Mouse Tissue Samples Research Articles

Electrogenerated chemiluminescence method for sensitive detection of hydroxylated double-stranded DNA through multifunctional polyadenine probe and hybridization chain reaction

5-Hydroxymethylcytosine (5-hmC), as the sixth base in the mammalian genome, is increasingly regarded as an epigenetic marker with crucial biological functions. Herein, a highly sensitive electrogenerated chemiluminescence (ECL) sensing platform for double-stranded DNA containing 5-hmC (5-hmC-dsDNA) in genome was fabricated, which based on polyadenine-gold interaction combining with signal amplification of hybridization chain reaction (HCR). This biosensing strategy could be executed to detect 5-hmC in the native double-stranded DNA, irrespective of the specific sequences of DNA. Firstly, DNA methyltransferase (M.HhaI) was introduced to chemically modify the hydroxyl of 5-hmC in double-stranded DNA, which converted 5-hmC-dsDNA to amino-derived 5-hmC-dsDNA. Subsequently, DNA sequence containing polyadenine (polyA) was used as an anchor probe to incorporate the derivatized 5-hmC-dsDNA by amide reaction to form compound DNA and then completely assembled onto the gold electrode surface through preferential polyA-gold interaction. Subsequently, two designed DNA hairpin probes which can alternately hybridize with each other were linked to the signal amplification part of multifunctional polyA probes, forming ultra-long nicked double helix structures. Finally, ECL indicator of Ru(phen)32+ molecules were intercalated into the groove of the nicked DNA double helices, resulting greatly increased ECL intensity. Therefore, the quantitatively analysis of 5-hmC-dsDNA could be obtained relied on the relationship between the ECL intensity and 5-hmC-dsDNA content. The detection limit of this method for 5-hmC-dsDNA was 0.0001 % (S/N = 3). Moreover, the biosensing platform was successfully applied to detect 5-hmC-dsDNA in mice tissue samples with satisfactory results, manifesting its great potential application in clinical analysis.

Mouse Quantitative Proteomics Knowledgebase: reference protein concentration ranges in 20 mouse tissues using 5000 quantitative proteomics assays.

The knowledgebase is available free of charge on http://mousequapro.proteincentre.com. Supplementary data are available at Bioinformatics online.

Bioinformatics Research Through Image Processing of Histopathological Response to Stonefish Venom

The present study utilizes coastal and environmental engineering to investigate the histopathological effects of Synanceia verrucosa venom on Albino BALB/c mice. S. verrucosa is the most hazardous venomous marine fish that belong to the family Synanceiidae, generally known as the "Reef Stonefish". Crude venom was collected from venom glands of the dorsal spines of stonefish samples taken from the Jordanian coastline of the Gulf of Aqaba, Red Sea. The mice were given intramuscular injections of the venom. Consequently, the research evaluated the acute toxicity and influence on selected serum biomarker enzymes, as well as possible histological alterations of the soleus skeletal muscles. The mice 24 h LD50 was 0.107 µg toxin/kg mouse body weight. After the treatment using venom sublethal dose, the serum biomarkers, including Lactate dehydrogenase (LDH) and Alanine aminotransferase (ALT), were significantly improved (P≤0.05). In addition, Lipid Peroxidation (LPO) contents were significantly increased (P≤0.05) after venom treatment. Moreover, we combined routine medical procedures and artificial intelligence-assisted image analysis for a rapid qualitative and quantitative diagnosis of stonefish injury, based on the histophotography of mice tissue samples during the observation period (1, 2, and 3 hours respectively). The novelty of our method is that we could detect severe and mild damage with an accuracy of 93% and 91%, respectively. The most histological abnormalities in muscles were the great variety in diameters, content, and widespread among randomly distributed muscle fibres. In addition, loss of the tissue's striated appearance was noticed in toxin-treated groups compared with the control group. Consequently, our findings indicate the Stonefish's harmful influences that may endanger human life and highlight the need for appropriate measures to be considered. This, in turn, can ensure beach safety in the Gulf of Aqaba.

