the use of these tools across the preclinical neuroimaging community.

Inter-Strain Differences in Default Mode Network: A Resting State fMRI Study on Spontaneously Hypertensive Rat and Wistar Kyoto Rat.

Genetic divergences among mammalian strains are presented phenotypically in various aspects of physical appearance such as body shape and facial features. Yet how genetic diversity is expressed in brain function still remains unclear. Functional connectivity has been shown to be a valuable approach in characterizing the relationship between brain functions and behaviors. Alterations in the brain default mode network (DMN) have been found in human neuropsychological disorders. In this study we selected the spontaneously hypertensive rat (SHR) and the Wistar Kyoto rat (WKY), two inbred rat strains with close genetic origins, to investigate variations in the DMN. Our results showed that the major DMN differences are the activities in hippocampal area and caudate putamen region. This may be correlated to the hyperactive behavior of the SHR strain. Advanced animal model studies on variations in the DMN may have potential to shed new light on translational medicine, especially with regard to neuropsychological disorders.

Genome-wide transcriptome analysis of hypothalamus in rats with inherited stress-induced arterial hypertension.

BackgroundThe hypothalamus has an important role in the onset and maintenance of hypertension and stress responses. Rats with inherited stress-induced arterial hypertension (ISIAH), reproducing the human stress-sensitive hypertensive state with predominant involvement of the neuroendocrine hypothalamic-pituitary-adrenal and sympathoadrenal axes, were used for analysis of the hypothalamus transcriptome.ResultsRNA-seq analysis revealed 139 genes differentially expressed in the hypothalami of hypertensive ISIAH and normotensive Wistar Albino Glaxo (WAG) rats. According to the annotation in databases, 18 of the differentially expressed genes (DEGs) were associated with arterial hypertension. The Gene Ontology (GO) functional annotation showed that these genes were related to different biological processes that may contribute to the hypertension development in the ISIAH rats. The most significantly affected processes were the following: regulation of hormone levels, immune system process, regulation of response to stimulus, blood circulation, response to stress, response to hormone stimulus, transport, metabolic processes, and endocrine system development. The most significantly affected metabolic pathways were those associated with the function of the immune system and cell adhesion molecules and the metabolism of retinol and arachidonic acid. Of the top 40 DEGs making the greatest contribution to the interstrain differences, there were 3 genes (Ephx2, Cst3 and Ltbp2) associated with hypertension that were considered to be suitable for further studies as potential targets for the stress-sensitive hypertension therapy. Seven DEGs were found to be common between hypothalamic transcriptomes of ISIAH rats and Schlager mice with established neurogenic hypertension.ConclusionsThe results of this study revealed multiple DEGs and possible mechanisms specifying the hypothalamic function in the hypertensive ISIAH rats. These results provide a basis for further investigation of the signalling mechanisms that affect hypothalamic output related to stress-sensitive hypertension development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0307-8) contains supplementary material, which is available to authorized users.

Comparative transcriptional profiling of renal cortex in rats with inherited stress-induced arterial hypertension and normotensive Wistar Albino Glaxo rats.

BackgroundThe renal function plays a leading role in long-term control of arterial pressure. The comparative analysis of renal cortex transcriptome in ISIAH rats with inherited stress-induced arterial hypertension and normotensive WAG rats was performed using RNA-Seq approach. The goal of the study was to identify the differentially expressed genes (DEGs) related to hypertension and to detect the pathways contributing to the differences in renal functions in ISIAH and WAG rats.ResultsThe analysis revealed 716 genes differentially expressed in renal cortex of ISIAH and WAG rats, 42 of them were associated with arterial hypertension and regulation of blood pressure (BP). Several Gene Ontology (GO) terms significantly enriched with DEGs suggested the existence of the hormone dependent interstrain differences in renal cortex function. Multiple DEGs were associated with regulation of blood pressure and blood circulation, with the response to stress (including oxidative stress, hypoxia, and fluid shear stress) and its regulation. Several other processes which may contribute to hypertension development in ISIAH rats were: ion transport, regulation of calcium ion transport, homeostatic process, tissue remodeling, immune system process and regulation of immune response.KEGG analysis marked out several pathways significantly enriched with DEGs related to immune system function, to steroid hormone biosynthesis, tryptophan, glutathione, nitrogen, and drug metabolism.ConclusionsThe results of the study provide a basis for identification of potential biomarkers of stress-sensitive hypertension and for further investigation of the mechanisms that affect renal cortex function and hypertension development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0306-9) contains supplementary material, which is available to authorized users.

Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats.

Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.

Standardization of a molecular method for epidemiologic identification of Leishmania strains.

Molecular studies of the evolutionary relationships among Leishmania species suggest the presence of high genetic variation within this genus, which has a direct effect on public health in many countries. The coexistence of species in a particular region can result in different leishmaniasis clinical forms and treatment responses. We aimed to standardize the kinetoplast DNA (kDNA) enterobacterial repetitive intergenic consensus (ERIC) sequence polymerase chain reaction (PCR) method for molecular epidemiological identification of Leishmania strains, and estimate existing inter-strain genomic differences and kDNA signatures using this technique. ERIC-PCR of genomic DNA revealed genetic polymorphisms between species, although some strains shared many DNA fragments. Leishmania guyanensis, L. amazonensis, and L. braziliensis clustered together in a dendrogram with similarities ranging from 42.0 to 61.0%, whereas L. chagasi grouped with these three species with a similarity of 28.0%. After amplification of kDNA, 780-bp bands were extracted from an agarose gel and purified for analysis of its genetic signature. kDNA ERIC-PCR electrophoretic patterns consisted of 100- to 600- bp fragments. Using these profiles, L. braziliensis and L. guyanensis grouped with a similarity of 26.0%, and L. amazonensis and L. chagasi clustered based on a similarity of 100%. The electrophoretic profiles and dendrograms showed that, for epidemiological identification by ERIC-PCR, genomic DNA had greater discriminatory power than kDNA did. More strains need to be analyzed to validate the kDNA ERIC-PCR method. The genomes of these strains should be sequenced for better epidemiological identification of Leishmania species.

Exploring Female Mice Interstrain Differences Relevant for Models of Depression.

Depression is an extremely heterogeneous disorder. Diverse molecular mechanisms have been suggested to underlie its etiology. To understand the molecular mechanisms responsible for this complex disorder, researchers have been using animal models extensively, namely mice from various genetic backgrounds and harboring distinct genetic modifications. The use of numerous mouse models has contributed to enrich our knowledge on depression. However, accumulating data also revealed that the intrinsic characteristics of each mouse strain might influence the experimental outcomes, which may justify some conflicting evidence reported in the literature. To further understand the impact of the genetic background, we performed a multimodal comparative study encompassing the most relevant parameters commonly addressed in depression, in three of the most widely used mouse strains: Balb/c, C57BL/6, and CD-1. Moreover, female mice were selected for this study taken into account the higher prevalence of depression in women and the fewer animal studies using this gender. Our results show that Balb/c mice have a more pronounced anxious-like behavior than CD-1 and C57BL/6 mice, whereas C57BL/6 animals present the strongest depressive-like trait. Furthermore, C57BL/6 mice display the highest rate of proliferating cells and brain-derived neurotrophic factor (Bdnf) expression levels in the hippocampus, while hippocampal dentate granular neurons of Balb/c mice show smaller dendritic lengths and fewer ramifications. Of notice, the expression levels of inducible nitric oxide synthase (iNos) predict 39.5% of the depressive-like behavior index, which suggests a key role of hippocampal iNOS in depression. Overall, this study reveals important interstrain differences in several behavioral dimensions and molecular and cellular parameters that should be considered when preparing and analyzing experiments addressing depression using mouse models. It further contributes to the literature by revealing the predictive value of hippocampal iNos expression levels in depressive-like behavior, irrespectively of the mouse strain.

The Influence of Infective Dose on the Virulence of a Generalist Pathogen in Rainbow Trout (Oncorhynchus mykiss) and Zebra Fish (Danio rerio).

Pathogen density and genetic diversity fluctuate in the outside-host environment during and between epidemics, affecting disease emergence and the severity and probability of infections. Although the importance of these factors for pathogen virulence and infection probability has been acknowledged, their interactive effects are not well understood. We studied how an infective dose in an environmentally transmitted opportunistic fish pathogen, Flavobacterium columnare, affects its virulence both in rainbow trout, which are frequently infected at fish farms, and in zebra fish, a host that is not naturally infected by F. columnare. We used previously isolated strains of confirmed high and low virulence in a single infection and in a co-infection. Infection success (measured as host morbidity) correlated positively with dose when the hosts were exposed to the high-virulence strain, but no response for the dose increase was found when the hosts were exposed to the low-virulence strain. Interestingly, the co-infection resulted in poorer infection success than the single infection with the high-virulence strain. The rainbow trout were more susceptible to the infection than the zebra fish but, in both species, the effects of the doses and the strains were qualitatively similar. We suggest that as an increase in dose can lead to increased host morbidity, both the interstrain interactions and differences in infectivity in different hosts may influence the severity and consequently the evolution of disease. Our results also confirm that the zebra fish is a good laboratory model to study F. columnare infection.