Mechanisms of ischemic skeletal muscle regeneration mediated by mechanically constrained human allogeneic mesenchymal stromal cells

Background and Hypothesis: Critical limb threatening ischemia (CLTI) is the end stage of peripheral arterial disease (PAD). CLTI presents a significant risk for lower extremity amputation, especially in diabetic patients with poor options for revascularization. Additionally, using a novel diabetic mouse model of CLTI, we have shown that IM injection of 3D cultured MSCs (spheroids) is more effective in promoting skeletal muscle regeneration of ischemic muscle than monolayer cultured MSC. We hypothesize that this result is due to the mechanical constrained nature of the MSC in spheroid form, which has been shown to alter the cellular phenotype. Alginate encapsulated cells are another form of mechanical confinement, and have additional benefits including resistance to immunological attack, making them a practical therapy for treatment of CLTI patients. 
 
 Project Methods: In this work, cells were encapsulated in 2% alginate using a centrifugation method. Media from these cells along with others were analyzed for IL-10 and IL-33 using ELISA. Mouse tissue samples were stained with WGA-555 antibody and analyzed using a scanning microscope. Effects on tissue perfusion were measured with Laser Doppler Perfusion Imaging. At the time of this abstract, the media from encapsulated and naked MSCs is being used to culture myoblasts, looking at cell growth. 
 
 Results: ELISA results did not show significant increases in IL-10 or IL-33 for encapsulated vs. non-encapsulated cells. LDPI has shown an increased perfusion rate for hindlimbs treated with encapsulated MSCs vs naked MSCs. Muscle fiber analysis is ongoing, but initial data appears promising. 
 
 Conclusion and Potential Impact: This experiment provides a starting point for improving and expanding cell therapy for critical limb ischemia, potentially resulting in better outcomes for diabetic patients and preventing lower limb amputations. The encapsulation process also has value in other types of cell therapy, as it could protect cells from host defenses and increase dwell time.

A lncRNA Gpr137b-ps/miR-200a-3p/CXCL14 axis modulates hepatic stellate cell (HSC) activation

Hepatic fibrosis is the wound healing response upon the liver tissue damage caused by multiple stimuli. Targeting activated hepatic stellate cells (HSCs), the major extracellular matrix (ECM)-producing cells within the damaged liver, has been regarded as one of the main treatments for hepatic fibrosis. In the present study, we performed preliminary bioinformatics analysis attempting to identify possible factors related to hepatic fibrosis and found that lncRNA G protein-coupled receptor 137B (Gpr137b-ps) and C-X-C motif chemokine ligand 14 (CXCL14) showed to be markedly upregulated within carbon tetrachloride (CCl4)-caused hepatic fibrotic mice tissue samples and activated HSCs. CXCL14 The silencing of lncRNA Gpr137b-ps or CXCL14 alone could significantly improve CCl4-induced fibrotic changes in mice liver in vivo and collagen I and III release by HSCs and HSC proliferation in vitro. miR-200a-3p directly targeted lncRNA Gpr137b-ps and CXCL14, respectively. LncRNA Gpr137b-ps relieved miR-200a-3p-induced inhibition on CXCL14 expression via acting as a ceRNA. In HSCs, the effects of lncRNA Gpr137b-ps silencing on collagen I and III release by HSCs and HSC proliferation were significantly reversed by miR-200a-3p inhibition, and the effects of miR-200a-3p inhibition were reversed by CXCL14 silencing. In conclusion, we demonstrated a lncRNA Gpr137b-ps/miR-200a-3p/CXCL14 axis that modulates HSC activation and might exert an effect on the pathogenesis of liver fibrosis.

Effects of lycopene on skeletal muscle-fiber type and high-fat diet-induced oxidative stress

Increasing studies report that many natural products can participate in formation of muscle fibers. This study aimed to investigate the effect of lycopene on skeletal muscle-fiber type in vivo and in vitro. C2C12 myoblasts were used in vitro study, and the concentration of lycopene was 10 µM. In vivo, 8-week-old male C57/BL6 mice were used and divided into four groups (n=8): (1) ND: normal-fat diet; (2) ND+Lyc: normal-fat diet mixed with 0.33% w/w lycopene; (3) HFD: high-fat diet; and (4) HFD+Lyc: high-fat diet mixed with 0.33% w/w lycopene. The mice tissue samples were collected after 8 weeks feeding.We found that lycopene supplementation enhanced the protein expression of slow-twitch fiber, succinate dehydrogenase, and malic dehydrogenase enzyme activities, whereas lycopene reduced the protein expression of fast-twitch fibers, lactate dehydrogenase, pyruvate kinase enzyme activities. Moreover, lycopene can promote skeletal muscle triglyceride deposition, enhanced the mRNA expression of genes related to lipid synthesis, reduced the mRNA expression of genes related to lipolysis. And high-fat diet-induced dyslipidemia and oxidative stress were attenuated after lycopene supplementation. Additionally, lycopene supplementation reduced the glycolytic reserve but enhanced mitochondrial ATP production in C2C12 cells.These results demonstrated that lycopene affects the activities of metabolic enzymes in muscle fibers, promotes the expression of slow-twitch fibers, and enhanced mitochondrial respiratory capacity. We speculated that lycopene affects the muscle-fiber type through aerobic oxidation, suggesting that lycopene exerts potential beneficial effects on skeletal muscle metabolism.

Toxicokinetics of methylmercury in diabetic KK-Ay mice and C57BL/6 mice.

We compared the toxicokinetics of methylmercury (MeHg) in KK-Ay type 2 diabetic mice and C57BL/6J mice to evaluate how metabolic changes associated with diabetes affect MeHg toxicokinetics. A single dose of MeHg (0.2, 1, or 5 mg mercury/kg) was administered orally to 12-week-old KK-Ay and C57BL/6J male mice. Total mercury concentrations in plasma, blood cells, whole blood, and tissues (brain, kidneys, liver, and pancreas) were measured after 4, 7, 11, and 14 days. The volume of distribution/bioavailability and the elimination rate constant per day were higher in KK-Ay mice, while the terminal elimination half-life was lower in almost all samples of KK-Ay mice. The area under the curve was lower in all blood and almost all tissue samples from KK-Ay mice. Total clearance/bioavailability was lower in all blood and tissue samples of KK-Ay mice at all MeHg doses. These results indicate that MeHg is more rapidly absorbed by, and eliminated from, the blood cells, brain, liver, kidney, and pancreas of KK-Ay mice under the experimental conditions. Different patterns of tissue-to-plasma and tissue-to-whole blood partition coefficients suggest that notable differences in MeHg transfer between plasma and blood cells affect its distribution in tissues of the two mouse strains. These findings are useful to understand the selective distribution of MeHg to target organs and the sensitivity to MeHg in pathological states.

CRAFT for NMR lipidomics: Targeting lipid metabolism in leucine-supplemented tumor-bearing mice.

Lipid profiling by 1 H-NMR has gained increasing utility in many fields because of its intrinsically quantitative, nondestructive nature and the ability to differentiate small molecules based on their spectral location. Most nuclear magnetic resonance (NMR) techniques for metabolite quantification use frequency domain analysis that involves many user-dependent steps such as phase and baseline correction and quantification by either manual integration or peak fitting. Recently, Bayesian analysis of time-domain NMR data has been shown to reduce operator bias and increase automation in NMR spectroscopy. In this study, we demonstrate the use of CRAFT (complete reduction to amplitude-frequency table), a Bayesian-based approach to automate processing in NMR-based lipidomics using lipid standards and tissue samples of healthy and tumor-bearing mice supplemented with leucine. Complex mixtures of lipid standards were prepared and examined using CRAFT to validate it against conventional Fourier transform (FT)-NMR and derive a fingerprint to be used for analyzing lipid profiles of serum and liver samples. CRAFT and FT-NMR were comparable in accuracy, with CRAFT achieving higher correlation in quantifying several lipid species. Analysis of the serum lipidome of tumor-bearing mice revealed hyperlipidemia and no signs of hepatic triglyceride accumulation compared with that of the healthy group demonstrating that the tumor-bearing mice were in a state of precachexia. Leucine-supplementation was associated with minimal changes in the lipid profile in both tissues. In conclusion, our study demonstrates that the CRAFT method can accurately identify and quantify lipids in complex lipid mixtures and murine tissue samples and, hence, will increase automation and reproducibility in NMR-based lipidomics.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Increased talin–vinculin spatial proximities in livers in response to spotted fever group rickettsial and Ebola virus infections