Abstract 5486: Identification of novel candidate genes associated with sorafenib cytotoxicity

Abstract Background: Sorafenib is an oral multikinase inhibitor that decreases tumor angiogenesis and proliferation. The antitumor efficacy and toxicity profiles of sorafenib vary among patients. Novel pathways and targets of sorafenib activity remain to be identified, and no predictive biomarkers of sorafenib activity exist to help guide clinicians. We aimed to identify novel genes associated with sorafenib activity by using an in vitro methodology based upon mouse genomics. Methods: We profiled primary mouse embryonic fibroblasts (MEFs) from 32 inbred strains for sorafenib cytotoxicity utilizing high content imaging and simultaneous evaluation of cell health parameters. The 32 strains were genomically characterized previously (PMID: 21623374). MEFs were treated with ten concentrations (0-300 μM) of sorafenib, incubated for 72 h, and then fixed and stained. Cytochrome C was immunolabeled in the fixed cells and imaged using an automated fluorescence microscope. An image analysis software assessed sorafenib-induced cytochrome C release from mitochondria. Dose response curves were generated from data, and EC50 values for each strain were identified using a Brain-Cousens model. Genome-wide association mapping, using the EMMA algorithm, was performed on cytochrome C EC50 values to identify quantitative trait loci (QTLs) associated with sorafenib cytotoxicity. Approximately 277,000 single nucleotide polymorphisms were tested, and genomic loci with p-values ≤ 1.0×10−7 were selected for additional analyses. Results: Interstrain EC50 variability among the 32 MEF strains was observed after 72 h (17.2-44.5 μM) sorafenib incubations. We identified one peak (chromosome 9 from 51-52 Mb; p = 1.0×10−8), which reached genome-wide significance and significantly associated with cytochrome C release. From this peak, we have identified candidate genes that may underlie variability in sorafenib-induced cytochrome C release from mitochondria. A total of nine genes expressed in MEF cells at mRNA level are present in this QTL. Of particular interest, the identified locus contains Rdx, a gene that encodes radixin. Radixin is a component of the ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) complex, and is conserved between humans and mice. In humans, radixin, as part of the EBP50 complex, has been shown to act as a tumor suppressor and to promote apoptosis in hepatocellular carcinoma by modulating β-catenin/E-cadherin (PMID: 23483729). It has also been shown to potentially enhance the metastatic potential of renal cell carcinoma (PMID: 20395446). Conclusions: Our high-throughput cellular genetics approach has detected robust interstrain cellular differences in sorafenib activity, and identified one region, which reached genome-wide significance, that potentially associates with sorafenib-induced cytochrome C release from mitochondria. Functional validation of Rdx and other promising candidates is currently ongoing. Citation Format: Daniel James Crona, Oscar Suzuki, O. Joseph Trask, Bethany Parks, Amber Frick, Timothy Wiltshire, Federico Innocenti. Identification of novel candidate genes associated with sorafenib cytotoxicity. [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 5486. doi:10.1158/1538-7445.AM2015-5486

The complex pattern of epigenomic variation between natural yeast strains at single-nucleosome resolution.