AbstractTalin and vinculin, both actin-cytoskeleton-related proteins, have been documented to participate in establishing bacterial infections, respectively, as the adapter protein to mediate cytoskeleton-driven dynamics of the plasma membrane. However, little is known regarding the potential role of the talin–vinculin complex during spotted fever group rickettsial and Ebola virus infections, two dreadful infectious diseases in humans. Many functional properties of proteins are determined by their participation in protein–protein complexes, in a temporal and/or spatial manner. To resolve the limitation of application in using mouse primary antibodies on archival, multiple formalin-fixed mouse tissue samples, which were collected from experiments requiring high biocontainment, we developed a practical strategic proximity ligation assay (PLA) capable of employing one primary antibody raised in mouse to probe talin–vinculin spatial proximal complex in mouse tissue. We observed an increase of talin–vinculin spatial proximities in the livers of spotted fever Rickettsiaaustralis or Ebola virus-infected mice when compared with mock mice. Furthermore, using EPAC1-knockout mice, we found that deletion of EPAC1 could suppress the formation of spatial proximal complex of talin–vinculin in rickettsial infections. In addition, we observed increased colocalization between spatial proximity of talin–vinculin and filamentous actin-specific phalloidin staining in single survival mouse from an ordinarily lethal dose of rickettsial or Ebola virus infection. These findings may help to delineate a fresh insight into the mechanisms underlying liver specific pathogenesis during infection with spotted fever rickettsia or Ebola virus in the mouse model.The authors observed an increase of talin-vinculin spatial proximities in the livers of spotted fever Rickettsiaaustralis or Ebola virus-infected mice when compared to mock mice. Furthermore, using EPAC1 knockout mice, they found that deletion of EPAC1 suppresses the formation of spatial proximal complex of talin-vinculin in rickettsial infections.

Safety profile of the transcription factor EB (TFEB)-based gene therapy through intracranial injection in mice.

Transcription factor EB (TFEB)-based gene therapy is a promising therapeutic strategy in treating neurodegenerative diseases by promoting autophagy/lysosome-mediated degradation and clearance of misfolded proteins that contribute to the pathogenesis of these diseases. However, recent findings have shown that TFEB has proinflammatory properties, raising the safety concerns about its clinical application. To investigate whether TFEB induces significant inflammatory responses in the brain, male C57BL/6 mice were injected with phosphate-buffered saline (PBS), adeno-associated virus serotype 8 (AAV8) vectors overexpressing mouse TFEB (pAAV8-CMV-mTFEB), or AAV8 vectors expressing green fluorescent proteins (GFPs) in the barrel cortex. The brain tissue samples were collected at 2 months after injection. Western blotting and immunofluorescence staining showed that mTFEB protein levels were significantly increased in the brain tissue samples of mice injected with mTFEB-overexpressing vectors compared with those injected with PBS or GFP-overexpressing vectors. pAAV8-CMV-mTFEB injection resulted in significant elevations in the mRNA and protein levels of lysosomal biogenesis indicators in the brain tissue samples. No significant changes were observed in the expressions of GFAP, Iba1, and proinflammation mediators in the pAAV8-CMV-mTFEB-injected brain compared with those in the control groups. Collectively, our results suggest that AAV8 successfully mediates mTFEB overexpression in the mouse brain without inducing apparent local inflammation, supporting the safety of TFEB-based gene therapy in treating neurodegenerative diseases.

Evaluation of the Biodistribution of Magnetoliposomes in a Tumor and Organs of Mice by Electron Paramagnetic Resonance Spectroscopy

Electron paramagnetic resonance spectroscopy has been applied for the first time to study the bio-distribution of magnetoliposomes formed with magnetite nanoparticles (Fe3O4) in tumors and organs of Lewis carcinoma-bearing mice in the absence and presence of an external magnetic field. The animals of the experimental group were subjected to an external magnetic field (0.6 T) in the tumor area after intravenous injection of magnetoliposomes at a dose of 7.56 Fe/kg. Analysis of the electron-spin resonance spectra of mouse organs and tissue samples showed that exposure to a magnetic field resulted in a two-fold increase in Fe3O4 accumulation within the tumor (p < 0.05) compared to the control; this makes it possible to recommend the obtained magnetoliposomes for use as a magnetically controlled carriers for targeted delivery of antitumor agents. A high concentration of superparamagnetic magnetite nanoparticles was detected in the liver in the absence and presence of an external magnetic field. The differences in the accumulation of Fe3O4 in the lungs and liver in the presence of a magnetic field were statistically insignificant.

Discovery of Chemicals to Either Clear or Indicate Amyloid Aggregates by Targeting Memory-Impairing Anti-Parallel Aβ Dimers.

Amyloid-β (Aβ) oligomers are implicated in Alzheimer disease (AD). However, their unstable nature and heterogeneous state disrupts elucidation of their explicit role in AD progression, impeding the development of tools targeting soluble Aβ oligomers. Herein parallel and anti-parallel variants of Aβ(1-40) dimers were designed and synthesized, and their pathogenic properties in AD models characterized. Anti-parallel dimers induced cognitive impairments with increased amyloidogenesis and cytotoxicity, and this dimer was then used in a screening platform. Through screening, two FDA-approved drugs, Oxytetracycline and Sunitinib, were identified to dissociate Aβ oligomers and plaques to monomers in 5XFAD transgenic mice. In addition, fluorescent Astrophloxine was shown to detect aggregated Aβ in brain tissue and cerebrospinal fluid samples of AD mice. This screening platform provides a stable and homogeneous environment for observing Aβ interactions with dimer-specific molecules.

Discovery of Chemicals to Either Clear or Indicate Amyloid Aggregates by Targeting Memory‐Impairing Anti‐Parallel Aβ Dimers

AbstractAmyloid‐β (Aβ) oligomers are implicated in Alzheimer disease (AD). However, their unstable nature and heterogeneous state disrupts elucidation of their explicit role in AD progression, impeding the development of tools targeting soluble Aβ oligomers. Herein parallel and anti‐parallel variants of Aβ(1–40) dimers were designed and synthesized, and their pathogenic properties in AD models characterized. Anti‐parallel dimers induced cognitive impairments with increased amyloidogenesis and cytotoxicity, and this dimer was then used in a screening platform. Through screening, two FDA‐approved drugs, Oxytetracycline and Sunitinib, were identified to dissociate Aβ oligomers and plaques to monomers in 5XFAD transgenic mice. In addition, fluorescent Astrophloxine was shown to detect aggregated Aβ in brain tissue and cerebrospinal fluid samples of AD mice. This screening platform provides a stable and homogeneous environment for observing Aβ interactions with dimer‐specific molecules.

Rapid and Low-Cost Culture-Based Method for Diagnosis of Mucormycosis Using a Mouse Model.