BackgroundEpigenomic studies on humans and model species have revealed substantial inter-individual variation in histone modification profiles. However, the pattern of this variation has not been precisely characterized, particularly regarding which genomic features are enriched for variability and whether distinct histone marks co-vary synergistically. Yeast allows us to investigate intra-species variation at high resolution while avoiding other sources of variation, such as cell type or subtype.ResultsWe profiled histone marks H3K4me3, H3K9ac, H3K14ac, H4K12ac and H3K4me1 in three unrelated wild strains of Saccharomyces cerevisiae at single-nucleosome resolution and analyzed inter-strain differences statistically. All five marks varied significantly at specific loci, but to different extents. The number of nucleosomes varying for a given mark between two strains ranged from 20 to several thousands; +1 nucleosomes were significantly less subject to variation. Genes with highly evolvable or responsive expression showed higher variability; however, the variation pattern could not be explained by known transcriptional differences between the strains. Synergistic variation of distinct marks was not systematic, with surprising differences between functionally related H3K9ac and H3K14ac. Interestingly, H3K14ac differences that persisted through transient hyperacetylation were supported by H3K4me3 differences, suggesting stabilization via cross talk.ConclusionsQuantitative variation of histone marks among S. cerevisiae strains is abundant and complex. Its relation to functional characteristics is modular and seems modest, with partial association with gene expression divergences, differences between functionally related marks and partial co-variation between marks that may confer stability. Thus, the specific context of studies, such as which precise marks, individuals and genomic loci are investigated, is primordial in population epigenomics studies. The complexity found in this pilot survey in yeast suggests that high complexity can be anticipated among higher eukaryotes, including humans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0019-3) contains supplementary material, which is available to authorized users.

The effects of the glial cell line-derived neurotrophic factor (GDNF) on the levels of mRNA of apoptotic genes Bax and Bcl-xl in the brain of mice genetically predisposed to pathological behavior

Similar to the other neurotrophic factors, the glial cell line-derived neurotrophic factor (GDNF) may inhibit apoptosis in vitro. However, the antiapoptotic GDNF effects in vivo are not known. We studied the effect of GDNF central administration of on the mRNA levels of genes encoding the proapoptotic protein Bax and the antiapoptotic protein Bcl-xl in the brain of ASC mice genetically predisposed to depressivelike behavior and mice of their nondepressive parental CBA strain. We found that the GDNF injection increased the Bcl-xl mRNA content in the hippocampi of mice of both strains (ρ < 0.05) and Bax mRNA (ρ < 0.05) in the hippocampus of ASC mice. Thus, we revealed both the antiand proapoptotic effects of GDNF in vivo. These effects substantially depended on the genotype of the animals. We also observed significant interstrain differences in the Bax and Bcl-xl mRNA levels. In ASC mice, the Bax mRNA levels were significantly higher (ρ < 0.001 and ρ < 0.01) in all investigated structures and the content of Bcl-xl mRNA was elevated in the midbrain. Our data demonstrate the activation of apoptotic processes in ASC mice and the substantial compensatory changes, probably directed to the rise of the threshold for neuronal apoptosis.

Identifying and assessing the impact of wine acid-related genes in yeast.

Saccharomyces cerevisiae strains used for winemaking show a wide range of fermentation phenotypes, and the genetic background of individual strains contributes significantly to the organoleptic properties of wine. This strain-dependent impact extends to the organic acid composition of the wine, an important quality parameter. However, little is known about the genes which may impact on organic acids during grape must fermentation. To generate novel insights into the genetic regulation of this metabolic network, a subset of genes was identified based on a comparative analysis of the transcriptomes and organic acid profiles of different yeast strains showing different production levels of organic acids. These genes showed significant inter-strain differences in their transcription levels at one or more stages of fermentation and were also considered likely to influence organic acid metabolism based on existing functional annotations. Genes selected in this manner were ADH3, AAD6, SER33, ICL1, GLY1, SFC1, SER1, KGD1, AGX1, OSM1 and GPD2. Yeast strains carrying deletions for these genes were used to conduct fermentations and determine organic acid levels at various stages of alcoholic fermentation in synthetic grape must. The impact of these deletions on organic acid profiles was quantified, leading to novel insights and hypothesis generation regarding the role/s of these genes in wine yeast acid metabolism under fermentative conditions. Overall, the data contribute to our understanding of the roles of selected genes in yeast metabolism in general and of organic acid metabolism in particular.

Interstrain differences of ionotropic glutamate receptor subunits in the hippocampus and induction of hippocampal sclerosis with pilocarpine in mice

Rodent strains used in epilepsy research have various neurological characteristics. These differences were suggested to be attributed to the diverse densities of the ionotropic glutamate receptor (iGluR) subunits. However, previous studies failed to find interstrain differences in the hippocampal receptor levels.We supposed that a detailed layer-to-layer analysis of the iGluR subunits in the hippocampus might reveal strain-dependent differences in their base lines and reactions induced by pilocarpine (PILO) between two mouse strains without documented ancestors.Levels of iGluR subunits in Balb/c and NMRI mice were compared using semiquantitative immunohistochemistry. The alterations in the neuronal circuitry were validated by neuropeptide Y (NPY) and neuronal nuclear antigen (NeuN) immunostainings.Immunohistochemistry showed interstrain laminar differences in some subunits of both the control and PILO-treated animals. The seizure-induced irreversible neuronal changes were accompanied by reduced GluA1 and GluA2 levels. Their changes were inversely correlated in the individual NMRI mice by Pearson's method. Increase in NPY immunoreactivity showed positive correlation with GluA1, and negative correlation with GluA2. The NMRI strain was susceptible to PILO-induced hippocampal sclerosis, while the Balb/c animals showed resistance.Basal levels of iGluRs differ in mouse strains, which may account for the interstrain differences in their reactions to the convulsant.

Immune cell-based screening assay for response to anticancer agents: applications in pharmacogenomics.

BackgroundInterpatient variability in immune and chemotherapeutic cytotoxic responses is likely due to complex genetic differences and is difficult to ascertain in humans. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at examining interstrain differences in viability on normal, noncancerous immune cells following chemotherapeutic cytotoxic insult. Drug effects were investigated by comparing selective chemotherapeutic agents, such as BEZ-235 and selumetinib, against conventional cytotoxic agents targeting multiple pathways, including doxorubicin and idarubicin.MethodsSplenocytes were isolated from 36 isogenic strains of mice using standard procedures. Of note, the splenocytes were not stimulated to avoid attributing responses to pathways involved with cellular stimulation rather than toxicity. Cells were incubated with compounds on a nine-point logarithmic dosing scale ranging from 15 nM to 100 μM (37°C, 5% CO2). At 4 hours posttreatment, cells were labeled with antibodies and physiological indicator dyes and fixed with 4% paraformaldehyde. Cellular phenotypes (eg, viability) were collected and analyzed using flow cytometry. Dose-response curves with response normalized to the zero dose as a function of log concentration were generated using GraphPad Prism 6.ResultsPhenotypes were quantified using flow cytometry, yielding interstrain variation for measured endpoints in different immune cells. The flow cytometry assays produced over 16,000 data points that were used to generate dose-response curves. The more targeted agents, BEZ-235 and selumetinib, were less toxic to immune cells than the anthracycline agents. The calculated heritability for the viability of immune cells was higher with anthracyclines than the novel agents, making them better suited for downstream genetic analysis.ConclusionUsing this approach, we identify cell lines of variable sensitivity to chemotherapeutic agents and aim to identify robust, replicable endpoints of cellular response to drugs that provide the starting point for identifying candidate genes and cellular toxicity pathways for future validation in human studies.

Comparative Analysis of the Relationship Between Trichloroethylene Metabolism and Tissue-Specific Toxicity Among Inbred Mouse Strains: Kidney Effects

Trichloroethylene (TCE) is a well-known environmental and occupational toxicant that is classified as carcinogenic to humans based on the epidemiological evidence of an association with higher risk of renal-cell carcinoma. A number of scientific issues critical for assessing human health risks from TCE remain unresolved, such as the amount of kidney-toxic glutathione conjugation metabolites formed, interspecies and interindividual differences, and the mode of action for kidney carcinogenicity. It was postulated that TCE renal metabolite levels are associated with kidney-specific toxicity. Oral dosing with TCE was conducted in subacute (600 mg/kg/d; 5 d; 7 inbred mouse strains) and subchronic (100 or 400 mg/kg/d; 1, 2, or 4 wk; 2 inbred mouse strains) designs. The quantitative relationship was evaluated between strain-, dose, and time-dependent formation of TCE metabolites from cytochrome P-450-mediated oxidation (trichloroacetic acid [TCA], dichloroacetic acid [DCA], and trichloroethanol) and glutathione conjugation [S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)glutathione], and various kidney toxicity phenotypes. In subacute study, interstrain differences in renal TCE metabolite levels were observed. In addition, data showed that in several strains kidney-specific effects of TCE included induction of peroxisome proliferator-marker genes Cyp4a10 and Acox1, increased cell proliferation, and expression of KIM-1, a marker of tubular damage and regeneration. In subchronic study, peroxisome proliferator-marker gene induction and renal toxicity diminished while cell proliferative response was elevated in a dose-dependent manner in NZW/LacJ but not C57BL/6J mice. Overall, data demonstrated that renal TCE metabolite levels are associated with kidney-specific toxicity and that these effects are strain dependent.