Prompt and targeted antifungal treatment has a positive impact on the clinical outcome of mucormycosis; however, current diagnostic tools used in histopathology laboratories often fail to provide rapid results. Rapid culture-based strategies for early diagnosis of Mucorales infections, which may influence treatment decisions, are urgently needed. Herein, we evaluated a microculture assay for the early diagnosis of mucormycosis in an immunocompetent murine model of disseminated infection, by comparing it with traditional diagnostic methods. The assay specificity was assessed using blood (n = 90) and tissue (n = 90) specimens obtained from mice infected with Rhizopus arrhizus using different inoculum sizes [1 × 104, 1 × 105, and 1 × 106 colony forming units (CFUs)/mouse] and blood (n = 15) and tissue specimens (n = 15) from uninfected mice. Surprisingly, 26 of 90 (28.9%) blood samples revealed positive results by microculture, whereas all blood samples were negative when assayed by conventional culture. The overall positive conventional culture rate for the mouse tissue (kidney) samples was 31.1% (28/90). The calculated sensitivity for kidney microculture was 98.8% [95% confidence interval (CI) 96.6–100], with an assay specificity of 100%. Hence, the microculture assay may be useful for rapid culturing and diagnosis of mucormycosis caused by R. arrhizus directly in blood and tissue samples. Hence, this method may allow for the timely administration of an appropriate treatment.

Podocyte Damage in Acute Hypertensive Mice; Involvement of TRPC6

In order to study the effect of changes in TRPC6 protein pathway on podocyte injury in hypertensive mice, 20 WT mice were randomly divided into the control group and the experimental group. Acute hypertension model was established by ligating abdominal aorta for 15 min in the experimental group mice. After successful establishment of the mouse model, in vivo freezing technology is used in all mice to prepare tissue samples for the experiment. H and E staining, SABC-DAB immunohistochemical staining, Western blot and calcium ion detections were made on kidney tissue samples of mice. Urine samples of mice were collected to test urinary albumin. The results showed that compared to the control group, the glomerular tubules and Bowman capsules in the experimental group were swollen, the expression of TRPC6 channels in podocytes increased, the concentration of calcium increased sharply and the concentration of albumin in urine also increased sharply. These results showed that hypertension could cause opening of TRPC6 channel to increase the calcium ion concentration leading to podocyte damage. The ability of damaged podocytes to deal with albumin is weakened and a large increase of albumin would cause further increase of Ca2+ concentration, that aggravated damage to podocytes this initiated a vicious circle. This study thus laid a foundation for exploring the mechanism of podocyte injury caused by hypertensions

Effect of tumor necrosis factor-related apoptosis-inducing ligand gene knock-out on the gut microbiota in mice with dextran sodium sulphate induced colitis