Computational modeling of nanoscale and microscale particle deposition, retention and dosimetry in the mouse respiratory tract

Comparing effects of inhaled particles across rodent test systems and between rodent test systems and humans is a key obstacle to the interpretation of common toxicological test systems for human risk assessment. These comparisons, correlation with effects and prediction of effects, are best conducted using measures of tissue dose in the respiratory tract. Differences in lung geometry, physiology and the characteristics of ventilation can give rise to differences in the regional deposition of particles in the lung in these species. Differences in regional lung tissue doses cannot currently be measured experimentally. Regional lung tissue dosimetry can however be predicted using models developed for rats, monkeys, and humans. A computational model of particle respiratory tract deposition and clearance was developed for BALB/c and B6C3F1 mice, creating a cross-species suite of available models for particle dosimetry in the lung. Airflow and particle transport equations were solved throughout the respiratory tract of these mice strains to obtain temporal and spatial concentration of inhaled particles from which deposition fractions were determined. Particle inhalability (Inhalable fraction, IF) and upper respiratory tract (URT) deposition were directly related to particle diffusive and inertial properties. Measurements of the retained mass at several post-exposure times following exposure to iron oxide nanoparticles, micro- and nanoscale C60 fullerene, and nanoscale silver particles were used to calibrate and verify model predictions of total lung dose. Interstrain (mice) and interspecies (mouse, rat and human) differences in particle inhalability, fractional deposition and tissue dosimetry are described for ultrafine, fine and coarse particles.

Enhancement of resting-state fcMRI networks by prior sensory stimulation.

It is important to consider the effect of a previous experimental condition when analyzing resting-state functional connectivity magnetic resonance imaging (fcMRI) data. In this work, a simple sensory stimulation functional MRI (fMRI) experiment was conducted between two resting-state fcMRI acquisitions in anesthetized rats using a high-field small-animal MR scanner. Previous human studies have reported fcMRI network alteration by prior task/stimulus utilizing similar experimental paradigms. An anesthetized rat preparation was used to test whether brain regions with higher level functions are involved in post-task/stimulus fcMRI network alteration. We demonstrate significant fcMRI enhancement poststimulation in the sensory cortical, limbic, and insular brain regions in rats. These brain regions have been previously implicated in vigilance and anesthetic arousal networks. We tested their experimental paradigm in several inbred strains of rats with known phenotypic differences in anesthetic susceptibility and cerebral vascular function. Brown Norway (BN), Dahl Salt-Sensitive (SS), and consomic SSBN13 strains were tested. We have previously shown significant differences in blood oxygen level-dependent fMRI activity and fcMRI networks across these strains. Here we report statistically significant interstrain differences in regional fcMRI poststimulation enhancement. In the SS strain, poststimulation enhancement occurred in posterior sensory and limbic cortical brain regions. In the BN strain, poststimulation enhancement appeared in anterior cingulate and subcortical limbic brain regions. These results imply that a prior condition has a significant impact on fcMRI networks that depend on intersubject difference in genetics and physiology.

ULTRASTRUCTURE OF SYNAPSES IN THE HIPPOCAMPUS OF RATS IN AGING

The aim of the study was to examine age-related changes in the ultrastructure of synapses in the CA1 region of the hippocampus of Wistar rats and premature aging OXYS rats.To assess changes of the sinaptoarhitectonics photographed on 15 randomly chosen fields of view of the pyramidal layer of the CA1 region of the hippocampus with five slices at a standard magnification of 10000. It was determined the amount of mineral contacts (size of the field of view is 50 μm2) and calculated numerical density of synapses per 100 μm2. The number of contacts with asymmetric and symmetric organization sistemasubsidiaries units, perforated, hypertrophic contacts are counted.The study showed that there are significant differences in the ultrastructural organization of the synapses between the lines, already 4 months old. The numerical density of snaps in this age group OXYS rats was significantly higher than that of Wistar. Ultrastructure of synapses in the CA1 region of the hippocampus of rats at the age of 18 months more bulupodadan pathological essentiaompetencies processes and membrane prematurely senescent rats in contrast to the control line. The numerical indicator of protect contents 18-month-old rats OXYS was significantly lower than in Wistar.Identified interstrain differences in the degree of changes of synapses can testify in favor of a different sensitivity to oxidative stress in rats investigated lines.