Objective To investigate the influence of tumor necrosis factor-related apoptosis-inducing ligant (TRAIL) deficiency on mice colitis and the gut microbiota composition by inclding the expermental colitis model in tumor necrosis factor-related apoptosis-inducing ligand gene knockout (TRAIL-/-) mice. Methods C57BL/6 TRAIL-/- mice and wild type (WT) mice were selected and assigned into TRAIL-/- control group (eight mice), TRAIL-/- colitis group (16 mice), WT control group (eight mice) and WT colitis group (16 mice). The mice of two colitis groups were oral administrated with 3.5% dextran sulphate sodium (DSS) in drinking water for seven consecutive days to induce experimental colitis model. The severity of colitis was evaluated by clinical appearance and histopathological examination. The colonic tissue samples of mice were collected and microbiota profile was analyzed by 16S rDNA sequencing method. USEARCH software and R language were used to analyze the difference of gut microbiota among TRAIL-/- control group, TRAIL-/- colitis group, WT control group and WT colitis group. T test and Mann-Whitney U test were used for statistical analysis. Results After modeling, the disease activity index (DAI) of WT colitis mice and TRAIL-/- colitis mice both gradually increased over time. Furthermore, compared with colitis mice, TRAIL-/- colitis mice developed body weight loss, diarrhea and hemafecia earlier. On the seventh day after modeling, the percentage of body weight loss of TRAIL-/- colitis mice and WT colitis mice was (28.98±2.84)% and (17.87±3.70)%, respectively; and the difference was statistically significant (t=9.53, P<0.01). The length of colon of TRAIL-/- colitis mice was shorter than that of WT colitis mice ((4.63±0.28) cm vs. (6.02±0.41) cm), and the difference was statistically significant (t=11.20, P<0.01). The DAI of TRAIL-/- colitis mice was higher than that of WT colitis mice (3.00±0.00 vs. 2.32±0.05), and the difference was statistically significant (t=54.40, P<0.01). The histological score of TRAIL-/- colitis mice was higher than that of WT colitis mice (6.19±0.25 vs. 3.87±0.22), and the difference was statistically significant (t=27.87, P<0.01). Under the microscope, colonic mucosal epithelial injury, crypt structure destruction and inflammatory cell infiltration were more obvious in TRAIL-/- colitis mice than in WT colitis mice. The alpha diversity of colonic flora was more significant in TRAIL-/- colitis group compared with that of WT colitis group. At the family level, the relative richness of Deferribacteraceae, Ruminococcaceae, Rikenellaceae, F16 and Paraprevotellaceae significantly increased in TRAIL-/- colitis group, but the relative richness of Enterococcaceae obviously reduced ((19.839±19.991)% vs. (7.224±11.241)%, (3.564±2.543)% vs.(2.861±3.821)%, (0.123±0.066)% vs. (0.068±0.049)%, (0.032±0.033)% vs. (0.006±0.011)%, (0.153±0.098)% vs. (0.062±0.054)% and (0.013±0.027)% vs. (0.054±0.121)%, respectively; U=51, 69, 53, 35, 49 and 69, respectively; P<0.01 and 0.05, respectively). In addition, at the genus level the relative richness of Oscillospira, Mucispirillum and Cytophaga in TRAIL-/- colitis group remarkably elevated, and the relative richness of Enterococcus significantly decreased ((2.363±2.147)% vs. (1.813±2.847)%, (19.839±19.991)% vs. (7.223±11.241)%, (0.104±0.153)% vs. (0.046±0.069)% and (0.076±0.049)% vs. (0.135±0.074)%, respectively; U=70, 51, 66 and 65, respectively; P <0.05 and 0.01, respectively). Conclusion TRAIL deficiency aggravate DSS-induced colitis, and increase the alpha diversity of colonic microbiota in colitis mice. Key words: TNF-related apoptosis-inducing ligand; Intestinal microbiota; Experimental colitis; Mice, knockout

TMOD-05. IN VIVO REPROGRAMMING OF POSTMITOTIC NEURONS INDUCES MEDULLOBLASTOMA

Abstract It is widely accepted that the “cell of origin” of tumors has to possess a proliferative capacity. Particularly for brain cancer, the transition of neural progenitors to differentiated postmitotic neurons is considered irreversible in physiological and pathological conditions. Therefore, postmitotic neurons have not been considered as suitable cell-of-origin for brain cancer. Here, we show that neurons reprograming may occur upon Shh activation and it leads to medulloblastoma (MB) formation in vivo. Shh MB is a cerebellar tumor, found in infants and adults that is thought to originate from cerebellar granule neuron progenitors. More recently, it was discovered that the two different forms of SHH MB are distinguished by different transcriptome/methylome levels suggesting that the adult SHH MB may originate from a different cell-of-origin. Relying on these data, we take advantage of a conditional Cre-Lox recombination system to recapitulate the human adult medulloblastoma pathogenesis in mice and demonstrate that post-migratory mature granule neurons can be reprogrammed in vivo. Furthermore, to define the contribution of chromatin changes in granule neurons dedifferentiation in response to Shh activation, we profiled changes in chromatin accessibility by ATAC-seq both in mouse tissue samples and also in human neurons. Upon Shh pathway activation we detected the presence of hyper-accessible chromatin regions corresponding to cis-regulatory elements specifically favouring activation of enhancers and super-enhancers. Our novel model of cancer development could explain the human SHH medulloblastoma onset in adult individuals where granule neuron progenitors are no more present. We strongly believe that our model represents an important starting point to study other tissues where postmitotic cells might originate cancer and therefore open a new field in cancer and stem cell biology.

Development and validation of a rapid and sensitive UPLC-MS/MS assay for simultaneous quantification of paclitaxel and cyclopamine in mouse whole blood and tissue samples.

The prominent stromal compartment surrounds pancreatic ductal adenocarcinoma and protects the tumor cells from chemo- or radiotherapy. We hypothesized that our nano formulation carrying cyclopamine (CPA, stroma modulator) and paclitaxel (PTX, antitumor agent) could increase the permeation of PTX through the stromal compartment and improve the intratumoral delivery of PTX. In the present study a sensitive, reliable UPLC-MS/MS method was developed and validated to quantify PTX and CPA simultaneously in mouse whole blood, pancreas, liver and spleen samples. Docetaxel was used as the internal standard. The method demonstrated a linear range of 0.5-2000 ng/mL for whole blood and tissue homogenates for both PTX and CPA. The accuracy and precision of the assay were all within ±15%. Matrix effects for both analytes were within 15%. Recoveries from whole blood, liver, spleen and pancreas homogenates were 92.7-105.2% for PTX and 72.8-99.7% for CPA. The stability was within ±15% in all test biomatrices. The validated method met the acceptance criteria according to US Food and Drug Administration regulatory guidelines. The method was successfully applied to support a pharmacokinetic and biodistribution study for PTX and CPA in mice biomatrices.

Abstract LB-328: Loss of TFF1 leads to activation of RAGE in mouse and human gastric tumorigenesis

Abstract Background: The receptor for advanced glycation end products (RAGE) is a central signaling molecule in the innate immune system and is highly expressed in various cancers including gastric cancer. RAGE is a pattern recognition receptor that binds to multiple ligands leading to its activation and onset of pro-inflammatory response. Trefoil factor 1 (TFF1) is a small secreted protein expressed in the gastrointestinal tract which plays a key role in protection of gastric mucosal membrane and provides protection from injury and damage, therefore acts as tumor suppressor gene. In this study we investigated the role of TFF1 in regulation and degradation of RAGE receptor in vivo and in vitro. Material and Results: Using quantitative real-time PCR, we observed significant increase of RAGE expression in gastric tissue from Tff1 knockout from non-dysplastic lesion; this increase of expression of RAGE was more notable in dysplastic samples, as compared to normal tissues (p&amp;lt;0.001). The Tff1-KO mice tissues with gastritis and low grade dysplasia (LGD) and high grade dysplasia (HGD) showed significantly progressive higher expression of RAGE, as compared to normal tissues (p&amp;lt;0.001). Analysis of human tissue samples demonstrated significant overexpression of RAGE, as compared to normal gastric tissues (p=0.01). Furthermore, we analyzed paired tumor and normal samples, and we found a significant up-regulation of RAGE mRNA expression in tumors compared to their corresponding normal samples (p=0.01). We also detected an inverse relationship between TFF1 and RAGE levels in mouse and human tissue samples. We next determined the levels of RAGE ligands, S100A2 and A4. The results showed significant increase in expression levels of S100A2 and A4 in human and mouse neoplastic gastric lesions, as compared to normal tissue samples (P&amp;lt;.01). To determine a causal relationship between TFF1 and RAGE, we used in vitro cell models. Using Western blot and RT-PCR analysis, the reconstitution of TFF1 in AGS cells showed a decrease in expression levels of RAGE, S100A2, and A4, as compared to control cells. Treatment of cancer cell lines (AGS, MKN28, and MKN45) with RAGE inhibitor Azeliragon led to significant reduction in cell viability with IC50 range AGS= 1.65µM, MKN28=1.22µM, MKN45= 1.37µM . Conclusion: Our data demonstrate, for the first time, that high levels of RAGE and its ligands S100A2 and A4 are mechanistically related to TFF1 levels. RAGE inhibitors demonstrated a therapeutic efficacy in pre-clinical in vitro models. Additional in vitro and in vivo studies are planned to determine the functional, mechanistic and therapeutic outcomes of RAGE in gastric tumorigenesis. Citation Format: Nadeem S. Bhat, Mohammed Soutto, Zheng Chen, Shoumin Zhu, Huma Naz, Ahmed Gomaa, Sen Wang, Barry I. Hudson, El-Rifai Wael. Loss of TFF1 leads to activation of RAGE in mouse and human gastric tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-328.